[Abstract Book Vol. 2, International Conference on AIDS (10th: 1994: Yokohama, Japan)]

Page  [unnumbered] Tenth International Conference on AIDS International Conference on STD The Global Challenge of AIDS: Together for the Future BOO Volume X s-Wednesday-Thursday Yokohama, Japan 7-12 August 1994 -1, C) - C 0 - N - L( - U

Page  [unnumbered] The Conference Organizing Committee thanks the Sasakawa Foundation for their generous contribution, which supported the production of this Abstract Volume.

Page  [unnumbered] TENTH INTERNATIONAL CONFERENCE ON AIDS International Conference on STD ABSTRACT BOOK Volume II Yokohama, Japan 7-12 August 1994

Page  [unnumbered]

Page  1 CO NTENTS ORAL ABSTRACTS Wednesday 10 AUGUST Plenary Sessions............................................................4 Tenth Anniversary Special Session.............................. 6 Abstract Sessions..................................................... 7 Special Session............................................................46 Thursday 11 AUGUST Plenary Sessions......................................................... 48 Abstract Sessions......................................................... 50 POSTER ABSTRACTS Wednesday 10 AUGUST & Thursday 11 AUGUST Track A.................................................................. 78 Track B..................................................................125 Track C..................................................................241 Track D..................................................................308

Page  2 ABBREVIATION OF SESSIONS The sessions are abbreviated as follows Plenary Sessions PS Round-table Sessions RT Abstract Sessions AS Poster Sessions P Tenth Anniversary Special Sessions TS Special Sessions SS Evening Special Sessions ES Special Recent Report Sessions SR KEY TO NUMBERING OF ABSTRACTS The presentations (oral and poster) are grouped by day and by thematic track as follows Volume I: Abstracts for Monday 8 August and Tuesday 9 August Volume II: Abstracts for Wednesday 10 August and Thursday 11 August The system used to number the abstracts identifies the track and the type of presentation. Examples: Oral presentation 001 B (Presentation #) (Track) Poster presentation P A 0001 (Poster) (Track) (Presentation 4)

Page  3 ORAL ABSTRACTS Wednesday 10 AUGUST Plenary Sessions Tenth Anniversary Special Session Abstract Sessions Special Session

Page  4 PS17 ':'bfigieia mon mW I!)L tiation of Antiretroviral Therapy" PA Volberding, MD, University of California AIDS Program, San Francisco Genetal Hospital PS18 GENE THERAPY FOR HIV INFECTION Wong-Staal, F., Yu, M., Yamada, O., Leavitt, M., Maruyama, A. Ho et al., UCSD, La Jolla, California, USA The optimal point to initiate antiretroviral therapy in HIV disease continues to generate controversy. Used in symptomatic HIV disease, zidovudine and other nucleoside analogues has reduced clinical progression, and prolonged survival. In asymptomatic stages of HIV infection, however, where the risk of progression or death are much lower, clinical benefits of antiretroviral therapy are less striking. This is the case despite a clearer rationale for such treatment, based on knowledge of the pathogenesis of HIV disease. Using more stringent definitions of progression (AIDS or death), zidovudine is active in those with CD4 cells below 500/mm3. Survival, however, is not additionally prolonged over that achieved with a delayed use of this drug. In those with CD4 cells above 500/mm', where the risk of clinical progression to AIDS or death is extremely slight, zidovudine has not shown clinical benefit in prospective clinical trials. The resolution of this controversy awaits the development of more effective and durable treatment strategies, but may be aided by virologic markers of response to therapy if clinical validation of these tests is successful. Faul A. Volberdihg, MD 995 Potrero Ave, Bldg 80, Ward 84 San Francisco, CA 94110 USA 415-476-4082/fax 476-9233 Objective: The goal of gene therapy for HIV infection is to repopulate the immune system with genetically altered cells that resist infection. Our group has been developing a hairpin ribozyme that cleaves HIV-1 RNA as a form of gene therapy, and have demonstrated in vivo efficacy in both lymphocytes and monocytes derived from progenitor cells. Methods: Peripheral blood lymphocytes were enriched for CD4 cells. Transduced cells were selected with G418 and challenged with HIV-1. Transduction of precursor cells was carried out with immunoaffinity enriched, prestimulated CD34 cells. Differentiation into monocytes occurred in the presence of GM-CSF. Results: Primary T-cells transduced with retroviral vectors containing the ribozyme were resistant to challenge with diverse strains of HIV, including clinical isolates. Hematopoietic stem/progenitor cells from adult bone marrow, fetal cord blood or mobilized peripheral blood transduced by the vector expressed the ribozyme efficiently and persistently in the CFUs. Transduction and ribozyme expression had no apparent deleterious effect on cell phenotypes or proliferation. The cultured stem cells differentiated into macrophages/monocytes and those derived from the ribozyme transduced stem cells withstood HIV infection in vitro. Discussion and Conclusions: Ribozyme gene therapy may be an effective preemptive strategy for interfering with HIV replication. A phase I clinical protocal to test the safety and function of the ribozyme in tranduced human PBL in vivo has been developed and approved by the NIH RAC. WONG-STAAL, Flossie, UCSD, La Jolla, CA, USA Telephone (619) 534-7957; Telefax (619) 534 - "O ", 0 PS19 WHO CARES? SYSTEMATIC AMPLIFICATION OF HIDDEN COMMUNITY HEALTH STRUCTURES RESPONDING TO AIDS IN SOUTHIIERN AFRICA. SABATIER. Renfe *; WILSON. D. ** * Director. Southern African AIDS Training Programme of the Canadian Public HIlealth Association: ** Chairman, Department of Psychology. University of Zimbabwe. Objective: To develop a programmatic approach to the amplification of community health structures capable of responding to AIDS in Southern Africa. Methods: The actual structures of community care comprise the pattern of agents, acts and resources available in the community to produce and protect the health of its members. The method employed by the Southern African AIDS Training Programme to identify, support and amplify these structures is described here as one example of such a mechanism. Results: Mechanisms can be devised to support and augment the actual structures of care which communities deploy to cope with the AIDS epidemic, and to interlink these with government services. Relatively small financial inputs, if accompanied by appropriate non-financial supports, such as local NGO skills-building and mentoring linkages, can achieve impressive results. Discussion: In the experience of the SAT Programme and its project partners, "who cares?" is a fundamental question which must be asked in order to achieve the broad community involvement needed in countries with an escalating burden of AIDS illness and death. Otherwise, community involvement remains a "black box", desired but not understood. Caring is shown to be an integral health-producing act based on respect for fellow human beings which at community level renders the distinction between "prevention" and "care" artificial: all good care incorporates elements of prevention; all sound prevention is caring. SAT's experience indicates that the structures of community care are an effective and sustainable entry-point into affected communities, and that responses grounded in care antidote the threat which communicable illness poses for human rights. SABATIER, Renfe, Director, SAT Programme, PO Box 390 Kopje, Ilarare, Zimbabwe. Tel: (2634)723-915/725-193 Fax:(2634)725-193. PS20 -u C,) (D CO) CO)

Page  5 PS21 Ms. Ana Filgueiras, Hand-in-Hand Network. Objective: to underline the urgent need for government and nongovernment collaboration in implementing holistic, inmrsectuorial, and culturally appropriate programmes able to electively reach, protect, and promote the health of out-of-school youth as part of HIV/AIDS prevention strategies. Methods: Millions of youth worldwide are outside of the formal school system, where they are at increased exposure to situations which make them extremely vulnerable to HIV/AIDS Infection and where they are outside the traditional reach of institutionalprogramme and prevendtonmessages. Studies in many countries also suggest that a higher proportion of young women than young men are out-of-school and at higher risk of HIV infection. A majority of both boys and girls living in developing countries are compelled to work for their own survival and/or contribute towards the family income. Data shows that these young people are increasingly sexually active early in life and consequently affected more by STDs and HIV infection because they do not have regular access to public health and services. Results: Rooted in the current experiences of these youth, and operating in their main settings, many innovative initiatives have been developed. Addressing issues from the perspecdtive of out-of-school youth such as gender. age. developmental stages, sexual preferences and other related factors, these programmes are progressively demonstrating how realistic and individual approaches might be implemented. Discussion and conclusions: The extent of the HIV/AIDS pandemic urgently requires pragmatic efforts from the perspective of Individual and social rights to ensure the protection and promotion of the sexual and reproductive health needs of out-of-school youth. Only through a multisectoral approach and the integration of government, NGO, and international agency responses can progress be made towards the goal of improved adolescent sexual health and HIV prevention. Ana Filgueiras. Hand-in-Hand Network, Caixa Postal 4884, Ag. Central, CEP 20 100. Rio de Janeiro - RJ, Brazil, tel/fax 55 21 227 4029. PS23 HIV variability and its implications for pathogenesis lasp Goudsmit, Human Retrovirus Laboratory, Academic Medical Center, Amsterdam, the Netherlands; participants of the Amsterdam Cohort Studies The natural history of HIV infection is characterized by extreme variability in the length of the symptom-free period. The length of the period from acute infection till the diagnosis of AIDS is determined by host and/or virus factors. Two determinants of the virus-host interaction appear to play a major role in the pathogenesis and natural history of HIV. Firstly the biological phenotype of the virus and secondly the replication level of the virus. Almost all new HIV infections are initiated by viruses of the non-syncytium-inducing (NSI) phenotype. During the acute infection the number of NSI viruses produced is in the range of 10'4/ml plasma or blood irrespective of the subsequent length of the symptom-free period. In the first few years of infection NSI progressors can be distinguished from non-progressors on the basis of stable and high numbers of virions relative to declining virus numbers in non-progressors. Syncytium-inducing (SI) viruses can be isolated from about half of the progressors and such SI progressors can be distinguished from non-progressors on the basis of CD4+ cell decline while the virus load does not differentiate SI progressors from non-progressors in the first years after seroconversion. Envelope antibody responses and variation in the HIV envelope appear to play an important role in these early pathogenic events. Enhancement of replication following acute HIIV infection may cause persistently high virus loads in NSI progressors. NSI virus neutralizing antibodies may cause a decline in viral burden in non-progressors, in at least half of these cases reversed by envelope mutations determining concomitantly decreased neutralization sensitivity and the SI phenotype. The phenotypic changes result subsequently in high virus loads, rapid CD4 decline and disease progression (the SI progressors). Results, based on the variation in genetic indicators of biological phenotype, number of virus replication cycles and neutralization resistance in the course of infection are represented confirming these notions. Jaap Goudsmit, Human Retrovirus Laboratory, Academic Medical Center, Amsterdam, the Netherlands, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands; tel 31-20-5664853, fax 31-20-6916531 PS22 SEXUALITY FROM THE PERSPECTIVE OF AN HIV-POSITIVE PERSON LEE, Karen Jannine, GNP+ OBJECTIVE To help erase the myths and problems that surround sexual intercourse with a person who is HIV/AIDS positive. Also to help PLWA's realise that they have a choice, and a right to love or be loved. METHODS The main criteria for this study was to make available as much information on Sex and the HIV person as possible, so that people can make an informed decision on what they should do in their own life and to help with the mental anguish that goes with this subject. RESOURCES Personal experiences and interviewing with people in support groups and other AIDS organisations. Also the statistics show that the percentage rate for HIV transmission is higher from man to woman where it is lower from woman to man. The World Health Organisation predicts that by the year 2000, 80% of HIV transmission will be through heterosexual transmission. RESULTS It is very hard to gauge the results on this particular subject. People are coming out more and talking on the subject of sex, which shows that there is a need for greater understanding and communication. It has also, through talking to people shown that one on one counseling is important for people to be able to discuss their sexual approach and fears. DISCUSSION AND CONCLUSION Everyone has the right to love and be loved and no-one should jeopardize what is a basic human right, even for those that are HIV/AIDS affected. Karen J Lee, Unit 5, 25-27 Peel St., Berwick, Victoria, Australia 3806 Tel/Fax: 61-3-7961164 "U CD C,) (D CD 0 CD C/) N "4 -o...) N| PS24 HIV DRUG RESISTANCE: MOLECULAR BASIS AND CLINICAL SIGNIFICANCE, MARK A. WAINBERG, McGILL UNIVERSITY AIDS CENTRE, MONTREAL, QUEBEC, CANADA Objective: It is now been over five years since the initial isolation of HIV variants resistant to AZT. Numerous investigators have now shown that HIV drug resistance can be selected against a wide variety of nucleoside and non-nucleoside antagonists of viral reverse transcriptase (RT), as well as against peptide inhibitors of HIV protease. These studies have been performed both by viral passage in increasing concentrations of antiviral drugs, as well as by isolation of resistant viruses from patients. Methods: Cloning and sequencing of relevant viral pol and protease DNA has identified a number of resistance-conferring mutation sites, that have been confirmed by site-directed mutagenesis. Many of these mutations map to the active sites of the viral protease and RT and, as well, to the nucleoside-binding domain of the latter. Results: In some cases, patterns of cross-resistance have emerged, such that a single mutation may confer resistance to more than one drug, e.g. as is the case for ddC, 3TC, and ddl. In other instances, introduction of certain resistance-conferring mutations into viruses that display resistance to AZT may actually increase sensitivity to the latter drug. Studies on mutated RT proteins have shown that they may no longer incorporate certain forms of triphosphorylated nucleoside analogs. Discussion: Resistance-conferring mutations can be directly identified in the plasma and cellular virus of patients receiving anti-viral therapy. Monitoring patients for total viral burden, as well as percentage of mutated viral genotypes, associated with drug resistance, may provide an important tool for evaluation of anti-HIV drugs during the course of antiviral chemotherapy. Mark A. Wainberg Lady Davis Institute-Jewish General Hospital 3755 Cote Ste-Catherine Road, Montreal, Qc, Canada H3T 1E2 Tel. (514) 340-8260 FAX (514) 340-7502

Page  6 = TS7 Mounting a successful response to the HIV/AIDS pandemic requires close collaboration among governments, non-governmental and multilateral organizations, with a recognition of the relative strengths and comparative advantages of each. The United States has played a lead role in providing financial and technical assistance in support of national governments' prevention efforts, conducting collaborative research and cooperating with other donor governments and multilateral organizations. Since the early 1980's, the United States has provided technical and financial support to over 70 countries in research and prevention programs. The United States' involvement builds upon its longstanding experience in health and development internationally, its collaboration with other donor governments, its extensive participation in the United Nations and multilateral system, its strong NGO/PVO community and its welldeveloped research capacity. The United States involvement in the international response to the HIV/AIDS pandemic is an example of the role of bilateral cooperation and demonstrates how it can complement the work of the host country, NGOs and multilateral organizations. Dr.oel&en enieAss )r., eners for Dieses ontr~ol&Prevren f ion~C)iwasnington, LUU independence ve...2 6 0stgton,DC 20201, HHH Bldg. 202-690-8598 TS8 INTERNATIONAL COOPERATION IN THE FIGHT AGAINST AIDS Lars 0. Kallings, Natl Inst of Public Health, Stockholm, Sweden Multilateral cooperation: It is a great challenge to the UN system to assist member states to respond to the increasing AIDS pandemic and to help raise the necessary funds.The need for multinational support was obvious already in 1984 when the global scale of the AIDS epidemic was realized.WHO assessed the epidemiology, modes of transmission and preventive measures.In 1986 a special WHO programme on AIDS was created, later Global Programme on AIDS(GPA).An unprecedented mobilization of funds and activities was unleashed.WHO raised public and political awareness and counteracted panic and discrimination.In less than 10 years most individuals on the globe know about AIDS and almost all countries have a National AIDS Control programme.When social and economic causes and consequences of HIV/AIDS as threat to development were recognized a WHO-UNDP alliance was established in 1988.Subsequently, other UN agencies contributed within their areas of competitive advantages. The WHO Global AIDS Strategy has been adopted as a UN strategy,upgraded in 1992.It is multisectoral and includes all aspects,eg the need to improve women's status and provide health care to the growing number of AIDS cases. To better coordinate the multinational cooperation the World Health Assembly in1993 initiated a joint and cosponsored UN programme on AIDS(WHO, UNCEF, UNESCO, UNFPA, UNDP and the World Bank)decided in July 1994 by ECOSOC.The success of the new UN programme at country level will be crucial to the future course of the pandemic.Leadership and a firm position on human rights will be decisive.The fight against AIDS is spearheading evolution of human efforts.The joint Programme will prove that the UN system is capable to create programmes which are truly collaborative. Prof. Lars 0. Kallings, Natl Inst of Public Health, Box 27848, S-l15 93 Stockholm, Sweden tel. +46 8 783 35 85, fax +46 8 783 35 86 ml Cl, ND TS9 International Cooperation in the Fight Against AIDS: the Role of NGOs Author: Jeffrey O'Malley, Executive Director, Intemrnational HIV/AIDS Alliance It is widely accepted that the majority of HIV/AIDS prevention and care activities around the world are organised and delivered outside of government, by individuals and by non-governmental organisations (NGOs). Despite this role of NGOs in service delivery, the institutions of international cooperation have been primarily dominated by governments and inter-governmental organisations, and have thus tended to serve the needs of governments rather than the voluntary sector. In response, there has been an increasing amount of international cooperation among NGOs involved in AIDS, through both direct contact between organisations and networks such as GNP+, ICASO, and the established development NGO networks. Like governmental cooperation, these NGO exchanges involve both financial resources and information, but NGOs have been more successful than other institutions at exchanging and adapting programme experience, prevention and care tools, and political strategy. Unfortunately, AIDS NGOs have been less successful than human rights or environmental groups at building broad public constituencies in support of increased resource mobilisation. NGOs also cooperate through (and in cooperation with) bilateral and multilateral governmental efforts, with a particular emphasis on ensuring public participation in policy development, and on encouraging the investment of public funds in the voluntary sector. An important innovation in this area is the International HIVIAIDS Alliance, which encourages governmental and other donors to co-mingle NGO support funds, and to allow NGO communities to determine their own programming priorities. Mr Jeffrey O'Malley, Executive Director, International HIVIAIDS Alliance Barratt House, 341 Oxford Street, London W1R 3BH, UK Telephone: +44 71 491 2000 Fax: +44 71 491 2)01 -I CD CO) C,, 0 O 00 CD 5.

Page  7 319A STRUCTURAL COMPARISON OF CLINICALLY RELEVANT INHIBITORS OF THE HIV-1 PROTEASE Kim, E.E.. Rao, B.G., Deininger, D.D., Baker, C.T., Dwyer, M.D., Naxia. MA., Thomson, JA., Tung, RD., et at Vertex Pharmaceuticals Incorporated 320A XM412: AN IMPROVED CYCLIC UREA INHIBITOR OF HIV PROTEASE FOR ORAL ADMINISTRATION 0Q0,.. M., Aldrich,P., Grubb,M., Shum, L., Erickson-Viitanen,S. et al, DuPont Merck Pharmaceutical Co., Wilmington, DE,USA We have solved the X-ray crystal structures of HIV-1 protease in complex with the clinically relevant inhibitors Ro 31-8959 (Roche), L-735,254 (Merck) and VX-478 (Vertex). VX-478 is representative of a novel compound class that is extraordinarily potent in vitro, orally available in vivo, and synthetically accessible. VX-478 evolved through the use of structure-based drug design, and is currently in advanced pre-clinical development. Crystals of the VX-478 complex diffract to 1.9A resolution in space group P61, with unit cell lengths a=b=63.5A and c=83.8A. An initial refinement of the structure of the complex yields an R-factor of 19.4%. All three inhibitor complexes crystallize isomorphously, facilitating a direct comparison of the structures. This allows us to rationalize the observed differences in relative inhibitory potency in light of the different patterns of enzyme interaction seen with the enzyme. We have, in turn, exploited these unique structural insights to better understand and anticipate the possible development of resistance by the virus to these compounds, and to design effective second generation inhibitors in response. Navia, M.A., Vertex Pharmaceuticals, Incorporated, Cambridge, MA 02139, USA Phone: (617) 576-3111; Fax, (617) 576-2109 321A AG1343, AN ORALLY BIOAVAILABLE NON-PEPTIDIC HIV-1 PROTEASE INHIBITOR. B. Shettyt. S. Kaldor2, V. Kalisht, S. Reicht, S. Webber'. 'Agouron Pharmaceuticals, Inc., San Diego, CA and 2Lilly Research Laboratories, Indianapolis, IN, USA. Objective: To evaluate AG 1343, a non-peptidic, water soluble inhibitor of HIV protease developed by protein structure based design for antiviral activity and oral bioavailability. Methods: Enzyme inhibition was measured against purified recombinant HIV-1 protease, and antiviral activity in acutely infected CEM-SS and MT-2 cells. Oral bioavailability and pharmacokinetics were evaluated in the rat, dog and monkey. Results: An inhibition constant of 2 nM was determined for AG1343; antiviral IC50 values were in the range 13-30 nM for HIV- RF infected CEM cells and approximately 60 nM when assays were conducted with strain IIIB in MT-2 cells. Antiviral activity was expressed at concentrations significantly below the IC50 for cytotoxicity; the therapeutic index was >1000 for assays in CEM cells and >400 in MT-2 studies. After i.v. administration, plasma levels of AG 1343 declined bi-exponentially with an average half life of 1 hour in rat and dog, and 1.5 hours in monkey. Absolute oral bioavailability was 52% in the rat, 31% in dog and 32% in monkey. Average maximum plasma concentrations of AG 1343 after oral administration of 50, 30 and 25 mg/kg AG 1343 in rat, dog and monkey, respectively, were 2060, 1810 and 1400 nM. Plasma levels which significantly exceeded the in vitro antiviral IC90 were sustained for 8 hours after oral dosing in all three species. The acute maximum tolerated dose was >250 mg/kg in mice and repeated dose administration of 100 mg/kg for 10 days was well tolerated. Discussion and Conclusions: AG 1343 exhibits potent antiviral activity coupled with promising pharmacologic characteristics and is therefore under development for Phase I clinical testing in humans. SHETTY, B., Agouron Pharmaceuticals, Inc. 3565 General Atomics Court, San Diego, CA 92121, USA. Telephone (619) 622 3000; Telefax (619) 622 3299 OBJECTIVE: Previously we described the design and synthesis of a novel class of nonpeptidyl inhibitors of HIV protease [SCIENCE 263:380-384 (1994)1. These cyclic urea inhibitors combined good antiviral potency with oral bioavailability in animals that was significantly enhanced relative to noncyclic inhibitors. Our objective was to discover cyclic urea inhibitors of HIV protease with improved oral pharmacokinetics and formulatability for development as oral therapeutics. METHODS: Antiviral activity was determined by standard methods in acutely infected cells. Pharmacokinetic parameters were evaluated after single p.o. or i.v. doses in rats and dogs. RESULTS: XM412 is a potent inhibitor of HIV protease (Ki = 0.3 nM) and of HIV-1 and HIV-2 replication in vitro. XM412 was equally effective against laboratory strains of HIV-1 and HIV-2 and against AZT-sensitive and resistant clinical isolates of HIV-1, with a mean IC90 against all HIV isolates tested of 0.12 +/- 0.08 M. Single oral doses (10mg/kg) of XM412 in solid or aqueous formulations produced peak plasma levels in dogs significantly higher than the antiviral IC90 (Cmax -10x IC90) Plasma levels were maintained above the IC90 for > 16 hrs. Oral bioavailability (F%) for the rat was > 50% and was 50%-100% for the dog, depending on formulation and salt form. CONCLUSION: XM412 is a potent inhibitor of HIV replication in vitro with good oral pharmacokinetic properties in rats and dogs. Clinical trials with this new cyclic urea await completion of additional preclinical studies. M.J.Otto, 500 S. Ridgeway Ave., Glenolden, PA 19036 USA, tel. 215-237-7764, fax 215-237-7926 322A STRUCTURE-BASED NON-PEPTIDIC HIVPROTEASE INHIBITORS. Thaisrivongs. Suvit, Tomich, P.K., Watenpaugh, KD., Romines, K.R., Howe, W.J., Chong, K.T., Schwende, F.J., Padbury, G.E., Ruwart, M.J. and Zipp, G.L., Upjohn Labs, MI, USA. Objective: To discover active, non-peptidic inhibitors of HIV protease with good oral bioavailability for clinical evaluation as anti-HIV therapeutic agents. Methods: A high-volume assay was developed to screen the Upjohn compound library for inhibitors of HIV protease. Crystal structures of inhibitor/HIV protease complexes were determined to direct the structure-based design effort. Compounds were further evaluated for antiviral activity in HIV-1,ds 1 infected MT4 and H9 cells. Oral bioavailability and pharmacokinetic properties were evaluated in rats. Results: From the initial screen, warfarin was found to be a competitive inhibitor, with an ICo value of 30 pM. Testing of similar compounds led to phenprocoumon with a K1 value of 1 pM. Beginning with the crystal structure of phenprocoumon/HIV protease complex, series of analogues were designed and led to much more potent, small-molecule inhibitors with K1 value in the nanomolar range. They were shown to inhibit p24 production in cell culture assays, and possessed good oral bioavailability and pharmacokinetic properties in rats. Discussion and Conclusion: Iterative cycles of crystallography-based modelling, analogue preparation, and biological assays greatly facilitated the preparation of active HIV protease inhibitors with high oral bioavailability. Selected compounds are under preclinical studies as potential anti-HIV therapeutic agents. Suvit Thaisrivongs, The Upjohn Company, Upjohn Labs, 7246-209-735.2, Kalamazoo, MI 49001, USA Phone (616) 385-6952 FAX (616) 385-5559 CD) r-U CD) CA) jD N

Page  8 323A DE NOVO DESIGN, CHARACTERIZATION, AND ANTIVIRAL EFFECT OF THE IRREVERSIBLE HIV PROTEASE INHIBITOR Kim, Sung Chun; Choy, N.; Lee, C.S.; Yoon, H.; Koh, J.S.; Son, Y.C.; Choi, H.; Park, C.; Kim, S. Lucky Biotech Research Institute, Dae Jeon, Korea Objective: The novel isostere including cis-epoxide was designed to inhibit HIV protease irreversibly. Optimization of the structure containing the designed isostere was performed to inhibit protease and HIV replication effectively. Methods: Designed inactivators based on enzyme mechanism were synthesized. The irreversible inhibition studies of HIV protease and the enzymological characterization studies were followed. Antiviral activities against HIV-1 in H9 and Sup T1 cell lines were obtained. Results: Compounds in the series inhibited HIV protease with time-dependent irreversible pattern, and the extensive dialysis after the enzyme inactivation could not revive the enzyme activity. The timedependent inactivation was partially blocked in the presence of the competitive inhibitor. Second order rate constants (kina/KI) of selected compounds ranged 109-1010 M-'min-1. Fifty % inhibition constants (IC50) ranged from 10 nM to 50 nM when assessed by syncytia formation and reverse transcriptase assay. Cytotoxicities were not detected up to 10 micromolar in the host cell lines. Discussion and conclusions: Compounds in the series showed the irreversible inhibition pattern to the protease, potent antiviral activity, and a high therapeutic index, indicating the high potential for the treatment of AIDS. KIM, Sung Chun, Lucky Biotech Research Institute, 104-1 Moonjidong Yusunggu, Dae Jeon, Korea Telephone(82)-42-866-2255;Telefax(82)-42-862-0332 324A ANTIVIRAL ACTIVITY OF HIV-1 PR INHIBITORS IN EARLY PHASE. Nagy, KAroly.1'2, Rayner, MM.3, Jadhav, P.K.3, and Oroszlan, S.. ABL-BRP, NCI-FCRDC, Frederick, MD1, Natl. Inst. for Dermato-Venereology, Budapest, Hungary2, DuPont Merck Pharmaceutical Co., Wilmington, DE3 Objective: We studied the antiviral activity of HIV-1 protease (PR) inhibitors during single cycle of infection. Methods: Antiviral activities of C2- symmetric inhibitors Q8024 and P9941 were quantitatively determined using the MAGI assay (Kimpton and Emerman, J. Virol. 66:2232, 1992) with HeLaCD4-LTR/p-gal cells. Infectivity assays were also carried out after pretreatment of infectious virus with Q8024. Results: Q8024 inhibited HIV-18 when added to cells 1 h prior to infection (IC50 = 1.15pM, ICoo = 4.48 pM). P9941 was less effective but at 13 pM it inhibited infectivity by -50%. No cytotoxicity of P9941 was seen at this concentration. TC50 of Q8024 was 83 pM. When HIV-111B (105 IU/ml) was preincubated in medium containing 2 pM Q8024, then diluted 50 fold as added to cells (0.1 m.o.i., 0.04 pM), >90 percent inhibition was obtained. Discussion and Conclusions: The results demonstrate the antiviral activity of two highly specific HIV-1 PR inhibitors, Q8024 and P9941, in a single cycle of infection. They corroborate our previous report (Nagy et al., J. Virol. 68:757, 1993) and strengthen the evidence that the viral PR has a crucial role in the early phase of viral replication. (Sponsored in part by NCI, DHHS, under contract NO1-CO-74101 with ABL.) CA) N, N, 0 325C COST-EFFECTIVENESS OF UGANDA BLOOD PROGRAMME Watson-Williams E. Jol, Kataaha P, Senyonga P, Kyeyune.D Ministry Of Health Nakasero Blood Bank, Kampala. Uganda. 326C BLOOD DONOR DEFERRAL BY HIV RISK FACTORS IS COSTEFFECTIVE IN ZIMBABWE. McFarland. William*; Kahn JG*; Katzenstein D**; Myere D***; Shamu R***. *UCSF, San Francisco, CA, USA; **Stanford U.; ***Zimbabwe BTS. Purpose and Background: In 1987 the National AIDS Control Programme initiated the reorganisation of Uganda Blood Transfusion Service (UBTS). Technical and financial support was by EC Community and the AIDS Task Force (ATF). We analyse costs and benefits of transfusion in all 92 Uganda hospitals during 1993. Methods: Blood was donated by altruistic volunteers, recruited by UBTS (57%0), and by relatives of hospital patients (43%). Data for transfused patients is from monthly hospital reports (509%6 compliance). HIV cost includes all costs of recruiting and counselling donors. Results: Assumptions; 33% of transfused patients die; 50% would die without transfusion; the general adult population (not blood donors) is 25% HIV and 6.3%I HBsAg positive. Patients transfused, mortality and potential virus transmission. Uganda 1993. Transfusions Survivors Infection prevented Cost X $1000 Patients Units Total Due to Tx. HIV Hepatitis B Blood HIV HBsAg Infants 11515 11515 7485 1727 1684 469 Others 8641 14689 6049 1296 1925 97 Total 20156 26204 13534 3023 3609 566 529 298 103 Conclusions: 1. Transfusion cost $308 for each life saved. 2. To prevent one transfusion transmitted HIV infection cost $80 for HIV and $182 for hepatitis B. 3. Giving unscreened blood would cause more HIV infections than the number of lives that would be saved. Objective: To assess the cost-effectiveness of deferring blood donors with risk factors for HIV infection prior to serological testing in Zimbabwe. Methods: We developed a decision analysis model to assess the cost per window period unit averted when blood donors with IIIV risk factors were excluded from donation before serological testing. A published analysis for Zimbabwe provided the costs to obtain a unit of blood. We obtained I1IV prevalence (19%), incidence (6%), and HIIIV risk factors among blood donors from a cohort of 969 male factory workers volunteering for blood donation in Ilarare, Zimbabwe. Results: The cost-effectiveness of donor deferral varied by the risk factor used for exclusion: Exclusion of donors Window period units Cost per window with history of: averted per 10.000 period unit averted Genital ulcer 17 Net Savings STD 31 Net Savings Paying for sex 39 $128 Multiple sex partners 62 $773 Exclusion of donors having a history of genital ulcer or any STD resulted in a net savings compared to serological testing alone because the savings from discarding fewer units testing IIVpositive was greater than the cost to question and recruit replacements for those deferred. Exclusion of donors with a history of paying for sex or multiple sex partners were not cost-saving because the high prevalence of these risk factors resulted in higher replacement costs. Conclusions: Blood donor deferral prior to serological testing is cost-effective and can be costsaving in Zimbabwe and sub-Saharan Africa when conducted in donor populations with high HIV prevalence and identifiable risk factors for IIIV infection. The cost-effectiveness of donor deferral is greatest when based on less common IllV risk factors with higher relative risks. William McFarland, MD, MPII&TM 74 New Montgomery San Francisco, CA. 94105 USA (415) 597-9303 (PI); (415) 597-9213 (FAX) E. Jolhn. Watson-Williams, c/o E.C. Delegation. P.O. Box 34871, Lusaka, Zambia. Tel. 260 1 253584 Telefax 260 1 250906

Page  9 327C RATE OF WINDOW PERIOD BLOOD DONATION IN BANGKOK,3 THAILAND. Kaewkunqwal, Jaranit,,Kitayaporn D,3 Bejrachandra S, Rungroung E, Chandanayingyong D, Mastro TD.14 1 HIV/AIDS Collaboration, Bangkok, Thailand;2 Rajamangala Institute of Technology, Bangkok; 3 Mahidol University, Bangkok; 4 CDC, Atlanta GA, USA. Oblectives To determine HIV seroprevalence and incidence among various blood donor types and to estimate the number of window period donors. Methods Retrospective data were analyzed from computerized records of all blood donors at a public university hospital in Bangkok. HIV-1 seroconversion rates were calculated assuming uniform distribution between the last negative and first positive tests. Results During 01/90 to 06/93, 97467 units were collected from 60483 donors, including 14492 (24%) repeat donors. Of the repeat donors, 79.4% were males. Summary results: Total(N) Voluntary Replacement Paid Other All Units 97467 67.1% 29.4% 2.9% 0.6% All Donors 60483 56.7% 41.9% 0.7% 0.7% Repeat Donors 14492 81.8% 14.8% 2.6% 0.8% Seroconverters 40 85.0% 2.5% 7.5% 5.0% Prevalence(/Units) 492 0.3% 1.0% 0.3% 1.3% Incidence calculated for all donor types was 191.3 per 100,000 person-years. Assuming a period of 45 days between onset of infectivity and the appearance of detectable antibody, the number of such "windowperiod" donors was 23.6/100,000 donors. Of all donated blood units, 5.7% were rejected due to HIV, HBsAg, or VDRL positivity. Discussion & Conclusions The expected number of window period donations among Thai repeat donors was relatively high compared to developed countries. Improved donor deferral criteria are needed in Thailand. Jaranit Kaewkunowal; HIV/AIDS Collaboration; 88/7 Soi Bamrasnaradura; Nonthaburi 11000; Thailand. Tel. 66-2 -5915445; Fax 66-2-5915443 329C.HemophiliHV Seroconversion Surveillance Project: Safety Of Blood Products Used In The Treatment Of Hemophilia L. Augustynialsk, A. Brownstein, S. Wiley, R. Simonds, W. Fricke, B. Evatt The National Hemophilia Foundation, New York, NY, USA OBJECTIVE: The Seroconversion Surveillance Project (SSP) was designed to monitor the risk of HIV transmission in the U.S. by clotting factor concentrates used to treat hemophilia. METHODS: From 1987-1993, we conducted telephone interviews 1-3 times a year with designated representatives of 138 hemophilia treatment centers (HTCs) across the U.S. to gather information on their patients, including type and severity of bleeding disorder and HIV status, and to determine if any patients had seroconverted within the past year. Collaborating agencies -- the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration, and the National Hemophilia Foundation - conducted epidemiologic and laboratory investigations on reported HIV seroconversions. RESULTS: We collected data on 18,651 persons with hemophilia, 11,646 of whom had been HIV tested, and identified and investigated 44 seroconversions. None of the 44 met the CDC operational criteria for probable association with factor concentrate; however, 9 patients investigated became HIV infected through infusion with concentrates made from plasma that had not been tested for HIV. Two HIV seroconversions identified through SSP were found to be cases of HIV transmission probably due to blood contact in the homes of patients on home infusion. CONCLUSIONS: Viral-inactivation methods and screening of donors has greatly increased the safety of blood products used in the U.S. Recent seroconversions underscore the importance of continued study and education of patients and their family members on the risks of HIV transmission in the home. Linda Augustyniak, RN, The National Hemophilia Foundation 110 Greene Street, Suite 303, New York, NY 10012 (212) 219-8180 ext. 3041 TEL (212) 966-9247 FAX 328C MONITORING ON HIV INFECTION AMONG THE GENERAL POPULATION ShimizuMSuzuki,T,Yoshizawa,,Sayama,T,Sekiguchi,S,Fuzisawa,K, KAMIYA,T, Tokyo Women's Medical College,Tokyo,Japan Objective: For the purpose of knowing the prevalence of HIV infection among the general population (blood donors in blood center and samples of patients in commercial laboratories) and high risk population(transfused patients and pregnant women in hospitals),we annually studied monitoring HIV antibodies. Methods:We examined HIV-1 antibodies by particle agglutination and confirmed by western blot. Moreover we examined HIV-2 antibodies when necessary. Results:The prevalence of HIV infection among blood donors in 1986 was 0.00035%. In 1988, the prevalence was 0.00013%,then increased steadily to 0.000472% in 1993.In Tokyo and 3 neighbouring prefectures,the prevalence was 1.53/100thousand donors.The prevalence of pregnant women in hospitals and HIV-2 infection among blood donors is nil. Among the general population, there is evidence to indicate that HIV infection by heterosexual transmission is increasing although the prevalence is low. Discussion and Conclusion: With respect to blood donors, there is a window period between the acquisition of the virus and the appearance of antibody to HIV.Thus, a negative HIV status may give severe risk to patients. Intensive targeted programs with enhanced anamnese to blood donors and testing need to be further supported and implemented.We need comprehensive health education that is the most important strategy to fight against AIDS epidemic. SHIMIZU,Masaru, Dept.Transfusion Medicine, Tokyo Women's Medical College,8-1 Kawada-cho,Shinjyuku-ku,Tokyo,162 Japan Telephone (81)-3-3353-8111;Telefax (81)-3-5269-7360 330C HIVINF CTIQNINRE PlNT QFB QDP Q CTSFROM DONORS WITH KNOWN DURATION O FETN 8 CTS FROn M Learmont Jt, Luo K', Wylie Bt, Stewart Gt & Kaldor J. National Centre in Epidemiology & Cinical Research', NSW BTSt, Westmead Hospital$, Sydney, Australia Objective: To examine the association between the rate of progression of HIV disease in blood donors with known duration of infection and the progression rate in the people who received HIV contaminated blood from these donors. Methods: A registry of people with transfusion acquired HIV infection (TAHIV) in New South Wales (NSW) Australia was established by the NSW Red Cross Blood Transfusion Service (BTS); annual follow-up was implemented in 1985. From the registry a group of recipients was identified for whom the duration of HIV infection in the corresponding donors could be established. Rates of progression to AIDS and survival were estimated for these recipients, and predictive factors, including the rate of disease progression in the donor, were assessed. Results: A total of 25 recipients, with 8 corresponding donors were included in the study. In multivariate analyses, longer survival (p-=0.01) was associated with the receipt of blood from donors who developed AIDS more than 10 years after HIV infection. Recipient age (<50 years) and a smaller number of transfusions were associated with better survival. Gender, zidovudine treatment and severity of illness at transfusion were not found to be significantly associated with survival time. Conclusions: Rate of disease progression in the donor appears to be an important predictor of survival in people with TAHIV, suggesting the role of viral pathogenicity. Lesley Ashton, National Centre in HIV Epidemiology and Clinical Research, 376 Victoria Street, Sydney NSW 2010 Australia. Tel: (02) 332 4648; Fax (02) 332 1837 C,) CA) 0 C)

Page  10 331BDESIGN AND DEMOGRAPHICS IN A MULTICENTER TRIAL OF CYTOMEGALOVIRUS (CMV) PROPHYLAXIS IN ADVANCED HIV DISEASE BrmosgrtC. Craig C, Louis TA, Hillman D, Costanzo L, Timpone J, Scott J, Nunley F, Stempien MI, for CPCRA, NIH, DAIDS, U.S.A. Objective: To evaluate the safety and efficacy of oral ganciclovir for prophylaxis against CMV retinal and gastrointestinal mucosal disease in lHliV-infected individuals with severe immune suppression. Methods:r The Community Programs for Clinical Research on AIDS (CPCRA) is an NIH/ NIAID sponsored clinical trials network. Since 1990, 5651 individuals with HIV infection have been enrolled and followed prospectively in an observational data base (ODB) and 10 experimental studies. For individuals with CD4 < 100 we studied the incidence of invasive CMV disease (retinal or gastrointestinal) using the Cox proportional hazard model to estimate relative risks for gender, race and HIV risk exposure. The estimated incidence rates were used to design a randomized, double-blind clinical trial of 3 grams of daily, oral ganciclovir versus placebo. Demographic associations were used to evaluate the demographics of enrollees relative to those expected to enroll. Resultas Two-year incidence of invasive CMV disease was estimated at 32%. As compared to white males, disease rates were substantially lower for injection drug users and non-whites. The two-year incidence rate coupled with 12-month accrual and a minimum of 12-months of follow-up per enrollee and a 2:1 allocation to the active treatment group produced a clinical trial sample size of 850 to detect with 80% power a 50% reduction in the relative risk of disease for the ganciclovir group. The trial started in April of 1993. Through January of 1994, 612 persons with CD4 < 100, with positive CMV serology but free of evident CMV disease have been enrolled. The mean age is 40; 96% men; 70% white, 22% black, 6% Hispanic, 2% other. Based on non-mutually exclusive categories, gay or bisexual males constitute 85%, IDU 12%, heterosexuals 38%. Mean Karnofsky is 85.5; 46% prior AIDS Ol/OM diagnosis; 63% CD4 < 50; follow-up is 90% after 3 months. 82% are participating in a nested quality of life study. Discussion and Conclusions: Patients at high risk of invasive CMV disease are being successfully enrolled and followed in a community-based CMV prophylaxis study. The high representation of gay white men is consistent with the background observational analyses of disease incidence. Carol Brosgart, MD East Bay AIDS Center, 3031 Telegraph Ave, #235 Berkley, CA 94705, USA Phone 510/204-1870; FAX: 510/848-9764 332B INTRAVITREAL TREATMENT OF CMV RETINITIS WITH AN ANTISENSE OLIGONUCLEOTIDE, ISIS 2922 Palestine. Alan G, Cantrill, H.L., Luckie, A. P. and Ai, E. Objective: The safety and preliminary efficacy of ISIS 2922, a phosphorothioate antisense oligonucleotide, were evaluated in a multicentered Phase I trial. Methods: AIDS patients with CMV retinitis who had developed intolerance or experienced progression while receiving either ganciclovir or foscarnet were entered. Patients received 50 pl intravitreal injections of either 75 jg, 150 jg, or 300 pg ISIS 2922, targeted to achieve final vitreal concentrations of 2, 4 and 8 pM, respectively. Doses were repeated on a weekly schedule for the first month followed by every-other-week dosing. Results: To date, 2 patients have received the 2 pM regimen, 3 patients have received the 4 pM regimen and 3 have received the 8 pM regimen. ISIS 2922 appears to be well tolerated by these patients who have histories of multiple progressions on both ganciclovir and foscarnet. A spectrum of reversible ocular events were noted which were not dose related and did not require intervention. Two of the 3 patients in the 4 pM group and all three of the patients in the 8 jM group have had prolonged positive responses confirmed by fundus photography. Discussion and Conclusions: Intravitreal ISIS 2922 was safely administered and the preliminary observations of efficacy support further study of ISIS 2922 in Phase II/III controlled trials. PALESTINE, Alan G,l 145 19th Street, N.W., Wash., DC 20036 Telephone (202) 833-1668; Telefax (202) 833-4698 CA) CA) CA) w 333B IMPLICATION OF HIV-I AND HHV-6 IN THE DEVELOPMENT OF AIDS RETINITIS.Oavi'.Hamida,Oreen',M.,Lewis',D.Hollinger',B.Pearson"G,and Ablashi",D.Baylor College of Medicine', Houston,Texas,Georgetown University", Washington D.C.,U.S.A. 334B JCV.DNA IN CNS OF AIDS PATIENTS WITH AND WITHOUT PHIML Vago L*, Cinque P*, Sala E*, D'Arminio Monlorte A*, Lazzarin A*, Costanzi Giulio * L. Sacco and *S. Raffaele Hospital, University of Milan, Italy Objective: Prior to death, majority of individuals with HIV-1 infection experience significant visual loss due to AIDS retinitis. As reported previously, the presence of HIV-I and HHV-6 antigens in retinas of AIDS patients in the absence CMV infection may have etiological implications in the development of AIDS retinitis. In the current study we have determined the frequency and proximity of HIV-1 and HHV-6 active infections in AIDS retinas in the presence or absence of CMV infection. Methods: Twenty AIDS globes with lesions of unknown etiology (gp1), ten AIDS globes with no visible lesions (gp2) and twelve AIDS globes with CMV lesions were analyzed for the presence or absence of viral antigens and transcripts using immunohistochemical staining and In situ hybridization. Results: Sections from 13 retinas (gpl) were positive for HIV-I antigens and transcripts. Three of the HIV-1 positive retinas were also positive for HHV-6 antigens and transcripts. Cells from two of these three retinas showed coinfection with HIV-I and HHV-6. In group 2, HIV-1 and HHV-6 transcripts were detectable in cells of all retinas which were mostly scattered in the ganglion cell layers. No CMV was detected in globes from gp 1 or 2. Sections from all 12 retinas (gp3) were positive for HIV-1 and CMV. Three of these 12 samples were also positive for HHV-6 antigens. Discussion and Conclusion: These data confirm our previous observation and support our hypothesis that HIV-1 and HHV6 alone or in combination may predispose retinal tissues to opportunistic infections including CMV at later stages of AIDS retinitis. Division of Molecular Virology, Baylor College of Medicine, Houston, Texas Telephone (713) 798-5583; Telefax (713)795-0524 Objective. To evaluate the presence of JC virus-DNA in the brains of AIDS patients with and without Progressive Multifocal Leukoencephalopathy (PML). Methods. Autopsy brain and spinal cord from 15 patients with AIDS and PML and from 16 patients with AIDS and no histopathological diagnosis of PML were examined. In all cases 5 sections from fronto-parietal lobes, basal nuclei, cerebellum and spinal cord were studied. JCV-DNA was demonstrated by (1) nested PCR on DNA extracted from paraffin-embedded sections of all tissue blocks examined, and (2) in situ hybridization (ISH) with a biotinlabeled DNA probes (ENZO, NY) in at least one section in PML+ cases and in all the PCR positive sections from control patients without PML. Results. PML patients: All the sections with histological evidence of PML were positive with PCR and ISH for JCV-DNA. JCV-DNA was also amplified in 60% of sections without histological features of PML. AIDS patients without PML: 44% (7/16) had at least one section with a positive PCR; of them only 3/7 (43%) were positive with ISH. On the whole, ISH was positive in 19% of this group of patients. Conclusions: JCV-DNA can be demonstrated with nested PCR in about half of the patients without histopathological evidence of PML. ISH is positive in only half of PCR+ cases; this confirms that in AIDS JCV can be present both in latent and minimally reactivated state. Work supported by ISS AIDS Project, 1993 Costanzi Giulio - V Cattedra Anatomeia Patologica Via G.B. Grassi 74- 20157 Milan, ITALY TEL: **39 2 35799348 FAX: **39 2 38200385

Page  11 3358 IIIV/AIDS IN THE CZECH REPUBLIC L. Machala*, J. Spala*, 11. Rozsypal*, N. Hearst**, J. Mandel** *Faculty Hospital Bulovka - Dept. of Infectious Diseases, Prague, Czech Republic **Center for AIDS Prevention Sciences, Univ. of Calif., San Francisco, USA Objectives: To analyze currently available data on the extent of the AIDS epidemic in the Czech Republic. Methods: National data on lIV antibody testing were tabulated and reviewed. Clinical records were also examined for patients treated by the Department of Infectious Diseases at the Faculty Hospital Bulovka in Prague, the Center where almost all Czech IIIV/AIDS patients receive medical care. Resulls: 3,409,610 blood tests for IIIV antibody have been conducted in the Czech Republic up until the end of 1993. Of a total of 254 HIIIV+ cases found, 84 were foreigners (68 from Africa) and 170 were Czech citizens (homo/bisexual men, 104; IVDUs, 2; hemophiliacs, 16; blood transfusion recipients, 14; heterosexuals, 19; unknown risk, 15). The total number of reported AIDS cases was 46; of which 27 are deceased. In the Czech capitol of Prague alone, there have been 100 H-IIV+ cases and 33 reported cases of AIDS. At Faculty Hospital Bulovka, serving the entire Czech Republic, 130 IIIV+ patients have been seen, of which 42 were diagnosed with AIDS. The most frequent opportunistic infections diagnosed were PCP (N=13), brain toxoplasmosis (N=9) and bacterial pneumonia (N=8). Other 1O's were less frequent. Up to the end of 1993, 24 persons have died of AIDS at Faculty Hospital Bulovka. The most frequent causes of death were bacterial pneumonia (N=6), brain toxoplasmosis (N=5), and generalized CMV infection (N=3). In five cases, PMLE was found at autopsy. Conclusions: Although the AIDS epidemic is in a relatively early phase in the Czech Republic, it so far seems to be following a pattern consistent with that of other European countries. Accordingly, rapid AIDS prevention efforts are both warranted and urgently needed. Ladislav Machala, M.D. Nad alejf 1735 162 00 Praha 6 Czech Republic FAX: 66 3 11506 66 3 539156 336B HIGH INCIDENCE AND RECURRENCE RATES OF BACTERIAL ENDOCARDITIS IN HIV + DRUG INJECTORS a ICatherine, Khoshnood, K., Alcabes, P., Friedland, G.H. Yale University School of Medicine, New Haven, CT USA Objective: To determine the incidence of bacterial endocarditis (BE) among injection drug users (IDU) and examine the role of HIV in the occurrence and recurrence of and mortality from drug-use-related BE. Methods: Records of all BE discharges from Yale-New Haven Hospital 1985-1992 were examined. Individuals with a drug-dependence diagnosis or medical-record evidence of IDU were selected. Multiple admissions were excluded for incidence calculation only if they occurred in the same year. Incidence rate was calculated by dividing IDU BE admissions by the estimated number of New Haven IDUs, 2,300. Results: A total of 212 drug-related BE cases occurred over the 8-year period. BE incidence among IDUs averaged 1%/year (95% CI 0.9 to 1.2%). Over 50% of BE cases were known HIV+. Year No. IDU with BE BE Incidence, % Known HIV+ BE, % 1985 1986 1987 1988 1989 6 19 30 30 22 0.26 0.83 1.30 1.30 0.96 50 32 47 57 68 1990 1991 992 All 24 30 23 184 1.04 1.30 1.00 1.00 54 74 52 56 75 HIV + patients (pts.) had 108 admissions and 87 HIV- pts. had 104 admissions. Although average length of hospital stay per admission was identical for HIV + and HIV- BE pts. (=30 days), 23/75 HIV + vs. 4/87 HIV- pts. had multiple BE admissions (p=.00001). In-hospital mortality risk was higher for HIV + (11/75) than HIV- (5/87), p=.059). Conclusions: Bacterial endocarditis incidence is very high among New Haven drug injectors, resulting in a substantial burden of acute-care hospitalization. Compared to HIV- IDUs with BE, HIV + IDUs have more frequent BE recurrences and are at higher risk of death while in hospital. Hardalo, Catherine, Yale University School of Medicine, New Haven, CT USA Ph:(203)737-2450 Fax:(203)737-4051 37B D INTEGRAL CARE OF PWA ND FAMI A MODEL FOR DEVELOPING COUNTRIES Navarro S*.Luna Ht,Garcia L*, Villegas M., Martinez J**. National Institute of Epidemiological Diagnosis and Reference *Mexican Foundation of Fight Against AIDS**. anRefer OBJETIVE: Training and supporting for the familiy, spouses and friends of PWA in the final stages of illness so they may perform basic activities that help improve their quality of life. METHODOLOGY: A integral program was carried out jointly with patient's family, spouses and friends of PWA living in Mexico City and its outskirts including the following topics: 1.- Provide clinical, couseling and basic information about HIV/AIDS in the home of the patient. 2.- Distribution of educational material about guidelines for clinical cars. 3.- Timely detection of infections.4.- Dynamic emotional, psychological and spiritual support for the patient, and family so as to reinforce ties of communication, affection and love. 5.- Couseling directed specfically to the family, spouse and friends during the final stages and death with the idea of reducing anguish and pain of separation. RESULT$:In 1993, 50 and 100 relatives, spouses and friends were recruited. The range of age of PWA was 19 to 40 years old, 25 men 30% of relatives living in the same household accepted to participate. Four patients were supported by friends. eighty porcent of the patients was infected by sexual transmission and 33% by transfusion, eighteen patients have died, and 3 are terminally ill. Among relatives, women (wife and mother) were more participant. CONCLUSION: Training for PWAS relatives and friends is an important alternative in the improvement of the quality-being, until a worthy death, either at home or at the hospital. Personal fears, and a better confronting with death ocurred.was achieved Active participation from the relatives and acceptance of pwa. Proposing a guide line is being prepared wich will be evaluatied with a larger number of families strategies to increase centered model are being identified. Participation of men and promotion of mutual support between paws and families. A COMPARISON OF PWA AND NURSE REPORTS OF SYMPTOMS / Holzemer, William L., Henry, S.B., Reilly, C.A. & Slaughter, R.E. University of California, San Francisco Objective: The aim of this study was to compare patient and nurse reports of symptoms experienced by patients who were receiving nursing care for an AIDS-related condition. Methods: As part of a longitudinal descriptive study examining the linkages among patient problems, nursing interventions, and patient outcomes; patient-nurse pairs completed a 56-item symptom checklist. Symptom severity was rated on a 4-point Likert scale ranging from not present (0) to severe (3). This analysis focuses on a convenience sample of 100 patient-nurse pairs who completed the checklist near admission to nursing care services in one of three settings, hospital, skilled nursing facility, or home care. Descriptive, correlational techniques were used to examine discrepancies between patient and nurse reports of symptoms. Results: Patients identified the presence of a symptom more frequently than the nurses. Fatigue, shortness of breath with activity and at rest, weakness, dry mouth, coughing, weight loss, headaches, thirst, and insomnia were the 10 most frequently reported symptoms with a mean intensity of 2.01, standard deviation of 0.79, and Cohen's kappa of 0.09. The agreement between patient and nurse ratings for all 56 symptoms was small as measured by Cohen's kappa statistic and ranged from 0.008 to 0.412. Conclusions: Many symptoms experienced by the patient were not Identified by the nurse caring for the patient. As symptom management Is an integral part of nursing care for persons living with AIDS, the Incorporation of a symptom checklist into a nursing assessment has the potential to identify problems requiring nursing intervention and to assess the effectiveness of nursing Interventions in symptom management. William L. Holzemer, PhD, RN, FAAN (415) 476-2763 University of California. San Francisco, N 39 San Francisco, CA, USA 94143-0604 FAX (415)476-9707 CA) CA) CA) 0 CA)

Page  12 FS 339B/D GENDER DIFFERENCES IN UTILIZATION OF HEALTH SERVICES AND INFORMAL CARE. Crystal,.Stephen; Sambamoorthi, U. AIDS Research Group, Institute for Health, Rutgers University, New Brunswick, NJ, USA. Objectives: To compare personal care needs, informal care, and utilization of formal health care services, for women vs. men with symptomatic HIV disease participating in a Medicaid home and community based care program in New Jersey, USA. Methods: Health care utilization data are from 1987-89 Medicaid claims of 623 men and 175 women. Data on functional status and personal care are from a random interview subset (218 men, 49 women); respondcnts were 44% non-Hispanic white, 40% non-Hispanic black, and 16% Hispanic; 50% of men and 49% of women had IDU histories; 83% of men and 80% of women had full-blown AIDS. Results: Women had significantly higher levels of impairment in activities of daily living (ADL) and instrumental activities of daily living (IADL) though they apparently did not enroll earlier in the course of disease. On average, women were impaired in 25% of ADL tasks and 42% of IADL tasks, as compared with 9% and 24% for men (p<.01). Help sources with such tasks were significantly different by gender: women were more likely than men to rely on formal sources and more likely to receive help from extended family members (especially daughters). Mean length of hospital stays was significantly longer (17.1 vs. 14.1 days); this difference remained with statistical controls for race, risk group, medical severity, and other variables. Hospital days to death, home care costs, and total cost of care were all higher for women. Conclusions: Both personal care needs and the structure of social support systems appear to differ by gender, such differences may help explain observed differences in utilization of formal services. In this population, utilization of formal home care services was higher for women, as was length of hospital stays and total cost of care. Women may be in particular need of formal services because of their functional status impairments or gaps in informal support systems available to them. More research is needed on women's needs both for medical care and long-term care services to address these needs. Stephen Crystal,Ph.D., Rutgers University, Institute for Bealth, 30 College Avenue, New Brunswick, NJ, USA 08903; (908)932-8579 phone; (908)932-6872 fax 341B/D TRADITIONALFCRUNSEU1 IR ESIC IN RUAL AREAS ARGR ASSR AANNOD M, NY R - UGANDA. Objective: Utilise the traditional counselling knowledge and skills of local advisors towards the AIDS pandeirc in rural areas of Rakai District. Methods: After trying the modern counselling model from Western countries, people's response in rural areas to the counsellors was still low. In the open-discussion meetings with a cultural-group of 35 elders, traditional skills and attitudes used by local advisors and wisemsen were identified. From 35 villages of approximately 700 people each, a group of 146 local advisors/wiseen was selected. Selection criteria was on past experience, people's trust, confidentiality, social class to serve and voluntarism. A formal training is given in the following - Traditional and modern counselling models and their intergration, and AIDS awareness and iane Care basics. Each local advisor serves over 150 people. Results: Each village has 4 trained local advisors, 2 youths, 2 elders, and each parish of 5 - 6 villages has 1 traditional witch craft practitioner, rendering services to over 22000 people in the one Sub-county. With their services, more people are responding to counselling, there is more acceptance of their status, and a higher degree of positive attitudes towards the epidenic and more preventive ideas from the commnity. Conclusion: With this increasing problen, counselling should be realistic and in relation to people's traditions and cultural aspects. It is equally important to utilise the local people's knowledge and give them the required skills through open discussions. This is a study worth trying in rural areas far from health centres. MARGARET SSEHJKASA CACRN - RAKAI - UGANDA P. O. BOX 1644 MASAKA UGANDA - EAST AFRICA TELEFAX NO 256/481/2014 340B/D THEATRICAL AND PSYCHODRAMATIC ELEMENTS AS PSYCHOTHERAPY FOR GAY AND BISEXUAL HIYV[+) MEN. Paido Moreno Mauticio; Liga Colombiana de LuchaContra el SIDA(LCLCS), Santate de Bogot., Colonbia. Objective To observe, describe and enalize the effect of a grupal psyc.hotherapy in gay and bisexual HIY(+) men, in relation to their psychological wellbeing degree, as well as the acceptance level of their sexual orientation and serodiagnosis. Methods: It included 16 gay and bisexual HIV(+) men, who attended the LCLCS and who had a previous psychiatric evaluation. Grupal therapy x.s conducted once a week in a three hours session duing four months, exploring items like bereavement, dead, sexuality, love, partner, life and future. Test for to measure depression, anxiety and autoacceptance levels were made al the beginning of the therapy and t 4ce in a month afteri. Results: Lowering in depression and anxietylevels were observed compared to those founded at the beginning, as well as a better acceptance of their sexual orientation and serodiagnosis. During the therapy there was a ten d encyto maintain themselves as a group, an this remain once it finished. Discussion and Conclusions: Grupal therapy.ith theatrical and psrhodnsnatic elements..s effective in this kind of patients; and i could be benefical in the treatment of similar groups including women, partners, family members and friends of HVi(+) patients. PARDO MORENO, Mauricio, LigaColfombiana de LuchaContra el SIDA, Avenida 32 No. 14-46 Sarntal de Bogota-Colombia, South America. Telephone 57-1-2454751; Telefax 57-1-2879392 342B/D COMPREHENSIVE PRISON HIV MEDICAL CARE AND PRERELEASE COUNSELING. Burzynski J, Flanigan TP, Kim J, DeCiantis ML. Brown University, Providence, RI. OBJECTIVE: To characterize the course of HIV infection among incarcerated men and women in a program which provides HIV testing, medical care, and pre-release planning to all state prisoners in Rhode Island. METHODS: Approximately 12% of women and 4% of men in the state prison are HIV+. Over 35% of all HIV+ persons reported statewide are tested in the state prison. Over 5 years, 276 persons were evaluated and followed every 3 months by the medical team; data on 136 persons has been analyzed thus far (101 men and 35 women). RESULTS: The average age was 35 yrs; 40% were white, 43% were African American, 17% were Hispanic. Risk behaviors were IDU 69%, multiple sex partners 19%, unknown 12%. Mean initial CD4 count was 527. CD4 counts declined a mean of 54/yr. The most common medical problems were thrush, genital herpes, and chronic viral hepatitis. In women the most common problems were gynecological (cervical dysplasia, trichomonas and candida vaginitis). 58% received antiretroviral therapy and 26% received PCP prophylaxis. Pre-release planning resulted in successful medical follow-up in 84% of inmates and drug treatment in 72% of inmates. CONCLUSION: For many HIVinfected inmates, an HIV care program is the first opportunity for HIV testing, comprehensive medical care, counselling, and referral to drug treatment. The state prison occupies a key position in HIV treatment and control in our community. Joseph Burzynski, M.D. c/o T.P. Flanigan, M.D. The Miriam Hospital, 164 Summit Ave. Providence RI 02906, (401)331-8500 FAX: (401)331-8501 CA) CA) 0 CA) w0

Page  13 343D "MARGINALS TO MARGINALS":EXPANSION OF PROGRAMA PEGACAO Longo,Paulo; N6cleo de Orientacao em Saide Social. Rio de Janeiro, BRAZIL OBJECTIVES:1)Expand the work of Programa Pegacao,an HIV/AIDS prevention project for male sex workers, working since 1989;2)A follow-up study, presenting new alternatives to the "peer education" model. METHODS:After more than 4 years working with male sex workers in Rio,many "boys" became involved in project's activities.Many(45)have been trai ned to work as peer educators with hustlers and other marginalized groups. Seuch activities have been regularly evaluated by project's team. RESULTS:The model of peer education has not been sucessful among hustlers.Many factors contribute to the failure such as power competition, lack of professional identity,barriers from the group attended.Therefore male sex workers has shown to be great educators among other marginalized groups,specially young female sex workers and street kids.A strong, permanent and interactive relatinship has contributed to the sucess, as well as the trustfulness from the target groups and a former experience from the hustlers themselves as being "apart". CONCLUSION:Peer education model is not the best for all groups.Generally it is incentivated by health care authorities to deny access to health care services and education("They educate and attend themselves").Therefore,marginalized people can help a lot other in the same condition,as the lack of basic needs and discrimination encourage a lot the REAL concept of SOLIDARITY 344D OUTREACH WORK WITH OFF-STREET FEMALE SEX WORKERS NATHAN, Tracey; Sheffield Centre for HIV & Sexual Health, 22 Collegiate Crescent, Sheffield, England OBJECTIVE: To provide women working as prostitutes in the off-street (Saunas etc.) sex industry with advice + information about HIV, STDs + sexual health. To provide easy access to the Department of GU Medicine. To monitor the work focusing on the uptake of GU services, reasons for attendance, STD rates and the take up of Hepatitis B vaccination. METHODS: (1) Recognising that women may not be using services because of embarrassment or fear of discrimination. (2) Outreach work to women at their place of work. (3) Widespread STD transmission is possible if target group is not practicing safer sex. (4) Monitoring STD rates + comparing this with rates pre-intervention (1989-1993). (5) Monitoring numbers of women attending clinics from saunas through analysis of records. RESULTS: Steady decline in numbers of women presenting with an STD e.g. Gonorrhoea (1989 - 17; 1990 - 17; 1991 - 14; 1992 - 6). DISCUSSION AND CONCLUSION: (1) Sensitive and respectful outreach work can reach the majority of women working in the off-street sex industry + can break down resistance to using GU services; (2) Positive results (incl. declining morbidity rates) are realised + possible to demonstrate using comparison between pre- and post-outreach data; (3) Agencies must put in long-term commitment to this work; (4) Suggested future research - experience of prostitutes in using sexual health services; Long-term research into outreach work encouraging attendance at GU clinics and its effectiveness in reducing STD rates. PAULO HENRIQUE LONGO R. Visconde de PirajA, 127 / 201 -Ipanema, Rio de Janeiro- BRAZIL - Fax: +55 21 227 5944 345D "100% CONDOM" use for Badii Sex Workers in Nepal:BHATT.P, Baltes,R, Swiss Development Cooperation/Nepal (Intervention in DOan District of the For Western Region of Nepal) Objective: "1002 condom use" by Badii sex-workers and their clients in Bang. etho: reeratio [f te whle B ii com un ty raining of Learders fnro the sex worers Educetio through - scnssons drama - songs - comic books - videofilms Establishment of o lcondom corner" SPreparation and supply of condoms SFollow up Results: Out of a group of 40 volunteer Badii sex-workers 6 self elected ebers were trained for the proposed prorassmme. SnKla Khola, Dng District, 30.000 condoms were distributed and 10 comic books. One task f the enders was the recruitment of other sex-workers. s a result o his task was the "1002 condom use" also introduced in 2 other self supporting areas with Radii sex-workers. Conclion:lhe involvement of Badii sex workers from the Bang District in a "1002 condow use" prgrae can be seen aa model. The trained Badiis "determination and skills" can be used for other places to extend the programme. Dr. Pushpa Bhatt, SDC/N.PO Box-ll3,Kathmancldu Nepal,Ph:522020 Fax:977-1-525358. 346D CHANGES IN SEXUAL BEHAVIOUR OF PROSTITUTES IN CALCUTTA S.Jana*,L.Khodakevich*", C.Larivee"**, I.Dey ***, N.Sarder *"[*All India Institute of Hygiene and Public Health,"**WHO/GPA-I,***'Health and Ecodefence Society (HEDS), *""Human Development and Research Institute (HDRI) Objective: To study the behavioural change in female sex workers in a red light area of Calcutta, under the influence of peer education and condom promotion. Methods: An HIVISTD prevention project was launched in September 1992 in the biggest brothel area of Sonagachi in Calcutta, hosting about 5000 female sex workers (FSWs). At the community level, the project activities included peer education and condom distribution. Sex practices of FSW were studied before setting up the project and after 13 months of the intervention; 463 and 627 randomly selected FSW were interviewed in the two surveys accordingly, and prevalence of STDs and HIV among them were studied. Results: The intervention has significantly changed at least two practices related to risk of HIV/STD transmission. The use of condoms increased significantly. The percentage of women who used condoms always or often rose from 2.7 to 67.8% (P <0.01). At the same time, probably due to the protection provided by condom, the proportion of female who used oral contraceptives always or often has declined from 16.7 to 10.3% (P<0.01). The proportion of females who never used condoms fell down 90.7% to 7.7% (P<0.001) Discussion and Conclusions: The three component programme implemented in the red light area during a year period has led to a dramatic change in sexual behavior among a relatively large target population at high risk. The project success at large depended on the close work with the community and provision of services, including treatment of STDs and condoms supply. JANA, S., ALL INDIA INSTITUTE OF HYGIENE & PUBLIC HEALTH, CALCUTTA, INDIA Telephone: (91)(33)(241 3954) Telefax (91)(33)(241 2539) CA).b CA) 0') 44

Page  14 347D aor oi!0 1A cwx~th'on is" f,,4~ ~VeY~it 348D EMPOWERMENT AGAINST AIDS IN THE SEX INDUSTRY. De loaya, E. Antonio: Ferreira, F.; Rosario, S.; Bello, A.; Mensajeras de Salud, COIN, Dominican Republic. Qblectives In April 1993.the ALCS has begin a study In the 'mllleu 'of masculine prostitution In both Marrakesh and Casablanca. this was done In order to acquire a better knowledge of the matter. of the habits and the factors of vulnerabillty of these population. The prncipal objective of the study Is to set the foundations of a specific action of pr6vention for STD's and AIDS Method: 2 speakers go regulary(3times a week)on the spot of work-parcs, bars. cafes,the streetand have sessions of Intervlews.ellher individually or collecticely with male prostituts In wich the principal subject of discussion is AIDS and ways for its prevention. But also of daily problems of a social-econoric nature wlch could have a direct or Indirect effect In the altertion of behaviour. The speakers take advantage of these sessions to distribute free condoms and Informative brochures. Results: Emerging from the preliirdnaries of this action that out of the 66 people(the youngest of whom Is 14 and the oldest 34)lntevlewed. 71%prostitule themselves regulary as professionals,and the threat of AIDS Is perceived only by a small number of prostitutes 6% and only 4.5% say they use condoms systematically.The weakest usage of condoms is found with popular prostitutes (60%) for they are the less scolarized and the less exposed to classic medla.The main obstacles to the frequent use of condoms seem to be the Night cost and the attitud of the customer. CONCLUSION:it clearly appears with Initiation of this action( the first In an arabo-muslm country) that despite the difficulty of access and the slowness, it Is possible but especially an Imperative to touch the masculine prostitutes In the context of a direct contact In their own mrileu and respecting their codes and their reality. An Information campaign run in parallal to that with the prostitutes should be led with the customers. the avalbiity of the condom is also a determinlng factors. R mi nc tOL4l4N*PrSB. Eeiseela.i,cde.e. Las. aCowC. se. 0 ag tC S. Tcr:lr4ek tcdkc. k, Wwe"ak 8 ser' s ac C a % owca toEocco Noe.111 t.t+ D.0. ros'L-A. 1.1.W'f Objective: To describe female sex workers (FSW) choices and achievements for enhancing self-esteem, solidarity and health education for AIDS prevention through a self-produced monthly newsletter. Methods: A feasibility study with a random sample of 89 FSW group leaders in 3 cities was conducted in:June 1993. Requisiteness, format/ content prefferences and willingness to participate were measured. The newsletter, La Nueva Historia (LNH), is being distributed gratis and discussed in FSW group meetings in larger Dominican cities. Results: Most (89%) FSW greeted the idea as a dream come true. Illustrated educational messages combined with news, jokes, poems and songs were preffered. Male participation was welcome; 82% were willing to write and 93% to read it to illiterate FSW. Circulation increased from 2000-5000 in 7 issues, with wide FSW and clients participation, mass media acceptance and public opinion support. Discussion & conclusions: LNH is becoming a leadinq mediun for enhancing AIDS prevention among FSW, clients, sex business owners and lower class women. The next step includes advertizing and commercialization throughout the sex industry. De Maya, E. Antonio; c/Arzobispo Meriio 505, Z. Colonial, Sto. Dgo. Dam. Rep.; 681-1515; 681-4827(809) 350A TAT-MINUS HIV-l REPLICATION CHANG, LUNG-JI UNIVERSITY OF ALBERTA, EDMONTON ALBERTA, CANADA Tat is an essential gene for HIV replication. The transactivation function of Tat is markedly reduced when the virus succumbs to latency. Therefore, it is informative to have a replicationcompetent, tat-minus HIV system for the study of virus-host interaction during chronic state of infection. A modified LTR expressing high basal level of promoter activity was constructed using the CMV-IE gene enhancer element. Tiple stop codon/deletion mutations were made in the tat gene. Several tat-minus HIV-l clones were made and genetically and biochemically studied. Replication and spread of tat-minus HIV-1 were observed only if the promoter activity was increased by more than 3-4 folds, and virus spread was only seen in MT4, C8166 and AA2 cells. Biochemical comparison of the tat-minus and wild type virions showed no difference in the protein profiles. However, all the tat-minus HIV constructs exhibited a significant reduction in Gag protein processing in different types of cells. This study demonstrates that HIV-I replication can occur without Tat, but virus spread is markedly reduced without Tat. In addition, analysis of a series of tat-minus HIV-1 suggests a connection between Gag protein processing and Tat function. This system is valuable for exploring non-transactivating Tat function. It is also useful for studying Tat-independent virusvirus interaction. CHANG, LUNG-JI 1-41 MSB, Dept. of Medical Microbiology and Infectious Disease, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7 Tel. 403 492-9821 Fax 403 492-9828 4 CA) 0 349A MECHANISMS OF TNFB ACTIVATION BY HIV-1 TAT PROTEIN. Buonaguro L., Buonaguro Franca M., Ensoli B.* and Giraldo G. I.N.T., "Fond. G.Pascale", Naples, Italy;*N.C.I., N.I.H., Bethesda MD, USA. Objective: The increased expression of Tumor Necrosis Factor 13 (TNFB) by HIV-1 Tat protein has been recently shown to be mediated by a "TARlike" structure, present in the TNFJ3 promoter region, which functionally and structurally resembles the HIV-1 TAR (L. Buonaguro et al., L.Virol. 1992 and in press). The objective of this study is to identify the biomolecular mechanisms and the minimal nucleotide sequence, within the context of the TNF3 "TAR-like" structure, necessary for such activation. Methods: Single nucleotide mutations have been introduced by site directed mutagenesis technique. TNF3 mutants are tested both in a transient co-transfection assay, with tat-expressing plasmids, and in an in vitro transcription assay, using cellular extracts from Tatexpressing cell lines (Jurkat-Tat). Results: The deletion of "TAR-like" structure from the TNF3 promoter abolishes the Tat-induced transactivation, even in the presence of the NFkB and Spl binding sequences. In addition, preliminary results indicate a critical role played by the nucleotide sequences homologous to those required for Tat-transactivation in the HIV-1 TAR. The complete panel of effects induced by single nucleotide mutations on Tat-mediated transactivation of TNF3 gene expression will be presented. Discussion and Conclusion: The results obtained in the model TatTNFB3 interaction suggest a novel mechanism of gene regulation, very similar to that involved in the Tat-mediated activation of the HIV-1 LTR. This observation has a potential biological relevance, given that the TNFB3 has been shown to be involved in the pathogenesis of AIDSassociated Kaposi's Sarcoma. BUONAGURO, Franco M., Div. of Viral Oncology Ist. Naz. Tumori "Fond. Pascale", Cappella Cangiani 80131 Naples Italy Tel./Fax 39.81.5451276

Page  15 351A MOLECULAR EVIDENCE FOR A BLOCK IN REV-FUNCTION IN CHRONICALLY HIV-1 INFECTED HUMAN GLIAL CELLS. Neumann.Markus*9. Kleinschmidt, A.*, Erfle, V.*, Felber, B. K., Brack-Werner, R*. and Pavlakis, G. N *. *GSF-Institute for Molecular Virology, Neuherberg, 85758 Germany. NCI-FCRDC, ABL Basic Research Proeram Frederick, MD 21702, USA Objective: The cloned chronically tHIV- lllb-intected human astrocytoma cell line TH4-7-5 shows only a very low level virus production. The cloned cells contain a single integrated provirus with strong sequence similarity to the Illb-related molecular clone HXB2 and resembles a replication competent isolate. Virus production is stimulated with phorbol esters (PMA) maximally threefold. Virus can be rescued from the glial cells by cocultivation with permissive cell lines such as myelomonocytic cell line RC2A further supporting replicative capacity. The phenomenon of viral latency in glial cells was analyzed. Methods: Viral gene expression was measured employing a quantitative RT-PCR approach using previously cloned HIV-I cDNAs as standards. Intracellular Rev levels were increased by transfecting Revexpressing constructs into the cells or by PEG-mediated fusion with Rev-producing cells, followed by measuring intracellular p24ga8 concentration. Rev localization was determined by indirect immunofluorescence. Rev and RRE sequences from the virus rescued in myelomonocytic cell lines were PCRamplified, analyzed by direct sequencing and compared to the original isolate. Results: The TH4-7-5 virus shows a phenotype of "blocked-early-stage-expression," a strong predominance of the Rev-independent, multiply spliced class of transcripts. Addition of Rev lead to a twoto four fold increase in p24gag production as compared to a hundred fold increase in Rev-competent HeLa cells. Rev protein showed a strong cytoplasmic localization indicating a block in correct transport to the nuclei. The Rev and RRE sequences showed 100% identity after virus rescue, further supporting that cell specific factors account for a lack of Rev function. Discussion: Our data suggest a glial-cell determined posttranslational inhibition of Rev function thus blocking virus replication at an early stage. This could be an important factor for understanding viral latency in cells of the central nervous system. Research sponsored in part by the National Cancer Institute, DHHS, under contract no. NOl-CO-74101 with ABL. NEUMANN, Markus, ABL Basic Research Program, Frederick, MD 21702, USA. 301-846-1474/301-846-5991 (fax) 352A REV-DEPENDENCY OF THE EXPRESSION OF GAG, ENV, AND NEF GENES OF HIV-1 FURUTA A. Rika, Hatanaka M., Kawamura M., and Adachi A., Institute for Virus Research, Kyoto University, Kyoto, Japan Objective: To investigate the Rev-dependency of HIV-1 gene expression, various reporter systems were developed and used to monitor the expression of three major HIV-1 mRNAs. Methods: Various viral clones carrying bacterial CAT gene in various sites were constructed and used as reporters. Reporter clones were transfected into several cell lines, and CAT expression was monitored. Results: The expression of gag, env, and nef genes was found to be differentially dependent on the Rev protein. This difference was not determined by almost all cis-elements in gag,pol, and env genes, but was lost when reporter constructs containing minimum elements for Rev-responsiveness were used for experiments. Conclusions: Our results suggest that fundamental structure of HIV-1 mRNA such as splicing signals is critical for the apparent differential regulation of gene expression by the Rev transactivator. FURUTA A. Rika, Institute for Virus Research, Kyoto University, Kyoto, Japan Tel 075-751-4010 Fax 075-751-3995 354A ACCURATE DIFFERENTIAL QUANTIFICATION OF HIV-1 tat, rev, nef AND pot mRNAs IN PATIENTS WITH EARLY-STAGE INFECTION. L Mo,. M. O'Shaughnessy, A. Merzouki, M. Estable, N. Vellani, S. Cassol. British Columbia Centre for.Excellence in IIIV/AIDS, St. Pauls Ilospital, Vancouver, Canada Obljective: To develop rapid competitive RT-PCR assays for the precise quantification of IlIV-1 mRNA transcription subclassses in peripheral blood mononuclear cells (PBMCs) and lymphatic tissue of patients at different stages of IIIV-1 infection and to evaluate these assays as markers of disease status and progression. Methods: To determine the relative abundance of specific HIV-I regulatory and structural transcripts in clinical specimens, we adapted competitive RT-PCR to a multicolor fluorescence format that yields quantitative data automatically. Varying known amounts of a genetically engineered competitive template containing equivalent copies of fat, rev, nef and pol internal standards in a 1:1:1:1 ratio, were added to replicate aliquots of a clinical sample containing an unknown amount of IIIV-I target RNA. After reverse transcription and amplification with fluorescent primers, samples were loaded directly on a fluorescence DNA sequencer. Individual products were separated and quantified automatically by the system's software. The use of a composite competitive template ensures that the amount of competitor is kept constant for all targets and that the ratio of PCR products reflects their ratio in the original samples. Results: Using this ratio approach, we were able to reproducibly measure 2-fold differences in the relative abundance of individual HIV-1 regulatory (tat, rev, nef) and structural (pol) mRNAs starting with as little as 0.2 ng of total RNA. Hligh levels of regulatory transcripts (2 to 160 transcripts/100 PBMCs) were detected in all patients, irrespective of disease status, but the pattemrns of expression varied. Multiply spliced tat mRNA was the most abundant species in patients with early asymptomatic infection and CD4 counts >600, whereas, in patients with CDC stage IV disease, multiply spliced tat mRNA was frequently undetectable. Transition to late-stage disease was associated with a significant down-regulation of tat and a concomitant rise in the rev:tat ratio in association with increased production of full-length (pol) mRNA. Conclusions: Rigorous and differential in vivo quantification of HIV-1 mRNA transcripts will provide a powerful tool for the study of HIV-1 pathogenesis, prediction of disease progression and measurement of patient response to therapy. Theresa Mo. BC Centre for Excellence in HIV/AIDS 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z 1Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. 353A REV-INDEPENDENT EXPRESSION OF HIV-1 George N. Paviakis, R. Schneider, G. Nasioulas, A. Valentin, A. Zolotukhin, S. Song and B. K. Felber NCI-FCRDC, ABL Basic Research Program, Frederick, MD 21702, USA Objective: To produce subgenomic vectors and genomic HIV-1 clones that are expressed independent of the Rev protein and its binding site on the viral RNA (RRE). Methods: Multiple point mutations were introduced in HIV-1 regions characterized as responsible for low expression (inhibitory/instability regions, INS or CRS). In addition, positively-acting RNA sequences able to substitute for the Rev-RRE regulatory system were inserted into HIV-1 RNA. Results: Elimination of the negatively-acting sequences present on the viral RNA in the gag region resulted in Rev-independent gag expression and the formation of viral particles. Contrary to the wild-type gag RNA, the mutated gag was also efficiently expressed in mouse and chicken cells. Therefore, these vectors have been successful for the efficient production of gag in many different cell types. This allowed the generation of retroviral vectors expressing authentic and truncated gag sequences in mouse and human cells in the absence of any HIV-1 regulatory factors. Expression vectors producing transdominant or interfering gag molecules are tested for the level of inhibition of HIV expression after gene transfer. Discussion: The Rev protein binding to RRE viral RNA is an important regulatory mechanism found in all lentiviruses. It may play an important role in the life cycle of lentiviruses, the long-term association of the virus with the host and the generation of chronic persistent infection. The high level of gag expression in many cells will allow the study of immune response to gag and the generation of simple vectors for gene transfer and immunotherapy. Research sponsored by the National Cancer Institute, DHHS, under contract no. NOI-CO-74101 with ABL. Pavlakis, George N., ABL Basic Research Program, Frederick, MD 21702, USA. 301-846-1474/301-846-5991 (fax) (J1 CA) 4I

Page  16 - 355B ZIDOVUDINE IN EARLY ASYMPTOMATIC HIV DISEASE:A CONTROLLED TRIAL IN SUBJECTS WITH GREATER THAN 500 CD4+ CELLS/pL Paul Volberding, SW Lagakos, JM Grimes, DS Stein, and the ACTG 019 Team. UCSF, California, Harvard School of Public Health, Boston, MA, NIAID, Bethesda, Maryland USA We report the results of the CD4+ >500 stratum of protocol 019 of the AIDS Clinical Trials Group. This double-blind study enrolled subjects with asymptomatic HIV infection and entry CD4+ cell counts above 500 cells/pL. A total of 1650 subjects were randomized to receive placebo, a 500 mg daily dose of zidovudine, or a 1500 mg daily dose of zidovudine. Subjects were retained on their initial treatment assignments until their CD4+ counts dropped to below 500 cells/pL, at which time they were offered the 500 mg daily dose of zidovudine. Subjects were followed up for survival and the development of AIDS-defining clinical events for a median of approximately 5 years. This study compares three distinct treatment strategies for the management of asymptomatic subjects with CD4+ cell counts above 500 cells/ L with respect to the effects on the development of AIDS, survival, and CD4+ cell counts. Paul A. Volberding, MD; 995 Potrero Ave., San Francisco, CA 94110 USA; 415-476-4082; 415-476-9233. 356B THE POST-CONCORDE ERA: CURRENT TRENDS IN ZIDOVUDINE UTILIZATION. Michael V. O'Shaughnessvy Ilogg RS; Strathdee SA; Le TN; Campbell D; Schechter Martin T; Montaner JSG. University of British Columbia and the British Columbia Centre for Excellence in HIV/AIDS; Vancouver, CANADA. Objective: To assess the impact of the publication of the preliminary analysis of the Concorde Trial and its associated publicity on zidovudine (ZDV) utilization in British Columbia (BC). Mgethods: The distribution of anti-retroviral drugs in BC is free of charge to eligible individuals through the provincial Centre for Excellence in IIIV/AIDS. In order for physicians to prescribe ZDV, they must complete a patient enrollment which also serves as the drug prescription. This application is to be accompanied by a CD4 count. IIIV infected individuals are eligible to receive ZDV from the program if they have at least one CD4 count below 500/tL. There is no other legal source of ZDV in the province. Linear regressions were performed to test the changes in ZDV requests and CD4 counts at enrolment into the program for the first 9 months of 1993. Reported p-values are two-sided. 150 Results: A total of 644 new ZDV requests were received during S1993. As shown in the figure, a decline in the number of new ZDV 100 Concorde requests was noted from 84 in January, to 26 in December 1993. Similarly, the proportion of new requests that involved ZDV declined S5from 0.74 to 0.43 (p-=0.004). Current CD4 counts at enrollment were.50 0 available in 561 individuals (with the remaining counts pending). The 0,wsw w,, mean CD4 count among new patients prescribed ZDV fell from 1 2 3 45 6 7 8 9 101112 236~54 /mm3 in January to 197~43 /mm3 in December (p=0.0856). Month Concluslons: Our data demonstrate a significant decrease in the number of patients started on zidovudine in this province during 1993. Although the numbers are small, ZDV appears to be increasingly reserved for patients with more advanced disease as indicated by lower CD4 counts. It remains unclear whether this change was based on thoughtful interpretation of the preliminary results of the Concorde trial, or on more sensational and inappropriate media reports stating that ZDV is useless. Dr Michael V. O'Shaughnessy. BC Centre for Excellence in IIIVW/AIDS 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z 1Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. 358B RESULTS OF LONG TERM FOLLOW-UP OF A DOUBLE BLIND STUDY OF ddl vs T 358 CONTINUED AZT AMONG INDIVIDUALS WITH CD4s 200-500/mm3. NataliLRedy; JSG Montaner; M Wainberg; A Rachlis; J Gill; R Beaulieu; W Schlech; C Tsoukas; M O'Shaugbnessy; J Raboud; A Thorne; L Smaldone; Martin T Schechter et al. Canadian HIV Trials Network (Canada) and Bristol - Myers & Squibb (USA). OBJECTIVE: To assess the long-term clinical effect of ddl vs continued AZT In HIV+ individuals with CD4 counts of 200 to 500/mm3. METHODS: Patients who had received > 6 months of prior AZT therapy (_ 500 mg/day) were randomized to standard doses of AZT or ddl and followed monthly with clinical and laboratory evaluations until 12/10/92. Clinical status was updated as of 1/12/93; an event was prospectively defined as the development of a new AIDS defining Illness or death. RESULTS: Out of 245 patients randomized, 66% were asymptomatic, 30% had ARC and 4% had AIDS. Mean baseline CD4 count was 324 and 337 (p=0.20) for ddl and AZT treated patients, respectively. The table below demonstrates that a change to ddl led to a sustained clinical benefit (p=0.020), as defined by the product-limit estimates of the probability of surviving free of an event. Change to ddl also led to a significant (p_<0.01), early (<2 weeks) and persistent (at least 36 weeks) increase in CD4s in the order of 50 cells. wk dd AZT While musculoskeletal pain (p<0.001), anemia (p=0.008), 64.97.92 thrombocytopenla (p<0.001), were more commonly associated 0 97.90with AZT therapy, hyperamylasemia (p<0.001), liver function test 80.96.90 abnormalities (p<0.001), gastrointestinal upset (p=.037), memory 96.93.89 loss (p=.031), glucose Intolerance (p=.012), hyperuricemia 112.90.78 (p=.004) and hypertrlglycerldemla (p=.017) were more 128.88.73 commonly associated with ddl therapy. CONCLUSION: Our results demonstrate that an early change to ddl led to a long term benefit in clinical outcomes; this remained for at least 128 weeks, over one year after the termination of the randomized portion of the study. Both medications were generally well tolerated in the context of this study. Ms.Natali Ruedy. Canadian IIIV Trials Network 200-1033 Davie Street, Vancouver, BC, CANADA V6E 1M7. Telephone (604)-631-5036; Facsimile (604)-631-5210. CA) 00 U'I 357B ZIDOVUDINE USE IN 104 PREGNANCIES: BIRTH DEFECTS. Kumar, Mona Rachana, Hughes, P. F., Khurranna, A. Dept. Obs/Gyn., FMHS, UAE University, Al Ain, UAE. Objective: To detect any major teratogenic effect of Zidovudine a) when administered to pregnant women infected with the human immunodeficiency virus; b) to analyse the cumulative data on birth defects to detect any possible trend in number or type of defect. Methods: Subject population comprised a group of 104 HIV-1 infected Indian pregnant women who had been exposed to Zidovudine at some stage of their pregnancies. Pregnancy outcomes were categorised as a) outcome with birth defects b) outcome without birth defects. Birth defects were defined as per CDC Guidelines for Birth Defects (1982). Results: In a series of 104 cases of intentional or inadvertent use of Zidovudine at different stages of pregnancy, there were 8 spontaneous abortions, 8 therapeutic terminations and 8 cases of foetal abnormality occurring in 88 women who delivered. Discussion and Conclusion: Analysis and correlation of antenatal data and drug therapy with individual cases failed to show any specific abnormality that could reasonably be attributed to Zidovudine therapy. While not proving safety, this data lends tenuous support to the use of this agent. KUMAR, Mona R., Dept. Obs/Gyn., FMHS, UAE Univ., Al Ain, UAE. Phone: (971) 3 669584, Fax: (971) 3 657134.

Page  17 359B VIROLOGIC RESPONSE TO DDI FOLLOWING PROLONGED AZT RX. Mayes. DL; Wagner KF; Chung RCY; Vahey MT; Weislow OS; Zhou S; Burke DS and RV43 STUDY GROUP. MMCARR, Rockville, MD., USA. Obi eis: To determine the virologic impact for patients (pts) with advanced HIV disease who switch to didanosine (ddl) after receiving prolonged zidovudine (AZT) monotherapy (rx) and whether any parameters, prior to the change in rx are predictive of the resulting change in plasma HIV RNA (pRNA). Methods: 22 pts in an observational cohort followed for 2 to 3 years with intensive virology have switched from AZT to ddl. Pts were evaluated every 3 months with CD4 counts and drug susceptibility of their HIV isolates. Primary HIV isolates were obtained on donor PBMC. Drug susceptibility testing was performed using the ACTG/DoD consensus assay. Mutations at RT codon 215 were detected using nested PCR or RT-PCR. Viral burden (pRNA) was assessed using RT-PCR of pt plasma. SI phenotype was determined on MT-2 cells.Virologic parameters were evaluated for HIV isolates obtained pre/post drug change. Results: The patients Initiated AZT at a CD4 cell count of 255~91 and switched to ddl at a CD4 cell count of 96~89 after 23+~9 months of AZT rx. pRNA decreased by > 0.5 loglo in 6 pts (27%), remained unchanged (pRNA A < ~ 0.5 loglo) in 10 pts (46%) and increased _0.5 logio in 6 pts (27%). None of the virologic parameters evaluated (ICso for AZT or ddl, mutations at RT codon 215, SI phenotype, pRNA, or p24 antigen) prior to initiating ddl therapy were predictive of the pRNA responses seen. Conclusions: One third of patients with advanced HIV disease experienced a significant decline in HIV pRNA (>. 0.5 loglo) during the 6 months following the switch from AZT to ddl. No virologic parameters evaluated were predictive of the individual responses observed. Douglas L. Mayers, M.D. Division of Retrovirology, Walter Reed Army Institute of Research, 13 Taft Court, Suite 201, Rockville, MD., USA 20850 Phone: (301)-295-6411; FAX: (301)-295-6422 361C Mobilization of Students for STDs/AIDS Prevention in Universities in Cameroon. Ella, M.'; Boupda, A.'; Fouda, A.'; Debuysscher, R3; 1. AIDSCAP, Cameroon; 2. Ministry of Health, Cameroon; 3. AIDSCAP Africa Regional Office, Kenya. Objective: To identify appropriate strategies to raise HIV AIDS awareness and promote effective condom use and behavioral change among students in five universities in Cameroon. Methods: Initiated May 1993: 1) Program for the prevention of the sexual transmission of HIV I AIDS in Yaounde universities implemented; 2) KAPB survey completed on a sample of 1,000 students selected from universities in Douala and Yaounde; 3) Five focus group discussions conducted in Douala and Yaounde; 4) Peer Health Educator (PHE) training seminars held for students and health personnel at university health centers; 5) Condom outlets identified around university campuses; and 6) Educational sessions planned. Results: To date: 1) three training seminars held for PHEs; 2) sixty PHEs trained in Yaounde and applications accepted for 100 more; 3) fifteen health care personnel attended a training seminar in information, education and communication (IEC) and counseling; 4) forty-eight educational sessions conducted on campuses between October and December 1993; 5) More than 1,600 students sensitized during formal and one to one talk sessions; 6) One hundred and five condom outlets identified around campuses -- an increase of 50%; 7) approximately 138,798 condoms sold between November and December 1993 -- an increase of 30%; and 8) an increase in educational materials distributed by PHEs. Conclusions: The follow up of PHE programs in Yaounde universities revealed that student PHEs are an important and effective means to reach their peers. The increasing number of students requesing information or applying to become P HEs shows that it is necessary to educate students by motivating existing student groups to form sustainable educational support groups. Also, the increase in the number of condoms sold around the campuses within the last two months indicates real progress in achieving behavior change. M. Ella; tel: 703.516.9779; fax: 703.516.9781 FHI/USA 3 60B FIVE YEAR FOLLOW-UP OF A PHASE-I STUDY OF DIDANOSINE (DDI) IN PATIENTS (PTS) WITH ADVANCED HIV INFECTION Robert Yarchoan. B-Y Nguyen, KM Wyvill, DJ Venzon, JM Pluda, H Mitsuya, and S Broder, NCI, Bethesda MD, USA Objective: To analyze the long-term course and assess predictors of survival in pts with advanced HIV infection entered on an escalating dose phase-I study of ddl. Methods: 72 pts were entered onto a study of ddl starting in July, 1988. Some were later given the option of switching to combination AZT/ddl. The cohort was followed until July, 1994. Follow-up is available on all pts who entered. Results: All pts had <400 CD4 cells/mm3 at entry (median 50 CD4 cells/mm3); 29 pts had AIDS at entry. For pts who entered prior to a diagnosis of AIDS, the median time to AIDS or death was 28 months (95% CI 17-41 mos). The median survival for all pts was 28 mos (95% CI 23-46 mos). Baseline CD4 counts, CD8 counts, hemoglobin, lymphocytes, sedimentation rates, a diagnosis of AIDS, and fever were (in decreasing order) predictive of survival. CD4 increases above baseline at 3 mos and 1 yr were significant predictors of survival when a correction was made for entry CD4 count. Pts who entered with 100-300 CD4 cells/mm3 had an estimated 80% survival after 4 yr. CD4 counts were sustained above baseline for 3 yrs in 5/16 evaluable pts who entered with 100-300 CD4 cells/mm3. Major toxicities were pancreatitis (6 pts) and peripheral neuropathy (11 pts). Discussion and Conclusions: Therapy with ddl can be tolerated for more than 4 yr in some pts and may have particular long-term utility in pts with moderately advanced immunosuppression (100-300 CD4 cells/mm3). YARCHOAN, Robert, 10/12N226, NIH, Bethesda MD 20892, USA. Telephone (301) 496-0328; Fax (301) 402-3645. 362C HIV/STD PREVENTION AMONG SEX WORKERS IN NICARAGUA Rivera Rosaura', M. Egger2, M. Paniagual, K. Volken', G. Davey Smith3 UNAN Le6n, Nicaragua'; Univ. of Bene, SwitzerfancP, Univ. of Glasgow, U.K.3 Objective: To develop and to evaluate an HIV/AIDS and STD prevention programme among sex workers in Corinto, the main port on the Pacific coast of Nicaragua. Methods: At baseline, levels of knowledge, attitudes and practices and STD rates were assessed. Condom use was promoted in workshops and via comic strips, posters and leaflets directed both at sex workers with clients. Free medical advice and condoms were provided at the local health center. A follow-up survey was started one year after initiation of the intervention. Results: Over time, the number of women participating increased. Fifty women were examined at baseline, and 55 up to January 1994 in the ongoing follow-up survey. Mean age was 25 years, over 90% of women originate from Corinto. Twenty-one women were examined on both occasions whose results were as follows: Knowledge & Practices: Baseline Follow-up Condom prevents transmission 45 % 91 % Always carries condom 73 % 100 % Condom use with last client 45 % 82 % Sexually transmitted infections: T. vaginalis 29 % 14 % N. gonorrhoea 12% 12 % C. trachomatis 15% 5% Conclusions: The data indicate that health education and condom promotion may have lead to increased condom use, decreased STD rates and decreased HIV/AIDS risk. Rosaura Rivera c/o Egger, Dept. of Social and Preventive Medicine, University of Berne, Finkenhubelweg 11, CH-3012 Berne, Switzerland, Fax +41 31 631 35 20 CA) 0

Page  18 = 363C OBJECTIVE: METHODS: CONDOM USE AND THE INCIDENCE OF HIV/STD AMONG STUDENTS. Kosia, A., STD Unit, Connaught Hospital, P.O. Box 1111, leetown, Sierra Leone. To determine the effectiveness of condom promotion and use among students. In June 1991, 450 college students registered in the STD Clinic and consented to participate in the study. They were educated on HIV/STD with emphasis on condom promotion, it's proper use and disposal. Base line level of condom acceptability and HIV/STD incidence were determined. 425 HIV negative students were followed-up for 18 months. Incidence rates of HIV/STD as indicators of effectiveness of condom use were recorded every six months. 364C RESULT: The incidence of HIV changed from 5.6% to 1.1% while those of STD from 15.5% to 4%. Condom acceptability increased from 20% to 80%. CONCLUSION: Effective condom promotion, proper use and disposal is a valuable intervention in the control of HIV/STD. It should be promoted among other high risk behaviours. Dr. Andrew M. Kosia, C/O Connaught Hospital, Freetown, Sierra Leone (22) 222002. 365C Introduction of Partner Referral & Treatment for Control of Sexually Transmitted Diseases in a Poor Haitian Community. Desormeaux, Julio.; Behets, F.2; Adrien, M.'; Dallabetta, G.3; Davis, H.'; Hoffman, 1.2; Cohen, M.2; Boulos, R.1. 1. Centres pour le Developpement et la Sante, 2. University of North Carolina at Chapel Hill, 3. Family Health International. Objective: To evaluate acceptability and strategies of partner referral and treatment (PR&T) as part of a sexually transmitted disease (STD) control program for antenatal (AN) women in a slum-based Haitian community. Methods: Focus group discussions were held with AN women and with male partners separately to eva uate STD knowledge and acceptability of AN STD control with PR&T. Subsequently, each detected AN STD patient was asked to refer partner(s) and asked if she agreed with tracing by community health workers (CHWs), if necessary. Partners presenting at the AN clinic were randomized for 1) prompt free treatment or 2) sent to the Centres pour le Developpement et la Sante curative center (CC) at usual cost. Men who were sent, but did not attend, were traced and interviewed. Results: Antenatal women and partners had some STD knowledge and endorsed the proposed STD program and PR&T. CHW referral was acceptable to 73% of STD patients and 47% of partners. For 384 treated STD patients, 331 reachable partners were named, 101 (30%) males presented referred by index; 38 (11%) were referred by CHWs. No adverse psychosocial effects were described. Fifty percent were lost. This was due to cost, time loss, self-reported "neglect," etc., rather than to distance or perceived quality of care. Conclusions: In this community, educational campaigns should enhance STD knowledge and prevention, emphasize the curable nature of non-HIV STDs, and expand understanding of vertical transmission. PR&T is possible in Haiti, and women were more successful than CHWs; however, the yield of both may be increased. Partners need to be treated promptly as they present. SYPHILIS SCREENING OF PREGNANT WOMEN IN KABAROLE DISTRICT, UGANDA. G.Sahlnmueller, W.Kipp, Deutsche Gesell.fTech. Zusammenarb.(GTZ), C.Masheiia, Ministry of Health, Fort Portal, Uganda Objective: A screening programme of syphilis was introduced as part of a district wide STD control programme for pregnant women, attending antenatal clinics in hospitals and peripheral health stations in Kabarole district, western Uganda. The aim of this intervention was to increase coverage of STD treatment in the district and to reduce the risk of HIV transmission. Methods: in three hospitals and 18 peripheral health stations health workers were trained to perform the Rapid Plasma Reagent (RPR) Test. Pregnant women, who came for routine antenatal services, were tested. External quality control of RPR testing was continuously done in the health units under supervision of the reference laboratory in the district capital Fort Portal. Mothers found to be RPR positive were treated with benzathin-penicillin and their male partners asked to come for treatment. Results: Over 90% of the pregnant women, attending antenatal clinics used the services. In 1993 a total of 7121 women were tested, 18% (1285) reacted positive and were treated. 78% of the RPR tests were correctly done by health workers. Wrong test results were mainly false positive. There was no difference of the results between qualified and non qualified health staff. Only 20% of the partners of infected women came for treatment. The screening programme covered 18% of expected pregnant women in the district in, 1993. Discussion and conclusion: It is feasible to establish a syphilis screening programme in rural areas in Uganda, aiming at covering the whole population. A critical factor for the programme is the low output of grossly underpaid health workers, use of STD drugs for other purposes and the lacking interest of male partners to visit the health station. A strong supervision system must be in place to guarantee the necessary quality of the work and to increase the morale of health workers involved. The cooperation of the pregnant women was excellent. J ' _.I T <tV r 7'( 9 c -'7 0) CA) 0) 0) C) 366C DRUGS FOR STDs IN DEVELOPING COUNTRIES Nieto Jorge', Casanova J.*, Fransen L.** *AIDS Task Force, C.E.C.,Brussels,Belgium **European Community, Brussels,Belgium Introduction: Today the lack of STD drugs is a major barrier in the control of STDs and HIV. The EC HIV/AIOS Programme has, together with other partners (WHO, UNICEF, etc.), actively developed interventions to make possible wide scale provision and use of STD drugs. Objective: With its interventions the EC H IV/AIDS Programme is demonstrating that inclusion of STD drugs in the national drug policy, and essential drug procurement in developing countries, makes these drugs more accessible, available, less costly and the intervention therefore is feasible. Methods: To reach the objective the following steps are taken: l)Analyses of the accessibility, the cost, the prescription practices and the availability of STO drugs in the country; review of the financial and human resources; assessment of the demand for STD drugs in the public and the private sector; analyses of the needs for treatment of STO of the population concerned. 2)Review of the different options to improve STD drugs availability and accessibility. 3)Asses the viability of each option at short and long term and to propose an optimal solution. 4)T1O support the implementation of the intervention, and the monitoring and evaluation of the results. Results: The EC HIV/AIDS Control Programme has implemented actions following this methodology in more than 24 developing countries in Africa, latin-America, and Asia. National STO Programmes have been created or strengthened if alredy existing, STD drugs have been included in the Essential Drug Policy and Essential Drug List of the countries After the first year of intervention prices for treatment have been ranging from 1.7 ECU in Mozambique to 3.7 ECU in Botswana. The availability and accessibility of STD drugs has been increased in the countries, specially in the areas of intervention. Depending on the policy of each country, some cost recoverynmechanisms have been for STD drug have been installed in the areas of intervention. Conclusion: The EC HIV/AIDS Programme has demonstrated over the last years that interventions focused on the provision of STD drugs are possible on a short term basis but longer term sustainable and integrated options are needed. The EC HIV/AIDS Programme has selected to support this approach through inclusion of STD drugs in the essenti als drug policy and procurement. The examples discussed demonstrate this option is feasible, cost-effective and plays a major role in making STD and HIV control a reality. Julio Desormeaux; tel: 703.516.9779; fax: 703.516.9781 FHI/USA NIETO,Jorge,AIDS Task Force,Brussels,Belgium Phone 32-2-2454390 Fax 32-2-2156747

Page  19 367D DEVELOPING AND IMPLEMENTING CURRICULA TO ADDRESS HIV/AIDS IN SCHOOLS IN 12 ASIAN COUNTRIES 368D HOW TO SPEAK ABOUTSEXUALIY IN THE SCHOOL IN MORROCO? Latifa IMANE, S.EL BERNOUSSI F.HADDAD, M.DERKAOUL R.VERBEKE * Association Marocalne de LUtte Contre le SIDA, Morocco. Authors: Barth6s, Anna Maria'; Baldo, M.2; Bahri, S. '; Jones, J.T.'; Chowdury, S.4 ' UNESCO, Paris, France; 2 WHO/GPA, Geneva, Switzerland; ' WHO/HED, Geneva, Switzerland; UNESCO, New Delhi, India Objective: To assist in the development and/or implementation of relevant national school curricula for the prevention of HIV/AIDS among young people in twelve Asian countries. Method: In January 1994 in New Delhi, UNESCO, in collaboration with WHO, conducted an Asian Planning Seminar on AIDS and Education within the School System. Over 50 persons from MOEs and MOHs participated in the seminar. Small group discussions between persons responsible for curriculum development were used to identify ways of developing relevant curricula for the prevention of AIDS. Results: AIDS education promotes responsible behaviour that can prevent the transmission of HIV and other STDs. In order to be efficient, AIDS education should be integrated into an appropriate topic and should be provided before the onset of sexual activity and before school dropout age. Discussion and Conclusions: The school system has an important role to play in education for the prevention of AIDS among young people, by promoting responsible behaviour that prevents the transmission of HIV. A curriculum dedicated to HIV/AIDS legitimizes and gives status and respectability to AIDS education. It is an important factor that encourages nation-wide provision of AIDS education to young people. Anna Maria Barth6s, UNESCO, 7 Place de Fontenoy, 75015 Paris, France Tel: 33-1-45-68-10-00; Fax: 33-1-47-83-27-10 Oblectilves: In April 1993,the ALCS has begin a KAP study among youth (15 /20 years) In the public school s In five regions in Morroco. The main objective of this study is to define the contents and the presentation, of a school booklet on AIDS and STD's prevention. Methods: The study is conducted among three populations: students, parents and teachers The interwlers have "focus group" sessions and individual Interwlews In which the main subject is "how to speak and prevent AIDS and STD's among youth?". An other subject is " how to speak about sexuality In school In arab and musllm context?" Results: Emerging from the preliminaries of this action that there is lack of communication between parents and youth specially on the sexual issues. Youth in their majority complain about the absence of sexual education on the school program. They feel very concerned by AIDS as adolescents and prefer to be Informed at early age. The discourse of prevention should be focused on scientifique facts and should respect the religious and the soclo cultural context of Morocco. The promotion of condom Is a very dellcat point. For the majority of parents, the diiscourse of prevention should be focused on abstinence and religious facts only, but the students want to receive a clear and realstic information. CONCLUSION: It clearly appears with initiation of this action( the first in an arab-musllm country) that despite the opposition of some muslim fondamentalist specially among teachers, the majority of parents, youth and teachers nterwleved have expressed the necessity to devellop a school program on sexual issues set on the new reality of Morocco. Latifa IMANE, ALCS, Facult4 de M6declIne, Casablanca,Morocco Phone:212(2)474040 Fax:293188 369D AIDS EDUCATION IN SCHOOLS - MULEY, DIWAKAR SITARAM, National AIDS Control Organization Larivee C., World Health Organization, New Delhi, India 370D DEVELOPING AIDS POLICY IN AN UNIVERSITY SETTING: A CANADIAN PERSPECITVE, Cox, Beverlee Ann; Professor, The University of Western Ontario; Faculty of Nursing, London, Ontario, Canada Objective: To produce a culturally sensitive co-curricular package on AIDS/STD, sex and sexuality for uniform use throughout India in schools. Methods: Through a Workshop, general framework of the package was developed. A core group from NACO, NCERT facility, WHO and UNICEF repackaged and edited materials collected from different sources. The package is being reviewed and field tested. Non-course approach is the major strategy. Results: NACO - NCERT package will be translated into more than 10 languages for adaptation by states. It will also trigger AIDS projects in these states. Discussion and Conclusions: Efforts to introduce AIDS education in school curricula is bedeviled by strongly held differences about what is and is not acceptable. Cultural and ethnic heterogeneity of Indian population results in divergent religious and secular viewpoints. Students desire and seek authentic information on sex and sexuality. But school curricula has not so far responded to these needs adequately. In India advocacy for AIDS education coincides with that of sex education. This makes issues still more complex. Though consensus is gradually emerging in favour of sex education, school curricula remains conservative and hesitant to incorporate sensitive issues like use of condoms. NACO with NCERT is developing a co-curricular package to address this problem. MULEY, Diwakar Sitaram, National AIDS Control Organization, Ministry of Health & Family Welfare, New Delhi, India Telephone (91)(11)(331 7519); Telefax (91)(11)(332 7660) OBJECTIVES - The many issues facing those concerned with the AIDS epidemic in campus settings are political, social, ethical, and legal in nature; these issues must be addressed in the formulation of an official policy that can meet the requirements of an university environment. The central objective described in this paper concerns the process and outcome of policy development in one particular Canadian university, The University of Western Ontario, over the past five years, and its end result, as compared with other policies across Canada. METHODS - The author describes an interdisciplinary process involving law, medicine, nursing, and applied health sciences on a Task Force that dealt with these major themes: l)confidentiality; 2)nondiscrimination; 3)mandatory vs. voluntary testing; 4)legal liability and responsibility; and 5)the need for ongoing education for the campus community. While the extended dialogue amongst the Task Force members was the major method for developing policy, consultation with experts in many fields was also utilized, along with ongoing discussion with professionals in clinical settings. RESULTS - The outcome of the Task Force deliberations was a Policy statement of 12 guidelines concerning the major issues listed above. This statement was unanimously adopted by the university's Board of Governors and has since been in a process of implementation. The Task Force continues to function as an advisory body on AIDS-related matters. DISCUSSION AND CONCLUSIONS - The policy development exercise described herein led to the formulation of an institutional framework for dealing with the complex, multi-faceted issues surrounding AIDS in a campus community. This policy represents a comprehensive document that is at the forefront of such statements by universities in Canada at this time. Dr. Beverlee Ann Cox, Faculty of Nursing, The University of Western Ontario, London, Ont. 519-661-3396 Tel Fa: 519-661-3928 Canada CA) 0) CA) 04 0 0

Page  20 371D ASSESSING EFFECTIVENESS OF SCHOOL-BASED HIV PREVENTION. D. Rug,*, J. Collins*, E. Sogolow*, S. Banspach*, A. Gilliam*, D. Kirby# *CDC, Atlanta, GA., USA; #ETR, Santa Cruz, CA., USA Objective: Since 1987, CDC has conducted evaluation research to determine "what works" in school-based programs designed to reduce HIV risk behaviors. Methods: Assessing intervention effectiveness involves three components: 1) short and long-term randomized studies, 2) synthesis of research literature using expert consensus, and 3) meta-analysis of an HIV education database. HIV-related risk behaviors are measured as the primary outcome. Subjects are youth ages 10-19 from various racial/ethnic groups who participate in school HIV programs across the U.S. Results: Data summarized across all three components show that well-designed programs: 1) do not hasten onset nor increase frequency of sexual intercourse, 2) can delay onset of intercourse among previously abstinent youth, and 3) can increase condom use among intervention youth compared to youth in comparison conditions (p <.05). Characteristics of effective programs include: 1) basis in social learning theory, 2) narrow focus on a specific behavior, 3) experiential activities to personalize risk information, 4) instruction on resisting negative social influences, 5)reinforcement of postive peer norms and values, and 6) activities to increase skills and confidence. Conclusions: Scientific evidence from multiple sources shows that school-based programs can be effective in reducing behaviors that result in HIV infection. It is essential that such findings inform the design of HIV prevention programs for youth. Rugg, Deborah L., CDC, 4770 Buford Highway, NE, MS-K33, Atlanta, GA, 30341-3724, (404) 488-5331, Fax (404) 488-5972 3 72D THE EFFECTS OF A SEXUALITY/AIDS/STD EDUCATIONAL PROGRAM ON KNOWLEDGE, ATTITUDES AND BEHAVIOURS OF CANADIAN GRADE 9 STUDENTS: A 3-YEAR STUDY. King. Alan J.C.", Warren, W.K.-, Beazley, Richard P.'; & Wright, N.P."; "SPEG, Queen's University, Kingston, Ontario; *Dalhousie University, Halifax. Background: In this presentation, the final results from a three-phase evaluation of a 20-hour educational program, called Skills for Heakhy Relationships (SHR) are presented. The program was based on a behavioural change model emphasizing skill development, responsible attitudes and motivational supports through peer msodeling and parental involvement. Objectives: The program was designed to meet the following objectives: participants will demonstrate significant changes in (1) knowledge of AIDS and other STD, (2) attitudes related to healthy sexuality, homosexuality, PLWAs and to condoms, (3) assertiveness and other communication skills, (4) sexual behaviours and, (5) condom use. Methods: A quasi-experimental, time-series design with a retest and three similar posttests were used. They were administered in four Canadian provinces to approximately 2000 students each in a demonstration group and in a comparison group. T-tests, chi-squares and analysis of variance were used to examine differences on pre- and posttest responses and regression analyses were done to predict sexual behaviours. Results: Demonstration group perceptions of SHR's impact on assertiveness and comfort in talking about condoms two years later were extremely positive. Their significant changes in AIDS knowledge (p <.001) and in some attitudes are other indicators that the program had a positive impact. Significant differences were found favouring the demonstration group with respect to individual behavioural intent and tolerance items. At Posttest 3, SHR seemed to have little practical effect on delaying vaginal intercourse. The sexually active males in the demonstration group were more likely to use condoms by PT2 but not by PT3. The less positive behavioural results after the second year suggest that SHR's content and approach need to be reinforced more than once, especially when the sexually active students also tend to engage in other risk behaviours and present a greater challenge to reach. CA) CA) 44h Dr. Alan J.C. KIing, social Program Evaluation Group Duncan McArthur Hall, Queens University, Kingston, Ontario, Cza;iadca. Tel: (G13) 545-G255; FAX: ((13)545-6584 MECHANISM OF ddC RESISTANCE OF THE K65R SUBSTITUTION IN HIV-1 REVERSE TRANSCRIPTASE. Zhengxian Gu, Eric Arts, Xuguang Li, Michael Parniak and Mark A. WainberQ, McGill AIDS Centre-Jewish General Hospital, Montreal, Qc, Canada 373A ROLE OF THE THUMB SUBDOMAIN OF HIV-I REVERSE TRANSCRIPTASE ANALYZED BY AN INHIBITORY MONOCLONAL ANTIBODY Chiba, Joe1, Yamaguchi, A.1, Wei, Z.1, Ohba, H.1, Saito, A.2, Shinagawa, H2 and Kurata, T.3: Dept. of Bio. Sci. and Technol., Science Univ. of Tokyo1, Res.Inst. for Microbial Dis., Osaka Univ2. and Dept. of Pathol., NIH3, Japan 374A Objective: Recent crystallographic studies have provided a much more refined structure of HIV-1 reverse transcripase (RT) molecule. However, the organization of enzymatically active motifes or roles of each subdomain of the enzyme molecule is still not completely understood. We report here function of the epitope on HIV-1 RT molecule recognized by the 7C4 monoclonal antibody (7C4) which completely inhibits RNAdependent DNA polymerase activity of RT. Methods: Epitope mapping was carried out by testing reactivity of 7C4 either with various trancated RT molecules expressed in E. coli cells or with synthetic peptides of defined sequences from the HIV-1 pol gene. Kinetic measurements of the RNA-dependent DNA polymetase activity of RT were carried out using poly(rA) oligo(dT) as template/primer and dTTP as deoxynucleoside triphospate substrate after incubation of the enzyme with varying concentrations of purified 7C4. Results: Epitope mapping of 7C4 revealed that the eptope existed on the thumb subdomain of RT molecule which had been determined by previous crystallographic analysis. Binding of equimolar 7C4 with RT resulted in complete inhibition of polymerase activity. The inhibition by Fab fragment of 7C4 was competitive-type with respect to the template/primer and mixed-type with respect to the deoxynucleoside substrate. Discussion and Conclusion: The epitope on thumb subdomain of RT, which recognized by 7C4 antibody, would be, or be closely located at, the template/primer binding site. Substitution mutations K65R and M184V in the HIV-1 RT coding region have been shown to be responsible for HIV-1 resistance to each of ddC, ddl and 3TC. We introduced these two mutations into both p66 and p51 subunits of the HXB2 HIV RT gene by site-directed mutagenesis and used an E.coli expression system to generate recombinant p66/p51 heterodimer RT proteins that were purified to >95% by FPLC. Steady-state kinetic parameters for each of Km and Kcat were determined for wild-type (wt) and mutant HIV-1 RTs under both processive and non-processive conditions using the template/ primer poly(rA)-(dT)12-18 and poly(rI)*(dC)12-18. A 2.2-fold increased Km value was observed for dCTP in the case of RT compared to wt RT. No significant changes were seen in Kcat for dCTP, or for either Km or Kcat for dTTP, between wt and mutant K65R RT. Inhibtion assays showed that the Ki value of K65R was 8.5-fold increased for ddCTP and 3.3-fold increased for ddTTP, in comparison to wt enzyme. However, ddCTP did not exert competitive inhibition effects on poly(rA). (dt) template/primer and dTTP substrates. We also assayed for incorporation of and chain termination by ddCTP, 3TC-TP, ddATP and AZT-TP during the synthesis of (-) strong-stop DNA using in vitro assays. Recombinant HIV RTs containing only K65R or both the K65R and M184V mutations yielded significantly more (-) strong-stop product in the presence of ddCTP, 3TC-TP and ddATP than did wt HIV-1 RT. A slight decrease in degree of chain termination was observed with each of AZT-TP and ddITP. Altered nucleoside-analog recognition and chain termination are likely involved in drug resistance mechanisms for K65R. Mark A. Wainberg, McGill AIDS Centre-Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Qc, Canada H3T 1E2 Tel: (514) 340-8260, FAX: (514) 340-7537 CHIBA, Joe, Dept. of Biol. Sci. and Technol., Science Univ. of Tokyo Noda, Chiba 278, Japan Tel. (81)-471-24-1501; Fax (81)-471-25-1841

Page  21 375A HIV-1 RESISTANCE GENOTYPES IN THE PLASMA RNA AND PBMC DNA DURING ZDV/DDI COMBINATION THERAPY. Eastman, P. Scott*, Boyer E*, Unles M*, Kolberg J*, Mole L** and Holodniy M**. *Chiron Corp., Emeryville, CA and **VA Medical Center, Palo Alto, CA, USA 376A CHARACTERISATION OF HIV ISOLATED FROM PATIENTS ENROLLED IN THE ALPHA DDI TRIAL. Zheng NN, Simasathiansophan S, McQueen PW, Hurren L, Evans L, Delaney SF, Penny R, Cooper DA. Uni NSW & St Vincents Hospital, Sydney, Australia. Objective: Evaluation of Zidovudine (ZDV) and Didanosine (ddl) resistance mutations in the reverse transcriptase gene (RT) of plasma HIV RNA and peripheral blood mononuclear cell (PBMC) proviral DNA with respect to viral and proviral load and CD4 counts after the addition of ddl in subjects who have received longterm ZDV therapy. Methds: Ten patients with longterm ZDV experience (mean 27 months) had ddl (400 mg/day) added to their regimen. Plasma and PBMCs were collected at baseline andl serially for up to 12 months after addition of ddl. Plasma virus RNA and PBMC proviral DNA were PCR amplified and changes in the codon 215, 74 and 135 natant (MUT) and wild-type (WT) populations were determined by differential hybridization using either a nonisotopic ELISA type format or an isotopic dot blot format. Plasma vinrus RNA was quantitated by the branched DNA (bDNA) Chiron QuantiplexTM HIV RNA assay. Proviral DNA was quantitated by a PCR-ELISA assay. Results: While ZDV genotypic resistance at codon 215 was observed in both the plasma RNA and PBMC proviral DNA of all subjects at baseline, dre in a decrease in plasma virus RNA was observed only in those patients with significant levels of WT RNA. No changes in proviral DNA levels were observed. After addition of ddl, relative levels of codon 215 WT RNA declined to undetectable levels, however, no significant change in the relative levels of codon 215 MUT and WT proviral DNA was observed. By 12 months of ddl therapy viral load again increased and CD4 cowrts declined in the responding patients. Genotypic resistance to ddl at codon 74 was not observed. Mutations at codon 135 were present prior to addition of ddl in both responders and nonresponders. Discussion and Conclusions: The addition of ddl results in a decline of HIV RNA levels and codon 215 WT RNA in the plasma of some patients. Proviral load and codon 215 MUT and WT proviral populations appear not to be affected. Furthermore, mutation at sites other than codons 74 and 135 must be responsible for the observed resistance to ZDV/ddl combination therapy. EASTMAN, P. Scott, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 USA; Phone 510/601-3048; Fax 510/655-7733 377A HIV-1 DEVELOPS A SET OF NOVEL MUTATIONS CONFERRING MULTIDRUG RESISTANCE DURING COMBINATION CHEMOTHERAPY. Shirasaka. Takuma: Kavllck, MF; Goo, W-Y: Kojima, E; Yarchoan, R; and Mitsuya, H. National Cancer institute, Bethesda, MD, USA Objective: To study the development of a set of novel pol gene mutations which confers on HIV-1 a reduced sensitivity to AZT, ddC, and ddl. Methods: HIV-1 was Isolated from a patient (ERS103) receiving a long-term chemotherapy of AZT and ddC In sequence at various time points. The 50% Inhibitory concentration (ICso) of AZT, ddC, and ddi against each Isolate was determined. Biochemical and genetic studies of viral isolates were also performed. Results: IC5o values of AZT, ddC, and ddl against virus Isolated before (HIV-1 ERS103pre) and after (HIV-1ERS103post) 41 months of therapy were 0.07, 0.1, 1.6 M and 12.6, 1.6, 47 pM, respectively. The Kis for AZTTP against the activity of reverse transcriptase isolated from HIV-lERS103pre and HIV-lERS103post were 0.8 and 27.6 nM, respectively. HIVI ERS103post had a set of 7 pal gene mutations: Ala62-Val, Va1ll75--lle, Phe77--Leu, Phel 16--Tyr, Val1 18-lle, Gln151-,Met, and Ile202-4Val. Gin 151-Met first developed after 16 months of therapy when viremla level suddenly Increased as assessed by quantitative PCR. Following the Gln15i-Met mutation, 3 mutations (Phe77-4Leu, Phel16-Tyr, lie202--Val) appeared by 27 months, and then Ala62-4Val, Vall75-+Ile, Val1 18-4ile appeared by 38 months. Viremia further increased with the development of this set of mutations. Discussion/Conclusions: HIV-1 can develop multi-drug resistance during long-term combination therapy with AZT/ddC by developing a set of novel mutations. This set of mutations has been seen elsewhere In patients receiving combination therapy with AZT plus ddl. The clinical significance of this set of mutations requires further research. SHIRASAKA, Takuma; Medicine Branch, National Cancer Institute, Bldg 10, Rm 5A1 1, Bethesda, MD 20892, USA. tel: (1)-301 -496-9238; telefax: (1)-301-402-0709 Objectives: To establish whether: there is in vitro evidence of reduced ddl sensitivity of HIV in infected individuals receiving ddl; the emergence of resistance to ddl is associated with any change in sensitivity to ZDV; the emergence of SI virus and resistance to ddl correlate with clinical progression of the disease; there is a relationship between SI/NSI virus and resistance. Methods: 175 people (ZDV intolerant) were recruited in the MRC/INSERM Alpha Trial to test the efficacy and safety of high-dose (750mg daily) or low-dose (200mg daily) ddl. Sensitivity of viral isolates to ZDV and ddl was determined. ZDV resistant mutation at codon 215 of the RT gene was detected by selective PCR. PCR products of the RT gene were sequenced. SI phenotype was determined using the MT-2 syncytia induction assay. Results: Mutation at codon 215 was found in 48 patients; 22 also showing wild type 215. 287 samples were SI phenotyped. 59% of the -2 week samples showed NSI virus. After 12 weeks ddl therapy NSI phenotype increased to 75%, declining again as therapy continued. Sequential data will be presented. In -2 wk isolates of 5 patients the virus was ZDV resistant and ddl sensitive. The sequential isolates of these 5 patients were ZDV and ddl resistant. Sequencing of these sequential isolates is underway. Discussion and Conclusions: Although all patients entering the trial were classified as ARC or AIDS, not all the isolates were SI. A high percentage of NSI isolates in week 12 samples suggests that ddI may be useful for partial containment of the expression of SI virus during the early stages of therapy. Although trends for individual patients varied, in general there was stability of ZDV sensitivity and an increase in ddl resistance with time. N N Zheng Dept of Biotechnology. University of NSW. FO Box 1, Kensington 2033 Australia. Tele 02 697 3872. Fax 02 313 6710. 378A LIMITING DILUTION PCR TO DETECT CHANGES IN THE RT OF HIV-1 FROM PATIENTS ON LONG TERM AZT TREATMENT Christian A Stein, P Levantis, B Goh, R Hillman, J S Oxford. LHMC. Objectives: To investigate any amino acid (aa) changes in the Reverse Transcriptase (RT) enzyme of HIV-1 in sequential samples (up to twelve/subject) from patients infected with HIV-1 and treated with AZT or ddl. To analyse the distribution of proviral DNA sequences, utilising a end point dilution polymerase chain reaction (PCR) method to detect single RTs with multiple samples at every time point. To relate changes in the aa sequence to the clinical status of a patient and to a crystallographic model of RT. Materials and Methods: We investigated the clinical and molecular history from five HIV+ patients since the onset of AZT treatment. Serial samples, taken every 3 months from treatment time 0 up to >2.5 years, were analysed. 10 ml of heparanised blood were taken and peripheral blood mononuclear cells (PBMC) separated by density centrifugation. DNA was phenol-chloroform extracted from the PBMC and amplified by a highly sensitive, high yield limiting dilution double PCR. The products, representing a singular HIV-1 RT molecule were then analysed by direct sequencing. Results and Conclusions: We observed the development of a plurality of mutations in a broad panel of different HIV-1 RT molecules in each patient over time and at each time point. Each patient carried a variety of different HIV-1 RTs in their genome at any time. The amino acid changes concentrated in two small domains on the RT, between aa position 63-90 and between position 180-220. It was of particular interest that AZT sensitive sequences were still detected after more than fifteen months of treatment. In addition to the most common mutations, we have observed a number of other changes that could be essential for the development of drug resistance. The aa changes have been fitted to the crystallographic RT model of the group of Arnold et al. Christian A. Stein, The London Hospital Medical College, Academic Virology, Turner Building, London E1 2AD, UK. Tel ++44-71-375 03 45, Fax ++44-71-375 25 97 CA)

Page  22 379A Characterization of E subtype HIV-1 from AIDS patients and M recent seroconverters in northern Thailand Zhe Wang. X. Yu, C. Beyrer, S. Chamboonruang, K. Nelson Jonhs Hopkins University, Baltimore. MD; Chiang Mai University, Thailand. Objective: Most of the information regarding HIV-1 transmission and pathogenesis to date has been obtained from North America and Europe where homosexual men and IV drug users are the main study subjects. The viruses that circulated in these areas are predominantly B subtype. We are interested in characterization of E subtype HIV-1 from AIDS patients and recent seroconverters in northern Thailand. Methods: Viruses and DNA sequences surrounding the V3 loop were obtained from AIDS patients and recent seroconverters in northern Thailand. All viruses were analyzed for replication and syncytiuminduction in PBMC, MT-2, SupT1, and H9 cells. A region of approximately 300 bps from gpl20 including V3 was sequenced using DNA from PBMC and, in the case of SI viruses, from MT-2 cells. Results: All viruses analyzed are E subtype HIV-1 based on phylogenetic tree analysis. Although amino acid sequences from some recent seroconverters were very similar to the previously published sequences, sequences from others were significantly different. Sequences from AIDS patients were highly divergent from the previously published sequences. Significant number of AIDS patients (65%) harbor SI variants. AIDS patients that had SI viruses were younger (avg. 24 yr) than those who had NSI (avg. 36 yr). Rules that use the V3 sequence to distinguish SI vs NSI for B subtype HIV-1 applied poorly for the E subtype SI HIV-1 (47%). Despite the predominance of GPGQ at the tip of V3 in previously published E subtype HIV-1 from Thaland, the majority of the E subtype viruses from the AIDS patients had GPGR and GPGH. A highly conserved N-linked glycosylation site within V3 in previously published E subtype HIV-1 from Thailand was preferentially mutated in viruses from the AIDS patients (71%), asp. in the SI viruses (100%). Conclusions: HIV-1 circulating in the infected population in northern Thailand Is significantly divergent from viruses characterized two years ago. Any vaccine strategy should consider the rapid diversification of HIV-1 in this new epidemic area. The appearance of SI variants was significantly associated with symptomatic AIDS patients in Thailand where E subtype of HIV-1 is circulating and heterosexual transmission is the major route of HIV-1 infection. Zhe Wang, Dept. Immunology and Infectious Diseases, Jonhs Hopkins School of Hygiene, 615 N. Wolfe Street, Baltimore, MD 21205, USA Tel: 410-955-3768 Fax: 410-955-0105 381A THE GPGQ STRUCTURE IN TIlE V3 LOOP DOMIAIN IN A JAPANESE HEMOPtHII.IAC MIURA.Takuma*, MEGURO.T**, YAMADA,K**, WATANABE,K***, IIONDA,M*** *Department of Pediatrics, Ilaga Red Cross hospital, Tochigi, Japan. **Department of Pediatrics, St. Marianna University School of Medicine, Kanagawa, Japan. ***AIDS Research Center, National Institute of lhealth, Tokyo. Japan. Objective:HIV-I isolates can be grouped into 6 clades (A-F), and they are divided into two major groups; the GPGR structure as North American/European type and the GPGQ structure as African/Thai type. Analysis of over 150 cases of Japanese hemophiliacs in NIII, Japan shows only clade B IIIV, North American/European type but no African/Thai type. We report a case with the GPGQ structure in the V3 loop. Case:A 16-year-old Japanese hemophiliac was admitted to our hospital because of convulsion. HIV was isolated and he was diagnosed to have HlIV encephalopathy. Results:The amino acid sequence of the V3 loop domain of the isolated virus revealed the GPGQ structure, but it was different from two genotypes in Thailand. Analyses of DNA homology and phylogenetic tree revealed that the virus belonged to the North American/European type. The anti-PND (principal neutralization domain) antibodies in this patient's serum had the highest reactions to MN type.Discussion: The isolated virus has the GPGQ structure, but its codon usage was similar to that of clade B virus. Interestingly, the codon usage of the GPGQ core sequence was identical with that of clade E consensus virus, but not clade B virus. The investigation of tropism of the virus and the follow-up will be critical for clearing up the particular change of the virus. TAKUMA, Miura, Department of Pediatrics, ltaga Red Cross Hospital, Mohka City. Tochigi, Japan Telephone (81)-285-82-2195; Telefax (81)-285-83-8790 380A VARIATION AND SHIFT OF HIV-I ENV FOUND IN IDUs OF DEHONG EPIDEMICAREA IN CHINA Shaoy.M 1,2; Wang,B.1';Zeng,Y.1; Hell,W.2; Wolf,H2 l.Institute of Virology, Beijing; 2, Institute of Medical Microbiology and Hygiene, Regensburg Objective: To investigate the genetic diversity and change of HIV-l strains in China's only HIV epidemic region, where about 70% of the country's total HIV infections were detected from the local injecting drug users (IDUs). Methods: Blood samples were collected from HIV infected IDUs, 30 in 1990 and 50 in 1993. Viruses were isolated by PMCs coculture method. HIV-l env genes were amplified by PCR from both cultured and uncultured PMCs and cloned. Sequencing was performed both manually and by using an automatic DNA sequencer (ABI, A373). Results: HIV- I subtype B like viruses were found in most of the uncultured PMCs samples in compression of their env V3 to V5 region with the HIV-1 env A to E subtypes. It is in agreement with our former report that the HIV infected persons predominantly reacted to V3 peptide of North American HIV- I strains (MN 82%,SF2 71%) and much less to that of other regions. The typical subtype B variants (all with a GPOR V3-tip tetrapeptide) constituted 8 of 10 samples (80%) in 1990, but only 12 of 21 samples (57%) in 1993. The proportion of subtype B/Thai genotype B (all with a consensus GPGQ motif) increased from 10% (1 Of 10) in 1990 to 29% (6 of 21) in 1993. Among the samples with GPGR V3-tip, the ratio for the terminal arginine codon AGA to CGA also increased from 1:7 in 1990 to 5:7 in 1993. In culture, all viruses isolated from 1990 samples could infect only PMCs with low titre and caused cell killing without syncytium formation. Whereas in 1993 culturing, one isolate grew to high titres in PMCs and in T cell lines. It could also cause syncysium formation in Jurkat cells but not in PMCs, MT4 and H9 cells. Discussion and Conclusions: The HIV-strains causing epidemic among IDUs in Dehong region are in the env subtype B group. The sequence data suggested a shift from GPOR to GPGQ on the V3-loop tip of local IIV-I strains with time. Because CGA is more close to the codon CAA of glutamine than AGA, the proportion of Thai genotype B viruses in this region might further increase in future. No definite conclusion could be drawn from the isolate of 1993 with more infectious properties, for the culturing condition was not identical in both cases. Further studies on follow-up samples and samples before, during and after culturing are progressing, focusing on env as well as other HIVI genes. SHAO, Yiming, Institute of Virology, 100 Ying Xin Jie,100052 Beijing,Tel.(861) 3038621 Fax.3013160; Present address:Institute of Medical Microbiology and Hygiene, Regensburg University Postfach 100662, D-93042 Regensburg, Tel. (49-941) 9446478, Fax. 9446402 CA) 003 382A CORRELATION BETWEEN GENETIC AND BIOLOGIC PROPERTIES OF AFRICAN AND EUROPEAN IIIV-I AT TIlE BIOLOGICAL CLONE LEVEL. Zhone Pie. Peeters M., Jutssens W., Fransen K.. Ileyndrickx L., Willems B., Piot P. and van der Groen G. Institute of Tropical Medicine, Antwerpen,. Belgium. OBJECTIVE: To study the correlation between the V3-loop amino acid sequence and biological properties (syncytium-inducing capacity, cell tropism) as well as neutralizing antibody capacity of African and European HIVI strains at the biological clone level. METIIODS: From 12 HIV-1 strains ( 5 European and 7 African ), thirty-eight biological clones were generated by the limiting-dilution culture technique. The syncytium-inducing capacity of all clones was measured in a MT-2 cellline. The V3 loop encoding region of biological clones was PCR amplified followed by direct sequencing. Thirty clones (13 SI and 17 NSI) obtained from 8 virus strains were studied for cell-tropism on different cell lines ( MT-2, Molt-4, CEM-SS, and U-937). The autologous sera of 8 patients were tested for the neutralizing antibodies against their biological clones. The clones were generated from HIV isolates taken at the same time, the sera were collected. RESULTS: 17/19 (6 of 17 from Africa) SI phenotypic clones contained a positively charged amino acid at position I 1 and/or 25 of the V3 loop, whereas 19/19 (8 of 19 from Africa) of the NSI clones contained a negatively charged or uncharged amino acid at these positions (p< 0.001). The 2 St clones with no positively charged amino acid at these positions were from 2 african HIV-1 strains. Significantly more SI clones (13/13) than NSI clones (2/17) could replicate in MT-2, Molt-4 and CEM-SS cell lines (p < 0.01). None of the clones could replicate in U-937. Only one of the eight patients had an autologous neutralizing antibody titer of 1/40 that could only neutralize the NSI clone. DISCUSSION AND CONCLUSION: This study found I.) the confirmation of significant correlation between V3-loop amino acid sequence at position I1 and 25 and syncytium-inducing capacity. 2.) no significant difference between European and African clones for their SI/NSI capacities related to the amino acid charges in the V3 loop. 3.) a strong association of the cellular-host range of biological clones with syncytium-inducing capacity. 4.) a lack of relationship between the unique V3 loop sequence of SI/NSI clones and the capacity to be neutralized by their autologous sera. Zhong Ping, Institute of Tropical Medicine, Department Infection & Immunity, Division of Microbiology, Nationalcstraat 155, B-2000 Antwerpen, Belgium. Tel: 32 3 247 63 32; Fax: 32 3 247 63 33.

Page  23 383A GENETIC CHARACTERIZATION OF HIV-I/2 ISOLATES FROM GHANA Osei-Kwasi, Mubarak#, Takehisa, J.*, Ayisi, N.K.#, Brandful, J.#, Ampofo, W.#, Magnusen, A.#, Hishida, O.*, Igarashi, T.*, Yamashita, M.*, Ido, E.*, Miura, T.* Hayami, M.* #Noguchi Memorial Institute for Medical Research, University of Ghana *Institute for Virus Research, Kyoto University, Japan Objective: To clarify the phylogenetic relationship of HIV in Ghana, 6 HIV-I and 5 HIV-2 isolates from ARC or AIDS patients in 1986, 1990 and 1992 were sequenced and analyzed. Methods: A part of pol region suitable for comparison among HIV/SIV groups and the env region of HIV- 1 were sequenced and analyzed. Results: HIV-1 is classified into five major subtypes from A to E. Four Ghanaian HIV1 strains belonged to subtype A, one strain to subtype D, and the other one strain remained outside of the current classification. Characteristic tetrameric sequences at the tip of V3 loop, GLGH, GPGH and GTGR are observed in Ghanaian isolates, which have not yet been reported before. Potential N-linked glycosylation site is generally well conserved, but Ghanaian strains lack two glycosylation sites. In HIV-2, one strain belonged to HIV-2b that is a subgroup different from the prototype HIV-2. The topology indicates that HIV-2b diverged at the almost same time of divergence of SIVsm/SIVmac and other HIV-2 strains reported previously. Discussion and Conclusions: In Ghana, there is unclassified HIV- I as well as HIV1 subtype A and D. Some of them have V3 tetrameric sequence different from those reported previously. Existence of HIV-2b as well as prototype HIV-2 was also confirmed. 384A MOLECULAR CHARACTERIZATION OF A HIV-1 STRAIN (HIV-IbNg) FROM NIGERIA Howard, Thomas M., and Rasheed, S., University of Southern California, Los Angeles, California, USA TAKEHISA Jun, Institute for Virus Research, Kyoto University Shogoin-Kawara-cho, Sakyo-ku, Kyoto 606, Japan tel: 81-75-751-3981, fax: 81-75-761-9335 Effect of Different Variables on the Results of Microbroth Dilution Method for In Vitro Antifungal Susceptibility Testing of Yeasts. Maurizio Del Poeta', Daniela Arzeni', Francesco Barchiesi' ', Giovanna Marinucci', Fausto Ancarani', Giorgio Scalise'. 1. Institute of Infectious Diseases and Public Health - University ofAncona, Italy. 2. Fungus Testing Laboratory, Department of Pathology, Univ. of Texas. Huth. Sci. Ctr. andAudie L. Murphy Mem. Veterans Hosp., San Antonio, TX, U.S.A. Objective: To characterize the overall genomic organization of a new strain of HIV-1 (HIV-1IbNg) from Nigeria. Methods: HIV-1IbNg was isolated from the peripheral blood mononuclear cells (PBMC) of a Nigerian individual using coculture techniques and phytohemagglutinin-stimulated PBMC from a HIV seronegative donor. Two different techniques were then employed to molecularly clone this virus from infected PBMCs. Firstly, the RT-PCR technique (reverse transcriptase, polymerase chain reaction) was used to amplify the HIV-11bNg genome using total, cytoplasmic RNA and resultant amplicons were cloned. Secondly, a phage library was constructed using unintegrated, circular viral DNA extracted by the Hirt procedure from infected PBMCs. Recombinant phage containing the HIV-1IbNg genome were identified using a probe generated from one of the recombinant plasmids containing the polenv coding region of the genome. All DNA sequencing was done by the dideoxy-chain termination method using double-stranded DNA templates. Results: A comparison of the nucleotide sequences of the gp120 coding regions of HIV-11bNg and other HIV-1 atrains revealed that this virus is most closely related to strains of HIV-1 belonging to the 'A' subtype. Significant differences in all five hypervariable regions, especially the V4 region, was noted when the deduced amino acid sequence of the gpl20 proteins of HIV-1IbNg and other viral strains grouped in clade 'A' were compared. The number and positions of the cysteine residues along with the number and positions of potential N-glycosylation sites, however, were found to be highly conserved amongst these strains. Discussion and Conclusions: This is the first known isolate of HIV-1 of Nigerian origin which has been molecularly cloned and characterized. Based on information obtained from the eny region of the viral genome, HIV-1IbNg clusters most closely with strains of HIV-1 of the 'A' subtype which have been isolated from inhabitants of central and western Africa. Characterization of HIV-1IbNg is important for the development of vaccines and diagnostics which may be used in regions of the world where HIV-1 strains of the 'A' subtype are endemic. HOWARD, Thomas, Laboratory of Viral Oncology and AIDS Research, University of Southern California, California, 90032-3626 USA Telephone 213-224-7135; Telefax 213-227-1840; email tmhoward@hsc.usc.edu 386B rouCONAZOL SOLUTION FOR FLUCONAZOLE-RRACIORY OROPH ARYNGEAL CANDIDIASIS IN AIDS: CORRELATION OF CLINICAL RESPONSE WTTH IN VITRO SUSCEPTIBILITY. Mahmood W, lHamann-Trou D, Phillips P, Zemcov SJ, Montaner JSG, Clarke AM. AIDS Research Program, St Paul's lospital, Vancouver, BC, Canada. OBJECTIVE: To determine the efficacy of itraconazole solution (ITR-SOL) in fluconazole-refractory oropharyngeal candidiasis (FLU-ROC), and to correlate clinical response with in vitro susceptibility. METHODS: Between 5/93 and 1/94, tllIV patients with FLU-ROC were prospectively evaluated including: clinical assessment; throat culture; ITR and FLU MICs (broth macrodilution) for Candida isolates. Patients were treated with ITR-SOL 200 mg daily x 2 wk, then 3x/wk. Disease activity (DA) was graded by a scoring system. FLU (at baseline) and ITR (at follow-up) serum levels were determined by bioassay. RESULTS:.16 cases (14 male, 2 female) were treated; all isolates were C. albicans. Patient characteristics included: median age 35 yr. (range 26-45); median CD4 count 1l0/mm3 (range 0-88); median interval from AIDS diagnosis 23 months (range 0-42); and median cumulative prior systemic azole therapy 12 months (range 3-30). Median peak ru e serum FLU level at baseline was 14 sng/ (range 4.7-40). ITR-SOL was generally well tolerated; one patient had ITR-related GI upset. Clinical responses (reduction in DA to <50% of baseline) were observed in 10/16 cases (63%), usually within 2 weeks. Relapse (DA >50 % baseline) was noted in 2 patients at 3-6 months of therapy. The median FLU MIC was 64 mg/l (range 8 ->64). The median ITR MIC was 1.25 mg/I (range 0.31- >10). ITR MICs of >4 mg/I were present in 4 cases (3/4 non-responders). Failed ITR-SOL therapy could be attributed to azole cross-resistance in 3/6, and possible drug interaction in 1/6 cases. CONCLUSION: Itraconazole solution was effective in 63% of FLU-ROC. In vitro cross-resistance to ITR (MIC >4 mg/I) was documented in 25% of cases, and appeared to account for failed therapy in 3 of 6 cases. No cases of FLU-ROC could be attributed to inadequate serum FLU levels; whereas all C. albicans isolates demonstrated resistance (MIC>8 mg/I) or borderline resistance to FLU. Dr. Wafeeq Mahmood. BC Centre for Excellence in IIIV/AIDS 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z 1Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. 385B Objective: We analyzed the effects of various assay conditions on the activity of fluconazole, ketoconazole, itraconazole, amphotericin B, and flucytosine against 30 strains of Candida spp. and 10 strains each of Torulopsis glabrata and Cryptococcus neoformans, isolated from AIDS patients, by a microbroth dilution technique. Methods: The variable parameters included: inoculum size (1,000 vs 1,000,000 CFU/ml), medium and buffer (RPMI 1640 buffered with MOPS vs BYNB buffered with sodium phosphate, both at pH 7), and length of incubation (24 vs 48 hr for Candida spp., and T. glabrata, 48 vs 72 hr for C. neoformans). The microplates were incubated at 35~C and agitated before reading. End-points were determined according to e the NCCLS 0 to 4+ criteria for the macrobroth dilution method. Results: Increasing the inoculum from 1,000 to 1,000,000 yeasts cells/ml raised the MICs for all azoles and flucytosine against all strains tested. Furthemore, the low inoculum permitted the separation between azole-susceptible and -resistant strains. Amphotericin B showed the best overall agreement with all time readings and the low inoculum sizes. The best agreement between the media was found with the low inoculum at the second time reading. Conclusion: We suggest that the proposed NCCLS parameters for the macrobroth assay also appears to produce the greatest agreement in this microbroth method. MAURIZIO DEL POETA M.D. INST. OF TNF. DIS. AND PUB. HLTH - UNIV. OF ANCONA-TEL.071-5963260 FAXO71-5963263 CA) Co CA) CA) CO 0) W

Page  24 387B HIGH MORTALITY IN AIDS PATIENTS WITH CRYPTOCOCAL MENINGITIS DESPITE FLUCONAZOLE THERAPY IN UGANDA. Kazunori Oishi*, R.Mugerwa**, S.Mitarai*, H.Mayanja**, K.Nalongo**, T. Nagatake*, K. Matsumoto*. Inst of Trop Med, Nagasaki Univ, Japan*, Dept of Med, Makerere Univ, Uganda". Introduction: Cryptococcal meningitis(CM) is a particularly common and fatal opportunistic infection, occuring to 30% of AIDS patients in Africa. Objective: To demonstrate the clinical features of CM in AIDS patients, and to establish the effective antifungal chemotherapy for this disease in Africa. Methods:40 HIV-seropositive patients who were positive for India ink staining and cryptococcal antigen test in CSF were enrolled. These patients were treated with oral fluconazole (FCZ) at doses of 200mg or 400mg for 2 months at random. CD4 levels and CD4/8 ratios in peripheral blood from 13 patients were determined using Dyna-beads T4-T8 Quant Kit (I)ynal). Results: The mean age and the ratio of male/female in these patients were 31.0 years and 2.0, respectively. Patients had headache (100%), prolonged fever (80%), oral candidiasis (70%), and abnormalities in consciousness (38%). The mean (+SD) of CD4 levels and CD4/8 ratios were 97 + 81 /mm3 and 0.35+0.23, respectively. 50% of patients died within two weeks after initial therapy. Similar clinical response rates (33%) were observed in patients who received FCZ at doses of 200 mg or 400mg at 2 months post- therapy Discussion and Conclusion: Frequency of abnormalities in consciousness before therapy were relatively high, and severe immunosuppression was observed. Early diagnosis and intensive therapyt were strongly desired for patients with CM. Treatment of CM with another anti-fungal regimen with FCZ at a dose of 200mg and flucytosine (150mg/kg) for CM is under developing. Kazunori Oishi, M.D. Dept of Intern Med,lnst of Trop Med, Nagasaki Univ. 1-12-4, Sakamoto-machi, Nagasaki 852, Japan. Tel:81-0958-47-2111(Ext:3776), Fax:81-0958-49-2395. 388B SURVIVAL, DEATH, AND DESENSITIZATION TO TRIMETHOPRIM/SULFAMETHOXAZOLE (TMPISMX) Kine. Christopher; Slaton. Amy; Okabe, Mitsunori; Conant, Marcus Conant Medical Group: Research, University of California, San Francisco, California Oblectlve: To evaluate the safety and efficacy of trimethoprim/sulfamethoxazole (TMPISMX) as a prolphylactic agent in individuals who, previously exhibiting hypersensitivity to sulfa-based drugs, attempted a standard TMP/SMX desensitization protocol. Efficacy in this study was defined as "successful" completion of the protocol and/or no development of an initial or recurrent episode of PCP or toxoplasmosis. Methods: Retrospective data collection and analysis of progress notes of 100 consecutive patients having attempted the standard TMP/SMX desensitization protocol. Patients had a documented history of at least one episode of hypersensitivity to TMP/SMX or other sulfa-based drug(s). All were attempting to initiate prophylaxis against Pneumocystis carinii pneumonia (PCP) for the first time, or change their current prophylaxis to TMP/SMX. All patients were included in this analysis regardless of prior PCP history or elevated toxoplasma titers (IgG,IgM). Results: In the sample of 100 patients, 84 (group A) (84%) were "successfully desensitized" (definmed as the ability to tolerate 80mg-160mg TMP and 400mg-800mg SMX every day without rash, fever, itching, flu-like or other symptoms attributed to TMP/SMX). Only one (1.2%) of these patients in group A, developed both PCP and acute toxoplasmosis (toxoplasma titers on this patient indicate acute disease one month prior to initiation of the protocol). Of the remaining 16 (group B) (16%) who were deemed protocol failures, eight (50%) subsequently developed PCP (and/or toxoplasmosis). Of these eight patients, two (25%) subsequently died of PCP. Data Analysis: In the original sample of 100 patients, 36 (36%) had a prior history of PCP (and/or elevated toxo titers). Of these 36 patients, 30 (83.3%) were successfully desensitized; of these, one patient developed PCP. Of the 6 (16.7%) who failed the protocol, 4 (66.8%) of these patients developed PCP or acute toxo. One patient is still living 30 months after an intial bout with PCP and has been maintained on prophylactic TMP/SMX for 22 months without recurrence of PCP. Conclusioann: This retrospective epidemiological study concludes that this standard desensitization protocol and subsequent therapy with TMP/SMX dramatically decreased the incidence of two life-threatening infections in those deemed a protocol success. As TMP/SMX remains the best prophylactic agent against the Pneumocystis organism, it follows that this desensitization protocol should be offered to all patients hypersensitive to TMP/SMX who are living with HIV infection. Christopher King; Conant Medical Group: Research 1635 Divisadero, Suite 601, San Francisco, CA 94115 Phone 415-923-0222; Fax 415-923-0237 0 CA) C0 0 w3 389B THE IMPACT OF PROPHYLAXIS GUIDELINES ON PNEUMOCYSTIS CARINHII PNEUMONIA (PCP) IN INFANTS, U.S.A. Simonds RJ*, Lindegren M, Thomas P, Scott 0, Connolly 0, Laraque F, Hanson D, PSD and PACTS Projects *CDC, Atlanta, GA, USA 390B IMPACT OF A COMMUNITY-WIDE OUTBREAK OF CRYPTOSPORIDIOSIS ON PATIENTS WITH AIDS. Gilson, Ian; Buggy, BP; Brummitt, CF; Busalacchi, M; Ivantic, K. Wisconsin Community-Based Research Consortium, Milwaukee, WI, USA. bjetiv: PCP in HIV-infected children occurs most often in infants (<1 year old) and can be prevented by chemoprophylaxis. However, although prophylaxis guidelines were published in 1991, no decline in PCP cases among infants has been detected. We evaluated the guidelines to determine why prevention efforts may have failed. Methods: We calculated PCP incidence among infants using national data from AIDS surveillance of children and HIV serosurveillance of childbearing women; we determined the timing of HIV and PCP prophylaxis evaluations by reviewing medical records of infants with PCP from 11 U.S. sites. Resuls: The incidence of PCP among infants born to HIV-infected women in 1989, 1990, 1991, and Jan-Jun 1992 was 2.7%, 2.7%, 2.1%, and 2.2%, respectively. We reviewed records of 223 infants (median age 4 mos) with PCP diagnosed 1/91-6/93. Of these infants, 169 (76%) had not received prophylaxis before PCP. Of the 169 non-prophylaxed infants, 105 (62%) had not been evaluated for HIV infection >30 days before PCP. Of the 64 infants who had been evaluated, 45 (70%) did not have CD4 counts measured. Fifteen (79%) of the 19 non-prophylaxed infants who had CD4 counts measured did not qualify for prophylaxis by the guidelines (no counts <1500 cells/pdl). For 42 infants (including 26 on prophylaxis) who had:2 CD4 counts before PCP diagnosis, the estimated decline in CD4 count during the 3 months before PCP was 937 cells/p l (95% CI 653-1221 cells/ l). Conclusions: The incidence of PCP among infants has not declined substantially since prophylaxis guidelines were published. Most recent cases can be attributed to lack of timely evaluation for HIV. Using CD4 counts as criteria for prophylaxis may be impractical for infants because counts may be difficult to obtain before the age of peak PCP risk (3-6 mos), and may decline rapidly before PCP. HIV screening and PCP prophylaxis strategies must be improved to maximally prevent PCP in infants. SIMONDS, R.J. Div HIV/AIDS, CDC, 1600 Clifton Rd, E45, Atlanta, GA, 30333 USA Telephone: 1-404-639-6133; Telefax: 1-404-639-6118 Objective: To study the impact of a community-wide outbreak of waterbomrne cryptosporidiosis (CS) on a cohort of AIDS patients, including morbidity, mortality, cost of care, and outcome of therapy. Methods: Retrospective cohort study of all IV-infected patients in two practices with CD4 count <200 at the time of the outbreak (a city-wide CS outbreak due to failure of a water filtration plant which resulted in - 400,000 cases of CS in Milwaukee, WI, in April 1993). Comparison of groups with defuinite (stool +, n=24) or suspected (clinical, n=9) CS (C) vs. no CS (n-=40) (N) at 6 months post-exposure. Therapy included paromomycin (PA) 500 mg qid and azithromycin (AZ) 600 mg qd or bid; responses were complete (CR), partial (PR), and none (NR). Results: Of 73 pts with CI4 <200, 33 developed CS (C) and 40 did not (N); incidence=45% (comparable to community incidence of 40%). The groups had comparable mean initial Karnofsky score (C 81, N 83, p=.7), mean CIX count (C 43, N 58, p=.13), and baseline CDC class C3 diagnosis (C 88%, N 68%, p=.08); baseline mean weight in pounds (C 162, N 151, p=.04) was higher in C. Overall 6 mo mortality rate was 26%. Mortality in C was higher than N (C=39%, N=15%, p=.05); CS was responsible for 68% of all deaths in the cohort, with a 6 mo excess mortality rate of 24/100 due to CS. 52% of C were either dead or terminally ill at 6 mo; 12/13 CS deaths occurred in pts with CD4 <50. Mean wt change (C -18, N -8, p=.05), increase in alkaline phosphatase (C 2.lx, N 1.2x, p=.05), decline in Kamrnofsky score (C -40, N -13, p=.0006), and TPN use (C 27%, N 5%, p=.02) were greater in C. Biliary disease occurred in 24% of C. Mean hospital days (C 12.1, N 1.1, p<.0001) were greater in C; 400 of 444 total hospital days, or 90%, occurred in C. Mean total care charges (C $36020, N $11908, p<.0001) were 3x greater in C; CS accounted for extra charges of $795,699 for the cohort. Response to PA (n=19) was 37% (1 CR, 6 PR, 12 NR); response to AZ (n=12) was 42% (1 CR, 4 PR, 7 NR). Conclusion: A municipal waterborne outbreak of CS resulted in a dramatic increase in morbidity, mortality, and costs in a group of exposed AIDS pts. Both PA and AZ were partially effective; further studies are indicated. Municipalities must properly monitor and treat community water supplies for cryptosporidia in order to protect this vulnerable population. Ian Gilson, MD, FACP 788 North Jefferson Street Milwaukee, WI, 53202 USA Phone: 414-276-1906 Fax: 414-276-4820 E-mail: 75210.1027@Compuserve.com

Page  25 391C INCIDENT HIV AND STDs IN DIRECT AND INDIRECT COMMERCIAL SEX WORKERS (CSWs) IN THAILAND. Chrs Beyrer, Khamboonruang C, Natpratan C, Celentano D, Nelson KE. Johns Hopkins Univ. Baltimore, MD USA, Chiang Mai University, and Ministry of Public Health, Chiang Mai, Thailand. Objective: We studied incident HIV and STDs and high risk behavior in cohorts of CSWs to assess their suitability for HIV vaccine trials and other interventions. Methods: Female CSWs were screened for HIV and seronegatives were followed q3mos. with behavioral interviews, physical exams (including pelvic exams), STD cultures and HIV serology. Direct (brothel-based) CSWs and Indirect (others) CSWs were compared for their risk behavior, followup rates, HIV and STD incidence. Results: Of 1068 CSWs screened 409(38.3%) were HIV pos at baseline; 396 HIV neg CSWs were followed. Direct CSWs (n= 176) were compared to indirect CSWs (n=220). Direct CSWs were younger (median age=20 vs 26 yrs; p<.001), had worked shorter time as a CSW (mean 2.3 vs 3.4 yrs, p<.02), were less educated (43% vs 7% no schooling, p<.001), and had more customers per night (median 4 vs 1, p=.001); the median highest number of customer per day was 9.0 (direct CSWs) and 4.0 (indirect CSWs, p<.01). Followup at 6 mos. was 58.5% for Direct CSWs and 75.5% for Indirect CSWs; 19 CSWs had seroconverted to HIV at 6 mos., 15 HIV seroconverters were direct CSWs (11.9/100 person semesters) at baseline; and 4 were indirect CSWs (2.0/100 person semesters), i.e. annual incidence of 24% and 4.0% respectively. Higher incidence of other STDs were seen in direct vs indirect CSWs at 6 mos; gonorrhea (7.1% vs 1.5%, p<.05), Chlamydia (6.3% vs 1.5%, p<.05) and Syphilis (5.0% vs 1.0%, p<.05). Conclusion: Both direct and indirect CSWs in northern Thailand have a high incidence of HIV and other STD infections despite current interventions, e.g. condom promotion. They could be suitable populations to evaluate other interventions such as HIV vaccines if followup could be improved. Chris Beyrer, MD, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand Tel: 6653221966; Telefax: 6653221849 393C THE INCREASE IN HIV-1 PREVALENCE IN COMMERCIAL SEX WORKERS (CSWs) IN THE GAMBIA, AND ASSOCIA TION WITH OTHER STDS INCLUDING CERVICAL ABNORMALITIES Method: From Dec. 1992 to Feb 1993 self-identified female CSWs were recruited at several sites in The Gambia. Each woman who agreed to participate was offered HIV testing and STD screening including cervical cytology. Results: 213 women were recruited with a mean age of 29.9 years. Overall, 8% were infected with HIV-1, 21.1% with HIV-2 and 5.6% were dually infected. These results were obtained from blood, saliva and blood spot eluate testing, and all results were consistent. There was a high prevalence of both ulcerative and non-ulcerative STDs, except Chlamydia trachomatis with which only 3.8% were infected 3.9% of women had CINl on cytology, 3.4% CIN2 and 2.4% CIN3 (4 of the 5 women with CIN3 had evidence of invasive carcinoma). Discussion: The prevalence of HIV-1 infection in CSWs in The Gambia has shown a four-fold increase between 1989 and 1992, whilst the prevalence of HIV-2 has remained relatively stable over this period. Given the previously demonstrated greater pathogenicity of HIV-1 in The Gambia, this increase in prevalence in a core-group of women represents a serious threat. The consistency of HIV results on testing blood, saliva and blood spots is significant for a population which has a high prevalence of both HIV-1 and HIV-2 infections. The high prevalence of abnormalities on cervical cytology in a young group of women emphasises the importance of incorporating methods to diagnose and treat this condition in STD control programmes in sub-Saharan Africa. The lack of an asscciation between cervical abnormalities and HIV serostatus hihlights the need for further studies in this region on the link between the 2 diseases. Hawkes, Sarah Dr. St Mary's Hospital Medical School, Norfolk Place, London W2 1PG, UK. FAX 071 402 2150 392C RAPID SPREAD OF HIV-1 INFECTION AMONG MALE COMMERCIAL STREET SEX WORKERS IN MIAMI, FLORIDA. Klaskala Wnslow. Lal S, Shor-Posner G, Sfaklanakl E, Gomez 0, Baum M. Dept of Epidemiology UnIv of Miami, Dade County Public Health Unit, Miami FL Objective: To determine prevalence, incidence and risks for HIV-1 in male commercial street sex workers (CSSW) in Miami, Florida. Methods: Male CSSW arrested for exchange of sex with men for money and/or drugs were tested for HIV-1 under mandatory legislated program. The incidence of new HIV-1 infection was examined in those men who were tested more than once. All sera reactive for HIV-1 were repeatedly tested with ELISA and confirmed with Western blot. Results: Of 165 CSSW tested from Nov 1987 to Dec 1991 for HIV-1 74 (44.8%) had HIV antibodies on their Initial test. Over this period a total of 115 men were found to be HIV positive, which represents period prevalence of 69.7%. Cumulative incidence (seroconversion rate) increased rapidly from 45.6 at the second HIV testing to 83.3% at the third (final) HIV testing. Median seroconversion time was 22.5 months (CI 18.1-28.7 at 95%). Age at the first HIV testing was significantly associated with seroconversion time. Adjusted for calendar age, country of origin, state of residence and race, those who were older than 35 years, were 9.2 times (CI 2.2-39.0 at 95%) more likely to become seroconverted compared to those who were younger than 35. Over 90% of the seroconverters had a history of contracting sexually transmitted diseases, mainly syphilis and gonorrhea. Discussion and Conclusions: Rapid rise of HIV-1 incidence among male CSSW suggests that among populations thought to have made significant advances in slowing the spread of HIV, such as men having sex with men, there is a possible slippage and increasing rate of new Infection. New, innovative, targeted Interventions are urgently needed to reduce HIV-1 transmission In this population. Winslow Klaskala, Ph.D. University of Miami School of Medicine Telephone: 1-305-547-4072 Telefax: 1-305-547-3328 394C RISK FACTORS FOR HIV SEROCONVERSION IN COMMERCIAL SEX WORKERS IN MOMBASA, KENYA: ROLE OF HORMONAL CONTRACEPTION & STDs Martin, Harold L.. Jr., Nyange PM, Jackson DJ, Mandaliya K, Holmes KK, Ngugi E, Ndinya-Achola JO, Plummer F, Kreiss J.; U. of Washington, Nairobi, & Manitoba, Coast Province General Hospital. Objectives: To define the incidence and risk factors for HIV infection in a high risk population of commercial sex workers (CSW's) and to establish a cohort for future prophylactic HIV vaccine trials. Methods: Prospective cohort study in women registered as CSW's in a municipal clinic. Results: Between February 1993 and February 1994, 1585 women were counselled and screened for HIV. Seroprevalence of HIV was 56.5%/, and 435 seronegative women were enrolled. Mean age was 28 years; mean duration of prostitution was 6 years. Mean sexual frequency was 1.6/week with 1 partner/week. Mean condom use was 63Y%, and the average charge for sex was KSh 638/= (US$9.5). Incidence of STD's was: gonorrhea 121/100 p-y, trichomonas 56/100 p-y, genital ulcers 20/100 p-y, chlamydia 16/100 p-y. Twenty-seven subjects seroconverted for HIV, an incidence rate of 16.2/100 py. Compared to women remaining uninfected with HIV, seroconverters had fewer sexual partners/week (0.6 vs 1.0, p=.02) and lower sexual frequency/week (1.0 vs 1.6, p=.01). There was no difference in age, mean charge for sex, years of education, or reported condom use. HIV acquisition was associated with the use of oral contraceptives (OR 2.6, 1.0-6.8) and depo provera (OR 3.7, 1.4 -9.6), presence of genital ulcers (OR 3.7, 1.4-9.9), gonorrhea (OR 2.2, CI 1.2-3.7), and vaginal discharge (OR 2.0, 1.2-3.4). Trends towards positive associations with HIV seroconversion were found with vulvitis (OR 1.9, 0.8-5.0) and cervicitis (OR 1.5, 0.9-2.4). Conclusion: In this prospective study, HIV seroconversion was significantly associated with the use of oral contraceptives and depo provera, genital ulcers, gonorrhea, and vaginal discharge. MARTIN, Harold L. Jr., University of Washington, ZX-32, Seattle, WA 98195 USA Telephone: (206) 223-8055; Telefax: (206) 223-8752 C.) 0 0

Page  26 I395C FEASIBILITY OF PREVENTIVE ACTIONS AMONG TRANSVESTITE/TRANSSEXUAL Q) PROSTITUTES IN PARIS. SerreAnne*. Cabral C, Castelletti S. De Vincenzi 1*. *European Centre for the Epidemiological Monitoring of AIDS, St-Maurice, *PASTT, Paris, France Objectives: To evaluate the pertinence and feasibility of preventive actions for STD/AIDS among the transvestite/transsexual prostitutes (TTP) in Paris. Methods: Two transvestites contacted other TTP at their work. At each contact, basic data (lifestyle, geographical origin, place of work) were collected. A sub-group of TTP completed a detailed questionnaire exploring transsexualism and prostitution, use of condoms, HIV testing (no test result was asked for in this feasibility study), drug use, and clients. Results: Between 1st April and 30th September 1993, 206 TTIP were contacted, mainly migrants originating from North Africa (35%), Latin America (20%), Southemrn Europe (11%) and Asia (8%), while 28% were from France. Transformists, who dress as women at night but live as men during the day (50% of the sample), were somewhat less marginalized than transsexuals who have undergone genital surgery (5% of the sample) or transvestites who present secondary female characteristics following medical treatment (45% of the sample). From 61 completed questionnaires, the average TTP was: 31 years old, of foreign origin, without social insurance, prostitute for 11 years, working 5 days a week with 6 clients per day. Most have had homosexual private partners and/or began prostitution as males. However, most clients considered themselves as heterosexual. Anal sex was frequent between TTP and their clients and 10% of TTP reported not always using condoms. Over a period of 5 years, 21% reported a history of STD and 82% underwent HIV testing. Conclusion: This population appears very heterogeneous. Risk of HIV infection and transmission to their clients (more than 6000 clients per week for the 206 TTP contacted) in the presence of high risk behaviour (unprotected anal sex) and difficulties linked to a rapid turn-over and illegal immigration status invalidating their access to health care, are all factors that indicate an urgent need for at present inexistant specific prevention programmes for transvestite and transsexual prostitutes. Anne SERRE - European Centre for the Epidemiological Monitoring of AIDS - 14 rue du Val dOsne - 94410 SAINT-MAURICE - FRANCE Tel: (33) 1 43 96 65 45 - Fax: (33) 1 43 96 50 81 396C 2-YEAR FOLLOW-UP STUDY ON IIIV, HEPATITIS B AND C VIRUSES AND SYPHILIS INFECTIONS IN A COHORT OF JAPANESE FEMALE SEX WORKERS KIHARA Masahiro1, IMAI, M.2, KONDOH, M.2, WATANABE, S.2, KIHARA, M.3, SODA, K.3(1Dept of Epidemiol, Kanagawa Cancer Center Res Inst, 2Dept of Virol, Kanagawa Prefectural Public Health Lab, 3Dept of Public Health, Yokohama City Univ School of Med, Yokohama) OBJECT: To investigate the prevalence of HIV, hepatitis B and C viruses and syphilis infections in Japanese female sex workers. METHODS: With a collaboration of a sex worker's association in Tokyo metropolitan area, blood tests were performed every 3 months since 1992, which covered 1410 persontests (436 net ), as of Dec.31,1993. Their experience of repeated skin-piercing treatments such as acupuncture and IDU were asked by questionare. Sera collected in 1992 from 300 female blood donors (aged 20s-40s) of the same district were assessed for comparison. RESULTS: None was found anti-HIV positive. Anti-HCV and TPHA positive rates were greater(p<001) in sex workers than controls. Only 10 out of 45 anti-HCV positives group and age n Anti-HIV Anti-HCV Anti-HBV TPHA received repeated Sex workers 20-29 yrs 163 0(0.0) 14( 8.6) 1(0.6) 25(15.3) skin-piercing 30-39 yrs 226 0(0.0) 27(12.0) 3(1.3) 30(13.3) treatment, mostly 40-49 yrs 47 0(0.0) 4( 8.5) 1(2.1) 3( 6.4) acupuncture, and Total 436 0(0.0) 45(10.3) 5(1.1) 58(13.3) none underwent Blood donors 300 0(0.0) 0( 0.0) 0(0.0) 0( 0.0) blood transfusion, suggesting sexual contact as a possible route of transmission. DISCUSSION AND CONCLUSIONS: HIV infection appeared not yet prevalent among Japanese female sex workers in Japan. They may be however highly vulnerable to HIV invasion since HCV which is rarely transmitted sexually is prevalent among them. KIHARA Masahiro, Kanagawa Cancer Center Research Institute, Yokohama 241,Japan. TEL:045(391)5761 FAX:045(366)3157 CA) co, tU C.) 397A A NOVEL EFFECTOR KINASE TO NFKB THAT ACTIVATES LATENT HIV: T.. OKAMOTO. T., HAYASHI, T., NAKANO, S., SAKURADA, Dept. Mol. Genetics, Nagoya City Univ.,Med. Sch., Nagoya 467, Japan. 398A REGULATION OF MAD3/ IkB IN HIV-INFECTED MONOCYTES-MACROPHAGES. McElhlnny, J.A., Bren, G.D., *Israel, A., Paya. Carlos V. Mayo Clinic, Rochester, MN; *Institute Pasteur, Paris, France Objective: NFKB is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including HIV. This study was attempted to identify a direct effector kinase associated with NFKB. Methods: NFEB/IKB complex was isolated from the cytoplasm of human primary T cells. DNA-binding activity was detected by EMSA. In vitro kinase activity, phosphoamino acid analysis and ATP-binding assay was performed according to the standard procedures. Results: We have detected a novel serine kinase of 43 kDa which closely associates with NFEB. This kinase, named NFKB kinase, phosphorylates serine residues of both subunits of NFKB, not IKB, and activates its DNA-binding activity. Serine/threonine kinase inhibitors, H7 and H8, did not inhibit the kinase activity. Discussion: Since NFB kinase is closely associated with NFKB/IKB complex in the cytoplasm, this kinase might have a central role in transducing various signals to NFEB. The upstream signaling pathway that activates NFxB kinase is currently under investigation. Understanding of novel activation pathways of NFKB may provide a clue to understanding of the molecular process of AIDS pathogenesis and to the development of biochemical intervention as alternative therapeutic modalities. Takashi OKAMOTO, Department of Molecular Genetics, Nagoya City Univ., Mizuho-cho, Mizuho-ku, Nagoya 467, JAPAN, TEL: +81-52-853-8204; FAX: +81-52-859-1235 Persistent HIV Infection of monocytlc cells and macrophages results In NF-kB activation. This mechanism may favor continuous virus replication In these cells. NF-kB Interacts with the inhibitory molecule IkBa (MAD3), resulting In its own cytoplasmic retention. Dissociation from the Inhibitor Is required for the nuclear translocation of NFkB. To further delineate the molecular mechanisms Involved In the activation of NF-kB in HIV-infected monocytes and macrophages, we have focused on the regulation of MAD3. MAD3 protein levels are decreased In HIV-infected human monocytlc cells and macrophages when compared to uninfected, as determined by Immunoprecipitatlon and immunoblottlng experiments. Conversely, total mRNA levels and new protein synthesis of MAD3 are Increased In HIV-infected monocytic cells. In both uninfected and HIVInfected cells, MAD3 Is a phosphoproteln with specific phosphorylation on serine. There are at least three different phosphorylated forms of this protein which can be Immunopreclpitated from monocytic cells. Each form exhibits a different phosphorylation pattern, with qualitative/quantitative differences. The Increase In RNA and MAD3 synthesis In HIV-lnfected cells Is transcriptionally regulated. Transcription of the MAD3 promoter Is significantly Increased In HIV-infected cells and Is NF-kB dependent. This suggests the existence of a triple autoregulatory loop in which persistent HIV Infection of monocytes and macrophages results In persistent NF-kB activation. This may occur via differential "HIV"-lnduced phosphorylation and subsequent degradation of the Inhibitory molecule IkBca (MAD3). NF-kB Is then able to Increase the transcription of HIV through the HIVLTR, of p105/p50 and of MAD3/IkB. PAYA, Carlos, Mayo Clinic, Rochester MN Telephone (507) 284-3747; Telefax (507) 284-3757

Page  27 399A MODULATION OF HIV TRANSCRIFPTION BY MULTIPLE FACTORS. Ishii, S., Maekawa, T., Yasukawa, T., & Sudo, T. (RIKEN Tsukuba Life Science Center) The long terminal repeat (LTR) of HIV is critical to regulate HIV transcription. We have analyzed multiple transcription factors that bind to the HIV LTR. By cDNA cloning, we have identified HIV-EP (also called MBP or PRDII-BF) that can bind to the HIV enhancer. A HIV-EP family has three member (HIV-EP1, 2, and 3), all of which encode large proteins more than 200 kDa containing 2 sets of metal-finger structure in their DNA-binding domain. Expression of HIV-EP is greatly induced by mitogen and phorbol ester treatment of T cells. Functional analysis of HIVEP will be presented. From human B cells, we have purified 39 kDa proteins, HIV-TFl, that binds to the specific site about 60 bp upstream from the HIV enhancer. HIV-TF1 stimulated transcription from the HIV promoter in vitro by incerasing the binding affinity of NFKB to the enhancer. Treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 is essential for DNA binding. The c-myb proto-oncogene product (c-Myb) can bind to the HIV LTR and stimulate transcription. We have identified two c-Myb binding sites in the HIV LTR. Recently, we have suceeded to identify the inhibitor of c-Myb (Lassin: leucine zipperaociated inhibitor) by cDNA cloning. Effects of Lassin on HIV transcription will be presented. Shunsuke Ishii, RIKEN Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Phone:0298-36-9031; Fax:0298-36-9030 401A INHIBITION OF NF-KB DNA BINDING BY ot-LIPOIC ACID. Suzuki. Yuichiro,I. & Packer, L. Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720 USA. NF-KB transcription factor regulates HIV activation. Natural and safe compounds which target NF-KB action may, therefore, be useful in AIDS therapy to support the actions of antiviral agents. We previously found that a-lipoic acid (6,8-dithio-octanoic acid) inhibits NF-KB activity induced by tumor necrosis factor (TNF), phorbol esters or HTLV Tax protein in cultured T cells. The present study compared the effects of lipoic acid homologues on NF-KB DNA binding activity in vivo and in vitro using band-shift assay. The addition of a-lipoic acid in the binding reaction mixtures containing nuclear extracts from stimulated T cells caused inhibition of NF-KB DNA binding in vitro. Octanoic acid which lacks the cyclic disulfide also inhibited DNA binding, suggesting that hydrocarbon chain participates in the mechanism of a-lipoic acid action. On the other hand, tetranorlipoic acid (2,4-dithio-butanoic acid), which lacks the hydrocarbon chain, was found to be more potent in inhibiting NF-KB DNA binding both in vitro and in vivo in TNF-stimulated Jurkat cells. Bisnorlipoic acid (4,6-dithio-hexanoic acid) was found to be less effective than tetranorlipoic acid; moreover, it never caused a complete inhibition of TNF-induced NF-KB activity in vivo. This suggests that tetranorlipoic acid affects NFKB action through a distinct mechanism which is suppressed by hydrocarbon chain. Thus, the a-lipoic acid molecule contains two structures which can inhibit NF-KB action: (1) hydrocarbon chain tail which requires high concentrations and (2) cyclic disulfide head which is potent and whose inhibitory action is suppressed by hydrocarbon chain. Since a-lipoic acid is metabolized to tetranorlipoic acid in physiological systems, the dual mechanism of a-lipoic acid action may constitute its potential for HIV chemotherapy. 400A CELLULAR FACTORS COOPERATIVELY INTERACT WITH THE REV AXIS OF HIV-1 AUTOREGULATION by Ming Jie Zhang, E.Dayton,& A.I. Dayton: LIR/NIAID/NIH Objectives: We are promoting a novel methodology to identify and clone cellular factors mechanistically involved in the Rev regulatory pathway. Mechods: We have formed Rev/RRE complexes in the presence of nuclear extracts to look for cooperative interactions. Ternary complexes formed by Rev, cellular factors and 32P-labeled RRE are stabilized by UV crosslinking and digested with ribonuclease to remove non-specific complexes. The complexes are then visualized by native gel electrophoresis. Results: At concentrations of Rev and RRE which otherwise form no complexes, the addition of nuclear extracts induces the formation of large amounts of a broadly retarded complex. The degree of retardation is proportional to the amount of added extract. Complexes are not formed with mutant RREs which are defective for Rev binding or with anti-sense RRE. Data will be presented on the purification of factors which form these complexes and on their cell-type distribution. Data will also be presented on regions of the Rev protein and regions of the RRE required for complex formation Conclusions: Nuclear extracts contain factors which cooperatively interact with Rev and the RRE. Andrew I. Dayton, 10/6A08 NIH/9000 Rockville Pike, Bethesda MD 20892 FAX:301-4024122 402A TAT- DEPENDENT AND TAT402A INDEPENDENT TRANSCRIPTIONAL ACTIVATION OF THE HIV-1 LTR BY THYROID HORMONE RECEPTOR. Desai-Yainik. Vandana and Samuels. H. H. Dept of Med., N.Y.U. Med. Ctr.. N.Y.. U.S.A. Objective: The HIV-l LTR interacts with several cellular transcription factors, including NF-KB and Spl. Since members of the steroid/thyroid hormone receptor gene family are known to regulate transcription from the LTRs of a number of retroviruses, we examined whether the HIV- I LTR could be regulated by members of this family. Methods: HeLa cells were cotransfected with a HIV-I-LTR-CAT +/- vectors expressing receptors for thyroid hormone (TR), retinoic acid. glucocorticoids, or Vitamin D +/- a tat expression vector. Cells were then cultured in hormone-depleted media +/- the respective ligand and CAT activity was determined after 48 h. Receptor binding to LTR sequences was determine by gel mobility shift assays. Results: Of the receptors studied, only TR activated transcription from the HIV-l-LTR. Purified TR binds to sequences within the NF-KB and Spl sites of the LTR. TR binds to these sites as monomers. homodimers and heterodimers with Retinoid-X-receptor. Mutations within the NF-KB motifs, which eliminated binding of TR. also abolished stimulation by T3, indicating that TR binding to the Spl region does not independently mediate activation by T3. The Spl region, however, is converted to a functionally strong thyroid hormone response element by tat, suggesting an interaction between TR and tat. A site-directed mutant of TR, which is inactive on a variety of promoters. is active on the LTR when tat is expressed, indicating that a different region of TR interacts with tat than with other transcription factors. Other studies revealed a direct physical interaction between TR and tat. Discussion/Conclusions: Our studies indicate that TR stimulates the expression of the HIV-1 LTR in both a tat- dependent and tat -independent manner and provide evidence that tat, which is essential for HIV-I replication and gene expression, directly interacts with TR. We are currently determnnining the domains in each protein essential for this interaction. DESAI-YAJNIK, V. NYU Medical Center 550 First Avenue TH 416 N.Y.C., N.Y. 10016 USA (p) 212 263-6279 (fax) 212 263-7701 CA) o 0

Page  28 403B Detection of M. tuberculosis andM. avium in AIDS patients through PCR: problems and promises. De Mirandal, AB, Grinstejn2, B, Veloso2, VG, Degrave1, WM and Suffys, PN1 DBBM1 and HEC2 - Oswaldo Cruz Institute - Fiocruz - Rio de Janeiro - Brazil 404B CYTOKINE PROFILES IN PATIENTS WITH PULMONARY TB AND HIV: EFFECTS OF ANTI-TB TREATMENT Morris Lynn, Makena SN, Tiemessen CT, Martin DJ, National Institute for Virology, Johannesburg, South Africa Confirmation of diagnosis of tuberculosis in AIDS patients is still hampered by the delay of culture and biochemical identification of M. tuberculosis and determination of its resistance towards antituberculosis drugs. Also, infection with M tuberculosis can be confused with infection with M avium, an organism thas is resistant towards most antituberculosis drugs. The polymerase chain reaction (PCR) is a technique with a potential for rapid and specific detection of parasite DNA, and, both for M. tuberculosis and for M avium, PCR systems have been described in literature. However, hardly any data are available for PCR-detection of these organisms in AIDS patients. We adapted the PCR system, detecting the IS6110 sequence of M tuberculosis, and adopted a specific amplification system for M avium. We also developed a processing protocol for blood, through isolation of PBMC and heat schock, allowing application of at least 1 tl of sample to a PCR reaction without inhibition. We encountered specific problems and have preliminary data on PCR detection of M tuberculosis in AIDS patients. We furthermore obtained M avium-specific amplification using pure DNA and are actually testing the PCR system on processed blood from AIDS patients. This research recieved support from the CNPq. Philin, Noel Suffys, DBBM, ICC, Fiocruz, Iv. Brasil 4365 - Manguinhos 21045-900, Rio de Janeiro, RJ, Brazil Tel 5521-5984.289 Fax 5521-2709997 OBJECTIVE: To determine the impact of HIV infection on TB-induced cytokine profiles and the effect of anti-TB therapy on these profiles in dual-infected patients. METHIODS: 45 patients with pulmonary TB were analysed of whom 30 were co-infected with HIV. The HIV/TB group were further divided into those with <200 (15) and those with >200 (15) CD4 cells/mm'. Peripheral blood mononuclear cells were stimulated in vitro for 3 days with phytohaemagglutinin and analysed for interleukin-2 (IL-2), IL-6 and tumour necrosis factor (TNF) secretion using specific biological assays and for interferon-y (IFN-y) secretion by ELISA. RESULTS: The levels of IL-2 and TNF were significantly reduced in both groups of HIV/TB patients compared to the TB only group, particularly in the <200/mm group for TNF secretion. In contrast the levels of IL-6 were elevated in both groups of HIV/TB patients compared to the TB only group. Longitudinal analysis of HIV/TB patients showed that there was a trend towards recovery of IL-2 secretion and a reduction in the levels of IL-6 secreted following treatment with standard anti-TB drugs. Analysis of the IFN-y levels showed that HIV/TB patients secreted similar levels of IFN-y, irrespective of their CD4 count, to the TB alone group suggesting that cells other than CD4 cells were responsible for the production of IFN-y in HIV/TB patients. CONCLUSIONS: HIV has a potent effect on the TB-induced cytokine profile. Alterations in IL-2 and IL-6 due to HIV co-infection can be reversed by anti-TB therapy, suggesting that such treatment improves the immune status of dual-infected patients and slows the progression to AIDS. Thus in sub-Saharan Africa where TB is endemic and HIV infection is increasing, TB chemotherapy and chemoprophylaxis may represent the most cost-effective method of controlling HIV disease. Morris L. National Institute for Virology, Private Bag X4, Sandringham 2131 South Africa Fax (011) 882 -0596 406B PRESERVED PPD REACTIVITY AND FREQUENT CAVITARY DISEASE AS INITIAL CLINICAL MANIFESTATIONS OF PULMONARY TUBERCULOSIS IN HIV-INFECTED UGANDANS. Nsubuga, Peter', Whalen C", Johnson JL", Byekwaso F', Okwera A', Mugerwa R', Ellner JJ". 'Makerere University, Kampala, Uganda and "Case Western Reserve University, Cleveland, Ohio, USA. TB is the most common serious opportunistic infection in HIV-infected individuals in East Africa. We evaluated the relationship between baseline CD4 lymphocyte count determined by flow cytometry and the initial clinical, laboratory and radiographic manifestations of sputum smear positive, culture positive pulmonary TB in 191 HIV-infected Ugandan adults (mean age 28 years) enrolled in a prospective cohort study of the impact of TB on HIV infection. TB occurred at a wide range of CD4 counts (median 346 l '; range 5 to 2900 il1); 30, 27, and 43% of patients had CD4 counts of 0-200, 201-400, and > 400,plt, respectively. Despite HIV infection, 93% of the patients had PPD reactions of > 5 mm. induration. Lower CD4 countswere associated with PPD anergy [21% (CD4 s 200pl') vs. 1.5% (CD4 > 200 plt); p <.05]. Cavitary disease was present in 54% of all patients and was associated with higher CD4 counts [67%, (CD4 > 401 ld '); 49%, (CD4 201-400 l '); 35%, (CD4 _ 200 pl ')] aswas radiographically more extensive disease. The location of pulmonary infiltrates (upper vs. lower lung field), pleural effusion, hilar and mediastinal lymphadenopathy, and miliary disease showed no relationship to CD4 count. Patients with higher CD4 counts also were more likely to manifest fever, anorexia, anemia, and leukopenia and have larger candida skin test reactions and higher serum fA microglobulin levels. Thus, larger skin test reactions and lung necrosis reflecting preserved delayed type hypersensitivity were present in patients with higher CD4 counts. In contrast to studies from industrialized nations, HIV-TB in developing countries with a high prevalence of TB more frequently presents with sustained PPD reactivity and typical radiographic features. Peter Nsubuga, MBChB, Uganda-CWRU Research Collaboration, P.O. Box 663, Kampala, Uganda PH/FAX 256-41-245-643 0 0 0 wn 405B PROSPECTIVE EVA.LUATION OF RISK OF TUBERCULOSIS IN HIVINFECTED PERSONS BY TUBERCULIN REACTION SIZE Girardi E, Antonucci G, Ippolito G, Anmignacco 0 and Gnppo Italiano di Studio Tubercolosi e AIDS (GISTA) '- Coordinating center:AIDS Unit USL RMi10 - Spallanzani Hospital - Rome, Italy OBJECTIVE: To determnnine the predictive value of different PPD skin-test cutpoints for active tuberculosis (TB) in HIV-infected persons. METIIODS: A mnulticentre, prospective cohort study was carried out in 23 italian hospital units of infectious diseases. 2695 tilV+ subjects (72% IVDUs) were enrolled and underwent skin tests with PPD by Muntotux procedure and with other antigens by Multitest CMI RESULTS: 83 TB were observed after a median follow-up of 21.2 months with an overall incidence of 2.6/100 p-y. Incidences of TB and relative risks by different PPD reaction sizes and response to other antigens (DTII) were: TB/TOTAL INCIDENCE/100p-y ItlAZ. RATIO* 95% C.I. PPD-/DTII+ 5/802 0.4 1 - ANERGICS 62/1579 3 3.7 1.5-9.6 PPD 2-4mm 1/54 1.2 2.1 0.2-18.1 PPD5-9mmn 5/128 2.8 5.8 1.7-20.2 PPD >l 10mm 10/69 9.7 20.2 6.8-60.3 'Adjusted fo:apec, eaUmaissionc atqory, CD4 cells cont, history of TB (Cox model) CONCLUSIONS: 5mm appears to be an adequate cutpoint for PPD positivity among HIV+ persons; however those with larger reaction size are at substantially higher risk for active TB. Further lowering of PPD cutpoint does not appear indicated. * Almi P, Angarano O, Babudieri 5, Bevilacqua N, Bini A, Bottura PBoumis E., Costigliola P, ChirianSni A, Cronato 1,Di Peri G, Esnante I, Fantoni M, Galli M, Ubanore M, Manzillo E, Minoli L, Narciso P, Pagano G, Pellizzer G, Rusooni S, Salmaso S, SSavalli E, Santero D, Suter F, Travenrso A, Viale P.. Work supported by Italian Ministry of tHelh- AIDS Project (grant n. 7203.08) Enrico Girardi, Unita' Operativa AIDS - Spallanzani hlospital, Via Portuense, 292 00149 Rome - Italy Tel. 39-6-58703002 Telefax 39-6-5826570.

Page  29 407B ATYPICAL CHEST RADIOGRAPHIC PATTERNS IN PULMONARY TUBERCULOSIS (PTB) PREDICT ADVANCED HIV INFECTION Wood Robin, Post FA, Pillay GP. Dept. of Medicine and Radiology, Univ. of Cape Town, South Africa. OBJECTIVE: To determine if chest radiographic changes of patients with PTB attending an HIV clinic are related to CD4 cell count. METHODS: Pretreatment radiographs of 105 patients with proven PTB were reviewed blinded. Upper zone infiltrates +/- cavitation were categorised as typical of reactivation PTB. Atypical radiographic patterns were defined by the presence of adenopathy, lower or midzone infiltrates, reticulo-nodular and miliary patterns or a normal chest radiograph. RESULTS: CD4 counts of patients with pleural effusions were significantly lower (p<0.01) than those with typical reactivation pattern. Atypical patterns occurred with further CD4 cell depletion (p<0.01) and had a 89% positive predictive value for defining patients with <200 CD4 cells. Radiographic Pattern Mean CD4 S.E.M. 95% C.I of Mean Typical reactivation n=14 446 41 391-501 Pleural Effusion n=24 222 40 179-265 Atypical pattern n=67 108 15 85-131 CONCLUSION: In an area of high PTB prevalence, atypical chest radiographic presentation of PTB is common and is a specific clinical predictor of advanced HIV infection. 408B AUTOPSY-PROVEN CAUSES OF DEATH IN HIV-INFECTED PATIENTS TREATED FOR TUBERCULOSIS IN ABIDJAN, COTE D'IVOIRE Greenberga. Alan E*/**, Lucas S***, Tossou O*, Coulibaly IM****, Coulibaly D****, Sidib6 K*, Ackah A*, DeCock KM*/**. *Projet RETROCI, Abidjan, CI; **CDC, Atlanta, GA, USA; ***Univ Coil London Medical School, London, UK; ****Centres Antituberculeux, Abidjan CI. Objective: To determine the causes of death in HIV-infected patients treated for TB in Abidjan, CI. Methods: A computerized listing of 496 patients enrolled in a 02-12/91 autopsy study of HIV+ persons in Abidjan was matched by name, age, and sex against a computerized listing of 9,523 TB patients seen in Abidjan's two large TB treatment centers from 07/89 to 12/91. The TB center records of all matching patients were reviewed to confirm the initial TB diagnosis and to assess the response to a standard 6-mo. regimen of isoniazid/rifampin (6 mos) and pyrazinamide (2 mos); all autopsy findings were reviewed. Results: 15 HIV+ patients who had been diagnosed with TB and who had later been autopsied were identified. 14 were adults aged 20-47 (M:F 12:2) and I was a two-year old boy; 11 were HIV-I+, 2 were HIV-2+, and 2 were dually reactive. 11 adults had been initially diagnosed with sputum AFB+ pulmonary TB (PTB): 6 were treated for 6 months and cured, but then died 1-4 months after completing therapy -- none died of TB (3 bacterial infections, 2 toxoplasmosis, and 1 CMV); 5 died 2-4 months after beginning therapy -- 2 died of TB, and 3 died of other causes (Kaposi's sarcoma, wasting syndrome, and nocardiosis). 3 adults had been initially diagnosed with extrapulmonary TB (EPTB): 1 died of TB 12 months after completing therapy; 1 died of TB 15 months after receiving 4 months of therapy; and I died of PCP after receiving 5 months of therapy. The child had sputum AFB- clinically-diagnosed PTB, and died of LIP after receiving 3 months of therapy. Conclusion: These data suggest that HIV + PTB patients who are successfully treated for TB but who die within 6 months of completing therapy are unlikely to die of TB; that HIV+ PTB patients who die during therapy may very well die of TB; and that HIV+ EPTB patients may require >6 mos of therapy. Alan E. Greenberg Projet RETRO-CI/CDC 01 BP 1712 Abidjan 01, Cote d'Ivoire; (225i 25-41-89 / FAX (225) 24-29-69 WOOD Robin, Dept of Medicine, Univ. of Cape Town, R.S.A. Telephone 021-402-6317, Telefax 021-402-6000 409C HIVISTD INCIDENCE AMONG TRUCK DRIVERS IN MOMBASA, KENYA: ROLE OF CIRCUMCISION STATUS Jackson. Denis Rakwar J, Bwayo J, Martin H, Mandaliya K, Moses S, Kreiss J. U. of Washington, Nairobi, & Manitoba; Coast Province General Hospital. 410C DECREASED HIV/STD RISK BEHAVIOUR IN SEXUALLY ACTIVE ADULTS. REKART MICHAEL L, Knowles LJ, Patrick DM. B.C. Centre for Disease Control, Vancouver, Canada Objective To measure HIV seroincidence and assess correlates of infection, especially STDs and circumcision status, in a cohort of men suitable for prophylactic HIV vaccine efficacy trials. Methods HIV seronegative men were recruited at trucking companies in Mombasa and followed prospectively at 3 month intervals. Results Among 874 men screened between March and December 1993, HIV-1 seroprevalence was 17.4%. Six hundred two seronegative men were enrolled. Sex with a commercial sex worker in the previous year was reported by 32% (of whom only 37% claimed consistent condom use) and 58% had 2 or more partners. Seven HIV-1 seroconversions were documented during 175 person years (py) of follow-up, an annualized seroincidence of 4%. Seventy five (12.5%) of the 602 enrolled men were uncircumcised. Uncircumcised status was not correlated with serologic evidence of past STDs or with incidence of gonorrhea, NGU, or HIV, but was associated with a significantly increased risk of genital ulcers (RR 3.5, 95% CI 1.6-7.9). Enrollment Prevalence of STD Antibodies Incidence of STD (per 100 py) Uncircumcised Circumcised RR Uncircumcised Circumcised RR T. pallidum 10.8% 6.7% 1.6 Gonorrhoea 12.1 22.7 0.6 H. ducreyi 26.4% 26.0% 1.0 NGU 24.2 27.3 1.0 HSV - 2 47.6% 41.1% 1.2 Genital ulcers 32.1 8.0 **3.5 C. trachomatis 40.5% 34.8% 1.2 HIV-1 4.0 4.0 1.0 Discussion Truck drivers in Mombasa had a high incidence of HIV and other STDs. Uncircumcised status was associated with a 3.5 fold increased incidence of genital ulcer disease. JACKSON, Denis, University of Washington, ZX-32, IARTP, Seattle, Washington, USA Telephone: (206) 223-8055; Telefax: (206) 223-8752 Objective: To assess changes in HIV/AIDS risk behaviour over the last four years in a stable, sexually-active, urban adult population. Methods: Patient behavioural data from a metropolitan STD/HIV clinic was examined retrospectively. Three areas of risk were compared from 1989 to 1993: condom usage (always, no, sometimes), number of partners in the last 2 months and sites exposed (rectal, genital, oral). Patient demographics, total visits and clinic variables were similar. Results: Consistent condom usage increased from 15% (891/5898) to 29% (1753/6047) - RR 2.21; 95% CI 2.05, 2.38. Patients who reported 2 or more partners decreased from 37% (2591/6983) to 31% (1603/5118) - RR.84; 95% CI.80,.89. Patients reporting oral sex rose from 26% (2452/9371) to 35% (3165/8929) - RR 1.38; 95% CI 1.32, 1.44 while genital sex decreased from 71% (6692/9371) to 60% (5356/8929) - RR.86; 95% CI.84,.87). The frequency of rectal sex did not change. Conclusion: In a stable, sexually-active adult population from an urban Canadian setting, three measures of HIV/STD risk improved significantly. These important changes reflect the combined success of community-based and public health education and prevention campaigns, media attention, advocacy and individual action. Renewal and intensification of these efforts will likely enhance success. Dr. M. L. Rekart, 828 West 10th Avenue, Vancouver, B.C. V5Z 1L8 Phone (604) 660-6170 Fax (604) 775-0808 O 4 0 44k I 0

Page  30 4 411C THREE YEAR IIIV SURVEILLANCE AMONG STD CLINIC PATIENTS IN PARIS. UtI~FRANCE Meyer Laurence*, Couturier E**, Brossard Y*** and the Prevaday Group. *INSERM U292, Bicetre, France, ** European Center for Surveillance of AIDS, St-Maurice, France, *** III St-Antoine, Paris, France. Qbjective: To estinate IIIV infection trends by transmission groups among STD patients. Mehods: Fromn 1991 to 93, during a 3 month-period, data on suspected STD, age, sex, birthplace, IV drug use, sexual orientation, known IIIV status were collected for all patients with suspected STD in Paris STD clinics. Sera collected for syphilis testing were tested for IIIVI-2 using an unlinked anonymous method. Results: Over the 3 years. no significant trends in IIIV prevalences were seen (see table). Year Number of patients Total Men 1113 Men Total non-IVDU Women other non-IVDU tested (% of women) Women born in pattern It Women 1991 1628 (32%) 9.9% 35.2% 1.4% 3.1% 0.3% 1992 1564 (34%) 8.2% 30.2% 1.7% 4.6% 0.6% 1993 1144 (34%) 8.0% 30.4% 1.3% 2.1% 0.4% IIIV prevalences were higher in men than in women. Among non-IVDU homolbisexual (JIB) men, IIIV prevalence was high, as was the proportion of known IIIV positive (24%, 21%, 22%). In 1993 in the HB group, there was a significant association between IIIV prevalence and age (10.5% among <25years; 29.2% 25-34 yrs; 41.5% >35 yrs). Only in 1993, non IVDU heterosexual men born in pattern II countries were more often IIIV infected than other non IVDU heterosexual mten (6.3% vs 2.3%,p<0.05). Among non-IVDU women, IIIV prevalences were higher in those born in pattern II countries than in the other women. The proportion of STD patients who were not tested for syphilis (and thus excluded from unlinked IIIV testing) was 5.7% in 1991, 3.6% in 1992, 6.8% in 1993; those patients were more often male and known to be IIIV positive than those tested for syphilis. Conclusion: The number of included patients decreased from 1991 to 1993; decreasing trends were also observed in the other French STD surveillance systems. The stability of IIIV prevalences as well as the rate of known positive among liDB men are similar to those observed in other european countries. They confirm that prevention strategies need to be improved. The higher rate of IIIV positive patients among those not tested for syphilis shows that sentinel IIIV surveillance systems in STD patients should not rely only on patients tested for syphilis. MEYER Laurence. Epidemiology Service, INSERM U292, Hospital of Bic8tre, Le Kremnlin-BDictre. France Tel (1) 45 21 23 34; Telefax (1) 45 21 20 75 412C UNLINKED ANONYMOUS SURVEY OF HIV PREVALENCE IN ATTENDERS AT STD CLINICS IN ENGLAND AND WALES, 1990-1993. Dr Christopher F Joyce, Dr AG Nicoll, Dr 0 Mercey, Mr I Simms, Mrs P Rogers, Dr M Catchpole, Dr ON Gill, CDSC, PHLS, London. "Academic Department of GU Medicine, Middlesex Hospital, London. Objectives: To monitor the prevalence of HIV infection by exposure category in patients attending STD Clinics. Methods: Residues of sera collected for first syphilis testing in 6 London and 8 provincial clinics were tested for HIV-1 and HIV-2 using an unlinked anonymous IUAI methodology. UA data on sex, age group, country of birth, sexual orientation, history of injecting drug use, whether known HIV infected, and summary clinical diagnosis were recorded. Results: Data from 104,678 specimens were collected (<1% objected to UA testing of their serum). HIV-1 seroprevalence was 19% for homolbisexual men in London, 4.6% outside London. For heterosexual women the figures were 0.7% and 0.1% respectively. Seroprevalence amongst those reporting injecting drug use was 7.1%. Comparison of those known to be HIV positive with their STD diagnoses indicated that numbers of HIV infected homolbisexual men continued to practice risk-taking behaviour. Preliminary analyses indicated a rise in level of infection amongst women not known to be HIV positive attending STD Clinics. Discussion: This survey has shown that UA methodology can be successfully applied in STD clinics within an industrialised country with adesvelopedSTD clinic system. The low objection rate indicated that the seroprevalence estimates would be unbiased. The main value of the survey will be for the detection of trends in HIV seroprevalence. Conclusion: Large scale unlinked anonymous HIV seroprevalence monitoring programmes can provide an invaluable method of monitoring the progress of HIV epidemics in industrialised countries. HIVISTO Division. Public Health Laboratory Service at the Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, United Kingdom. Telephone: (44) 081 200 6868, Fax: (44) 081 200 7868 414C PREVALENCE OF HIV INFECTIONS AMONG THAI414CLANDERS ATTENDING TO AN STD CLINIC IN TOKYO METROPOLITAN AREA SHIMIZU. Motoyuki1, KIHARA, M2, KIHARA, M.3, NAKAHARA, T.4,SODA, K.2 (1Shimizu Clinic, Tsuchiura, 2Dept. of Public Health, Yokohama City Univ. School of Med. Yokohama, 3Dept. of Epidemiol., Kanagawa Cancer Center Res. Inst., Yokohama, 4Dept. of Public Health Administ., Inst. of Public Health, Tokyo) OBJECTIVE: To explore the prevalence and the trend of HIV infection among female Thailanders in Tokyo metropolitan area of Japan. METHODS: Medical records of female Thailanders between1989-1992 were surveyed at an STD clinic, located in Tokyo metropolitan area, which reported 1/10 of foreign female cases to the HIV/AIDS Surveillance in Japan. Results of laboratory tests for HIV, syphilis, gonorrhea and chlamydial infection were investigated. RESULTS: The study revealed (1)that over 1000 Thailanders attended the clinic during the study period with a marked increase from 1990 to 1991, (2)that among the tests only anti-HIV testing rate markedly elevated by 2-3 folds from 1990 to 1991 and (3)that 39 HIV positives were identified with overall positive rate around 4%, being fairly constant during 1990-1992 and highy prevalent(14.0%) in females aged<20. DISCUSSION AND CONCLUSIONS: The present study suggested that HIV infection rate of female Thailanders in Japan is much lower than the reported rate for female prostitutes in Thailand. It also suggested that the sudden increase in foreign female cases seen in the HIV/AIDS Surveillance in Japan from 1990 to 1991 is likely due to the increase in anti-HIV testing rates as well as in the number of people attended the medical institutions but unlikely due to the increase in the HIV positive rate among the female Thailander population in Japan. SHIMIZU Motoyuki, 13-1 Arakawaoki-cho, Tsuchiura,Japan., TEL:0298(42)5467 FAX:0298(43)1152 40 lI 44h -f 44h 0 413C THE AETIOLOGY OF SEXUALLY TRANSHITTED DISEASES IN BOTSWANA. Mofat Hovyard,* Rahman M,* Ballard R,** Dangot Y,** vd Veen F,***. *AIDS/ STD Unit, Botsvana; **SAIHR, Johannesburg; ***AIDS Task Force, EC. Itkground: S'IDs are a major problem in Botswana. Nearly 10% of all clinic attendances are for symptoms of STD. This study was done to obtain fase-line data on the aetiology of STDs in Gaborone. Objectives: 1) To determine the aetiology of aenital. infections in symptomatic pat ient.s. 2) To determine the genital microbiological flora in asymptomatic sexually active women. 3) To determine antimicrobial resistance. 4) To determine the prevalence of syphilis and FIIV infection in the study groups. Methods: One hundred consecutive patients in each of the following four groups were investigated: a) vaginal discharge; b) women attendina for family plannina (FP) advice; c) urethral discharge in men; d) aenital ulcer in either men or women. Results: 16% of women with vaginal discharae had gonorrhoea as opposed to 7% in the FP group. Otherwise there was no difference in the rates of genital infection in the two groups. Mixed infections occurred in 71% of the women making clinical diagnosis impossible. 63% of men with urethral discharge had gonorrhoea. Over 40% of all isolates of N. gonorrhoea were resistant to pn icillin. 26% of genital ulcers harbotred H. ducreyi and 24% herpes simplex. Positive syphilis serology occurred in 56% of subjects. 41% of those with genital ulcers were HIV positive. These findings have importait implications and support, inter alia, the concept of the syn dromic approach to STD management in the absence of a laboratory. D)r H.J. Moffat, Princess Marina Hospital, Pu Box 258, Gaborone, Botswana. Tel: (267) 353221 Telefax: (267) 306147

Page  31 415D KNOWLEDGE, ATTITUDES AND BEHAVIOR AMONG NEWCOMERS OF COLLEGE IN PORTO ALEGRE, STATE OF RIO GRANDE DO SUL, BRAZIL. 'Corea CC, 'a~mpeseMS, #Barcelos NI, "Bo'ako MP,#Gomes A, #Rodrigues ACS, *Soibelmnan M, #Wiers 0, #SRamos MC 'Hospital Cd ices Porlo Aleogre #RGS Slate Ieelth Secretariat 416D KNOWLEDGE AND SAFE SEX PRACTICE AMONG HETEROSEXUAL TERTIARY STUDENTS AS AN OUTCOME OF SOCIOCULTURAL CHANGE. Crawford, June, Kippax, S and Rodden, P. National Centre for HIV Social Research, Macquarie University NSW Australia. QObjectives: To evaluate knowledge, attitudes, behavior (KAB) and sources of information related to AIDS among 492 students just entering college in Porto Alegre. Brazil. Methods: Newcomers of two major universities (Medicine.Dentistry. Pharmaceutics, Biology) were surveyed anonymously using a guided code Identification procedure on a self-administered questionnaire in February 1994, in Porto Alegre. RS, Brazil Results: The response rate was 61.80 % (N=492/796) 84.2% of students were between 17-20 years old (18.9) and the first Information about AIDS was obtained between 10-15 years old (75.6%). mainly through TV and newspaper (70.3%). The sample consisted of 62.2% female and 37.8% male. Regardingo n o AIDS transmission the following Items were correctly answered: blood transfusion (97.8%); unprotected sexual Intercourse (97.0%); sharing needles (96.7%); WC (84.5%); mosquitos (70.1%); blood donation (51.0%). Among 455 completed questionnaires 85,4% of males and 53,5% of females had first sexual intercourse between 14-19 years old and 62% had unprotected sex at that time. Only 0.8% reported previous IV drug use compared with 91.9% for alcohol consumption 4.7% of the respondents were HIV tested and all results were negative. Conclusions: Despite getting early information, most of students has not adequate knowledge about AIDS Unprotected sex during sexual initiation is alarming and demands adequate management among young students at college In Porto Alegre. Marcia Zampese, MD - Hospital de Clinicas de Porto Alegre R. Dr. Timoteo, 8161210; Porto Alegre-RS, BRAZIL, 90570-041 Phone: (55) 51-2225312 FAX: (55) 51-3328324 417D Knowledge and attitudes to HIV and AIDS and sexual practices amongst University students in Lueaks, Zambia. R.Beggley*,**, J.Mukosha***, D.Chipanta***, M.Phirt***,M.Teabo+, P.Oodfrey-Fauesett**,*****Kara-Zambsrt Project, Lusaka **London School of ygilene and Tropical Medicine ***Kara Counselling and Training Trust, Lusaka,****Zambart project, Department of Medicine, Universit ty Teaching Hospital, Lusaka,+UNZA Counselling Centre. Lusaka Objectives: 1. To determine the HIV knowledge and attitudes of students attending the University of Zambia (UNZA) to aid the planning of an HIV education programme. 2. To assess sexual practices and condos use amongst the students. Methods: Anonymous self-administered, pro-tested questionnaires were distributed to students at UNZA during August 1993. The questionnaires were distributed to the halls of residence by the counselling department and the students were asked to fill them in at their leisure and return them completed to the counselling centre. Results: 946 of the 2 000 questionnaires were returned. Knowledge regarding transmission was only moderately good: 92% knowing that HIV was transmitted in semen, 87% in blood and 85% in vaginal fluid. 15% thought that emosquitoes and 46 that saliva transmitted HIV. Only 32% of people knew no one with HIV. 38% had had a relative die of HIV and 401 had a friend or acquaintance with HIV. Despite this there were still many people with negative attitudes to those with HIV: 8K felt that people with HIV had led immoral lives, 15% felt that they should be isolated and 40% did not like the idea of sharing cups with HIV positive people. 79% felt that people with HIV should have equal opportunities. Only 235% felt that they were definitely at risk from HIV, with 23% not being sure. 1% had had I end 71% had had more than I sexual partner. 62% had ever used a condom 17% using always with a regular partner and 32% with a non-regular partner. 75% felt that condoms should be more easily available at UNZA and 51% said they would use them if they were. 88% said that there was not enough information about HIV at the University and 71% felt that HIV counselling should be available. Conclusions: I. Basic knowledge about HIV was only moderately good and the students at UNZA felt that they were not receiving enough education about HIV at the University and that HIV counselling should be available. 2. A large percentage of those questioned have contact with people with HIV and programmes should be directed at improving attitudes to those who already have HIV and reducing the stigma of HIV. 3. The majority of the students were sexually active but regular condom use was low. Condom use and safer sex advice should be made msore available to students at UNZA. Rachel Baggley, Kara-Zasbart Project Kara Counselling and Training Trust Po sBox 3755, Lusaka Zambia Phone +260 1 229847 Pax + 260 1 22984$ Introduction: KAP studies of young heterosexual students have usually found little correlation between knowledge and practice when data are collected at one point in time. Objectives: Data were collected in each of four years and analysed to investigate (i) whether knowledge and practice of 18-19 year old tertiary students change over time (ii) whether there is evidence for a relationship between knowledge and practice when this relationship is conceptualised as a sociocultural rather than an individualistic phenomenon. Method: From 1990 to 1993 self-report questionnaires were completed by a total of 2236 (almost all heterosexual) students in their first week at university. One index of 'accurate' knowledge of HIV transmission was the absence of a distinction between 'regular' and 'casual' partners when judging safety or risk of seven sexual practices. Sexual safe practice was defined as either no penetrative sex or consistent use of condoms. Results: Analysing data only from the sexually active, it was found that for the four samples combined, accurate knowledge was associated with safe practice. Those with accurate knowledge (i.e. making no distinction) were more likely to use condoms consistently with 'regular' partners (p<0.0001). Those making a distinction were more likely to avoid 'casual' partners (p<0.0001). Accurate knowledge and safe practice both increased over time. It was found using logistic regression that the best predictor of safe practice was time, not knowledge. Conclusion: Accurate knowledge and safe sex are associated to some extent, but practice may produce knowledge as well as vice versa. By examining a different cohort each year, strong evidence was found that change in practice needs to be understood as a function of sociocultural change, not individual change. Dr. June Crawford, National Centre for HIV Social Research School of Behavioural Sciences, Macquarie University 2109 Australia +61 2 805 8112 418D THE IMPACT OF AIDS EDUCATION ON UNDERGRALUAE MEDICAL STUDENTS. OsP. AGGAR WAL AND A.K. SHARMA Univ. Coll. Of Med. Sci, Delhi-95, India Ob ecive s to evaluate the gain in knowledge on AIDS of Medical students during four and a half years of their under graduate education. Method: The level of knowledge of the final year medical Students was assessed using a multiple response questionnaire. Questions were asked with respect to knowledge of general aspects, Pathology, Microbiology, Epidemiology, mode of transmission, blinical features, diagnosis, management and prevention. For analysis, findings were compared with that of the new entrants to the medical college. Res t & i,,sCUSion: The level of knowledge of final year students in every aspect was significantly higher than that of the new entrants. However, on the whole lacunae in knowledge were observed regarding the distribution of the disease, magnitude of the problem and management of cases of AIDS. The reasons for this lacunae could be ever changing picture of AIDS in India and the attached GTB Hospital not having a special ward for AIDS patients.,nclusion s A detailed knowledge on all aspects of AIDS needs to be emphasized in medical under graduate curriculum, Dr, O.P. Aggarwal, Head, Deptt. of P.S.M., Univ. Coll. of Med. Sci, Jelhi-110095, India f.No.Res:2214616, Offices2282971 Ext. 251 44b U' N~ 00b

Page  32 C 419D MEDICAL STUDENTS' ATTITUDES IV ABOUT HIV-RELATED ISSUES CHANGE AFTER 1 CLERKSHIP MacArthur Rodger D, White D, Kosmyna J, Lipton L, Asbury P, MacArthur RG. Medical College of Ohio; Toledo OBJECTIVE: To determine whether medical students' attitudes toward HIVrelated issues change after 1 clinical clerkship. METHODS: We developed a 26-item questionnaire designed to measure medical students' attitudes about HIV-related issues affecting them during training and susequently. The questionnaire was administered at the end of the second year, and again 3 months later to students whose first clinical clerkship was either medicine or surgery. RESULTS: 138/146 students (95%) completed the initial questionnaire; 64/70 students (91%) completed the second questionnaire. Students responded more favorably to HIV-related issues after completing 1 clerkship. Students choosing medicine as the initial clerkship responded much more favorably than did students choosing the surgery clerkship. Responses of those who participated in the care of HIVinfected persons did not differ from the responses of those who did not. CONCLUSIONS; Medical students' attitudes about HIV-related issues persons can become more favorable with minimal clinical experience. Students choosing medicine as the inital clerkship appear to view HIVrelated issues more favorably than students choosing surgery. 420D RELIGIOUS AND CULTURAL NORMS VERSUS SAFE SEX PROGRAM FOR SCHOOL CHILDREN: The case of Tanzania. Viv MARI, HIV/AIDS UNITECH PROGRAM. Rodger D. MacArthur, M.D. Medical College of Ohio 3000 Arlington Avenue - Toledo, 419-381-4328 FAX: 419-382-0354, Ohio 43614 OBJECTIVES: To investigate the extent to which sex education is given to children at family and school levels. To find out whether some girls drop out of school because of pregnancies and thus to establish the fact that children practice pre-marital sex contrary to cultural norms; and to assess their knowledge on HIV/AIDS & safe sex. MRTHOD: Between 1990 and 1993 we interviewed 3000 school children aging from 14 to 17 on the following:- level of sex education at home & school; magnitude of the problem of pregnancies among school girls; level of knowledge on safe sex and how they regard religious and cultural norms that prohibit them from pre-marital sex as well as condom use and the dangers of HIV infections. RESULTS:15% talk freely about sex with their parents; 100% admit that family life education is taught at school but it is inadequate; 10% were expelled because of pregnancies;: 90% admit to be practising sex; 40% have had sex with more than one partner;: 100% are informed of HIT but only 60% have used condoms more than once; 95% don't see why religious/cultural norms should prohibit use of condoms;80% requested for condom accessibility. CONCLUSI1N: If children are to be protected from HIV/AIDS infections, adequate safe sex education must be included in the school curriculum as well as to propagate the change of obsolete cultural/religious norms such as those which prohibit condom use. 4, MEARI, HIVAIDS UNITECH PROGRAM, 0/o Medical Aid Poundation, P. o. Bbx 1430, Dar es Salaam, Tansania. Tel. 36207' S Retroviral zinc finger chelate as a new target site for,IV chemotherapy. E. Kun, J. Mendeleyev, Alaeddin llakam and WO. Rice, Octdamer Research Foundation, Romberg Tiburon Centers. San Francisco State University, PO.Box 915,lTiburon CA 94920 USA, and Lab of Antiviral Drug Medhanism, Program Resources ItncJlDynCorp, NCI- Frederick Cancer Research and Development Center Bltdg. 431 T-B., P.O.Box 21702, Frederick MD 21702 USA We have demonstrated that the retroviral zinc finger, specifically the CCHC zinc chelating amino acid residues present in HIV-1 p7NC are specific targets for certain mild oxidizing agents: aromatic C-nitroso compounds, which by generating disulfides eject zinc from these sites (Nature 361, 437, 1993; PNAS 90, 9721, 1993), resulting in complete abrogation of the infectivity of the HIV virus. This effect is highly specific and selective, since none of the other steps of the infectious cycle are affected by these drugs. Mutations in RT, producing drug-resistance do not interfere with the antiviral action of CNO drugs. Since C-NO molecules are too unstable we developed highly stable and nontoxic (in vivo tolerated dose = 200 mg/kg) halogenated nitro"pre"-drugs which are enzymatically reduced to active C-NO within cells and in vivo. Results demonstrating the chemotherapeutic efficacy of these "pre"drugs will be presented. 44~h 421A ANTI-HIV AND ANTI-APOPTOTIC ACTIVITY OF THE WHEY PROTEIN CONCENTRATE: IMMUNOCALTM Sylvain Baruchel', Rend Olivier2., Mark Wainberg.' 'Montreal Children's and Jewish General Hospital, McGill AIDS Centre, Montreal, Quebec Canada and 2 AIDS and Retroviruses department, Pasteur Institute, Paris, France. OBJECTIVES: The in vivo glutathione (GSH) promoting activity of undenaturated Whey protein concentrate (WPC) has already been demonstrated. Here we demonstrate the anti HIV and anti Apoptotic activity of a WPC product termed IMMUNOCALTM and its relation with GSH synthesis. METHODS: IMMUNOCALTM is produced in linear fashion in order to maintain proteins in a non denaturated form and to preserve their glutamyl cysteine residues. We tested the in vitro anti-HIV activity on cord blood mononuclear cells and MT 4 cells by studying each of reverse transcriptase (RT) activity, p24 antigen production, and syncytium formation. GSH was measured by spectrophotometric recycling assay. Apoptosis was evaluated by flow cytometry on PBMC from HIV infected individuals (cells were stained with acridine orange and ethidium bromide)(n=6). RESULTS: An anti HIV activity was found at WPC concentrations between 100pg/ml and 500pg/ml. Inhibition of syncytium formation occurred with a 1C50 of 150pg/ml. PBMCs cultured with these WPC concentrations (n= 3) had a statistically significant increase in GSH synthesis when compared to untreated cells, 9.6~1.5 vs 5.4 ~ 0.4 nmoles/107 cells, p= 0.01. HIV infected PBMCs cultured in the presence of 100lpg/ml of WPC were less prone to die of apoptosis than untreated cells, 15% ~2.6 vs 37% ~2.4 p<0.001. CONCLUSION: IMMUNOCALTM (WPC) possesses antiviral and anti-apoptotic activities which may be related to its glutathione promoting activity. A clinical trial is currently going on with children with AIDS and wasting syndrome. Dr. Sylvain Baruchel Montreal Children's Hospital 2300 Tupper, Montreal, Quebec Tel: 514-934-4322 Fax: 514-934-4301 Ernest Kun, Octamer Research Foundation, Romberg Tiburon Centers, San Francisco State University, P.O.Box 915, Tiburon, CA 94920, USA. Tel: 415-435-8851, FAX: 415-435-8853

Page  33 423A AN INHIBITOR OF LIPID SIGNALING SUPPRESSES HIV EXPRESSION. Leung. David. Peterson P*, Gekker G*, Chao C', Bursten S, Bianco J, & Singer J Cell Therapeutics, Inc., Seattle & University of Minnesota*, U.S.A. 424A VARIATIONS AT AMINO ACID 82 IN HIV-1 PROTEASE (PR) CONFER REDUCED SENSITIVITY TO STRUCXIURALLY DISSIMILAR HIV PR INHIBITORS Winslow, D.L., Anton, E., Horlick, R., Zagursky, R., Tritch, R., Scarnati, H., Ackerman, K., Otto, M., Bacheler, L. DuPont Merck Pharmaceutical Co., Wilmington, DE, USA Objective: To test whether an inhibitor of phosphatidic acid (PA) generation, CT-2576, would suppress the expression and replication of HIV in cells. Methods: A reporter gene expression system under the control of the HIV-LTR promotor in 293 -EBNA cells was set up as a surrogate marker to screen compounds that inhibit HIV-LTR directed expression through blocking of PA. The selected compounds were then tested for their capability of inhibiting HIV expression in the chronically HIV infected promonocyte U1 cell line. Results: CT-2576 inhibited the activation of HIV-LTR promotor by tat or TNF-a with an IC50 value of about 5 tM with minimal cytotoxicity on 293-EBNA cells. CT-2576 was also effective in inhibiting CMV and SV40 early promotor activity but had little effect on the housekeeping gene, phosphoglycerate kinase, promotor activity. CT-2576 suppressed the constitutive expression as well as the TNF-a or IL-6 mediated induction of HIV-1 p24 antigen in U1 cells with an IC50 value of around 1 pM. Discussions and Conclusions: CT-2576 inhibited the cytokine-induced and tat-directed expression of HIV with minimal cytotoxicity. CT-2576 inhibited HIV expression by interfering with the intracellular signaling phospholipid PA which appears critical to cell activation. CT-2576 and other compounds that can suppress PA generation should be of therapeutic interest for delaying or preventing the onset of AIDS and certain other viral diseases in HIV-infected patients. David W. Leung, Cell Therapeutics, Inc. 201 Elliott Ave. W., Suite 400, Seattle, WA 98119, U.S.A. Telephone (206)-282-7100; Telefax (206)-284-6206 425A ALX40-4C: ANTI-HIV, CELL UPTAKE AND PHARMACOKINETIC ANALYSES Sumner-Smith, M., Dabek, B., Presseault, S., Yungblut, P., and Climie, S.C. Allelix Biopharmaceuticals Inc., Chen, I.S.Y, UCLA AIDS Institute, and Conway, B., Ottawa General Hospital. Objective: ALX40-4C (N-a-acetyl-nona-D-arginine amide acetate) was developed as a competitive inhibitor of the binding of the HIV tat protein to its RNA target TAR and therefore has the potential to inhibit HIV transactivation by a unique mechanism, distinct from that of other 'Tat inhibitors'. Transactivation inhibition in model systems has been demonstrated, as has inhibition of HIV in culture. We wished to extend the analysis of the anti-IV activity of ALX40-4C to a range of HIV strains in different cell types, as well as to assess the cellular, tissue and organ uptake of the drug. Results: Replication of HIV in culture is inhibited by the compound. IC50 values ranging from 10iM to 5 sM were measured in various combinations of HIV strains (clinical and laboratory) and cells (primary and transformed). Cell uptake analysis, employing '4C-labelled ALX40-4C, demonstrated that within 24hr, approximately one quarter of the compound in culture was cell-associated, and that 50-80% of that fraction was nuclear-associated. This result is important, because the nucleus is the site of transactivation and therefore the required site of action of the drug. Preliminary studies using 14C-labelled ALX40-4C in mice showed rapid distribution and uptake of drug into tissues, and slow clearance. An ELISA assay for unlabelled drug was developed and employed for pharmacokinetic analysis of the compound in dogs. Discussion and Conclusions: ALX40-4C shows promise in the treatment of HIV infection. Phase I safety studies with HIVpositive, asymptomatic, human subjects were recently completed in Canada. SUMNER-SMITH, Martin, Allelix Biopharmaceuticals Inc., 6850 Goreway Dr., Mississauga, Ontario L4V IV7, Canada. Telephone (905) 677-0831 x302; Telefax (905) 677-9595 CBJETIVE: Previously we reported the selection in tissue culture of HIV-1 variants with substitutions at amino acid 82 in PR with reduced susceptibility to C2 synnetric diols (PNAS 90:7543-7547, 1993). Our objective was to insert defined mutations into the PR gene of an infectious molecular clone of HIV-1 to determine the in vitro susceptibility of recovered virus to structurally dissimilar PR inhibitors. METHODS: Site-directed mutagenesis was used to create mutations in the PR gene of HXB2. Virus was recovered after transfection into Mr-2 cells and susceptibility to PR inhibitors was determined by an RNA hybridization assay and the ACTG PBMC consensus assay. RESULTS: V82A and V82F variants displayed reduced sensitivity to P9941 (C2 syarnetric diol), A80987, and XM323 but not to R031-8959. V82I was as sensitive as wild type virus to all 4 inhibitors tested. CONCLUSICONS: Variants with substitutions at amino acid 82 display reduced sensitivity to structurally dissimilar HIV PR inhibitors. D.L. Winslow, P.O. Box 80026, P26/1154, Wilmington, DE 19880-0026 USA Tel. 302-892-7224, FAX 302-992-5195 426A DESIGN AND OPTIMIZATION OF POTENT, ORALLY ACTIVE IIIV ASPARTYL PROTEASE INHIBITORS. Tun.R., Livingston, D.L, Rao, G.B., Kim, E., Pazhanisamy, S., Delninger, D.D., et. al. Vertex Pharmaceuticals, Incorporated The integration of structural information into the design of enzyme inhibitors, particularly of the HIV aspartyl protease, has helped to create a new paradigm for the rapid optimization of novel structural classes of compounds. In this talk we will discuss our application of a cycle of biophysical analysis, chemical synthesis and evaluation of biological activity to address the issue of attaining oral bioavailability while retaining biochemical potency and synthetic accessibility. We will review details of the interactions made by clinical lead compound VX-478 with the HIV-1 aspartyl protease with an emphasis on specific modifications designed to modify pharmacological parameters. We will also compare the binding interactions of VX-478 with that of several other highly potent inhibitors recently under clinical evaluation. Tung, R.D., Vertex Pharmaceuticals, Incorporated, Cambridge, MA 02139, USA Phone: (617) 576-3111; Fax, (617) 499-2437 44~h CA)

Page  34 .a 427B THANATOLOGICAL HOME-ATTENTION: AN ATTENTION MODEL TO FAMILY, FRIENDS AND/OR PARTNERS OF PAIENTS WITi I AIDS TERMINALLY ILL. Javier Mattnez Badillo*, *, Guerra A.*, Ramos M.*, SalameE.** *Mexican Foundation of Fight Against AIDS. *INDRE. SCD. Secretariat of Healt, Mexico. 428B REDUCING LENGTH OF STAY (LOS) FOR HIV+ PATIENTS AT AN URBAN MEDICAL CENTER. Holmes l., Alcabea P., ZapanoC., Selwyn P., FriedladO.H, Rawlings J. Yale-New Haven Hospital and Yale School of Medicine, New Haven, CT, USA. Objetive: To teach and support family, friends and/or partners of people with AIDS terminally ill in order to accomplish basic activities that can give and provide quality of life, dignity and confort during death process. Methods: In order to give attention to HIV infected subjects, great efforts has been realized in Mexico City and bordering areas since 1989. Our purpose is to reduce the anguish, fear, guilt, etc. of those who have a terminally ill patient. We try to teach different social sectors giving as much as we can, integral care and tiping to create resposible and cooperation attitudes toward terminally ill HIV/AIDS persons. Result: Professional advice with the following issues was given to familie members: HIV/AIDS basic information Personal Hygiene of HIV/AIDS patients Cure material Death process 30 patients andl150 family members were recruited. 30 % of all family members joined the attention team. 50 % were partners and 20 % friends. Patients rank of 16-60 years old. 24 men and 6 women. Discussion and Conclusions: During the process of attention to AIDS patients we are constantly confronting death. That is why it is necessary to create commitment not only with the family and partners but with health professionals of gobermantal institutions so they can teach and support this Kind of attention model. CARLOS CRUZ PROL. CARPIO 470, COL. STO. TOMAS, DELEG. MIGUEL HGO., C.P. 11340 MEXICO, D.F. TEL.: (525)341-40-46 Ext. 35 FAX: (525)341-32-64 4 29B C OF LIVE INMHIVA TIC PATIENTS WITH LESS TA 429B o50.i Izazola-Licea JA, del RIO C, Valdez-Garcia M, Orti z R, Flores M. OONASIDA, MEXICO. becive. Evaluate Quality Of Life (QOL) in a cohort of asyptanatic patients, franom patients' perspective. Methods. A cohort of 159 HIV asysptanatic patients with less than 500 od4+ cell counts, receiving 500 mgr/day of AZT, was followed for one year. Ten percent are women, and the rest gay and bisexual men. An standardized questionnaire of QOL was self-administered. Univariate and factor analysis were used. Results. Physical aspect: 9.5% showed difficulty in doing exhausting exercise and 7% in doingmoderate activities, suchas climbing stairs, biking, etc. Thenmstreported synptomse, not diagnostic of AIDS, are pain and rabnese in hands and feet (8%), itching and/or skin disorders (4%), pain in muscles, bones and/or joints (4%), difficulties in swallowing and eating (4%), mneory problems (15%). Only 3% of patients had to renain in bed instead of going to work, 4% interrupted daily activities (school or work), and 5% interrupted social visits to friends or relatives. 9% reports sadness, 6% depression, 10% nervousness, 9% difficulties to be concentrated and 28% lack vitality. In regard to social interactions 45% does not have a partner, and of those who have one, report it to be good or excellent in half of the cases. 15% receives little support froman his/her family, and 12% froman friends. 12% of relatives and 6% of "best' ' friends ignore that they are HIV positive. 18% of patients do not receive "enough"' social support, at least to speak about personal problems with anybody. 82% are currently involved in what they consider important projects. SeKual libido has importantly decreased in 3% of patients, 4% reported not to have had an organm in the last five years. 43% reported complete satisfaction with their lives since they knew they were seropositive, cmpared with 74% who considered so before they knew. Conclusionas. These patients report a low numbnter of health disorders, however none of than diagnostic of AIDS. The majority of problemse are related to lack of social and emotional support, and sme "minor"', but constant disorders. Lic. Raul Ortiz Mndragon Insurgentes Sur #1397 2o. Piso. Col. Insurgentes Mixcoac. M8xico City. Phone (5-25) 598-1145 FAX (525)5544202 Objective: To determine the impact on acute hospitalization and HIV care costs of enhanced HIV services in an 800-bed urban hospital providing inpatient and outpatient care for a diverse population of HIV infected patients, most living at or below the U.S. Federal poverty level. Methos: Enhancement of services began in 1990, intended to reduce dependence on acute-care hospitalization, and included: a designated outpatient HIV/AIDS clinic with dedicated multidisciplinary staff; designated inpatient HIV/AIDS service; improved coordination of inpatient and outpatient HIV/AIDS services; inpatient social services staff to expedite appropriate discharges; and formal liaisons with community-based agencies to aid discharge planning. Review of hospital discharges and outpatient visits for Fiscal Years (FY) 1990-1993 by ICD-9-coded discharge diagnoses. Results: Adult average length of stay dropped by 26.6 %, even though the number of HIV discharges at YNHH increased by 78.6 %. During this period, outpatient HIV/AIDS visits increased 86 %, from 6,524 to 12,137. For FY 1993 alone, the 4-year reduction in average LOS from 17.3 to 12.7 days resulted in a savings of 3,695 hospital days for the 818 discharges, and $4.06 million in costs. Fiscal Year: 1990 1991 192 1993 HIV Discharges: 458 556 684 818 Total Inpt. Days: 7966 8548 9702 10456 Average LOS: 17.3 15.3 14.1 12.7 Conclusions: Specific enhancements and coordination of inpatient and outpatient care services can result in reduced acute-care hospital stays and costs associated with HIV care. This experience has relevance for other large urban hospitals. Friedland, Gerald Yale University School of Medicine, AIDS Program, 135 College St. New Haven, Ct. 06510-2483 Fa20 33342851 '4h 0 W 430B MENTAL STATUS OF JAPANESE HIV/AIDS PATIENTS AND THEIR MAJOR CONCERNS Ishihara Miwa'2; Kimura, S.'; Oka, S.' and Shimada, K.'. 'Institute of Medical Science, University of Tokyo,Tokyo, Japan. 2Japanese Foundation for AIDS Prevention, Tokyo, Japan. OBJECTIVE: To assess HIV/AIDS patients' degree of depression, and to identify their major concerns in their daily life. METIIODS: An anonymous questionnaire had been distributed to all of 75 HIV/AIDS out-patients of our hospital from December 14, 1993, through February 14, 1994. A total of 60 subjects (80%) returned this questionnaire by mail (33 hemophiliacs, 27 STDs). Measures of Mental Health Status were examined based on the Center for Epidemiologic Studies-Depression Scale (CES-D). Additional scale has been used in order to identify out-patients' major concerns. RESULTS AND CONCLUSION: Using the conventional definition of depression (CES-D>l6), 55% of out-patients are classified as depressed (N=33), and 45% as non-depressed (N=27). The rate of depression among hemophiliacs was 70%, and 37% among STDs (x 2=4.95 p<0.05). According to these results, more than half of Japanese HIV/AIDS out-patients were suffering from depression. CES-D score correlated to score of major concerns. Major concerns of Japanese HIV/AIDS patients were identified as: no treatment for HIV disease; community's lack of understanding of the HIV disease: uncertainty for future; being a burden to family; and so on. There were differences between hemophiliacs', and STDs' major concerns. Miwa Ishihara, Dept. of Infect.Dis., Inst. of Med Sci., Univ. of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, JAPAN. TEL: 03-3443-8111, FAX: 03-3443-6259

Page  35 431B NUTRITIONAL STATUS AND QUALITY OF LIFE *Turner John *Muurahainen N *Terrell C *Graeber C & +Kotler C *Phila FIGHT PA +St Lukes Hosp NY NY USA Objective: To evaluate the effect of malnutrition (decreased body cell mass) on quality of life in HIV infection after controlling for degree of immune suppression (CD4 helper lymphocytes). Methods: Between 3/93 and 2/94, 67 HIV+ outpatients without AIDS-defining illnesses were recruited from HIV caregiving sites in Philadelphia and underwent evaluations of body cell mass (BCM) by bioelectrial impedance analysis (BIA-RJL 101-Q, Fluid States 2.0). Subjects also completed the Medical Outcomes Study (MOS) Short Form Health Survey and provided other history by questionnaire and interview. Body cell mass (BCM) was expressed as BCM for height (BCMht: the BCM of each subject [kg] divided by height in meters). Pearson product correlations and multiple regression analyses were used to examine relationships between MOS subscales (quality of life - outcome variables) and BCMht, and CD4 counts (predictor variables). Results: The HIV+ sample included 88% men and 12% women; 49% were white, 43% black, and 8% other. CD4 counts were determined 3.4 (+/- 0.7) months prior to study and ranged from 60 to 500 (mean 263 +/- 132) cells/mm3. Body cell mass (BCMht) was independently associated with diminished quality of life for the MOS subscale for physical functioning (r=.28, p=.0194) after controlling for CD4 counts. Conclusion: In clinically stable HIV+ persons, decreased body cell mass is associated with diminished quality of life (decreased physical functioning), independent of the degree of immune suppression. Further research is needed to determine whether nutritional repletion will improve quality of life in persons with early HIV infection who have decreased body cell mass. TURNER John Graduate Hosp 1800 Lombard St Pepper 505 Philadelphia PA USA 19146 Ph (215)893-2714 Fax 893-7363 432B GROWTH HORMONE THERAPY OF AIDS WASTING. Schambelan M*, LaMarca At, Mulligan K', Grunfeld C', Kennedy St, Breitmeyer Jt, Daar E~. UC San Francisco', Therafirst Ft Lauderdalet, Cedars Sinai Los Angeles~, Serono Laboratories$. Objective: Increased caloric intake in patients with AIDS-associated weight loss (AWL) may produce weight gain without restoring body protein stores. We have previously reported (JCE&M 77:956, 1993) that treatment with mammalian cell-derived recombinant human growth hormone (rhGH) for 7 days induced weight gain and nitrogen retention while decreasing protein oxidation and increasing lipid oxidation in patients with AWL. The present study was designed to evaluate the effects of extended rhGH therapy in patients with AWL. Methods: A 12-week, phase III, multicenter, double-blind, placebo-controlled trial of rhGH (SerostimTM, 0.1 mg/kg/d, avg. 6 mg/d) in persons with AWL (N=179) has completed enrollment. Assessments include weight, body composition by dual-energy X-ray absorptiometry (DEXA), endurance, strength, quality of life, immunological parameters, and safety. Results: Preliminary analysis has been performed without unblinding in 78 subjects from 3 sites who have completed the 12-week placebo-controlled study. Weight change ranged from -12.3 to +9.1 kg; in the 41 subjects who gained >1 kg (mean +3.7+0.3 kg), increased body mass by DEXA was >90% fat-free mass (FFM). These results are consistent with the proteinanabolic effect of rhGH noted previously. Weight change in the remaining 37 subjects averaged -2.5+0.4 kg. Analysis of plasma viremia, p24 antigen, and CD4 levels shows no deleterious trends. Therapy with rhGH/placebo was well tolerated with common side effects limited to arthralgias, edema, and carpal tunnel syndrome which resolved with dose reduction. Discussion and conclusions: The preliminary results of this study suggest that rhGH therapy in AWL is safe over extended periods. Although results are still blinded, the increases in FFM noted in approximately half of the participants suggest a therapeutic effect of rhGH and may present an important therapeutic advantage over nutritional support or appetite stimulation. Morris Schambelan MD, Division of Endocrinology, San Francisco General Hospital, San Francisco CA 94110 USA. Tel (415) 206-5820 fax (415) 826-3381. 434B TWO-YEARS EXPERIENCE IN ENROLLMENT AND RETENTION OF PREGNANT WOMEN IN AIDS CLINICAL TRIALS: NEW CHALLENGES, NEW PERSPECTIVES. Bd,Arlene, Grandchamp, J., Picardi, J., McSherry, G., Connor, E.,, Dept of OBJECTIVES: To describe sn eno soD oga mC ance Wi protocol of HIV-infected pregnant women enrolled in Perinatal AIDS Clinical Trials Protocols. METHODS: Medical records were reviewed. Soclodemographlcs, clinical and Immunological status of potential candidates for enrollment were abstracted. Patients were classified as either eligible or Ineligible for enrolment. The reasons for Ineligibility or declining enrollment were documented. Compliance with study visits and drug Intake was done by study coordinators. Ranking score Included: poor, fair, good and excellent RESULTS: There were no significant difference In racelethnicity, financial status or history of substance use between potential enrollees and enrolled patients. The acceptance rate for particlpation In trials was fourteen percent (16/118), twenty-five percent refused and sixty percent were Ineligible (72/118). A trend toward decreased refusal to participate In trials and Increased number of Ineligible women was noticed over time. Period Potential case Enrolled Refusal Ineligible Disqualfied 3/91-6/91 19 2 8 9 7/91-12/91 28 3 10 13 2 1/92-6/92 28 3 9 16 7/92-12/92 16 4 1 11 1/93-6/93 19 2 1 15 7/93-8/93 8 2 0 Two enrolled patients had previously HIV-infected children. Patients enrolled were clinically asymptomatic with mean age, absolute CD,, CD, of 29 ~ 5, 705 + 171, 1096 + 817, respectively. The average length of time In study was 11 weeks, compliance was good in seventy-eight percent (11/14) of the participants. CONCLUSIONS: HIV-infected minority pregnant women are well motivated to participate in cllnlcal trials and demonstrate good compliance with current protocols. The major hindrance In enrolment In Perlnatal trials Is the limited avallability of protocols geared toward the Immunosuppressed pregnant women. Availability and Implementation of Perinatal trials in community at drisk is feasible and should be encouraged. Arlene D. Bardeguez, M.D., Department of Obstetrics and Gynecology New Jersey Medical School, 185 South Orange Avenue, Rm E506, Newark, NJ 07103 12011 982-5482 - Fax number [201) 982-4574 433B EVALUATING AUTOMATED ELIGIBILITY DETERMINATION FOR HIV CLINICAL TRIALS. Sison, J.*,**, Miller, E.*, Tu, S.*, Carlson, R.*, Deresinski. Stanley*,**, Musen, M.* *Stanford University, CA, **Santa Clara Valley Med Ctr, San Jose, CA, USA Qbectives: Determining patient eligibility remains a critical task in the accrual of patients to clinical trials. We previously developed a computer-based system for evaluating patient eligibility. In this study our objectives were (1) to validate the conclusions of our automated eligibility system and (2) to determine the reasons for non-enrollment of patients deemed eligible by the system. Methods: We reviewed the charts of 60 HIV patients at a county hospital. Data from the charts were entered into our computer system which was designed to match patient data with the inclusion/exclusion criteria of 17 protocols open during the 7-month study period. For those patients deemed eligible for enrollment to specific clinical trials, an independent physician not involved in the design of the system reviewed the charts to determine the validity of the system's conclusions. Results: Twelve of the 60 patients were eligible for at least one protocol according to the computer system. In ten cases, the reviewing physician agreed with the system. Only one patient was enrolled. In two of twelve cases, the system made the wrong determination because of incomplete data in the database or insufficient knowledge about opportunistic infections. In the remaining nine cases, the chart showed no indication that the physician had considered the patient for protocol enrollment. Four patients were seen by non-HIV specialists who may have no knowledge of open clinical trials. Two of the nine patients had problems (acute infection and cocaine abuse) that were not specified as formal exclusion criteria, and the physicians may have determined these as factors adversely affecting the patient's status and ability to comply. These nine patients represent possible enrollment opportunities that were missed. Conclusions: Our findings imply that (1) physicians may not have all the Information necessary to determine patient eligibility; (2) given appropriate data, our computer system was accurate in determining patient eligibility; (3) an automated system can enhance accrual to clinical trials by providing early notification of patient eligibility to these trials. Stanley Deresinski, 77 Birch Street, Suite A, Redwood City, CA USA, phone: 415-366-0519, telefax: 415-364-9001 44) W. w 44h W 44b W

Page  36 435B A COMMUNITY BASED, OPEN ARM, RANDOMIZED STUDY OF ZDV/ddl VS ZDV/ddC - SHOULD WE EMBRACE NEW DRUGS WITH OPEN ARMS7? Leila Stou; Montaner, J.S.G.; llogg, R. S.; Mandigo, K.; Barber, C.G.; O'Shaughnessy, M. V; Schechter, M.T., et al. B.C. Centre for Excellence in IIIV/AIDS and the Canadian Trials Network. QBJECTIVE: To assess the impact of an open arm in a randomized study of ZDV/ddl VS ZDV/ddC. MEIHODS:IIIV+ patients tolerant of ZDV >400 mg/d were eligible if they were ddl and ddC naive and had CD4 counts of 50 to 350/mm3. Eligible patients could select between the open arm of the study, namely choosing between ZDV+ddl and ZDV+ddC, or randomization to either regimen in full doses on an open label basis. Drugs were made available free of charge for both arms of the study. Patients were stratified according to whether they had previously treated with ZDV and to CD4 count at baseline (5100 and 2100/mnm3). Randomized paticnts were seen at 1, 2, 3, 5 and 7 months for assessment of clinical status, adverse effects, CD4 count, P24 antigen and quantitative plasma lIIV-RNA PCR. RESUIJS.L As of JanJ94, 442 patients were receiving 500 Open arm combination therapy in B.C., 234 on AZT/ddl and 208 on; 400 AZT/ddC. A total of 491 patients started this therapy through the Sa 300 present study since Nov/93, 333 on AZT/ddl and 158 on - AZT/ddC. However, of these, only 91 (18.5%) were referred to c 200 Randomized arm the randomized arm of the study (see figure). As well, while 57 S100 physicians prescribed combination therapy in the open arm, only 0 17 physicians have referred patients for randomization with 69.2% 0 5 10 15 of these being referred from a single practice. Month CONCLUSION: Our results illustrate the challenge posed to recruitment into clinical trials by the existence of an open arm. This is despite the non-coercive, open label, community based nature of this protocol and high priority given to it by a variety of community organizations as well as by the Canadian HIV Trials Network. It is clear that an increased commitment by all will be required if we are to ensure the viability of open anas. Supported in part by NHRDP 436B A STRATEGY FOR THE EVALUATION OF MULTIPLE PROPHYlACTIC AGENTS FOR OPPORTUNISTIC INFECTIONS AMONG HIV-INFECTED PATIENTS E, SadrW, Wentworth D, Cohen D, Gordin F, Brosgart C, Costanzo L, Walker J, Hafner R, and CPCRA. NIAID, NIH, Bethesda, MD, USA Background: HlV-infected patients are at risk for a number of opportunistic infections (Os). Since patients may take many preventive treatments for these Ols, studies of the use of multiple prophylactic drugs on 01 incidence, toxicities, and survival are needed. Methods: The Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA) has initiated a number of 01 prophylaxis protocols. Data collection plans for studies have been designed to facilitate co-enrollment. Currently 17 centers are enrolling patients in studies to evalutate preventive treatments for PCP (2 studies), CMV, MAC, Tb (2 studies), and fungal diseases. One of the studies of PCP prophylaxis which has enrolled 1548 patients and is scheduled to enroll 2500 patients provides the foundation for this approach to multiple opportunistic pathogen prophylaxis studies (MOPPS). Based on co-enrollment to date and enrollment projections, specific research questions related to drug interactions have been formulated. Results: Through January 1994, 2554 patients have been enrolled in one or more O prophylaxis studies. Of these patients, 462 (15%) are enrolled in two studies; 63 (2%) are enrolled in three or more 01 prophylaxis studies. The average CD4+ count of patients enrolled in at least one study is 217 cells/mm3. Demographic characteristics are representative of patients infected with HIVY in the U.S. 23% of patients are women; 42% are black and 21% are Latino; 35% reported use of injecting drugs. Conclusion: This approach of designing multiple studies for OI prophylaxis and encouraging co-enrollment is a practical and efficient method for addressing drug interaction questions. Since prophylaxis is initiated at different CD4 levels for different Ols, this approach both mimics usual clinical practice and is less restrictive than MOPPS designs that require simultaneous initiation of different prophylactic treatments. Wafaa EI-Sadr, MD, MPH Harlem AIDS Treatment Group/ Harlem Hospital, MLK Pavilion, Room 3107 506 Lenox Ave, Bronx, NY 10456, USA Phone: 212/939-2940; FAX: 212/939-2968 44h CA) 00h W Dr. Leila Srour 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z 1Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. 437B BARRIERS TO PRISONER PARTICIPATION IN AIDS-RELATED CLINICAL TRIALS IN THE U.S. Kelly, Eileen T., AIDS in Prison Project, Correctional Assoc. of NY; New York, NY, U.S.A. OBJECTIVE: Is the enrollment of U.S. prisoners in AIDS-related clinical trials feasible, legal and ethical? Why does the virtual moratorium on such research persist? What is the experience of clinical researchers who have enrolled prisoners in clinical trials? METHODS: Through a review of the literature and applicable law, as well as interviews with U.S. government officials, correctional officials and clinical researchers, seek to measure barriers in policy and law, describe research which has incorporated prisoners, and discuss obstacles posed by the correctional setting. RESULTS: U.S. federal law permits the inclusion of prisoners in research, while imposing safeguards to protect prisoners from exploitation. Barriers include a widespread misperception that such research is barred under federal law, a policy banning clinical research in all federal prisons, as well as bans under state laws and correctional policies. Prisoners have enrolled in clinical trials in several states, notably where research institutions provided inmate health care prior to enrolling prisoners in clinical trials. Despite obstacles, interviewed researchers who enrolled inmates in clinical trials remain committed to this research. DISCUSSION AND CONCLUSION: U.S. prisoners are barred on an official or de facto basis from clinical trials in all federal and most state prisons. Research which meets federal safeguards should be open to prisoner enrollment. Providing access to clinical trials is a component of providing first-rate care to prisoners living with AIDS/HIV. The underrepresentation of ethnic minorities and IV-drug users in AIDS-related clinical research, and their disproportionate presence in U.S. prisons, makes the inclusion of prisoners in clinical trials valuable to researchers seeking comprehensive data on treatments for this disease. Eileen Kelly, Coordinator of Research/Special Projects AIDS in Prison Project, Correctional Assoc. of NY 135 E. 15th St., New York, NY 10003, U.S.A. Phone: 212 254-7500; Fax: 212 473-2807 438B INTERACTION BETWEEN BIOMEDICAL/SOCIAL FIELDS AND NGO'S IN RIO DE JANEIRO VACCINE CENTER F.Sutmoller, C.Bastos, T.Penna, C.Souza Objective: To analyze the social factors involved in the preparation for preventive IIIV vaccine efficacy trials. Methods: Evaluation of the first phase of a project on HIV incidence in a seronegative open cohort which includes the impact of preventive interventions and evaluation of sociobehavioral aspects involved in volunteering for vaccine efficacy trials in Rio de Janeiro, Brazil. Results: Divergences between different parts involved (NGOs, social scientists, biomedical specialists) arose mostly due to different conceptual traditions and prevention implementation methods: a background mistrust about the possible abuses of medical experimentation in developing countries enhanced disagreement, in spite of the common interest of the different parts involved in the preparation for vaccine trials. Discussion: Articulation between the medical and social spheres was fundamental for the accomplishment of this vaccine preparation project. Expanded ethic committees, "conceptual translators" and liaisons between the different parts was used to negotiate divergences and which suggested possible changes in the research protocols. Frits Sutmoller, Evnadro Chagas Hospital FIOCRUZ. Av. Brasil 4365, 21040 Rio de Janeiro, RJ - Brazil (021) 590-9988 537-0224

Page  37 439D Assessing Government Strategies for Organizing and Managing the HIV/AIDS Epidemic. Objective:This study assesses government strategies for addressing the HIV/AIDS epidemic in terms of: i) problem identification; ii) focus of government program control; iii) extent of nrogram activity; and iv) health and related outcomes. This study is conducted with specific reference to Asian countries who are init inting government program interven t ions. Methods:Case studies from Asia and also Africa and Latin America assessing each country's HIV/AIDS program effort according to the above criteria. Results from the case studies are summarized according to these criteria. Results: Emerging findings appeal: to be: i) government perception of the HIV/AIDS problem influences jurisdiction interventions; ii) Vestihng leadership in MOH focus most interventions in public health institutions; iii) couiiLries with other leadership modalities seek greater use of private and NGO involvement; iv) program coordination is difficult in any type of managerial setting, but where the leadership supports community involvement, interventions tend to have the greatest health and development results. Discussion & Conclusions: While interministerial coordination is difficult to achieve, where it is given a chance to work, program interventions are more diverse, and outcomes more sustainable and far reaching. Dr. Michael Porter, The World Bank tel (202)458-2525 1818 H. Street N.W. Washington D.C. 20433 fax (202)522-1226 440D INNOVATION, TOLERANCE AND PRAGMATISM: THREE ESSENTIAL TOOLS IN DEVELOPING A PARTNERSHIP APPROACH. MARILYN CHALKLEY, EDUCATION UNIT, AIDS/CD, C'WEALTH DEPT OF HUMAN SERVICES AND HEALTH, AUSTRALIA, DAVID FOWLER, AIDS BUREAU, NEW SOUTH WALES DEPT OF HEALTH, FELICITY YOUNG, AUSTRALIAN FEDERATION OF AIDS ORG'S. OBJECTIVE: Through partnership between Federal Health, State and Territory governments and with organisations representing communities and individuals infected and affected by HIV/AIDS, to provide leadership coupled with a flexible approach so that a rapid response can be made to any changes in the HIV epidemic in Australia, and appropriate education/prevention strategies put in place. METHODS: To evaluate the previous national strategy on HIV/AIDS(1990-93) in consultation with affected communities and State and Territory governments, and then to develop an agreed national strategy for 1993-96. Part of this process involves evaluation of education/prevention programs. RESULTS: In relation to education, a scaling down of large mass media campaigns aimed at the general community, and the development of more closely targeted education and prevention programs often involving extensive community development models aimed at the populations mostly at risk, including gay men, men who have sex with men, HIV positive people and IV drug users, and Aboriginal and Torres Strait Islander people. DISCUSSION AND CONCLUSION: Australia's education and prevention programs have been widely regarded as successful to date, and are distinguished by a pragmatic and innovative approach in partnership with affected communities. There has been a substantial decrease in the number of HIV infections in Australia since 1987 with a plateauing of infections more recently. Nevertheless, Governments must be constantly vigilant and able to assess programs that were once successful but are no longer appropriate, and replace them with new ones. Marilyn Chalkley Dir.EducationUnit, AIDS/CommunicableDiseases Branch, Dep't Human Services & Health,PO Box 9848 ACT 2601 Australia.Ph:61 6 2896911, Fx: 118WVI 3 441D THE PIlLIPPINE NATIONAL AIDS FRIVENTION AND CONTROL PROGRAM: 1988-1993 Hernandez, Enrique R.t, Villanueva, J.H.F.:,PHIL-AIDS Foundation, Manila, Philippines 442D SOCIETAL RESPONSIBILITY AND RIGHTS OF THE HIV-INFECTED Mrs Elizabeth Wong Chin Chi-lien, ISO, JP HONG KONG GOVERNMENT, HONG KONG OBJECTIVE: To organize a rational, enlightened and non-sensational though determined, long-tern societal response to the threat of AIDS which was deemed potentially disastrous and certain to continue for many years to come. METHODS: Use of mass media; multi-sectoral consultations and creation of linkages; upgrading the Health Department's leadership capability in prevention, Ionitoring, diagnosis and care; provision of financial and technical support to other organisations; advocacy for appropriate prevention strategies and the basic rights of individuals and groups affected; formal creation of a national AIDS council; organization of local city-based AIDS committees; external review of the implementation of the national program's first nediun-term plan; HIV/AIDS-related legislation. RHSULTS: Awareness of its widespread implications made addressing the AIDS threat gain a high priority in government, and the AIDS program truly national with a strong and active multi-sectoral response. Despite the numerous diverse interests that energed, foundations were laid, Including a comprehensive AIDS Prevention and Control Act soon to be passed by Congress, to help ensure effective implementation of the national program in the future. As a result, potentially costly mistakes were avoided, limited resources available to AIDS programs were multiplied and the use of these aximized. CONCLUSION: Understanding the nlti-dinensional aspects of HIV/AIDS, multi-sectoral participation in both levels of policy development and program implementation, culture-sensitive and indigenous approaches, the leadership role of the country's highest office, government support to arouse multisectoral interest, and the strength of law, are all necessary to initiate and ensure the organization of a societal response. Dr. Enrique R. Hernandez 307 Bernabe St., Pasay City, Metro Manila 1300, Philippines Tel: (632)581186 Pax: (632)721-1266 Any responsible and compassionate society should value and protect its more vulnerable members. Such protection may be enshrined in legislation, thus providing a benchmark for societal response. However, anti-discrimination legislation alone cannot dispel ignorance and prejudice. Often it provides at best an arduous, time-consuming and potentially expensive avenue of legal redress. A change of attitude cannot be imposed upon a society society has to be involved in the change. The Hong Kong Government considers wide community involvement essential in providing a more caring and supportive atmosphere for the HIV-infected. Accordingly, it has established a HK$350 million (US$45 million) AIDS Trust Fund for, inter alia, funding community projects providing publicity and education or direct medical and support services to the HIV-infected. The response from community-based organisations has been encouraging. Wong Mrs Elizabeth, Secretary for Health and Welfare Health and Welfare Branch, 7/F Central Government Offices, Lower Albert Road, Hong Kong. Tel (852) 810 2550 Fax (852) 840 0467 CA) 440 44

Page  38 443D INTERNATIONAL HIV/AIDS ALLIANCE: LINKING ORGANIZATIONS FOR TA & FUNDING TO LOCAL NGOs Authors: Crane, Susan F', Calica C", Dahourou R'" Intl HIVIAIDS Alliance, London; "PANSuP, The Philippines; 'PSBP, Burkina Faso OBJECTIVE To establish a locally run agency in the Philippines and Burkina Faso able to provide techrical assistance (TA) and funding to local non-governmental organizations (NGOs) working in HIV/AIDS prevention, care and community support. To devolve NGO funding and TA support decisions and capabilities to the national level. METHODS Local groups in the Philippines and Burkina Faso were canvassed about existing mechanisms for TA and funding to local NGOs. NGO representatives decided on the type of new structure required. The Alliance worked with each group to develop a three year strategy for identifying NGO TA and training needs and providing funds to meet these. Initial small grants were made to test this approach. RESULTS Representatives of health, development, women's and HIV/AIDS NGOs met together with Alliance members and established committees to work as Alliance linking organizations. Country-specific systems were established in the Philippines and Burkina Faso. Each group set priorities to determine funding mechanisms and TA provision. Each organization provided small grants to several projects in early 1994. Subsequently, both groups prepared 3 year plans for funding local NGO projects and providing TA. DISCUSSION & CONCLUSION The experience in both countries showed that a new type of mechanism for funding and identifying TA needs for NGOs is required and can be quickly established. The Alliance used this experience to establish linking organisations in 7 more countries in 1994. NGOs have a strong role to play in HIV/AIDS prevention, care and community support and the Alliance believes that it has developed an appropriate means to support their vital work. 444D HIV/AIDS Grassroots Policy Action Team Anne Donnelly, David Lewis - Project Inform Project Inform Treatment Action Network (TAN) provides members with background information regarding federal HIV/AIDS research policy issues that affect their lives and the lives of those close to them. TAN alerts also provide suggestions of grassroots actions that members can take to ensure that their voices are heard at a federal level. The Treatment Action Network (TAN) was established in early 1991 to provide people concerned about HIV/AIDS treatment policies and issues a vehicle to Join with others around the country and make their numbers and concerns known to their elected officials. The recognition of the need for such a vehicle grew out of discussions with members of Congress which revealed that in some states, the numbers of local people requesting HIV/AIDS treatment information from Project Inform far exceeded numbers of AIDS cases reported to the Centers for Disease Control for that state. Many of the same people who had chosen to be proactive about their own treatment Information could also be a powerful voice in the halls of Congress in demanding the leadership and the resources needed to fight this life-threatening pandemic. Since 1991, TAN has grown to include over 600 members residing in 47 states and the District of Columbia. TAN has not only grown in membership but has proven itself to be a highly responsive, articulate and effective team. Members of TAN have taken action on issues concerning regulatory policies, HIV/AIDS legislation and appropriations by writing letters, making telephone calls, and visiting their legislators on Capitol Hill and in their home districts. Members are contacted via "TAN alerts" sent through the mail or by fax. TAN also maintains a dedicated telephone line, where members can call in to access a prerecorded message which is changed regularly. TAN is administered by Project Inform's Public Policy Department and staffed by volunteers. Anne Donnelly, David Lewis, Project Inform, 1965 Market Street Suite 220, San Francisco, CA 94103 415.558.8669 415.558.0684 (fax) TAN Action Line 415.626.7231.rh 44 0) 445D THAI PROSTITUTION: ECONOMIC, SOCIAL AND CULTURAL FACTORS Supanya Lamsam* ** Mechai Viravaidya* *Population and Community Development Association, Bangkok, Thailand **Thailand Business Coalition on AIDS, Bangkok Thailand Objective: The objective of this operations research is to gain insight into the various factors that influence women from northern Thailand to enter the commercial sex industry (CSI) and based on the findings of the research, design and implement strategies to reduce the number of women choosing thlis option. Methods: A baseline survey using questionnaires, in-depth interviews and focus group discussions was conducted in ten representative villages in Chiengrai and Phayao province. Villagers were surveyed on their knowledge, attitudes, behaviour and practices regarding sex, prostitution and AIDS and were asked to identify reasons why women from the north enter the CSI. Girls of the same socioeconomic background were surveyed to ascertain factors why they chose not to enter the more lucrative CSI Results: The results of the research indicate that not only economic but also social and cultural factors, serve to foster prostitution. Increases in income disparity and information accessibility via the media have caused increased awareness and yearning for wealth and material comforts elusive to the majority. Education and labour laws serve to exacerbate the problem. A cultural environment has evolved where there are only limited social repercussions and few disincentives for working in the CSI. Discussion and Conclusions: Previously, when tackling the prostitution problem in Thailand, the emphasis has been placed on the women working in the industry and not enough attention and effort has been made to decrease the demand for commercial sex. The strategies designed for implementation in this project aim not only to provide women with immediate short-term economic and educational alternatives to commercial sex work but also to explore long term strategies to addressing the issue of prostitution in Thailand Lamnisam, Supanya, Thailand Business Coalition on AIDS, 270 Raintree Office Garden, 2nd Floor, D2 Bldg., Rama 9, Bangkok 10310 Telephone (662) 719-6450 Telefax (662) 719-6453 446D A CO IAE*IYR SPUDY O05 OfNICIAL 83M I1WUS I1 Al! AIDS; Sutasim P i aInlth Office of arli; tit Norati. TCitra Ueadbca Ineneoia. o0WC!TVM and m ErU S A wies ft obs rvltinfintervie w have beai onanot to collect nder emaos m kc fnaotors welated to IDS in three proincial capit*ls t Bli. Nest Java md Suntheast Sulawesi in 199: Pindings were then disassm d in two i-w i *aheps and wre finally nilysod doeoriptively. Remsults will be used as inputs in devo pin g AIDS Cencaention Schemoe.' RESIRS and ISCSR8IONJ; - The rai nature of oemaretoial ena in adustries is far aw a f disio i ploley mt*'os thoughts. This itial point of AIDS diaseosintion should be understood bopa id aonul tteontios but should not c orte a masking f etot" by noglecting other w yof AIDS aissomination. - C"mm re~l sec wakers developed ateh mroe saccoafl social.nricoting sohee than other sectors involved in A IDS prvation ctivitie. -Southeast Sulawes is ensid aroed as a ln priority proince m f tO speeood up AIDS preovention activitie:tht then have meam big fishery industries a ol ying foreigners amd a ertain tradition Tl frionahip dan e pterford publicly, encourage premisuityr among sone of the participants: Once if AIDS affeoting this provincej diseeninatien asng lcali pooploes will be match fastor than in other pr"vinoee - Further anthropelgnl studies on sirlar situatio"n is then noodod. Sutriasna 56 oaui Stroot DI(PASAR 80233 Indonesia, phone a (0561) 222602

Page  39 447D, =w W mes CoI8IINpls SEI (i151l 4i47 D'N I E.N.,, Kau P.1, Nyabola L.,onald.3,: Voluntary oaai Rhabilitation aInstitute (YOURII), University of Nairobi and VOWI,Nairobi 3 University of banitoba.Canada.4 University of Nairobi.h Kenya UIj. To mi lisa 'ewas la sex Idustry wiling to exit prostitution, educate and support this in anangeent of nil bsineses, and monitor their umodificeation in seial habits. i. Fifty four wenl,0 from arhakos Tore and K4 from Nairobi who bad earlier indicated that they are seeking altenative muns of inome wee recruited. lndepth interview with trained interviewers and also participatory observations re aed to complement qualitative data. The wmen ware then trained in msagesent of small business as well as costing. Reinfotrcemnt of SIDIAIDS, nafe/safer sex and fertility education as part of capability building. Finally, between Ksh.2,500/* and 25,000/ was disbursed according to their meeds. i Theim age of these wmen was 151 to 45 years frot several parts of the country and practising prostitution in hoses, hostels, streets and bars. They hlave i-8 children with an average of 4. A quarter of then have HIl infection and 55 have AIDS. 21 are child prostitutes and of then is already a mother at 15 and a half. They have started a variety of business in the arkets e.g. vgetable. fruits, sirsa, grain and are saking handcrafts. Two are in training for weavinga ad tailoring respectlvely. 6.21 got sarried, 43 have so seul partners at aloil and others have reduced sexual partners from 25 per eek to 15. 45% are keeping a regular sexual partner or lover. Those whe still lhave 2 are insisting on condo use to 605 now from 405% before. Group therapy takes place once a week and all of then are eperienclng a sense of sell being and pride that they do not have to sell oa and e pose teselves to the deadly AIDS to survive. Uben they do, they insist e n condo use. They have increased sense of accoeplishnent. AI U i This is a sure way of empowering wonea in core groups to realn free of STD/LRIV as well as project other sexual partners; thus breaking the chain of HiV transission. MitS. MARGARET NJOI IRURI, VOLUNTARY WOMEN RH.ABILITATION INSTITUTE (VOWRI) BOX 14401,NAIROBI TEL. 718895, Fax No. 712007 448D COMMEtCIAL SEX WORKERS' RISK TO HIVY IN PUNE. Mawar Nita, Divekar AD, Tripathy SP, Bagul RG, Swamy M, aner]ee K* & Rodrigues JJ. NARI (ICMR) P.Box1895, PUNE 411 026, INDIA: *NIV (ICMR) P.Box 11, PUNE 411 001. Aims: A pilot study aimed to understand diversity of CSW inrelation toHIV risk in their work setting. Methodology: 191 CSWs from Pune were covered using a semi-structured questionnaire. Results and Discussions: Majority of sexworkers are females (170)& the rest 21, eunachs. Most are unmarried illiterate (64%) speaking Kannad (53%) Telugu (16%) & other languages indicating diversity of origins. They are Hindus (86%) or Muslims. Their sources of entertainment & information are through video (63%) or cinema (57%). Only 18% used some family planning method, despite institutional delivery (46%) & abortion (25%). Many entered into prostitution through the devdasi system,theogamy,specially those from Karnataka & Andhra Pradesh. Through this system the young girl is married to the temple god, which gets them a ritual sanctity,later, enter into commnercial sex, sometimes at the cost of their own health. Although 62% claimed using condoms sometimes,this is not a regular practice; 1/5th reporting sex even during menstruation to make a living. The FCSW enter this occupation to earn enough money for own maintenance, children & others they support through this trade which puts them at a great health risk including HIV. Conclusions & Reconnendations:The significance of the socio-economic & cultural factors should be understood for the dyad of this interaction - the CSW & her clients, requiring an indepth study using qualitative data to understand the varied types of sexwork, only then effective interventions for desired impact of risk reduction through behaviour change be planned. ER. NITA MAWAR, Senior Research Officer, NATIONAL AIDS RESEARCH INSTITUTE, Plot # 73, 'G' Block, M. I. D. C., Bhosari, P.Box1895, PUNE-411 026, INDIA. Tel. 91 212 791210. Telefax: 91 212 791071. 449D An European Obsorvatory for Sanitary and Social Researches on sex Workers L. Braggiotti, M. El Amri, A. Cantero, N. Boutet and the wole team of Bus des Femmes 450D SEX WORK, HUMAN RIGIS AND HEALTH. WHAT ARE ITHE ONNECTIONS AND WHAT IS GOING WNG? Overs, C. Network of Sex Work Related HIV/AIDS Projects, London, United Kingdom Objectives: Adapt to five European countries (Spain, Netherlands, Belgium, Germany. Italy) the Bus des Femmes research and action field methods, as evaluated in Paris since 1990. Methods: By carrying out exchanges with institutional organizations, field workers and health and social care professionals, allow in each country the: a) Breaking barriers between sex workers and researchers, between the field and institutions. b) Training preventive teams (Both sex workers and researchers collect, analyse and use sanitary and social information to update epidemiological studies and public health activities). e) Exploitation of information to optimize the use of sanitary and social networks. Results: The creation and support of 25 preventive sex workers in Europe. Awarcness compaigns (Spain, Netherlands. Belgium, Germany, Italy) and training sessions (Spain, Belgium) between professionals and sex workers. Participation of two sex workers supported by the Bus des Femmes in an injection equipment exchange progam in Mackid and Anvers. Conclusion: The implication of sex workers, their exchanges with researchers and sanitary and social professionals, created a dynamic current that brought a rapid strengthening of the sex workers preventive behavior towards HIV. L. Braggiotti The Bus de Femmen, Paris, France Telephone (33)-I 45 085660. Telelax (33) -I 45 08 56 60 This paper will explore the reasoning behind the claim that human rights violations and persecution of sex workers increase vulnerability to HIV and sexually transmitted diseases. The first perspective is about practical matters. It is now a familiar idea to many people that legal and social persecution limit effective condom promotion programs and clinical services to prostitutes. But lack of services and "access" to prostitutes by health professionals is not the major issue. It is of far greater importance that systematic violation of human and industrial rights creates and maintains working conditions which exacerbate health risks to sex workers. The second area of interest here is examinations of formulations of human rights as they apply to sex workers. It will be argued that existing concepts of human rights are inadequate because they are based on mistaken understandings and analyses of work, gender and sex. The paper will conclude with concrete recommendations for change. OVERS, C. Network of Sex Work Related HIV/AIDS Projects, 10 Danbury Street, London N1 Tel: 44-71-3541664 Fax: 44-71-3541664 44h 44h 0

Page  40 451A H AIoEI E N F A1 Q E INAS A. IBaldwin, C.R. More, G. hxietmn, and M.L. Kalish. Canters for Disease C ntrol and Prevmtion, Atlanta, Cerrgia 30333. Objective: To investigate the transmission of HIV-1 involving two brothers with hemophilia, using three different regions of the HIV-1 genome. Methods: DNA fragments of the env V3 and flanking regions, pol protease, and gag p17 were amplified from PBMC by a nested PCR and cloned into an M13 vector. Both direct PCR and cloned DNA products were sequenced. To determine the relatedness of the viral strains, nucleotide divergence was examined and phylogenetic analysis was performed using the neighbor joining bootstrap algorithm. Results: Phylogenetic analysis of all 3 gene regions was consistent in grouping the two brothers. The strongest phylogenetic support for grouping the viruses from the two brothers was seen by comparing either the p17 or the V3 and flanking regions, with bootstrap values of 98% and 96%, respectively. The weakest support was seen in the protease gene with a value of only 59%. By sequencing clones from the V3 region, we observed that two variants of HIV-1 were shared by the two brothers. One variant was predominant in the older brother while the second variant was predominant in the younger. Unlike the env region, no distinctive variants were detected in the gag p17. Discussion and Conclusions: The high degree of genetic relatedness and the strength of bootstrap support is consistent with HIV transmission occurring from one brother to the other. The optimal regions for studying transmission in this case were the gag p17, and env V3 and flanking regions. These findings have important implications for future investigations involving HIV-1 transmission. Chi-(hag LI, Divisim IVl/AMS (012) Canters for Disease Ccntrol Atlanta, GA 30333 IA Srrne (Llt) 639-3231, Fhax: (404) 639-260 453A CHARACTERIZATION OF A CASE OF INTRAFAMILIAL INFECTION OF HIV-1 SUBTYPE E Sato, Hironoril; Taniguchi, Kiyomil; Tomita, Yasuhirolt; Miyakuni, Tuyoshi2; Takebe, Yutakal 1 AIDS Research Center, National Institute of Health, Tokyo, Japan; 2 Naha Prefectural Hospital, Okinawa, Japan Objective: To examine the sequence variability of HIV-1 subtype E viruses upon transmission through heterosexual contact and the transmission from mother to infant Methods: We have analyzed a case of intra-familial infection of HIV-1 subtype E. The father was likely to be infected by sexual contact with commercial sex worker(s) in 1989 or 1990. His spouse and their child (16 month old) were later found to be seropositive. The C2/V3 region (324 bp) of proviral DNAs were amplified by PCR and the nucleotide sequences were determined. Results: We have compared the nucleotide sequence divergence of env C2 region (125bp), V3 loop (105bp) and post-V3 region (94bp) among the patients. The V3 loop sequences of the mother and the infant were less diverse than those of the father (mean value of % similarity~SD= 99.1~0.8, 98.3~1.2 and 91.2~4.3 respectively). In contrast, no significant difference in the diversity was observed in the C2 and post-V3 region. Interestingly, the V3 loop sequences of the mother (virus recipient) exhibited greater similarity with HIV-1 subtype E consensus sequence than those of the father (virus donor) (mean value of % similarity~SD = 95.8 ~ 0.5 and 91.4 ~ 2.1 respectively). Discussion and Conclusion: There seems to be a selection pressure to conserve V3 loop nucleotide sequences upon heterosexual transmission of HIV- 1 subtype E. SATO, Hironori, AIDS Reasearch Center, Tokyo, Japan Telephone (81)-3-5285-1111, Telefax (81)-3-5285-1177 452A SELECTIVETFEATURES OF HIV-1 THAT INFLUENCE MATERNAL: CHILD TRANSMISSION Kliks Srisakul,Contag C*,Wara D,Levy JA,Univ. of Calif San Francisco CA USA;*Stanford Univ.,Stanford CA USA Objective: To evaluate the biological, immunological and genetic properties of HIV- I that are selective for vertical transmission of infection from mother to child. Methods: HIV-l isolates from 10 non-transmitting mothers, 5 transmitting mother and their 5 infants were evaluated for growth characteristics in macrophage. PBMC, and T-cell lines. The isolates were tested for susceptibility to neutralization and antibody-dependent enhancement (ADE) by autologous maternal or infant sera. V3 sequences of all the isolates were determined from multiple clones of PCR amplified products of the infected peripheral blood mononuclear cells (PBMC) used in the propagation of the virus. Results: A selection of efficient replicating strains of HIV-1 as defined by the "rapid/high" or "high" replication pattern in PBMC and ability to infect T-lymphocyte cell lines was found associated with vertical transmission. Neutralization by autologous maternal sera appeared to play a role in protection (8/10 among non-transmitters vs 2/5 among transmitters). Moreover, four maternal isolates and one infant isolate showed resistance to neutralization and sensitivity to ADE respectively. V3 sequence analysis showed a higher degree of differences between paired mothe/infant isolates that showed discordance than those that showed concordance in their reactivity to maternal sera. Conclusion: Vertical transmission of HIV-1 may be favored by an efficient replicating virus that is further selected by the virus resistance to neutralization or sensitivity to ADE by autologous sera. This immunological selection can be reflected in V3 sequence differences between the paired mother/infant isolates. Kliks, Srisakul, University of California, San Francisco, CA USA 94143-0128 Telephone (415) 476-4071 Telefax (415) 476-8365 44bh uJ U' 454A Characterization of HIV Isolates From a Set of Twins With Different Disease Courses Zhou,Yi; Hutto, Cecilia; Hue, Jun; Scott, Walter; Charles Wood. University of Miami School of Medicine, Miami, Florida USA Objective: This study was carried out to examine the biological and molecular characteristics of HIV isolates recovered longitudinally from a pair of perinatally infected twins with marked differences in their course of disease. The biologic properties and genomic sequence variations of viruses isolated from each child longitudinally were studied to determine how they contribute to diverging disease courses. Methods: HIV was isolated from the peripheral blood lymphocytes of each child initially at 6 weeks of age and then at approximate 6 month intervals. Four isolates from each twin have been characterized. The viruses were tested for their ability to form syncytia, their replicative titer, and their ability to infect macrophages and established T cell lines. The env gene of each isolate was cloned by PCR and the sequence of the V3 was determined for each virus. Results: Both twins were asymptomatic when first seen at 6 weeks of age with heights and weights >50% but the CD4 # and (%) for twin A and B were 1882 (33) and 699 (25), respectively, and the most recent at 18 mo. of age were 1011 (17) and 64 (3), respectively. Twin B had clinical criteria for AIDS at 7 mo. of age, has had numerous infections requiring hospitalization and has FTT and encephalopathy while twin A meets P2 criteria with hepatosplenomegaly and lymphadenopathy only. The viruses isolated from both children at various time points grow equally well in PBMC. They were non-syncytia inducing and varied in their ability to infect macrophages and established T cell lines. Sequence variations within a 400 bp region of gpl20, including the V3 loop, were observed between the viruses isolated from the twin at each time point. Discussion and Conclusion: The viruses isolated longitudinally from each twin appear to display differences in biological properties and viral genomic sequence. It is possible that differences contribute to the marked differences in the clinical manifestations observed in this set of twins. Charles Wood, Ph.D., Associate Professor of Neurology and Microbiology Univ. of Miami School of Med. (D4-5), P.O. Box 016960 Miami, FL. 33101

Page  41 455A MAPPING CD4 T-LYMPHOCYTE EPITOPES IN A LONG TERM SURVIVOR. Ratto Silvia, Sitz KV, Robb ML, Frey SC, Moriarty R* Redfield RR, Birx DL. WRAIR and *NNMC, Washington, DC. OBJECTIVE: Distinctive immunologic and virologic characteristics of long term survivors may contribute to the development of therapeutic and preventive vaccines. A clinically and immunologically stable (CD4#>500 x 4 years) adolescent who was perinatally infected with HIV 13.5 years ago is undergoing extensive evaluation of viral phenotype, genotype and virus specific immune responses. METHODS: CD4 T-lymphocyte lines were established from a single time point and were used to map the reactivity to various HIV envelope peptides. Peripheral blood mononuclear cells (PBMC) obtained by ficoll density centrifugation were subjected to 3 cycles of stimulation, 15 days apart, with rcmgpl20 IIIB and rcmgpl20 MN. Each line was tested for specificity using a 3 day proliferation assay. In addition, the lines were challenged with various HIV envelope proteins and reactivity was assessed using a 3 day proliferation assay. Viral burden was measured with quantitative PCR and env sequences were determined by PCR cloning of a 736 nucleotide fragment containing V I-V3. RESULTS: Viral burden in the PB at age 13.5 years was at the lower limit of detection (1-10 copies/100,000 cells). Env sequences were highly restricted with less than 1.5% total diversity. CD4+ T cell lines were successfully established with rcmgpl20 IIIB and rcmgpl20 MN with lymphocyte stimulation indices (LSI) of 49 and 62, respectively. The gpl20 IIIB line crossreacted with the gpl20 MN molecule (LSI=14) while the gpl20 MN line cross-reacted with the gpl20 IIIB molecule (LSI=20) as well as a northern Thai strain, gp160 CM, (LSI-=4). The gpl20 IIIB line also reacted to peptides in the C2 and C5 region when challenged with 1I peptides partially covering the gpl20 sequence. CONCLUSION: Preliminary mapping of the CD4 T lymphocyte gpl20 epitopes from a long term survivor were determined. The gpl20 lines cross-react with gpl20 molecules of different viral genotypes and further epitope mapping reveals reactivity to a previously undescribed region of C2. Dr. Silvia Ratto Suite 200, 13 Taft Court, Rockville, MD 20850, USA Telephone: (301) 762-0089 FAX: (301) 762-4177 456A INTRA-PATIENT VARIABILITY OF HIV-2 ENVELOPE V3 LOOP AND DISEASE PROGRESSION. Sankal Jean-Louis, Sallier de la Tour R, Renjifo B, Marlink R, Mboup S*, Essex M, Kanki P. Cancer Biology, Harvard University, Boston, USA. *Bacteriologie-Virologie, Universit6 C.A. Diop, Dakar, Sfnfgal. Objectives: To study intra-patient variability of HIV-2 envelope in sequential PBMC samples from seropositive individuals and disease progression. To compare HIV-2 intra-patient V3-loop sequence variation to HIV1. To evaluate the variation of the V3-loop sequences between PBMC viral DNA and cultured virus. Methods: We used a hemi-nested PCR to amplify a 469 bp fragment containing all the V3 loop. The PCR product was purified, cloned and sequences of the entire V3 loop (102bp) were determined by chaintermination method. 5-11 clones were sequenced per sample. Sequential uncultured DNA samples were obtained from 2 asymptomatic females (A and D), a healthy seropositive male that experienced a drop in CD4 levels during the 54 months observation period (B), and 2 AIDS patients (C and E). We also obtained viral sequences from viruses isolated from 3 of these individuals. Results: Sequence heterogeneity in a given sample ranged from 0% to 1.4% and increased with disease progression. The average intra-patient variation was 0.7 in asymptomatic individuals and 1.2 in symptomatic ones. HIV-2 intra-patient V3-loop sequence heterogeneity showed a similar pattern but was less than in HIV-1. In 2 of 3 cases, the cultured virus was identical to the PBMC from which it was derived. Conclusion: These data indicate that intra-patient variability of the HIV-2 envelope V3 region in asymptomatic individuals may be relatively low. HIV-2 intra-patient V3-loop sequence variation is less than HIV-1. V3-loop sequence variation correlated with disease progression. Variation in cultured virus was higher in the individual with end-stage AIDS. Further studies are needed to evaluate the role of genetic variation in the pathogenesis of HIV-2 disease. JEAN-LOUIS SANKALE, Harvard School of Public Health, Departmnent of Cancer Biology, 677 Huntington Avenue, BI-903, Boston, MA, USA, Telephone: 617/432-1482; Telefax: 617/739-8348. 457C INCREASE IN ENDOCERVICAL CD4 LYMPHOCYTES IN WOMEN WITH NON-ULCERATIVE STD. Levine. William C', Pose V*, Bhoomkar A**, Bullard J*, Zaid A*, Tambe P, et al. CDC & FCHD, Atlanta, USA. 458C RISK FACTORS FOR GENITAL. ULCER DISEASE IN STD CLINIC ATTENDERS. 'NasioM. 'Nagelkerke NJD). 'Mwnatha A. '"Moses S, 'Ndinyn-Achola JO. "'hs,-muer FA. Objective: The mechanisms through which non-ulcerative STDs (gonorrhea, chlamydia, trlchomonas) may enhance HIV transmission In women have not been fully clarified. Because CD4 lymphocytes are susceptible to HIV infection, we sought to determine If these cells occur more commonly on the cervix or In vaginal secretions when these STDs are present. Methods: Women were enrolled at an STD clinic If they were contacts of men with urethritils or had vaginal discharge. A saline vaginal wash, ectocervical scraping, and endocervical brushing were collected; the latter two were suspended in saline. Using flow cytometry, CD4 lymphocytes were measured as a proportion of the total number of cells In suspension. Patients were considered to have chlamydia if detected by polymerase chain reaction or culture, and gonorrhea or trichomonas If detected by culture. Differences In median CD4 lymphocyte count per 10,000 cells were assessed with the Kruskal-Wallls non-parametric test. Results: Of 42 patients enrolled thus far, 20 had an STD: 11 had cervIcal gonorrhea, 8 chlamydla, and 4 trlchomonas; 3 patients had 2 of these InfectIons. The median number of CD4 lymphocytes per 10,000 cells In endocervical specimens was significantly higher among patients with STDs: Median and range of CD4 lymphocytes ter 10.000 cells by Infection with non-ulcerative STD S miIn infected (n=20) Not Infected (n=22) _n-value Endocervix 497.6 (39-1587) 255.8 (0-972) 0.005 Ectocervlx 4.0 (0-454) 2.5 (0-592) 0.9 Vaginal Wash 0 (0-123) 0 (0-48) 0.9 Conclusion: Non-ulcerative STDs were associated with an Increase in endocervlcal CD4 lymphocytes. These STDs may Increase susceptibility to HIV through this mechanism. LEVINE, William C, DMsIon of STD/HIV Prevention, Centers for Disease Control and Prevention (MS E02), Atlanta, GA, USA 30333. Telephone 404-639-8368; Telefax 404-639-8610. 1 University of Nairobi, 2 University of Manitoba Objective: To explore differences in HIV prevalence, behavioral factors and circumcision status between a group of genital ulcer disease (GUD) male patients (cases) and a group of urethritis patients (controls) attending the same STD clinic in Nairobi, Kenya. Methods: 614 GUD patients and 201 urethritis patients were enrolled into the study. Behavioral risk factors and circumcision status were ascertained by a standard questionnaire. Specimens were collected from the base of the ulcer and the urethra for microbiological diagnosis. Serum was collected for RPR, TPHA, and HIV serology. Logistic regression was used for multivariate analysis. Results: Of the 614 ulcer patients 269 were diagnosed as chancroid, 82 as syphilis and 263 as 'other ulcer'. Of ulcer patients 31% were HIV-1 positive and 77% were circumcised while among urethritis patients these percentages were 16% and 85% respectively. Their risk behaviour as regards putative source of STD were similar. Both HIV and noncircumcision were independent risk factors for GUD. Discussion: Circumcision was protective against both HIV and GUD. Most STDs were reportedly acquired recently making it unlikely that HIV infection resulted from the same contact. The association between GUD and HIV therefore cannot be explained by the theory that GUD exclusively facilitates HIV transmission. Exploration of additional causal pathways may be needed to explain the impact of HIV on GUD. Alternatively much of the association between the two could be attributable to an unknown confounder which behaves similar to circumcision. t 2fV S J r c U r-OP C P et e( "t'eV- 0. iMJIIoS ka to v G93 FYie 7lnoL.fi To.e 71546. lts will F iv '7 15-74-5 Ta. 7 i 9 s5i Un ~,n o 0

Page  42 459C PREVALENCE OF STDs AMONG HIV+ AND HIV- PATIENTS IN AN * STD CLINIC IN HAITI. Mellon, LR; G4lin-Charlot, C; Grand'Pierre R; Mevs,M; and Liautaud, B. Objective: To determine and compare the prevalence of STDs among HIV+ and HIV- patients. Methods: From 02/91 to 10/93, 374 HIV+ and 548 HIV- patients were referred to our clinic for signs and symptoms suggestive of STDs. A standardized questionnaire was administered to each one and a complete physical examination performed. Based on presenting syndrome, a specific laboratory workup was done and all patients were treated according to modified CDC/WHO algorithms. Results: The HIV+ patients differed from the negatives mainly by 3 syndromes/presentations: positive serology for syphilis [16.3% vs. 3.5%; OR= 5.43 (3.10-9.59)], genital warts [11.4% vs. 5.1%; OR= 2.41 (1.43-4.08)] and GUD [30.7% vs. 26.6%; p=ns]. When comparing etiologic diagnoses, syphilis was by far the most common in HIV+ patients [30.6% vs. 16.8% in males; OR= 3.51 (2.34-5.29) and, 21.4% vs. 8.0% in females; OR=3.13 (1.56-6.37)]. A similar finding was noted for condyloma [16.3% vs. 4.3% in males; OR= 4.37 (2.25-8.56) and, 8.1% vs. 5.7% in females; p=ns]. Conclusion: Among STDs, syphilis and condyloma are strongly suggestive of concomitant HIV infection among patients in Haiti. Bernard Liautaud, Institut National de Laboratoire et de Recherche, No. 33, Blvd. Harry Truman, Port-auPrince, Haiti 460C HIV TRANSMISSION TO FEMALE PARTNERS OF HIVPOSITIVE BLOOD DONORS IN CHIANG MAI, THAILAND Suriyanon, V.'; Nagachinta, T."; Duerr, A."'; Rugpao, S.'; Khamboonruang, C.'; Nelson, KE.'"" *Chiang Mai University, Chiang Mai, Thailand, **CONRAD, Atlanta, GA, ***CDC, Atlanta, GA, ****The Johns Hopkins University, Baltimore, MD. Objectives: To describe factors associated with HIV infection in regular female partners of HIV-infected blood donors at Chiang Mai University Hospital, Thailand. Methods: Between 3/92 and 11/93, a total of 217 regular female partners of HIV-positive male blood donors were interviewed, examined (including pelvic) and cultured for STD. Women with risk factors other than sexual contact with the index male were excluded. Gynecologic infections, including BV, vaginal candidiasis, trichomoniasis, gonorrhea, chlamydia infection, and syphilis were assessed in addition to HIV status and CD4 level. Results: Ninety-six (44.2%) women were HIV-positive. HIV+ women did not differ from HIV-negative women with regard to demographic characteristics, mean age at first sexual intercourse and number of lifetime sex partners. HIV+ women reported higher frequency of fever, rash, diarrhea, weight loss, oral thrush in the previous 6 months (p=NS), were more likely to have a history of genital herpes (p=.02) and positive HSV-2 serology (p=.05). On physical exam, HIV+ women were more likely to have abnormal vaginal discharge (OR=3.3 p<.01). However, there was no difference in frequency of BV and other STDs by HIV status; STD history or prevalence in the male index cases was unrelated to HIV positivity in the women. Conclusions: We found extensive spread of HIV and other STDs to spouses of HIV infected men. HIV positivity in the partners was associated with genital herpes and vaginal discharge. Vinai Suriyanon, RIHES, Chiang Mai University, Box 80 CMU Chiang Mai, 50002, Thailand Facsimile: 66 53 221849 462C ROLE OF GARDNERELLA VAGINALIS AS A COFACTOR IN HIV INFECTION G I T G. HAVM & CHRHATA R. SURVE. G.S.I.C. & K.E.M. HOSPITAL BOIBAT, INDIA. Objective Sexually Transmitted Diseases play an Important role in transmission of HIV. Gardnerella associated vaginosis is a very common STD & it may act as a cofactor in HIV infection. However there are no reports on association of HIV & G. vaginalis infection. Hence the study was carried out to evaluate the role of G.vaginalis as a cofactor in HIV infection. Method!! Three different groups vere included in the present study. (llprostitutes ICommercial Sex Vorkers(CSUll - 150. (2)Patients attending Gynaec OPD with a complaint of leukorrhoea - 200. (3)Patients attending family planning clinic - 150. The vaginal swabs were processed for the identification of G. vaginalis using standard procedures. HIV antibody testing was performed as per VHO protocol using commercially available kits. Results: A significant difference was observed in isolation of G. Vaginalis from three different groups i.e. Prostitutes - 54.0%, Gynaec OPD patients - 20.5%, Family Planning group - 7.3%. The prevalence of G.vaginalis infection in 80 HIV positive prostitutes was 65.0%, while in 70 HIV negative prostitutes it was 41.4%. The difference was statistically significant (P<0.05). None of the patients from Gynaec OPD & Family Planning Clinic were HIV positive. Discussion ad Conclusions: Good correlation was observed between HIV status isolation of G.vaginalls, presence of clue cells & pus cells. Presence of G.vaginalls infection may increase the Infectivity of the HIV carrier and may play an important role in transmission of HIV. The early diagnosis & treatment of this STD will help in the prevention of HIV transmission. As G.vaginalis infection is a common STD in prostitutes, it may also be taken as a marker for risk behavior. 44b <D 0 0 N nm 461C DETECTION OF HIV ANTIBOI)Y AMONG CHLAMYDIA TRACHOMATIS INFECTED PREGNANT WOMEN IN JAPAN Objective: Reliable HIV surveillance system among general population has not yet been established in Japan.Chlamydia trachomatis infected persons will be a proper target for examination of HIV infection since both HIV and Chlamidia infections occur through sexual contact. Methods: Senrum samples were collected from pregnant women attending obsteric clinics of 5 different prefectures in north eastern region of Japan. l)Detection of anti-chlamydia trachomnatis antibody was conducted by ELISA using HITAZYME (Hitachi Chemical Ltd.), and anti-HIV-1 and lIlV-2 antibody was conducted by agglutination using SERODIA (Fujirebio) and by Western blot for confirmation. Results: Total 2,41 1 sera were collected between January 1993 and February 1994. 479 sera of them were positive for chlamydia antibody. Chlamidia antibody positive rate was between 21% and 30% among the 3 clinics showing bigger figure than that of Japanese average of 10-15%. In one clinic it was 17% and rest was 13%. All the 479 sera were negative for both HIV-1 and HIV-2 antibody. Discussion: The result that all 479 chlamydia antibody positive sera were negative for HIV antibody was acceptable since HIV prevalence among general population seems to be still low in Japan as reported that accumulated HIV infected persons is less than 10,000. A collaborative network established among 6 obsteric clinics in north eastern region of Japan will provide a model to develop HIV surveillance system among general population in Japan. UMENAI, Takusci', SASAKI,T., SATO,N., ENDO,C., hONDA,S., HIROI,M., ITO,C., NAKAGOMI,O., OSAKA,Y., The University of Tokyo', Tokyo, Japan Dr.Geeta Bhave,358,Hunicipal Tenements,Govt.Dairy Rd. Vorli, Bombay400 018 Ph.No.off. 91-22-4110997, Res. 91-22-4934937, Fax 91-22-4143435

Page  43 463C OUTBREAK OF HIV IN A SCOTTISH PRISON. Taylor, Avril; Goldberg, D; Cameron, S; Emslie, J. Ruchill Hospital, Glasgow, UK. Objiectives:To assess spread of HIV infection among inmates in Glenochil Prison in Scotland. Methods:In response to an outbreak of acute hepatitis B and diagnosis of two apparently recent HIV infections, counselling and testing for HIV were offered to all inmates over a 2 week period in July 1993. Prisoners were asked about drug injecting practices, sexual behaviour and previous HIV testing. Resuls:Of a total 378 inmates, 227(60%) were counselled and 162(43%) tested for HIV. Twelve (7%) of those tested were HIV-positive and 12 in the window period. One third (76) of those counselled had ever injected drugs of whom 33(43%) had injected in Glenochil; all 12 sero-positives were found in this latter group. Two further inmates diagnosed as HIVpositive two months previously also reported injecting in the prison. Comparison of dates for last HIV-negative test, arrival in Glenochil and first HIV-positive result, indicated that at least 8 transmissions had occurred within the prison in the first half of 1993. 32/33 of Glenochil injectors had shared needles/syringes there; 31 always cleaned equipment prior to use but mostly with ineffective methods such as cold/hot water. Discussion:This is the first documented report providing evidence for an outbreak of HIV infection occurring within the prison setting. Needle/ syringe sharing was undoubtedly the responsible behavioural practice. Dr Avril Taylor, Scottish Centre for Infection and Environmental Health, Ruchill Hospital, Glasgow, UK., Tel. 44-41-946-7120 Fax 44-41-946-4359 465C HIV-1 INFECTION IN ADULTS AND YOUNG OFFENDERS IN ONTARIO, CANADA. Calzavara, Liviana'; Major C"; Myers T'; Schlossberg J; MIIIson M; Wallace E; Fearon M"; Rankin J. *Faculty of Medicine, University of Toronto. "Ontardo Ministry of Health, Ontario, Canada. Objective: To determine rates of HIV-1 infection in those entering jails, detention, and youth centres. Methods: Left-over urine specimens routinely collected from adults and young offender (YO) males and females admitted to all 42 Ontario remand facilities between Feb. & Aug. 1993 were screened using a modified HIV-1 EIA kit (Cambridge Biotech) and confirmed by western blot after concentration. In addition, all reactive, grey zone and 5000 randomly selected negative specimens were tested using Murex GACELISA. Descriptive information on gender, age and history of Injection drug use (IDU) were also collected. Results: Urine specimens were available on 87.9% (12,561/14,284) of new entrants. The rates of HIV were: 0.99% (95% Cl, 0.79, 1.19) for adult males (n=9,201); and 1.23% (95% CI, 0.63, 1.83) for adult females (n=1,302). None of the YOs (n=1,331) were found to be positive. History of IDU was highest among adult females. 20.4% of adult females, 12.5% of adult males, 3.1% of YOs had a history of IDU. HIV rates were highest among IDUs. Rates were 3.6% (95% Cl, 2.6, 4.7) for adult males (n=1,184) and 4.2% (95% CI, 1.8, 6.6) for female IDUs (n=262). Corresponding HIV rates for nonlDUs were 0.6% (95% CI, 0.4, 0.8) and 0.5% (95% CI, 0.1, 0.9). Rates of HIV-1 were 6.3 times higher than in the general population. History of IDU is a strong predictor of infection, and explains much of the gender differences. Conclusions: For the first time in Canadian history, officials have an estimate of the number of HIV-infected admitted to Ontario prisons. A comprehensive strategy for dealing with HIV is needed. Research on rates of seroconversion and modes of transmission among prisoners is needed. Dr. Liviana Calzavara, Preventive Med. & Biostatistics, University of Toronto, 12 Queen's Park Crescent West, Toronto, ON, Canada M5S 1A8. Tel: (416) 978-4643 Fax: (416) 978-8299 464C HIV-RELATED RISK REDUCTION AMONG WOMEN OFFENDERS IN JAIL AND IN THE COMMUNITY. Schillinq, Robert F.; Ivanoff, A.; EI-Bassel, N.; Borne, D.; Kabas, F. Columbia University School of Social Work, New York, NY 10025 Objective: Women drug users account for a growing proportion in jails and prisons, and HIV seroprevalence rates of incarcerated women are higher than those of their male counterparts. This study tests the efficacy of a group intervention, combining skillsbuilding and social support, and designed to reduce HIV risk behavior among drug-abusing female offenders. Method: Derived from socio-behavioral theories and from developmental work with the target population, the intervention consists of 8 group sessions in prison and 8 individual post-release sessions in the community. Women with recent drug abuse, approaching release from a 3-12 month sentence, are recruited in Rikers Island jail, New York City. Result: More than 300 women have thus far been randomly assigned to the skills-building/social support intervention or to a single AIDS information session. Measurement includes baseline, and follow-up at 1, 3, 6, and 12 months. In a logistic regression model controlling for demographic variables and number of sessions attended, preliminary findings (N=159) favor skill/social support participants on indicators of condom use improvement (OR=3.92, p<.07) coping skills improvement (OR=2.14, p<.l), and emotional support improvement (OR=2.42, p<.05), but not sex trading. Conclusion: Early results lend some support to group skills-building interventions to reduce HIV risk behavior among female offenders. Robert F. Schilling Columbia University School of Social Work 622 West 113th Street, Rm 715 NewYork, NY10025, USA Tel: 1(212) 8543149 Fax: 1(212) 854s549 466C HOW DO PRISIONERS USE CONDONS IN BRAZILIAN JAILS? Oliveira, Sylvio; Longo, Paulo; Damsceno, J.L. N6cleo de Orientacao em Sadde Social. Rio de Janeiro- BRAZIL OBJECTIVES:1)Identify how condors are used within prisions in Brazil; 2) Create estrategies to asure that condons are properly used. METHUDS:Interviews with 450 prisioners attend by PROJETO TEREZA, a regular project on HIV/AIDS prevention in prisions in Rio de Janeiro, to verify how condons distributed by the project have been used. Discussions aiming to create estrategies for a proper condon use. RESULTS:It was possible to identify at least 9 different uses for condons: personal hygienne;money keeper;carrying drugs;as a belt;in exchange for several goods;and some others.It is clear that, due to the lack of many basic needs make condons an important substitute to such goods. Discussions has shown that pression must be made,so the State must provide such needs. Requests for donations (soap,tooth paste,toilet paper) to labs and factories has been sucessful in many cases. CONCLUSION:Due to the strong lack of conditions in brazilian prisions, condoms provided tend to be used in many situations, instead of the correct use. There is an "hierarchy of necessities" which make condoms more used for other purposes. An integrated work between project's team and prisioners has found estrategies to attend such needs, as well as a continued and systematic education process. SYLVIO DE OLIVEIRA R. Visconde de PirajA 127/201 - Ipanema- Rio de Janeiro- BRAZIL Fax: +55 21 227 5944 0) C) n 0) 0

Page  44 4467C HIV INFECTION IN PRISON IN MEXICO "Mavis Carlos.*, Del Rio A.*, Gonzalez G.*, Garcia ML*, Valdespino JL*, Sepulveda J.** * Instituto Nacional de Diagn6stico y Referencia Epidemiolbgicos, Mexico, ** Subsecretario de Salud, Mexico Objectives. To describe the relation between HIV infection and prisons in Mexican men. Methods: The 1990-1993 6146 men sentinel database was analyzed. 1065 men with antecedents of imprisonment were selected. Sociodemographic profile, practice of risk factors to HIV were analyzed and compared to the seroprevalence with confirmation by western blot. Results: The general seroprevalence in this group was 3.7%. Mean for age: 29.57 standard deviation 11.93. Singles 54.5%. 39.65 had children. 64% had basic education and 10.4% had professional studies. 83.6% had relations with women 21.45% had relations with men and 14.3% had relations with women and men. Risks factors: sex with men outside the prision odds ratio 10.54 Cornfield 95% confidence interval (CI) 4.90<or<23,.10.OR 5.37 CI 2.65<or<10.870R 5.37 CI 2.65<or<l0.87; receptive sex with men OR 2,36 CI 1.15<or<4.81; practice of commercial sex OR 3.69 CI 1.68<or<8.05. Use of intravenous drugs OR 2.21 CI l<or<4.77. sex with men inside prision OR 8.14 CI 3.91<or<16.89; Use of intravenous drugs inside the prision OR 3.87 CI 1.36<or<l0.39. Homosexual and bisexual practices were more prevalent outside the prision 21.4% than inside the prision 10.3%. Intravenous drug use was more prevalent outside the prision 15% than inside the prision. Sharing needles was more prevalent outside 13% prision than inside 4.33% Discussion: Until 1993 HIV infection is more associated to sexual relations with men than to the use of intravenous drugs. Both practices are more prevalent outside prision. Postal address: Carlos L. Magis Rodriguez Carpio 470, Colonia Santo Tomas Mexico City, C.P. 11340, Telephone: 3414953, Fax:3413264 468C COMPULSORILY DETAINED HIV PATIENTS IN SWEDEN Alexius Birgitta, Wistedt B. Karolinska Institute, Danderyd Hospital, SWEDEN When HIV infected patients in Sweden fail to comply with Sweden's Prevention of Infectious Diseases Act and there may be a risk for disease transmission the medical officer of Environmental Health and Infectious Diseases Control Department can apply for compulsory detention. Objective. This study describes psychiatric diagnosis for 34 HIV infected compulsorily detained patients. Methods. All patients isolated in an institution in Stockholm from February 1986 to December 1993 were included. Patients from the entire country are referred to this special unit for compulsory detention of HIV cases. Psychiatric data were collected from the patients' psychiatric reports. Results. Twenty-one patients were intravenous drug users (IVDUs), ten women and eleven men. Seven of these patients were psychotic and had difficulties internalizing that they were HIV infected. Two men were alcohol addicts with severe organic brain syndromes due to alcohol. One man had an organic delusional syndrome due to cerebral toxoplasmosis. Ten male immigrants from countries outside Europe had adjustment disorders or post-traumatic stress disorder. Twenty-one patients were discharged from isolation in December 1993. Dicussion and Conclusions. The compulsorily detained HIV infected patients had mental disorders but were not so seriously disturbed that they could be committed in psychiatric hospitals. Some patients with several diagnoses; for example, IVDU and psychosis, needed institutional care for long periods, eventually for the rest of their lives. Birgiltta Alexius, Department of Psychiatry, Danderyd tlospital, 5-182 88 Danderyd, SWEDEN. Tel:46 8 6555000 470D ADOLESCENTS AS SKILLED AND COMMITTED HIVISTD PEER EDUCATORS IN JAMAICA: Hue, L; Bain, B; White, B; Richardson, A; Petitgirard, A; and Fee, N; Intern' Fed. of Red Cross and Red Crescent Societies & Jamaica Red Cross Society. OBJECTIVES: To enable 13 - 18 year old peer educators to use participatory methods, e.g. role play, games, interactive strategies and condom demonstration to undertake HIV and STD education with groups of adolescents, in order to stimulate behaviour change and safer sex practices. METHODS: Peer educators were trained in a five-day workshop, then worked in groups of 2 or 3 to undertake a set of 3 educational sessions with groups of 10 young people. Key elements were, the selection of PEs by young people; a clear PE role definition; development of a set of group-based games and exercises; and active supervision of the PEs by local adult volunteers. Research activities included pre and post test surveys (intervention and control areas) and qualitative methods. RESULTS: 23 PEs reached 250 adolescents over 6 months. PEs gained knowledge about STDs, skills in group facilitation, and developed more positive attitudes towards PWAs through training. PEs showed ability, commitment and skill in facilitating group education. Adolescents eagerly participated in the educational sessions, and preferred peer-led sessions, and group - and game - based learning. CONCLUSIONS: Adolescents want to learn from each other, and to help others to learn about HIVISTD prevention. There are good educational and practical reasons to promote active groupbased learning for younger adolescents. Training and supervision have produced eager and skillful facilitators. Lessons learned from this project will be incorporated into a larger nationwide peer education project. Mrs Lois Hue, Jamaica Red Cross, National Head Quarters, Central Village, St Catherine, Jamaica. Tel: (809) 984-7860-3; Fax: (809) 984-8272 0) "4h 0 V -4 V 0 469D AN EVALUATION OF AN AIDS EDUCATION PHOTO-COMIC FOR THE YOUTH OF SOUTI AFRICA. Reddy P. Everett K, Mathews C, Swanevelder S. Medical Research Council, South Africa. Two thirds of those South Africans who will be infected with HIV by the year 2000 are currently under the age of 20. This paper reports on an evaluation of a community based AIDS education intervention targeting youth. The intervention consisted of an AIDS education photo-comic that was developed with the participation of the intended readers and the evaluation was undertaken in a culturally diverse school in Cape Town. Objectives included: I. Evaluating changes in knowledge and attitudes to HIV and AIDS after exposure to the comic. 2. Testing comprehension of the storyline and IIIV prevention messages. 3. Assessing the acceptability and appeal of the comic medium for AIDS education. Method The study was a repeated measures, experimental design. The experimental group received the comic and were surveyed before, immediately after the intervention and 6 weeks later. The control group comprised 334 students. Results The majority of students evaluated the photo-comic positively and 92% felt that the comic assisted them in making a decision about protection. A single reading of the comic was associated with improved knowledge about the relative risk of certain behaviours, the effectiveness of lubricated condoms as a means of preventing tlIV infection and vertical transmission. Students indicated greater confidence in using condoms and more positive attitudes towards condoms in the post test. Conclusion The data suggests that an intervention which has high appeal and acceptance among youth can succeed in changing knowledge and attitudes. Priscilla Reddy, Medical Research Council, National AIDS Research Programme, PO Box 19070, TYGERBERG 7505 South Africa. Tel: (021) 938-0372; Fax: (021)938-0342

Page  45 471D SCHOOL BASED COMMUNITY --71 ACTION FOR AIDS/STD PREVENTION.- TANiSKUL,, Prasert, Ministry of Education Thailand. Funded by the EC AIDS Programme Objective: To establish a network for peer counselling to encourage behavior change for AIDS/STD prevention among out of school youth in rural commurnities. Methods: Baseline data on risk behavior and condom use is gathered and skills oriented training methodology will be developed in detail. A group of student volunteers recruited and trained by the teacher colleges in the pilot area will be dispatched to the target communities to provide peer education training. Out of school youths recruited from the target communities will be trained as peer educators to conduct informal AIDS/STD education among youth in the communities. The training will provide skills for informal peer counselling and interactive education in the village environment. An impact assessment will be carried out looking at peer education skills and target group behavior. Result: It is expected that with adequate number of properly selected and trained peer educators along with continuous support and encouragement from the teacher colleges in the area, the reevaluation of values and practices concerning safer sex will be initiated. As a result, safer practices regarding sex and IVDU will be introduced. Discussion and conclusions: The experiences and methodology gained from the pilot phase of this study will be assessed before further expansion to cover wider areas. The findings will also be used to guide the development of sustainable, simple and effective behavior modification models for AIDS/STD prevention in Thai communites. Tanskul Prasert, Senior Specialist, Department of Teacher Education, Ministry of Education, Bangkok, Thailand 10300 Telephone (662) 2800885; Telefax. (662) 2804909 473D GENDER-SPECIFIC CORRELATES OF MULTIPLE SEXUAL PARTNERS AMONG 473DAFRICAN-AMERICAN ADOLESCENTS IN A tRGI RISK URBAN ENVIRONMENT. Harer. Gary*, Lodico, M., DiClemente, R., & Coates, T.*. *Center for AIDS Prevention Studies (CAPS), University of California, San Francisco, USA. OB.JECTIVE: Sexual intercourse with multiple partners is a key factor which increases the potential for HIV infection. This study identified gender-specific psychological correlates of the lifetime number of heterosexual sex partners among African-American adolescents living in public housing projects. METHODS: Field recruiters referred African-American adolescents living in two public housing projects in San Francisco for interviews with same-ethnicity research staff. We interviewed 97 sexually active adolescents between the ages of 12 and 18 years. Predictor variables included psychological characteristics and alcohol use prior to sex. The outcome variable was the lifetime number of vaginal sexual partners. RESULI& Respondents' mean age was 14.6-51% were female and 49% were male. Separate multivariate linear regression models were constructed to develop gender-specific psychological profiles of adolescents who had reported multiple sexual partners. After adjusting for years of sexual activity, increasing number of lifetime sexual partners was associated with loneliness and isolation (=.48, p=.0004) and alcohol use prior to sex (6=.29, p=.01l31) for females; and aggressive and antisocial behavior (6=.60, p=.0005) for males. The full multivariate model accounted for 53% of the variance for females (p<.0001) and for 49% of the variance for males (p<.0001). CONCLUSIONS: The findings suggest that the psychological factors associated with economically disadvantaged African-American adolescents' decisions to participate in sexual activity with multiple partners are gender specific. Prevention efforts aimed at decreasing this IIV risk behavior among females should emphasize the role of loneliness and isolation in sexual decision making, and should explore alternative forms of coping with these feelings. In addition, the potential relationship between alcohol use and increased sexual activity should be stressed. IIIV prevention programs for males may benefit from identifying the relationship between sexual activity and aggressive/antisocial behaviors, and should help to explore prosocial activities and non-risk behaviors in which these males can participate. GARY W. HARPER. Ph.D. 74 NEW MONTGOMERY ST., SUITE 500 SAN FRANCISCO, CA 94105 USA 415/597-9208 (P11); 415/597-9213 (FAX) 472D SOCIAL MARKETING OF 'AIDS EDUCATION FOR YOUTllH'THE INDIAN EXPERIMENT DR. SHAHABUDDIN Y. QURAISHI NATIONAL COORDINATOR, YOUTH ACTION ON AIDS (INDIA) OBJECTIVE: To discover the extent of youth awareness level on AIDS. To develop participative communication package for peer-education. To apply principles of social marketing in promoting AIDS awareness among youth. MEI3DS: Youth being the target high-risk group, methods and campaign strategies were identified through base line, situation study and inprocess evaluation. Questionnaires were administered to assess attitude, knowledge and impact generated. Other tools used were interview, training modules, message-design guide, media-reach-packets, focus group, Quiz, public-rallies, folk-media and advocacy materials. RESULTS: The Study-cum-campaign population covered 8,00,000 youth of wich samples of 40,000 respondents were analysed. IEC materials developed by target-groups themselves. Ehvironment of social-support for community mobilisation was created. Participative peer-involvement Induced self-evaluation of behaviour and created a sense of 'Youth ownership' of programme. Concurrent research pin-pointed grey-greas to improve future plans. DISCUSSION AND CONCLUSION: It was realised that Social marketing methods can prove effective in making AIDS Edlucation Programme low-cost and sustainable. Need to overcome information gaps, to involve parents and teachers and to introduce sexuality and AIDS Prevention education in curriculum was felt. DR. SHAHABUDDIN Y. QURAISHI, NATIONAL COORDINATOR, YOUTH ACTION ON AIDS, DEPTT. OF YOUTH AFFAIRS 6 SPORTS, SHASTRI BHAWAN, NEW DELHI, INDIA Tel: 384152. Fax:011-387418 474D OFS"P "'V/AlIPS Wo9R EfEIEsGc.r OF AS4 4tp5 PREVaEtN,O Afp CP~R ROE AM fAOR, OMFLEs5 "VO r t, Al-9p SD LT5 I "1s a ses T.A DE.~ URBAt, A.Tw eeEsA, e.I...tAR Ft.. KAAt tYAT 14, PA+ -t t."Ao PttQ t Ie O Ak v-A I vA tt.* t PP tN. Work on HIV/AIDS began as early as the time the virus causing AIDS was given a name. Numerous success stories on various aspects of the work has been shared in big conferences which have helped encourage people to do or go into similar ventures. With the growing concern shown by the increasing number of organizations - GOs, NGOs, CBOs and private engaged in the struggle to stop the spread of HIV infection all over the world, it is high time that the banes of doing HIV/AIDS work be exchanged and shared. Lessons learned and actions taken to resolve hazards of implementing a community-based HIV/AIDS Prevention and Care project among sex workers in a country such as the Philippines, may have some value to areas where similar situations are evident. Likewise, the process lends to listing of effective strategies that consider cost-benefit in the light of dwindling funds available to support efforts in trying to cope with the problems caused by HIV/AIDS. This paper therefore, will seek to paint a true picture of the challenges an organization faces in conducting community-based efforts among sex workers in the Philippines. H4A. THERe.SA A. URPatJ S64( 3-Mg,4LD. AGrtGlQRE S-. t-..GAS(P VitLAC M HAATI Ptv f Ptu s S 8 13- 47 FAY.1 - 8 435'.rh 44h

Page  46 $ SS6 SS7 C,, 0l) Cl) C,) mD SS8 SS9 THE NEED FOR FEMALE-CONTROLLED HIV PREVENTION Weiss, Ellen, Rao Gupta, G. ICRW, Washington, DC, USA Objective Women currently constitute approximately 40% of the global total of adult HIV infections worldwide. Current HIV/AIDS risk-reduction strategies (sexual partner reduction, mutual monogamy, consistent condom use, and appropriate treatment for STDs) do not address the broader economic and sociocultural factors within which sexual behavior takes place. In order to provide a more comprehensive understanding of how these factors influence women's risk of HIV infection, findings from 17 studies conducted by the Women and AIDS Research Program of the International Center for Research on Women will be presented. Methods In order to provide insights into the realities of women's and mek's sexual lives, the studies supported through the Women and AIDS Research Program utilized a variety of qualitative methods, including focus group discussions, individual interviews, sexual life histories, and participant observation. Results Data from the studies show that women in a variety of life circumstances are at risk of HIV infection, and face numerous constraints in adopting HIV risk-reduction behaviors. Cultural norms which encourage men to have multiple partner relationships also expect women to be ignorant and passive in sexual interactions. Economic vulnerability not only contributes to women exchanging sex for goods and services, but also prevents them from leaving high-risk sexual relationships. Discussion and Conclusions Policy and program recommendations which emerged from the studies--including the need to develop female-controlled technologies--will be presented. WEISS, Ellen, ICRW, Washington, DC, USA Telephone (202) 797-0007; Fax (202) 797-0020 CD 0 C) CD m. O"

Page  47 ORAL ABSTRACTS Thursday 11 AUGUST Plenary Sessions Abstract Sessions

Page  48 PS25 BUILDING EFFECTIVE PARTNERSHIPS BETWEEN COMMUNITIES AND GOVERNMENTS PROF DENNIS ALTMAN, IA TROBE UNIVERSITY, AUSTRALIA This presentation argues that the social, economic and political requltements to effectively deal with HIVIAIDS demand meaningful partnership between a wide range of national and inteniational players, of whom the pivotal are community/non-governmental organisations and governments. The difficulty is to make such partnerships meaningful, given the disparities of resources and legitimacy available to the different parts of such a partnership. An examination of successes and failures in both developed and developing countries will help determine what factors are critical in ensuring what factors are critical in ensuring that there is optimum mobilisation including the empowerment of thosern most affected. There is a need to question the meaning of the term "community" and its relevance to different cultural and epidemiological situations. A meaningful partnership must also involve other key players, such a health professionals, international agencies, earers, business and education. Within and between these sectors there will be critical divisions, which need be addressed. Genuine partnership requires a recognition of legitimate differences as well as the creation of common goals and programs. Responses to HItV/AIDS have become a model of international and intranational policy making, which have implications for large ireas of development, health and social policy/ Getting our responses right has large implicatlons for our ability to deal with other crises of modernity. Prof. Dennis Altman, School of Politics, La Trobe University, Bundoora 3083 tel:479 2699 fax: 479 1997 PS26 Controlling the Emergence of Tuberculosis (TB) in HIVinfected patients (Pt).Gatell Jose M. Hospital Clinic. Barcelona. Spain. Since mid 80's the incidence of TB is no longer declining, in western world. Moreover, outbreaks of multidrug resistant tuberculosis (MDRT) have been reported, mostly in USA, together with a mild increase in the baseline level of primary resistance to first line antituberculosis agents. There is a close temporal relationship between the HIV epidemic and the increasing incidence of TB. A percentage of the "unexpected" new cases of TB occur in already known HIV Pt. Apart from reactivation of latent forms, a substantial number of the cases of tuberculosis including MDRT in HIV patients may be relatively rapid progressions of recently acquired infections and also reinfections. Main step to control the emerging problem of TB including TB in HIV Pt and MDRT is early diagnosis, isolation measures while the sputum smear is positive (focusing in health care facilities), tracing contacts (houses, shelters, prisons), and starting soon the adequate treatment (even supervising it and making the necessary drugs available). On the other hand a PPD test should be performed in all HIV Pt. If positive chemoprophylaxis with Isoniazid is strongly recommended and has been proven effective, although the duration of the protection is yet to be determined. Chemoprophylaxis may also be considered in selected HIV Pt with skin anergy mainly when the CD4 cell count drops below a given threshold. It also remain to be tested if the recently recommended prophylaxis for MAC with Rifabutin will also cross protect for TB. Jose M. Gatell. Infectious Disease Unit. Hospital Clinic. Villarroel 170. 08036 Barcelona. Spain. Telf. Int+34+3+4534261. Fax. Int+34+3+4514438. -u (0 Co) PS27 KNOWLEDGE ATITrUDES AND PRACTICES OF MOROCCAN MEDICAL RESIDENTS TOWARDS HIV INFECTION Hakima Himmich, Chraibi S., Heikel J.,Chakib A. Division of Infectious Diseases,University hospital Center, Casablanca. Objectives: To evaluate knowledge attitudes and practices of moroccan medical residents towards HIV infection. Methods: In june 1993, we conducted a survey based on the interview of all medical residents in Morocco. The technic was based on a face to face interview during which each resident had to fill a standardized questionnaire. Results: A total of 240 medical residents were interviewed among 300 (80%). The two main reasons for not interviewing 60 residents are related to their annual vacancy and to their training abroad. Among the most interresting results, we notice the fact that 79.2% of the residents don't know all the mode of transmission of the HIV virus; and 98.3% are unaware of the case notification procedure in the country. Concerning their clinical practice, 42.9% of the interviewed residents suggest to put a red stick on the medical records and the laboratory list of AIDS patients. In fact 33% propose to limit or reduce laboratories exams for such patients. Discussion and conclusion: This study underlines the urgency of informing and educating medical residents in Morocco. This sensibilization should have three goals: improving the scientific knowledge, changing a health care behavior and integrating ethical aspects in the care setting. Hakima Himmich,Division of Infectious Diseases,Faculty of medicine; 19, IbnTarik street, Casablanca.Tel (212-2) 204333; Fax(212-2) 293188 PS28 CD CD C,,

Page  49 PS29 FUTURE STRATEGIES REGARDING CHILDREN AFFECTED BY AIDS. Mazuwa Banda, Churches Medical Association of Zambia, Lusaka, Zambia. PS30 The Impact of HIV/AIDS Stefano M. Bertozzi Global Programme on AIDS, World Health Organization Impat of HIV/AIDS on children:The IIIV/AIDS pandemic has had far reaching implications on many population groups world over, but more especially in developing countries. Children have not been spared by the pandemic. In Africa particularly, an increasing number of children are getting infected with HIV, being orphaned and experiencing numerous stresses from the effects of ilIV/AIDS. Data on the current situation and future projections concerning HIV and children are reviewed. HIV/AIDS impacts on children is in many ways. Generally HIV disease progresses much more rapidly in children that are infected with lIIV compared to adults. There is profound emotional and psychological effect on children affected by IIV/AIDS. Other consequences include loss of schooling opportunities, lack of material support and greater vulnerability to being exploited. Current elTfforts:The paper identifies some approaches being followed to address the problem children affected by HIV/AIDS in Africa. These approaches are classified as institutional and non-institutional. Institutional approaches mainly involve the provision of care and support through institutions such as orphanages or children's homes. Non institutional approaches include the extended family system, foster parenting and adoption, skills training initiatives, day care centers, support through older siblings and peer group support. Future strategies:Suggestions for future strategies for support to children affected by IIV/AIDS are offered. Three basic principles are identified in the development of strategies and these are: (1) the need to identify the most crucial needs of the children in a particular situation or locality, (2) the necessity of innovatively utilising existing resources and systems as much as possible and (3) the need to invest in children in the HIV/AIDS campaign. Mazuwa Banda, P.O.Box 34511, Lusaka, Zambia. Phone (2601) 223297/229702, Fax: (2601) 223297 As discussion about the HIV/AIDS pandemic swings from epidemiology to impact, the vocabulary swings from risk behaviours to orphans and sectors of the economy. The impact on the people-withHIV/AIDS themselves has too often been skipped as if it were self-evident-perhaps because it is assumed that it falls within a different category, not social or economic but medical. Because of the unique characteristics of this virus, its impact, especially on the individual, is difficult to quantify; incompletely captured by measures such as years of life lost or lost earnings. As HIV spreads, its impact mounts as does the pressure to characterize the impact -- but characterize how, for whom and to do what? To mobilize prevention efforts? Or to guide efforts to alleviate impact? If to mobilize employers, governments and donors to increase their prevention efforts, then we should seek out the examples of impact most compelling for advocacy and magnify them. If to alleviate impact, then we need to identify those who bear the greatest burden. Foremost among these will be infected people and their households- not employers or sectors of the economy. And we need to identify their greatest needs, which for many will be water, food and shelter. When the impact can be broadly characterized as an increase in poverty, then intervention should be in the context of broader efforts to alleviate poverty and assistance should be targeted to those in greatest need. Interventions specific to HIV/AIDS should focus on alleviating impact specific to the epidemic, such as: for infected individuals, addressing terminal care and counselling needs (poorly addressed by medical systems oriented toward acute curative care); for households, helping them plan for the future with their person-with-HIV/AIDS to minimize the impact following his/her death; and for all affected people, combating HIV/AIDS-related discrimination, stigmatization and isolation. Stefano M. Bertozzi, Global Programme on AIDS, World Health Organization, 1211 Geneva 27, Switzerland. Tel: (41) 22 791 4679, Fax: (41) 22 791 0107, bertozzi@who.ch - "D PAC 1< CO) CD) O 0 Co I -o CD

Page  50 CD 475C USER ACCEPTABILITY OF FEMALE CONDOM AMONG FEMALE SEX WORKERS IN SINGAPORE ChanRSoon Terns Dept of STD Control Siappore. Objective: To assess the user acceptability of Femidom among female sex workers in Singapore. Methods: The study was conducted in the STD clinic in October and November 1993. Twenty-nine sex workers were recruited into the study. They attended a workshop comprising of a talk, video and demonstration to introduce the condom. Personal and demographic data were collected. Each was given 8 or 9 condoms to try over a period of one week, after which they returned for a second interview. Results: Out of the 29 sex workers, only 21 managed to use the condoms in the study period. Of these 52% liked the product, 76% thought that it was acceptable as a protection against infections, 43% found the product easy to use, 62% found greater ease with subsequent use. Two-thirds took under 30 seconds to insert the condom. The majority either never or only rarely experienced any discomfort during or after intercourse. Tearing of the condom was experienced by one sex worker, the majority did not notice the condom slipping out or wrong entry of the condom. The major complaints by sex workers was that the condom was complicated to use, clients objected, that the condom was too large, and that it looked ugly. Most did not report any loss of sensation by their clients. However, the majority had problems persuading their clients to accept the female condom, their main reasons were that they preferred male condoms, female condoms looked ugly, and that female condoms reduced sensation. Ond quarter of the sex workers said they would use the female condom exclusively as protection against infection. Discussion: The female condom is a useful and timely addition to the methods to prevent transmission of STD/AIDS. We found that it was readily acceptable by sex workers. Difficulties encountered were mainly with their clients and to a lesser extent with the construction of the product. Department of STD Control, Blk 31, #02-16, Kelantan Lane, Singapore 0820. Tel: 2939716 Fax: 2994335 Promotion of Condom Use 47 C for AIDS prevention-Experience in the Uganda Muslim Community. AUTHORS:Y.WALAKIRA, M.KAGIMU, R.MUNYAGWA. OBJECTIVE: The Islamic Medical Association of Uganda is carrying out a pilot project to educate the Muslim Community about AIDS, HIV transmission and its prevention, condom use is one of the methods promoted for prevention. METHODS: The project is carried out in two of the districts of Uganda namely: Iganga and Mpigi. These have high Muslim populations. Imams and their Assistants including community Family AIDS Workers were trained to give AIDS education for behaviour change through persistent family visits. RESULTS: Condom promotion was intially met with strong resistance from Muslim religious leaders. The Muslim community welcomed the idea of condom education for AIDS prevention. The IMAU continued dialogue with the religious leaders. Misconceptions on the condom were identified and corrected through giving true facts. The Muslim leaders understood the AIDS problem and agree on a quiet promotion of the condom for special circumstances. CONCLUSION: While the Muslim religious leaders are still shy, community positive response about condom use encourages the IMAU to continue promoting it in the community. Through this way the community will learn facts about its proper use, indications and short comings. Dr. Y.M. Walakira, P. O. Box 2775, Kampala, Tel. 272812/271159 Kampala. 4a~ C) C) nD NU C, ONI 477C GLOBAL PROJECTIONS OF CONDOM REQUIREMENTS FOR STDIAIDS PREVENTION, 1994-2005--Patrick Friel and Eva Lustig, WHO/GPA, Geneva, Switzerland 478C THE ONE HUNDRED PERCENT CONDOM PROGRAMME IN THAILAND, AN UPDATE. ROJANAPITHAYAKORN, WIWAT. DEPARTMENT OF COMMUNICABLE DISEASE CONTROL, BANGKOK, THAILAND. Objectives: To estimate indicative quantities and costs of condoms required by National AIDS Programmes (NAP) in developing countries for STD/AIDS prevention over the decade between 1994 and 2005; to share this information with other agencies, donors and NAPs; and, to stimulate greater awareness amongst all parties of the need to quantify risky sexual behaviours, set realistic targets and generate resources to cover the costs. Methods: Social marketing sales, donor shipments and NAP-generated estimates were used to infer a 1993 condom distribution baseline and assumed number of men using condoms in risky sex. Projections for 1994 to 2005 were based upon the assumed male population 15-59 years of age who engage in risky sex in 1993, the annual increase of users due to promotion, their coital frequency and their condom use. Costs were based upon 1993 prices for public sector provided condoms. Results: Quantities of condoms for STD/AIDS prevention required by countries in 1993 are 525 million. The cumulative low and high projections of condoms requited between 1994 and 2005 are 8.6 billion and 19.6 billion units, respectively. The cumulative condom procurement cost to agencies that purchase in bulk for STDIAIDS prevention between 1994 and,2005, using the high projection, is estimated to be US$ 1.28 billion. Discussion and Conclusions: Data gathering revealed that fewer than one in five NAPs are prepared to estimate long-term condom requirements. Few data exist to quantify the size of the population that engages in risky sexual behaviour, their coital frequency and condom use. The estimated procurement costs do not include other expenses associated with condom programming such as logistics management training and promotion. To strengthen the planning process, copies of the analysis were shared with other agencies, donors and NAPs. Periodic review is needed to update the projections. Dr Patrick Friel, Scientist, The World Health Organization, Global Programme on AIDS, Division of Technical Cooperation. Prevention Unit, 1211 Geneva 27, Switzerland, Tel.: 41-22-791 4742, Fax: 41-22-791 03 17 Objective: To prevent sexual transmission of HIV by increasing utilization of condoms in sex establishments to achieve 100% condom use. Methods: Meetings are organized among government authorities and owners of sex establishments in the provinces in order to instruct or require their sex workers to use condoms in all sexual encounters. If their customers refuse to use a condom, the sexual services will be withheld, and the customers will be refunded. Condoms will be supplied free of charge to all sex establishments, and a monitoring system will be established using epidemiological data from STD clinics. Efforts will be put to ensure that the measure will be taken by all sex establishments in the area so that sex seekers will not be able to purchase sex services without condom use. Results: The programme, initiated in 1989 and expanded nationwide in 1991, has been very effective in increasing condom use, from under 50% in June 1989 to 94% in June 1993. As a result, the national STD incidence has decreased drammatically, from 6.5 per 1,000 population in 1989 to 4.48, 3.31, 2.07 and 1.64 in 1990 to 1993 respectively. Discussion and Conclusions: The programme has succeeded in raising a remarkably high level of condom use among sex workers and their clients, the main sources of HIV transmission in Thailand. ROJANAPITHAYAKORN, Wiwat, Department of Communicable Disease Control, Devavesm Palace, Samsen Rd., Bangkok 10200 Thailand. Tel (662)2826620-3; Fax (662)2828351

Page  51 479C HOT RUBBER-THE CONDOM: Marketing lifestyle, improving preventive behaviour, generating money for NGO's: THE SWISS EXPERIENCE. (M. Hausermann, C. Roggli, H. Riedener, A. Stahli, Swiss Aids Foundation) 480C PREDICTORS OF CONDOM USE WITH CASUAL PARTNERS AMONG MEN IN KAMPALA, UGANDA M.R. Kamya*, A. Ssali*, R. Busulwa*, B.V. Marin**, N. tlearst" *Makerere University, Kampala, Uganda; **CAPS, Univ/Calif, San Francisco, USA Objective: The Swiss Aids-Foundation demonstrates an effective possibility to link national efforts in HIV/STD-prevention to fundraising for NGO's by marketing high-quality condoms. Methods: Creating an own Condom brand for distribution whithin the gay community in Switzerland. Adapting the successful Hot Rubber Concept for gay men to a larger public, especially young people, in Switzerland and Europe. Marketing The Hot Rubber Image. Measuring at the same time preventive behaviour among young Swiss as well as the rising market shares of The Hot Rubber. Results: Distributing condoms and lubricant within main target groups demands high financial investments. By marketing The Hot Rubber, Swiss Aids Foundation's own high quality condoms, these investments are being covered after only 12 months of sales. The growth of market shares leads the Swiss Aids Foundation, as well as other cooperating NGO's, to net revenues and, of big importance during economic recession, a larger independency of public funds. Conclusion: The Hot Rubber Concept is an effective and realizable strategy for national NGO's to stand to gain by using synergies with public prevention campaigns and to provide a high impact on HIV-/STDprevention, fundraising, PR, international networking. HAUSERMANN, Michael, Swiss Aids Foundation, Box 141, 8031 Zurich phone:*41-1-273-42-42 fax: *41-1-273-42-62 481B DETERMINANTS OF PROGRESSION IN A HEMOPHILIA COHORT FOLLOWED FOR 14 YEARS. Hatzakis Angelos*, Karafoulidou A,**, Touloumi G*. PolychronakiH*, GialerakiA**, Kapsimali V** *,Economidou 1***, MandalakiT* *. *Athens Univ. Med. Sch., **Laikon, ***Evangelismos Hosp., Athens, Greece Objective: To describe the progression of HIV infection and to assess the infuence of age at seroconversion and prevalent cytomegalovirus (CMV) infection in a hemophilia cohort. Methods: 158 hemophiliacs (149 hemophilia A, 4 hemophilia B knd 5 Von Willebrand's disease) seroconverted to HIV between 1980 and 1985. The presumed date of seroconversion was calculated by smooth models based on spline functions (Kroner and Rosenberg, 1993, submitted). The mean (SD) age at time of seroconversion was 26.2 (15.4), range 1.3-86.0 years. The mean (SD) duration of follow up time from seroconversion was 9.1 (2.4). median 9.8, range 1.1-12.7 years. Results: By November 1993 40 patients had developed AIDS, 91 HIV Disease (CDC Group IV) and 110 AIDS or CD4s200/mm3. The Kaplan Meier estimation of progression risks are shown. Years from seroconversion 3 4 5 6 7 8 9 10 11 12 13 Risk of: AIDS(%) 0 1 3 6 10 15 24 28 32 36 36 HIV Disease(%) 1 4 10 20 29 43 55 60 64 71 81 AIDS or CD4<_200(%) 1 8 15 25 42 59 72 77 79 83 89 Cox proportional hazard analysis using AIDS as outcome indicated that age at seroconversion was a highly significant variable (p =0.009) CMV infection was not a statistically significant variable in models including or not including age. Further analysis of age suggested that only patients aged above 40 yrs progressed faster to AIDS (RR=3.0, 95% Cl 1.3-6.7. p=0.009). Similar results were obtained using HIV disease and AIDS or CD4s_200 as outcomes. Discussion and Conclusions: 1. Despite the risk of progression to AIDS remains relatively low, the risk of HIV disease or advanced immunodeficiency is high 13 years after seroconversion. 2. Prevalent CMV nfection seems not to be a determinant of progression. 3. Age at seroconversion remains the only important determinant of progression but its effect is evident in patients aged above 40 yrs. HATZAKIS Angelos, Dept. of Epidemiology, Athens University Medical School M. Asias 75. Athens 11527, Greece Tel 301-7719725, Fax: 301-7704225 Obiectives: To identify factors related to condom use with casual partners among young men living in a low SES urban village of Kampala. Methods: A cross-sectional sample of 301, 18-45 year-old men was drawn by a two-stage (zones and then subzones) cluster sampling technique. Interviews were conducted by trained social scientists in May and June, 1993. The questions included: knowledge and beliefs about condoms, risk perception and risk taking behaviors, self-efficacy, and social-cultural constraints. [Condom use was defined as always or sometimes using condoms, or used condoms at last sex with a casual partner. Casual partner was defined as sexual partner a man does not fully provide for financially.] Multiple logistic regression was used to identify the independent predictors of condom use with casual partners. Results: Median age of the men was 28 years. The frequency of reported condom use with casual partners was: Always using 31%; Always or sometimes using 50%; Used at last intercourse with casual partner 46%; Intends to use condoms in future 78%. The independent predictors of condom use were: Less embarrassment to buy condoms (OR = 5.5, CI 1.6-19.8, P = 0.01), Knowing a place to purchase or obtain condoms (OR = 8.5, CI 1.1-64.6, P = 0.04), imagined or actual experience of a condom breaking (OR = 0.2, CI 0.1-0.8, P = 0.02), and a report that "I would use condoms even if I were drunk" (OR = 3.7, CI 1.3 -11.2, P = 0.02). Conclusions: Condom promotion efforts should emphasize increasing comfort with buying condoms, advertising places to buy condoms, and teaching proper use and other modalities to prevent condom breakage. Moses R. Kamya, MD Department of Medicine, Mulago h lospital, 4th Floor Post Office Box 7051, Kampala, Uganda Tel. 256 41 530188 FAX: 256 41 259130 c/o James Kasavubu 482B PROGRESSION TO AIDS AMONG HIV-POSITIVE HEM OQPHILIACS Alesnd Ghirarini, F Chiarotti,.,M Puopolo, G, Mancuso,o. Zanetti, I IerugiOw t( Aircerii,,F, Scarag, nd th Italimn Group oj,.ongep tIl Coaguro9.es (GI'~ Istitup, t prlore dia Sanmta, Rome; emoplia Cen re o 1 orino, a rermo,ai; Italy. Objective: To evaluate the progression to AIDS among the Italian cohort of HIV positive hemophiliacs. Methods: 742 HIV-positive hemophiliacs were the study subjects, representing 90.5% of the infected subjects referred to the national registry of hemophilia. 520 of patients were affected by hemophilia A, 191 by hemophilia B, and 31 by other types of hemophilia. HIV seroconversion was calculated by considering the median point, under a Weibull distribution, between the last HIV negative and the first HIV positive test. The cumulative AIDS incidence was estimated by Kaplan Meier method. A hazard regression model was then applied to identify factors independently associated with progression to AIDS. Results: The cumulative AIDS incidence 10 years after seroconversion was 40.4%. Hemophiliacs with age at seroconversion >35 years exhibited an AIDS incidence of 52.6%, compared with 39.7% for those aged 13-34 years, and 35.2% for those < 13 years (p=0.003). Subjects with severe B hemophilia had a faster progression to AIDS than severe A hemophilia, 49.5% vs 34.5% (p=0.005). Also considering all types of severity of hemophiliacs, hemophilia B subjects had a significantly faster progression to AIDS than the other subjects, (p=0.003). The proportional hazard model showed that older age at seroconversion (R.H.=2.1) and hemophilia B (R.H.= 1.6) were independently associated with a faster progression to AIDS. Discussion and Conclusions: 10 years after the beginning of exposure to HIV, the cumulative incidence of AIDS was 40% among HIV positive hemophiliacs. Older hemophiliacs show the faster progression to AIDS. The study outlines that HIV positive B hemophiliacs have a higher risk of developing AIDS than the others hemophiliacs. Alessandro Ghirardini, Istituto Superiore di Sanith Lab. Epidemiology and Biostatistics Viale Regina Elena 299, 00161 Rome Italy, tel +39-6 -4460124, fax +39-6-4460120 (0 nE 44 W~

Page  52 Ci 483B CHANGES IN VIRAL LOAD IN HIV-1 INFECTED HEMOPHILIACS. S.Y.Horikoh*,J.Mimaya*,T.Tonouchi*,K.Amano*,T.Morita**, H.Naitou4, *Division ofHematology/Oncology,Shizuoka Children's Hospital,**Graduate School of Nutritional and Environmental Sciences,University of Shizuoka. Objective: We amplified HIV-1 RNA by RT-PCR from sera in HIV-1 infected hemophiliacs to determine whether quntitative methods reflect viral load or not.Methods: Subjects were 8 HIV-1 infected hemophiliacs(AC;2,ARC;4, AIDS;2).HIV-s particles was collected and extracted from 1OOul of sera. HIV-I RNA was reverse-transcripted into cDNA and diluted from 10to 109 level.RT-PCR was carried out with primer pairs of SK145/SK39 for 1st PCR and SK145/SK1O1 for 2nd PCR.PCR products were analyzed by dot blot hybridization with digoxigenin-labeled probe.Results:HIV-1 RNA sequences was detected from lOto 1O'dilution range of 8 patients.In one ARC patient treated with AZT and ddC,viral load decreased with detected range from 106 to 10i dilution.In another AC patient became fatigued,viral load increased with detected range from 10 to 10isdilution and so we changed anti-viral agents.Also one AC patient with almost nomal CD4 count has viral RNA level with detected range of 10 dilution on several times.Discussion and Conclusions: Detected range of HIV-I RNA was changed with disease progression and treatment.This methods may reflect viral load and may be useful as a marker of drug efficacy and progression to AIDS.The AC patient with nomal CD4 count had a lot of viral load and needs to further examination of viral characteristics and host defense mechanism.Small change of viral load will be observed by competitive PCR methods. Yasuo Horikoshi,Shizuoka Children's Hospital,860 Urushiyama,Shizuoka,Japan, Telephone(81)-54-247-6251;Telefax(81)-54-247-6243 484B CONDITIONS OF HIV INFECTED HEMOPHILIACS IN JAPAN Ishida Yoshiaki*, lenishi S*, Ogasawara K, Kawamura M. *Friendly Society of Hemophiliacs in Kyoto Objectives: We have researched on hemophiliacs, how much they were damaged and socially affected by the HIV infection. It was in July '85 that the Japanese government permitted the use of heat treated blood products. Because of such slow countermeasures, many hemophiliacs got infected with HIV. At the point of our research (Dec.'88), 88.6% of HIV infected people in Japan were hemophiliacs, and now 56.0%(1728/3084) are. Methods: We delivered 1000 questionnaires through the hemophiliac groups, collected 284 sheets anonymously by mail (Oct.-Dec.'88) and analyzed 255 of them. The second survey is now ongoing, and we will report it on the workshop. Results: Of the respondents, 83 (32.6%) were HIV positive, 48 (18.8%) were negative, and 124 (48.6%) weren't told whether positive or not. Among 207patients (48negatives were excluded), only 59 (28.5%) were under treatment, and 87 (42.0%) weren't treated for HIV disease. 59 in total had experienced medical care refusals in clinics of dentistry, internal medicine, pediatrics, orthopedics and surgery. 13 were rejected (or claimed HIV negative proofs) at school or company. We will report other results in the workshop. Discussion and Conclusions: What keeps doctors from truth-telling? Why are patients left without treatment? What to do to get rid of discrimination? Some problems have come into view. The second survey, currently under way, will reveal the present condition. ISHIDA Yoshiaki. Friendly Society of Hemophiliacs in Kyoto, Kyoto, Japan. Tel 075-643-6301; Fax 075-643-6119 486B HIV INFECTED HEMOPHIULIACS TREATED WITH ANTIRETROVIRALS: Santarelli Maria, Perez Bianco R., VarnavA D., Tezanos Pinto M. Inst. Inv. Hematol6gicas 'Mariano R. Castex', Buenos Aires,Argentina. OBJECTIVE: to assess the benefits of antiretroviral agents in the treatment of hiv infected hemophiliacs (HE). MATERIAL: a retrospective review of the charts of HE patients (pts) treated in our institution between 1984 and 1992 revealed a total of 103 pts who received antiretroviral therapy. Median age was 18 years, range (4-68); HE A 86 pts, B 17 pts; mild 10, moderate 29 and severe 64; initial stage (ST) (WHO Staging System): ST 1: 98 pts, ST 2: 3 pts and ST 3: 2 pts, mean initial CD4 count 505 ~ 282.59. Life tables were constructed by the method of Kaplan-Meier. RESUL: 103 pts were treated with zidovudine (AZT), alone; in combination, or alternating with dideoxynosine (DDI) 45 pts, and/or dideoxycytidine (DDC) 24 pts. Median time to therapy was 50 months (mo) range (1-96). 43 pts progress to AIDS: 9 pneumocystis carini pneumonia, 8 toxoplasmosis, 6 herpes simplex virus, 5 cryptococcosis, 5 candidiasis of the oesophagus, 2 cryptosporidiosis, 2 HIV encephalopathy, 2 cytomegalovirus and with 1 pt: atypical mycobacteriosis, multifocal leukoencephalopathy, disseminated mycosis, extrapulmonary tuberculosis. Median time to progression (TTP) to AIDS was 93 mo, estimated probability of TTP at 8 years was 54%, 22/43 pts who progressed are dead, the estimate overall SV was 70% at 90 mo. CONCLUSIONS: we compare this treated group (TG) of pts with a control group (CG) without therapy: TTP for TG was 93 mo vs 78 mo for CG (p=0.0013), median SV was 98 mo for CG and not reached for TG (p=0.0000). This results show better SV associated to retroviral agents suggesting that antiretrovirals offers benefit for the treatment of HIV infected HE. Sonsored in part by 'Alberto F. Roemmers' Foundation. Santarelli Marfa, Instituto de Investigaciones Hematol6gicas "Mariano R. Castex" de la Academia Nacional de Medicina, P. de Melo 3081 (1425) Buenos Aires, Argentina. Telephone (541) 792-3328,Telefax (541) 792-3328. w 40h 00 0) 485B COUNSELING FOR HIV-POSITIVE HEMOPHILIACS IN JAPAN YANAGA Yuriko, ONO O, SHIRAHATA A. Hemophiliac Center, Univ. of Occup. & Educ. Health, Japan. Objective: Description of current status of counseling for patients who are HIV positive at a Hemophiliac Center in Japan. Methods: Clinical experience with 23 hemophiliacs (aill males, age range 16 -56 years, mean 29 years), 22 family members (8 couples of parents, one grandmother, 5 siblings), and 9 sexual partners (5 wives and 4 girlfriends) during a 46-month period between April, 1990 and February, 1994 is detailed. Methods of counseling consisted of individual counseling in 32 cases, couple counseling in 8 cases, and family counseling in three cases. Results: Indications for counseling consisted of 1) fear of or shame over the community's awareness of the illness (37 cases), 2) concern about the impact of HIV infection on the family (21 cases), 3) risk of transmitting HIV to sexual partners (12 cases), 4) uncertainty about medical prognosis and fear of death and dying (12 cases), 5) career and family life in the future (7 cases), 6) interest in treatment options, (5 cases), 7) daily life style, including how to maintain health, and how family members can live with patients (10 cases), 8) parents' feeling of guilt over the patient's infection (5 cases), 9) lack of knowledge of hemophilia, HIV infection and AIDS (7 cases), and 10) bereavement (two cases) Discussion and Conclusions: Our experience clearly verifies the importance of psychoeducational counseling for HIV-positive hemophiliacs and their family or sexual partners in Japan. YANAGA Yuriko 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Japan Telephone (81)-93-691-0323; Telefax (8 1)-93-691-0324

Page  53 487D IMPRISONED WOMEN, APOSITIVECHANGE Lic. Leonardo Perelis, Patricia Perez. SOLDAR IMPRISONED WOMEN, A POSITIVE CHANGE Principals: Since the work of Argentine Networh of Women living with HIV/AIDS, women under detention have being Identified as the more affected. Added to this difficulties, prison conditions restrict better way of living for these women. Caractheristics: 1.-her own care;2.- as an Imprisoned mother living with o without her children; 3.-Care of the positive or negative children;4.-relationship with family outside the prison. Objectives: 1.-Research on the women living with HIV/AIDS In argentine prisons. 2.-Workshops in: a.-knowledge and care of concerned women Illnesses, b.-Therapeutic work with children of imprisoned mothers: (1.- if children live Inside prison, 2.-outside, 3.- If they are HIV +, 4.if negatives.), c.-Therapeutic work with rest of the family:(1.-while deteined, 2.-ready to freed,3.-care in freedom), d.-selfcare conscience devellopment. Methods: 1.-Programmed work with prison authorities. 2.-Count quantity of women HIV + and Identification of those who want to be Involved, 3.-Inclusion of prison staff, 4.-workshops supervised by coordination, 5.-self work started by trainers, 6.-Form multipliers in or outside the prison. Results: 1.-Count of large groups inside prison, 2.-Objective evaluation of the relationship with children and others after one year of work, 3.- better condiclon for children of imprisoned women, 4.-Publish of a newsletter about experiences, 5.- create a solidarity network for freed women living with HIVIAIDS. Lic. Leonardo Perelis, Alsina 833 6', (1087) Buenos Aires. TE/Fax (54-1) 343-6186 or 342-7603 488D Effective HIV/AIDS policies and programs for the prison system Mr Gino Vumbaca, Prison AIDS Project, Department of Corrective Services Objective: To minimise the transmission of HIV in prisons. Methods: Implement a comprehensive HIV/AIDS strategy that includes policies and programs on bleach availability, HIV testing, education for staff, peer education for inmates, a unit for HIV positive inmates, specialist videos, AIDS committees and awareness days. Collect and analyse data on operation and effects of these policies and programs. Results: In the past 18 months less than 5 inmates have seroconverted whilst in prison. In the past 12 months 42% of staff and 66% of inmates have participated in some form of HIV education (AIDS Committees, OH&S program, peer education etc). A recent evaluation of the HIV peer education program, of which 8% of inmates attend each year, has shown an increase in the inmates' HIV knowledge and awareness, positive changes in attitude, and a reduction in prejudices towards HIV positive people. In addition to this over 30 inmates have completed the Lifestyles Unit voluntary residential program for HIV positive inmates and a policy to have liquid bleach available for inmates was introduced 16 months ago Discussion: Whilst the debate on prison and AIDS issues has always tended to focus on the availability of condoms and sterile injecting equipment for inmates, there are other crucial factors that also need to be addressed if effective management, particularly in the future, is to occur. In effect recognition of three important factors in prison must occur when developing a comprehensive strategy; 1.The prison environment or culture is created by staff and inmates, both of whom require their needs and concerns to be addressed. 2.Only through education and knowledge can any real behaviour change occur. 3.That programs based on peer education principles are far more effective in addressing the needs and concerns of a target group. This paper will provide a descriptive analysis on the range of llY/AIDS policies and programs currently available, and planned for the future, in the NSW prison system. Mr Gino Vumbaca, Prison AIDS Project, Department of Corrective Services, GPO Box 31, Sydney, NSW, 2001, ph:612-289-1468 fax:612-289-1563 490D HOMOSEXUAL CONTACTS AMONG MALE PRISON INMATES IN RUSSIA. Albovy Aleksei P.*. lssaev D.D.**, *Ministry of Internal Affairs; **Sexological Center, Bekhterev Institute, St. Petersburg, Russia OQjective. To determine the prevalence of homosexual contacts and condom use among jail interments. Methods, A survey was conducted on 1 100 male prison inmates aged 18 tb 80 years old, that had been in prison for 1,5-10 years. Results. Only 10-15% of the inmates had no sexual contacts while serving their term. There was a group of inmates (8- 10%), so-called "the offended", that had regular homosexual contacts as passive partners. 50% of this group were under 20 years old and 30% more were under 30 years old. 33% of all "the offended" were convicted for rape. Most of them had sexual contacts with anyone; they had oral and anal sex with 30-50 partners concurrently. There were persons, who "served" only a privilege group of inmates (10-15 partners) that practically did not change during quiet a long period. 5-7% were involved in a longstanding homosexual relationship. Such relations were strictly kept in secret. Revealing this secret would mean shame and so-called "putting down". 2% reported having sex with men in group sex being on leave of absence. Oral and anal sex seems to be extended equally. The overall sexual practices were high risk: condoms were never used in all homosexual contacts; according to self report condoms were used by 3% with their wives just for contraception and never used with occasional female partners. Condoms are not practically available in prison; they are withdrawn while searching the parcels. "The offended" is the only group that regularly has medical examination, is tested for HIV and STDs. Discussion and Conclusions. Male prison inmates are the group of the highest risk. The problem of AIDS is ignored by the prison staff. Multiple homosexual contacts and lack of condoms could result in sudden AIDS-epidemic in prisons. ALBOV Aleksei P., The Dep. of Reformatory Affairs; Ministry of Internal Affairs; St. Petersburg, Russia; Tel. (7)- 812- 235-7556: Fax (7)-812-567-7727 489D SID/ItV Peer Fducatirn Icarcerated: Maltirredia mdel Scott Cozza, LCS, AC3I, California [edical Facility, Vacaville. Objective: HIV/SID risk reduction for the incarcerated cpiplaticn: To decrease and/or eliminte the trananissimon and/or reinfection of HIV; to increamrse txderstatndirg and carcassicn towards thee incarcerated who are living with HIV; to create a mnn devaluirg high risk behavior; to inprove the quality and quantity of the lives of those livirg with HIV. Methods: Miti redia sdel/peer educatcrs (drama, video, slides, posters, role play, parhlets, cmtests, audio, and HIV Library). Reslts: The prtam has had a positive inact on influencirg behavioral and attitrudiraml chre amcrng the incarcerated populatin. Disacssicn and Conclusins: The (VF HIV Peer Education has been na tiomally reacgized by the U.S. Centers for Disease Control and Prevention as an effective risk reductin prg-am for the incarcerated pplation. The HIV/SID risk reduction prta is a hIelth and safety pr nsfor the incarcerated and benefits the cxn ity at large. I recas-no that we have a ratincal and globtal HIV prevention pr gan for the incarcerated. Scott Cozza, L134 (707) 448-6841 ext. 2413 California Mdical Facility, Vacaville P.O.Box 200, Vacaville, CA 95696 FAX (707) 453-7009 0

Page  54 S491D SITUATION OF HIV/AIDS INMATES WITHIN THE FRAME-WORK OF INTERNATIONAL RIGHTS, WHO DIRECTIVES AND CHILENA LEGISLATION, ORTIZ F.,BUSTO(S P., CHILD R. NATIONAL COMISSION OF AIDS-CHILE. OBJETIVES AND METHODS: To anaLize the situation of inmates affected with HIV/AIDS in Chile in reference to the International Pact of Civil and Politics Rigths (U.N.,1966),the American Convention of Human Rights (A.S.A.,1969), the International Pact of Economics, Social and Cultural Rights(U.N.,1966),Directives about HIV/AIDS in Prisons (W.H.O.,1993) and National Law. RESULTS: There is no mandatory HIV testing by Law in Chile. Convicted inmates are proposed to be tested in massive screening applied annually, processus acompannied with information, counselling and educative programs. HIVpositive inmates so detected or inmates known as HIV positive when coming to jail are isolated in a special built unit in the only national. Penitentiary Hospital; this in order to avoid violence an discriminatory behaviour towards homosexual or IVDU men, HIV positive in this case. Also, the objective of isolation is to give a better quality of life to HIV/AIDS inmates, taking in account the general situation of prisoners in Chilean prisons. There is no special Legislation and no jurisprudence about advanced Liberation in benefit of AIDS prisoners. There exists a National Program of AIDS Prevention in Prisons, developed by the Ministry of Justice,with the support -not always in coincidence- with the Ministry of Health.There are experiences of condoms distribution inside prisons as part of education for STD/AIDS prevention.There is no education with IVDU neither distribution of syringes. CONCLUSIONS: Cultural and legal facts affect the way each country and their organizations face HIV/AIDS epidemic. The way HIV/AIDS prisoners are treated in chilean prisons - within the obvious limitations they carry - agrees with human dignity. Dealing HIV/AIDS prisoners in advanced liberation (observed freedom) is a next defy. 492D People in detention, protagonist of change. Lic. Nilda Vazquez, Sr. Daniel Barberis. FUNDESO PEOPLE IN DETENTION, PROTAGONIST OF CHANGE. Objectives: Since 1987, FUNDESO have been developping and working on training programms and on information and prqvention campaigns in the field of HIVIAIDS; addressed to the people who are convicts, and the objective is to train those who can be able to spread the work in every prison, to their social relationships and to their families as well. Methods: 1.- Scheduled workshops with the people under arrest of every prison, to have idea of the needs and a diagnosis of the difficulties. 2.- Establishment of thematic workshops, including convicts who are invioved in HIVIAIDS problem, and qualified professionals in each area. 3.- Meeting where to evaluate the contacts, dicussions over difficulties, up to date seminars to allow the continuity and proyect empowerment. 4.- Publishing of a newsletter, prepared by the contacts, distributed inside and out the prison. Results: 1.- Better approach with the jail language. 2.- It's eassier to teach and prevent because the contacts themselves are recognised by their equals. 3.- In this way, we obtain the near family interest. Conclusions: 1.- Effective programs and strategies are possible, being the main social actors willing the change. 2.- Integration between positive and negative persons inside the same prison. 3.- Coordinated programms, including convicts and qualified organizations. This programm is intended to be worked In different argentine jails. 4b co Jr~ 44 Lic. Nilda Vazquez, Alsina 833, 6'. (1087) Buenos Aires, Argentina TE/Fax:(54-1)343-6186 or 3j,2-7603. 493B MOLECUIAR DETECIONOFIVINCERVICALSCRAPINGS RashaL Suraiya*Li,2.; XuD.andovasA I University of Southern California, Los Angeles, CA, USA Objective: To define the extent of HIV infection in cervical tissues of HIV-seropositive women. Methods: 67 samples each of blood and cervical scrapings from HIV-seropositive women and 9 samples each from HIV-seronegative controls were examined for HIV by culture and PCR. The cell-free virus in the cervical secretions was evaluated by culture of filtered medium in which cervical swabs were soaked for > 2 hrs. Cellular DNA from cervical cells was tested by PCR using HIV primers SK38/39 and confirmed by hybridization with SK19 probe. The amount of cell-associated and cell-free virus in the peripheral blood mononuclear cells (PBMCs) and plasma was quantitated by culture. Results: Our results indicate that 45 of 62(72.5%) HIV-seropositive women have up to 3X103 TCID50 cell-associated tus titers per 10' cells. The plasma virus was cultured from 18 of 46 (39%) samples (1- 3X10' TCID50). Only two of the filtered cervical secretions yielded HIV in culture. However, HIV DNA was detected in 21 of 43 (45.6%) of DNA samples from cervical scrapings. None of the HIV-seronegative control samples were positive by culture or by PCR. Discussion and Conclusions: Our data indicate a significant correlation (83%) between virus isolation from PBMC and the presence of HIV in the cervical cells. However, high cell-associated or cell-free virus titers in the blood were not associated with the presence of HIV in the DNA of cervical cells. The absence of HIV in the cervical DNA of (17%) women who had significant amount of virus in the blood suggests that mucosal immunity, cytokines and other factors produced locally may be responsible for the amount of HIV present in the cervical cells. Rasheed, Suraiya, Laboratory of Viral Oncology and AIDS Research, 1840 N. Soto St., Los Angeles, CA 90032-3626 Telephone (213) 224-7415 Fax (213)227-1840. 494B HIVMODIFIES SYPHILIS: SECONDARY RASH ANDCHANCREINHIV+PATIENTS Carlos Cruz Palacios. Garcia ML, L6pez A.. Loo E., Martinez BJ.. Villasefor HM., Valdespino JL, Sepdilveda J. INDRE, SCD, Secretariat of Health, Mexico. Objective. To compare clinical characteristics of syphilis in patients HIV positive and HIV negative. Methods. Homosexual men attending an HIV clinical in Mexico City are offered diagnosis of STD: Clinical examination and lab are performed: HIV (ELISA, WB) syphilis (VDRL, FTA-abs). Service is free and confidential, treatment is provided. Results. Seven hundred patients were seen HIV: 38%; syphilis 17%. Clinical and serological diagnosis of syphilis was equally frequent between HIV+ and HIV- (19% v.s. 16%). Among the 120 patients with syphilis,HIV+ patients more frequently had secondary lesions: HIV+ n=50 28% HIV n=70 3% OR IC 95% P Secondary syphilis 13.22 (3-123) <.01 Chancre was common among HIV+ with secondary syphilis (10/14, 71%). None of the 2 HIV- patients with secondary syphilis had chancre. Conclusions: HIV infection modifies natural history of syphilis, secondary rash and chancre are more common among these patients. These manifestations are probably associated to immunodeficiency. Follow up studies are necessary to evaluate progression and treatment. CARLOS CRUZ PROL. CARPIO 470, COL. STO. TOMAS, DELEG. MIGUEL HGO., C.P. 11340 MEXICO, D.F. TEL: (525) 341-40-46 Ext. 35 FAX: (525)341-32-64

Page  55 495B EOLOGY OF COMMON STD RELATED SYNDROMES OBSERVED AT A CUIIC IN WIND OE. AE 9 9 8 ARSEARCH STUDY FOR THlE DEVELOPMENT OF STD CLINICALDALGOIUHMS.IWNDOK Gk-CINELLI Msass1ZO N' YAMBO S. ERNANOEZ D. 01K P" DANGCR Y. BALLARO R"' CEC'STD ACVISER TO NACP. "NACP MIMSTRY OF HEALTH, WINCHOEK-NAMIBIA ""5T0 RESEARCH UNIT. SAJMR. JCHANNESBURG-SOUTH AFRICA In the process of developing a National STD Control Programmne, an operational research study was initiated in November 1993 as a Clinic in the capital city of Wiidhoek. targeting STD patients. Objectives -Identify some of the aetiological agents responsible of the most common STD-related syndromes. -Provide the rationale for STD management guidelines based on syndromic approach & treatment at PHC level. -Describe demographic. social feanures,-personal medical history, sexual practice & behaviour of STD patients. Methods Consecutive male & female STD patients at their fist presentation were admitted into one of the three series. namely vaginal discharge, urethral discharge and genital ulcer. Following inorsed consent and administration of a questionnaire, clinical examination and sampling was done at the Clinic. Specimens were inoculated either onto transport or growth media. L.aboratory tests included direct microscopy, culture for C.albicans. T.waginalls, N.gonorrhoeae. Mhominis, U.realyricum, C.trachomatis,r. HSV and H.ducreyi and serology for syphilis, HIV and C.trachomaris. Counselling, follow-up and treatment of contacts were part of the routine activities. Data are being entered in Dbase and analysis of results will be done by Epiinfo. Results More than 300 STD patients were studied: amongst cases of urethral discharge, Ngonorrhoeae isolation rate was 68.5, and U.urealricum 32%. In females with vaginal discharge Candida species were isolated in 49% ofthecases, T.aginalis in 14.4%,M.homsnisin:0.3%. U.urealyricunin 2.4% and.V.gonorrimoeaeein 11%. Amongst patients with genital ulcers T.pallidum was identified in 18.5% and H.ducrei isolated in 38.6% of cases. Chlamydial and herpes cultures, as well as serology are currently in progress. Discussion Though preliminary, results are clearly showing that the aetiology of STD syndromes in Namibia is far more complex than usually believed. This is having important implications in the design of clinical algorithms, supporting the principle of multidrug approach. In the fast changing STD environment, regular surveys are strongly recommended to maintain management and treatment guidelines as specific and effective and possible. GHIDINELLI MASSIMO NICOLA phone: 264/61/203.21.98 fax: 264/61/22.41.55 496B IEALTH SEEKING BEHAVIO R AMONG3 MALE STD PATIENTS OF IE, INDIA: GA GAKIE[KAR RAMAN, Mehendale S, Quinn T*, Mawar N, Rodrigues J and Bollinger R*, NARI (IKTR) P. B.1895 Pone 411 026 INDIA, JHU, Baltinore tEA. Objective - To study variables influencing early health seeking behaviour asrr the male SID atta.se Itihd - Early health seeking behaviour (defined as visit to a STD clinic within 10 days of onset of a STD) was assessed among 600 male patients attending a STD clinic in Pune in an ongoing longitudinal study. Results - Health seeking behaviour (HSB) was found not to be associated with age, marital status, education, no. of sex partners ever/3 months and HIV serostatus. Early HSB was noted in patients with gonorrhoea (90%), herpes (77.3%), chancroid (64.8%): significantly more with painful GUDs vs GDs tended to show early HSB. About 10% patients were tested for HIV before. Conclusion - Reducing the spread of HIV by STD control through early HSB and complete treatment may be more cost effective than by patients learning through pain and past STDs. Extensive education efforts and strengthening of counselling facilities in STD clinics is mandatory. Dr. Raman R. Gangakhedkar, Senior Research Officer National AIDS Research Institute (ICMR): 73,G MIDCBhosari, P.B.1895 Pune 411 026 (MS) INDIA. Ph. 91-212-791210 & FAX 91-212-791071. 497B TheIntegration of STD/HIVservices as effective and interest example for developping countries in AID'S Prevention IBRA NOOYE* - M.L. SA1HOS - E. BENGA* - M. TARDY* - L.C. SARR* - I. DIAW* - S. NBOUP* - *: National AIDS Committee Senegal 498B THE ALGORITHMS FOR SYNDROMIC APPROACH OF STD IN ADOPTION BY THE BRAZILIAN MINISTRY OF HEALTH (MOH. RodriguesLGMLim a JRC, Bueno H, Comes F National ST/ rogram, MOW, Brasilia OF, RAZIL. jeoti: Demonstrate that integration of STD/HIV services is a necessary strategy which will be used in all poor ressources countries. titlso: Since the outset of HIV epidemic in Senegal, STD and AIDS programes are integrated. This country now contitutes one of the best examples of integrated HIV and STD services in the world. - 80X of components of STD and Aids programmes are integrated in all levels of Primary health care - 3 biannual reviews have attested the Interest of such a choice. Bliag: 240 trainers trained in STD and HIV services have developped their training for all medical and IEC manpower in 45 districts. Sentinel survey of HIV and STD shows in 1993 a prevalence of HIV (1X) and Syphilis (7,52) in pregnant women and respectively 121% and 5 X in prostitutes compared to the results of 1986: prevalence of HIV (0,85X) and syphilis (12X) in pregnant women and respectively 15% and 25 X in Prostitutes. - 3 ST syndromic managment (urethral and vaginal discharge, genital ulcer) are tested and used in all medical centers without laboratory since 1988. The integration of STD an AIDS has facilitate the introduction of Aids matter in school programms, has promoted the use of condoms and has cancelled the stigma of Aids. All IEC actors: N00, religious leaders etc... use integrated STD and AIDS messages to promote changing behaviour. Discussion and conclusion The positive results in Prevention of Aids in Senegal confirm that the example of AIDS and STD program of Senegal is an effective models of STD and Aide services. This experience of 8 years in STO and HIV integrated services must to be promoted in all poor ressources countries, in account of the effectiveness the accessibility and the low cost of that choice. Objective: Present the proposed algorithms for management of four STD syndromes. Methods: Three flow charts developed by national and international experts for the Brazilian MOH are presented. The algorithms cover the following syndromes: genital ulcer, urethral discharge, vaginal discharge and pelvic pain (the last two combined in one flow chart). Discussion and conclusion: The algorithms are being implemented for use at health centers throughout the public health services network of Brazil. They do not depend on laboratory tests for diagnosis, permitting treatment at the first visit of patients with suspected STD. The implementation of the algorithms is being suported by additional measures such as: - Validation of the algorithms through a multicentric and transversal study. - Periodic determination of the sensibility of N.gonorrhoeae and H.ducreyi to the different antibiotics, and the proportion of PPNG in different regions of the country. - Publication of a manual and posters about the syndromic approach. - Training of health professionals of the public system on how to use the algorithms. The proposed algorithms will help to standardize STD diagnosis and treatment at the primary level of care, contributing to the national effort to control STD. 44h (D w OD ar auN (652 4; F1X (- ZS gBRAZIL

Page  56 0- 499A THE CARBOXYL-TERMINAL REGION OF HIV-1 Net PROTEIN IS A CELL SURFACE DOMAIN WHICH INTERACTS WITH CD4+ T CELLS IKUTA, Kazuyoshi1, OTAKE, K.2, NAKAYA, T.1, NISHINO, Y.1, ZHONG, Q.1, FUJINAGA, K.1, KAMEOKA, M.1, OHKI, K.1, and FUJIl, Y.2 1Institute of Immunological Science, Hokkaido University, Sapporo, 2lnstitute for Laboratory Animal Research, Nagoya University School of Medicine, Nagoya, Japan Objective: Our previous studies revealed that the HIV-1 Net antigen is expressed, at least in part, on the surface of inlected cells. In addition, the function of the Net protein was also shown to be suppressive on the growth of human naive CD4+ T cells. Here, we examined cell surface domain of Net and its function to CD4+ T cells. Methods: Cell surface domain of Net was determined by epitope maps of several anti-Net monoclonal antibodies (mAbs) reacting with the surface of HIV-1-infected cells. Several entire and truncated forms of Net were expressed as fusion proteins. Baculovirus-expressed truncated Net was also used. Results: Two mAbs (E7 and E9) recognizing Net amino acid residues 192-206 and 158-206 could react with the surface of infected cells, whereas the other two (Fl and 4H4) recognizing 148-157 and 1-33 did not. The Net protein displayed high affinity to the surface of uninfected CD4+ T cells and this binding was specifically blocked by the E7 and E9, but not by the F1 and 4H4. HIV-1-induced syncytium formation was also specifically blocked by E7 and E9 mAb. Discussion and Conclusion: The carboxyl-terminal domain of Net which was expressed on the surface of infected cells might play an important role for the interaction between infected and CD4+ uninfected T cells. 500A CD4+ T HELPER CELLS WITH A THO/TH2-LIKE FUNCTION DURING HIV INFECTION: EXPANSION AND INFECTION OF THE CD7 -SUBSET - Autran B., Blanc C., Legac E.,Gorochov G., Debr6 P. Lab. d'Immunologie, CNRS URA 625, H6p Piti6-Salpetrire, Paris, France IKUTA, Kazuyoshi, Section of Serology, Institute of Immunological Science, Hokkaido University, Sapporo, Japan Telephone and Telefax (81)-11-707-6837 ALTERATIONS IN PHENOTYPICALLY-DEFINED SUBSETS OF CD4 AND CD8 CELLS IN ASYMPTOMATIC HIV+ PATIENTS Benito JM,Zabay Jose M,Sanchez E,Fernandez-Cruz E. Dept. Immunology,Hospital Gregorio Marafion,Madrid,Spain Obiectives: To further assess the differentiation, helper cell function and HIV-infection status of the CD4+CD7- memory T cell subset expanded during HIV Infection. We previously reported the progressive amplification and in-vivo activation of this subset which is characterized by a low CD3-triggered cell proliferation and a THOITH2 profile of cytokine production. Methods: The CD4+CD7- T cells from healthy normal individuals and 30 HIV-seropositive patients at distinct stages of the disease were studied. Their phenotype, proliferation, cytokine secretion and reactivity, as well as their in-vivo HIV-infection were analyzed after cell-sorting and stimulation of the CD3, CD28, PKC pathways or generation of T cell clones and lines. Results: The CD4+CD7- T cells maintained their phenotype in long term culture. The CD4+CD7- cells from normal donors produced significantly lower levels of IL-2 (mean CD4+7 -/CD4+7+ ratio: 0.6+0.3) than the CD4+CD7+ control cells, and higher levels of IL-4 and IL-10 (mean CD4+7-/CD4+7+ ratios: 5.4+4 and 20+23 respectively) whereas the IFNg production was similar in both subsets. In HIV-infected patients, we observed a similar, although impaired, profile of cytokine production with a preferential production of IL-4 and IL-10 in CD4+CD7 -cells. Their CD3-triggered-proliferation was preferentially enhanced by addition of IL-4, IL-7, IL10 but not by IL-12. A similar proportion of HIV-infected cells was observed ex vivo in both subsets by in-cell PCR. Conclusions: The CD4+CD7- memory T cells represent an end stage of differentiation which share characteristics of THOTH2 CD4+ T helper cells and might increase during HIV infection as a result of exposure to TH2 cytokines or continuous T cell activation. Patrice DEBRE, Lab. Immunologie Cellulaire et Tissulaire, URA CNRS 625, CH Piti-Salpetrire, 83 bd de I'h6pital, 75013 PARIS, France Tel. - Fax 502A VIRO-IMMUNOLOGICAL STUDIES IN ACUTE HIV-1 INFECTION Roos Mariike T.L., de Leeuw N.A.S.M, Huisman H.G., Meyaard L., Schellekens P.T.A., Claessen F.A.P.', Schuitemaker H. and Miedema F; CLB & Lab. Exp. Immunol., Univ. of Amsterdam; Dept Int. Med., Academic Hosp. Free University, Amsterdam, The Netherlands. Background: A patient who presented with symptomatic acute HIV-1 infection was monitored for virological and immunological parameters over seroconversion (sc), in relation to the clinical course. AMethods: Virological studies included determination of frequency of productively HIV-1 -infected PI3MC and viral RNA-load in plasma and p24 antigenemia. Immunological studies included the analysis of T-cell subsets, the expression of activation markers, the expression of CD45RO and CD45RA antigens, the frequency of cells programmed for death (PCD), and T-cell function. Results: Before sc high plasma titers of p24 and HIV-I RNA were observed. The number of productively HIV-l-infected PBMC peaked during this period coinciding with CD4+ T lymphocytopenia. From sc onwards, CD8+ T lymphocytosis was observed, the expanded cell population being of the CD8+CD38+, CD8 + CD27+ and CD8+CD28- phenotype. CD8+ T lymphocytosis was paralleled by a high percentage of PCD. T-cell function was severely depressed at the time of sc. Ten days after sc PHA-induced proliferation was restored to normal levels while responses to the CD3 mab only showed a partial restoration. Following sc, concomitant with the rise of activated CD8 + T cells, p24 Ag levels and viral load of both serum RNA and the number of HIV-producing PBMC steeply declined in 2 weeks time. Conclusion: These findings demonstrate HIV-1-induced abnormalities before seroconversion followed by signs of a strong immune response believed to be responsible for efficient control of viral replication following seroconversion. M. Roos, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Tel. 31-20-5123114. Fax: 31-20-5123310. cD 0 501A OBJECTIVE:To define the subsets of CD4 and CD8 T cells (memory,naive.cytotoxic,suppressor) and its activation status in asymptomatic HIV+ patients. METHODS:Forty patients at the clinical category A of HIV infection,and 22 healthy controls,were studied.T cell subsets were quantified using 3-colour flow cytometry (FACScan.B&D). RESULTS:A)Subsets of CD4+ T cells.Percentage of CD45RO+ cells was significantly increased only in the group of patients in category A3 (79~6.2,p<0.05),as compared to controls (61~3.4).Alsothe % of the memory activated subsets [CD45RO+CD38+(24~2.5,p<0.05), CD45RO+DR+(17~2.7,p<0.001), and CD45RO+CD38+DR+(13~3 7,p<0.05)] were markedly increased, as compared to controls (14~4.2;5~1.1;and 4~1.9,respectively).The % of the naive activated subsets [CD45RO-DR+(1~0.4,p<0.05) and CD45RO-CD25+(4~0.8,p<0.05)] were also increased in patients,as compared to controls (0.2~0.2;and 1~0.4,respectively).B)Subsets of CD8+ T cells.% of CD45RO+ cells was markedly increased in patients in categories A2 (70~4.2,p<0.001) and A3 (70~5.5,p<0.001),as compared to controls (40~2.9). Also, % of memory activated subsets [CD45RO+CD38+ (41~2.9,p<0.001), CD45RO+DR+ (41~2.5,p<0.001), and CD38+DR+ (44~2.5,p<0.01)],were markedly increased in patients, as compared to controls (7~1.1;8+1.4;and 6~1.1, respectively).The % of the activated cytotoxic subsets CD 11b-DR+(44~2.7) and CD 11b-CD38+(45~2.9) were also increased (p<0.001). Clncreased levels of the activated memory subsets of CD4+ and CD8+ cells did correlate well with low levels of CD4+ T cells,and with increased levels of (-microglobulin and p24 Ag Levels of activated cytotoxic CD8 cells correlated negatively with levels of CD4 cells (r=-0.33). And levels of p24 Ag correlated negatively with the % of naive CD4+CD45RO-CD25+ cells (r=-0 78). CONCLUSIONS:Asymptomatic patients showed a marked increase in activated memory CD4 and CD8 T cells. that was associated with parameters of immune impairment and viral replication. Zabay Jose M.Dept.Immunology,Hospital Gregorio Maraion. Dr.Esquerdo,46 28007 Madrid, Spain Telephone number:34-1-5868460.Fax number:34-1-5868018.

Page  57 503A VI, REPERTOIRE OF y8 T CELLS IN HUMAN PERIPHERAL LYMPHOCYTES. INFLUENCE OF HIV-INFECTION SBoullier. M. Cochet. F. Poccia, M.L. Gougeon. Unite d'Oncologie Virale, Institut Pasteur, Paris, France. ObjectIives:a To define if the peripheral expansion of the VII yI T cell subset observed in the course of lilVinfection is the consequence of a specific antigenic stimulation. Methods: Peripheral yI T cell subsets were investigated for the expression of the Vy and VS chains using specific mAbs and FACScan analysis. The VII T cell gene rearrangements were analysed by polymerase chain reaction (PCR) using specific VI1 and CS oligonucleotides. TCRs rearrangements and CDR3 size distribution of the VSI+ subset were analysed on an Automatic DNA Sequencer using fluorescent JS's oligonucleotides. Phenotypic analysis of VSl+ T cells was performed using specific mAbs (CD8, CD25, C1)69, CD38, CD45RO. CD45RA) and FACS analysis. Results:li In healthy donors 60 to 70% of the yI T cells (I to 10% of human CD3+ T cells) express the Vy9-V52 chain association and 20-30% express VII chain (usually associated with Vyl chain). We have previously shown that the proportion of y8T cells expressing the VSI epitope was significantly increased in IIIV infected individuals as early as stage II and was sustained in AIDS patients. We show here that the increase of this VII subset is not associated with a particular Vychain expression. The repertoire analysis of VI1-CI transcripts indicated no major difference in the CDR3 size profil between normal donors and IlIIV-infected patients. This was confirmed by sequencing the VII-JSt junctions. Ex-vivo expression of activation markers indicated that the VI subset from normal donors harboured a resting cell phenotype. A similar phenotype was found in patients' VSI1+T cells except that they expressed CD38, a knsown activation marker associated with disease progression. This was correlated with the ability of these cells to spontaneously respond in vitro to IL-2. Conclusion: CDR3 analysis of the human VII 7yI T cell subset in IIIV-infected individuals indicated that the repertoire of this subset is unrestricted and not significantly different from that of healthy donors. It suggests that the enhancement of this subset in the course of the disease is not correlated to a peripheral antigenic selection. The influence of IIIV on the specific expansion of this subset has to be considered. BOULLIER Severine, Unit d 'oncologie virale, Institut Pasteur, Paris, France. Tel. (33) 1 45688914; Fax (33) 1 45688909. 504A RAPID FLOW CYTOMETRY DETECTION OF T CELL SUBSET ACTIVATION IN AIDS. Maino.Vernon C.*, Suni, M.*, Ruitenberg, J.*, Holodniy, M.**; *Becton Dickinson Immunocytometry Systems, San Jose, CA., **Palo Alto VA Medical Center, Palo Alto, CA. Objective: Disease progression in AIDS is characterized by diminished cell mediated immunity as reflected by a reduced in vitro T lymphocyte proliferative response to antigens and mitogens. The purpose of this study was to assess cell mediated immune function parameters in AIDS patients using a rapid flow cytometry based assay measuring the expression of an early activation antigen, CD69 on CD3+ cells following stimulation by specific antigen or polyclonal activators. Methods: A 3-color flow cytometric assay was developed to monitor the expression of an early activation marker (CD69) with whole blood or PBMC samples after 4 h incubation with specific antigen (e.g. Candida albicans) or polyclonal activators including pokeweed mitogen (PWM), phytohemagglutinin (PHA), and anti-CD2/CD2R. T cell responses were measured as a function of the percentage of CD4+ or CD8+ T cell subsets expressing CD69. Results: The response to various stimuli as determined by the percentage of specific T cell subsets expressing CD69 after 4 h activation was significantly diminished in PBMC isolated from HIV positive individuals with identified CD4 counts above 200/mm3 compared to a panel of normal donors. This difference was most significant in the CD8+ subset of T cells. Interestingly the level of cell mediated response to T cell stimuli as measured by CD69 expression did not correlate with absolute CD4 counts in these patients. Discussion and Conclusions: This study demonstrates that a rapid 4 h multiparameter flow cytometric assay based on the expression of an early activation antigen, CD69, can be utilized to assess T cell function in AIDS. Maino, Vernon C., Becton Dickinson Immunocytometry Systems, 2350 Qume Drive, San Jose, CA. 95131, USA Telephone: 408-954-2487, Fax: 408-954-2506 506A TRANSACTIVATION OF THE HIV LTR BY IE2 PROTEIN OF HUMAN CYTOMEGALOVIRUS IS DOWNREGULATED BY THE RETINOBLASTOMA GENE PRODUCT. K.S. Choi, S-J Kim, and Sunyoung Kim"Seoul National University, Korea The IE2 gene product of human cytomegalovirus (HCMV) is one of the few viral regulatory proteins expressed immediately upon infection of the host cell. This regulatory protein is a potent activator of the HIV LTR. Deletion analysis of the HIV LTR indicated that IE2 does not appear to need a specific cis sequence to activate the LTR, consistent with the finding by others that IE2 probably interacts with factor(s) involved in the general transcription machinery, such as TFIID. Surprisingly, the retinoblastoma susceptibility gene product (Rb) dramatically suppressed this IE2 transactivation of the HIV LTR. In contrast, Tat transactivation of the LTR was not significantly affected by coexpression of Rb, suggesting that Rb suppression is highly specific to IE2. This suppression was not specific to the HIV LTR, because transactivation of other promoters by IE2 was also highly suppressed. Unlike another tumor suppressor protein p53, Rb did not have any significant effect on basal levels of transcription, suggesting that Rb specifically interacts with IE2 rather than other cellular factors involved in the general transcription machinery. We will present data suggesting the direct interaction between IE2 and Rb. Sunyoung Kiln: Building-105, IMBG, Seoul National University, Seoul 151-742, Korea, Tel: 82-2-880-7529, FAX: 82-2-875-0907 505A HHV-7 USES CD4 AS A RECEPTOR AND INTERFERES WITH HIV INFECTION IN CD4+ T-CELLS AND MONONUCLEAR PHAGOCYTES Lusso, Paolo, Secchiero, P., Crowley, R.W., Berneman, Z.N., and Gallo, R.C. LTCB, NCI, NIH, Bethesda, MD, USA Objective: The cellular membrane receptor for HHV-7 and the interactions between HHV-7 and HIV were studied to evaluate the possible role played by HHV-7 in AIDS. Methods: Fluorocytometry, infection inhibition by monoclonal antibodies and soluble CD4, radiolabelled virus binding, and HIV-1 p24 antigen capture. Results: We found that HHV-7, like HIV and SIV, uses CD4 as a cellular membrane receptor. CD4 is progressively downregulated in the course of HHV-7 infection. Diverse monoconal antibodies to CD4, as well as recombinant soluble CD4, induce a dose-dependent inhibition of HHV-7 infection. Moreover, CD4 cells acquire the ability to bind HHV-7 and become susceptible to HHV-7 infection after transduction of human CD4. Data will be presented regarding the CD4-binding protein of HHV-7, that is currently being characterized in our laboratory. A reciprocal interference was observed between HHV-7 and HIV-1. In particular, exposure of primary CD4+ T cells or mononuclear phagocytes to HHV-7 (live or UV-light inactivated) markedly inhibited infection by several HIV-1 isolates, including primary isolates. Conclusions: HHV-7 behaves as an effective natural inhibitor of HIV infection, which could be exploited to devise novel therapeutic approaches to AIDS. LUSSO, Paolo, Laboratory of Tumor Cell Biology, NCI, NIH, Bethesda, MD, USA Telephone (301) 402-3031; Telefax (301) 496-8394 J1 0 CA) 0 O

Page  58 507A FREQUENCY OF HHV-6 INFECTION IN HIV INFECTED PATIENTS Ablashi Dharams; Kaplan M2;Salahuddin SZ3; and Pearson GR1 Gtown U, DC; 2No.shore U Hosp, NY; 3Univ of So Cal, USA. Objective: Because of the in vitro interaction between HIV-1 and HHV-6 (Human Herpesvirus-6), and the dissemination of HHV-6 in tissues of AIDS patients, we investigated the frequency of HHV-6 reactivation in HIV+, AIDS and ARC patients. e hods: The peripheral blood mononuclear cells (PBMC) from patients and healthy donors were PHA stimulated and cultured in the presence of IL-2. The cells were examined for HHV-6 with Mabs. The plasma or sera were titered for HHV-6 antibody. Results: The PBMC from 25% of HIV infected asymptomatic individuals expressed HHV-6 antigens. PBMC from 42.1% of ARC patients had HHV-6 antigens, whereas 74.3% of AIDS patients' PBMC expressed HHV-6. Only 10% of controls' PBMC had HHV-6 expression. No correlation between HHV-6 1gG antibody and HHV-6 expression in vitro was observed. In 70% of patients/controls, however, detection of IgM antibody correlated with antigen expression. AIDS patients showed significantly increased HHV-6 antibody titers after treatment with AZT. Conclusions: A progressive increase in HHV-6 reactivation in HIV infected patients was observed. The significantly higher HHV-6 antibody titer in AZT treated AIDS patients suggests that AZT is killing CD4+ cells which may be infected with both HHV-6 and HIV, releasing HHV-6 to stimulate B-cells. The correlation of IgM and antigen expression suggests HHV-6 reactivation. Thus reactivated, HHV6 in HIV infected individuals may contribute to enhanced disease manifestation. Ablashi, Dharam V., 4117 Barnsley Lane, Olney MD 20832 Telephone 1-301-774-7046 Telefax 1-301-570-4258 509A BK VIRUS INFECTION IN PATIENTS WITH AIDS SATA TetsutaroI, MATSUKURA T2, KURATA T3, WAKABAYASHI T4 (Laboratory of Pathology, AIDS Research Center', Dept. of Virology 112 and Pathology3, NIH, Dept. of Clinical Pathology, Institute of Medical Science, University of Tokyo4, Tokyo, JAPAN) Objective: Human polyomavirus JC was infected to develop PML in patients with AIDS as an opportunistic infection. BK virus is supposed to infect AIDS patients, but its infection has not been obscured. We explored the cases of BK virus infection in autopsies with AIDS in Japan. Methods: Autopsy cases were examined histopathologically to detect inclusion-bearing cells in kidney. The sections with nuclear inclusion bodies were applied by immunoperoxidase method and in situ hybridization. The extracted DNAs from frozen tissues were analyzed by Southern blot hybridization and dot blot hybridization. Results: Among 80 cases with AIDS, we found one case with BKV infection in kidney, which showed interstitial nephritis with amphophilic or eosinophilic nuclear inclusion bodies in tubular epithelia. These cells were positive for immnunohistochemistry and in situ hybridization. Southern blot hybridization detected BKV genome in kidney, lymph node, bone marrow, spleen, heart, brain and liver, except for a complicated malignant lymphoma. BKV was isolated in HEK cells from kidney and urine, which were confirmed by immunofluorescence and electron microscopy. Discussion and Conclusions: BKV is also considered as one of the opportunistic infection in AIDS. Virus-infected cells could be identified only in kidney, but the virus genome disseminated through blood stream to several organs. BKV infection is also under examination by PCR for earlier detection in PBMCs from HIV infected patients. 508A HHV-6 SALI-L ORF-1 REACTIVATES HIV-1 PROVIRUS. ROSENTHAL. Leonard, Sadaie, M. R., & Kashanchi, F. Georgetown University, Washington, DC, USA Objective: To identify the HIV-1 transactivator within the HHV-6 Sall-L fragment and assess its ability to reactivate latent HIV-1 provirus. Methods: Subcloned Sall-L ORFs were tested for their ability to up-regulate HIV1 LTR CAT expression. Reactivation of HIV-1 from HLM1 cells was determined by measuring p24 antigen released. Results: Both ORF-1 and Sall-L transactivated the HIV-1 LTR 7-9 fold, whereas ORFs 3, 6, & 7 had no effect. Purified ORF-1 protein also transactivated the HIV1 LTR by electroporation in vivo and by transcription in vitro. Finally, the ORF-1 gene was sufficient to reactivate HIV-1 provirus from latently infected cells. Conclusion: The transactivator of HIV-1 within HHV-6 Sall-L was identified as ORF-1. Furthermore, ORF-1 activated latent HIV-1, raising the possibility that HHV-6, through expression of ORF-1, induces viral production from latently infected T-cells in asymptomatic HIV-1 carriers, ultimately contributing to the development of AIDS. ROSENTHAL, Leonard, GEORGETCWN MEDICAL CENTER, Wash., D.C. Telephone: 202-687-1140; Telefax 202-687-1800 N 0.0 510A ASSOCIATION OF MYCOPLASMA PENETRANS WIT1 IIIV INFECTION. GrauI B. Slizewicz2. P. Tuppin',. V. Launayt, A. Lafeuillade3, E. Bahraouil. A. Blanchard. W. Rozenbaum4 and L. Montagnierc Institut Pasteur, Paris; 2Diagnostics Pasteur, Marnes-La-Coquette: 3tt6pitha Chalucet. Toulon; 4H6ipital Rothschild. Paris - France Objective: To assess our hypothesis of a possible role of mycoplasmas as co-factors in AIDS pathogenesis, we have determined the prevalence of antibodies directed against Mycoplasma penetrans, a newly discovered Mycoplasma species isolated from the urine of AIDS patients. Sera from 156 HIV-positive and 290 IllV-negative individuals (250 blood donors, 40 patients attending STD clinics) were tested. Methodls: ELISA and western blot tests specific for M. penetrans were developed by using membrane associated antigens purified from M. penetrans by Triton X-1 14 selective extraction. Results: Thirty-one point four per cent of the IIlV-infected patients were M. penetrans seropositive, versus 1.0% (3/290) in IllV-negative controls (p<l0-5). Among HIV-negative, MAl. penetrans positive individuals, two were patients attending STD clinics and one was a blood donor. The M. penetrans seropositivity was neither associated with one of the risk groups, nor with CD4 count, stage of the disease and Kaposi's sarcoma formation. In addition, we have detected DNA from M. penetrans in urine from several M. penetrans-seropositive patients by using a specific PCR assay. Discussion and Conclusions: The remarkably high seroprevalence of M. penetrans in lIlV-positive individuals vs IllV-negative subjects strengthens the notion that this mycoplasma could play a role of co-factor in AIDS pathogenesis. M. penetrans does not behave like an opportunistic agent, since it was detected not only in the latest stages, but also in the earliest stages of the disease. In addition, we could not find a correlation between M. penetrans seropositivity and homosexual behaviour or Kaposi's sarcoma fonnation. SATA, Tetsutaro, AIDS Research Center, NIH, Tokyo, Japan Telephone (81)-3-5285-1111 ext. 2627; Telefax (81)-3-5285-1150

Page  59 5118 EFFICACY OF NEVIRAPINE ADDED TO ZIDOVUDINE IN P24+ HIV-1-INFECTED PATIENTS WITH CD4+ COUNTS <500/mm3. Cart Andrew, St Vincent's Hospital, Sydney, for the Nevirapine Australian-Italian-Dutch Study Group. Objectives: To assess the virologic activity of, and immunologic response to, nevirapine (NVP) 400 mg/day added to zidovudine (ZDV) monotherapy in p24 antigenaemic, nucleosideexperienced, HIV-l-infected patients with CD4+ lymphocyte counts less than 500/mm3. Methods: 49 patients were randomised to continue ZDV 500-600 mg/day monotherapy or to receive additional therapy with NVP 200 mg/day for 2 weeks followed by 400 mg/day (NVP/ZDV) for 6 months. ZDV patients were allowed early addition of NVP where there was sustained evidence of ZDV failure. Baseline characteristics were: Group Age CD4+ (cells/mm3) ICD-p24 antigen (pg/ml) Prior ZDV (weeks) NVP/ZDV 32 195 137 84 ZDV 32 211 66 70 Responses markers were ICD-p24 antigen, CD4+ cell counts and CD44 percentage. Results (all medians) were analysed by expressing treated observations as changes from baseline. Treatment groups were compared using Rank-sum tests (2-tailed, os<0.05). Results: The maximum response (median 28 days) in the NVP/ZDV group was an increase of 104 CD4+ cells/mm3 (versus 35 cells/mm3 in the ZDV group; p<0.00) and a decline in ICD-p24 antigen of 74% from baseline (versus 36% in the ZDV group; p<0.01). At weeks 12 -16, median ICD-p24 antigen remained 39% below baseline in the NVP/ZDV group (versus 29% above baseline for the ZDV group); the CD4+ change in each group was -32 cells/mm3. Data for 6 months NVP therapy will be presented. Treatment-limiting toxicities of NVP occurred in 20% of the NVP/ZDV group, including rash (16%) and liver function abnormalities (4%). Discussion: NVP causes a rapid reductionin viral load and improvement in CD4+ lymphocyte counts. When added to ZDV monotherapy, NVP 400 mg/day results in sustained p24 antigen suppression for at least 12 weeks in the majority of patients. CARR, Andrew, Centre for Immunology, St. Vincent's Hospital, Sydney 2010 Australia. Telephone 61-2-3617700; Telefax 61-2-3612391 513B EVALUATION OF THE EFFICACY AND TOLERANCE OF R18893, R89439 (loviride) AND PLACEBO IN ASYMPTOMATIC HIV, PATIENTS. Staszewski Schlomo*, Vandercam B.**, De Vuyst H.**, Colebunders B.**, Clumeck N.**, Stille W.*, Peeters M. ** Andries K. ***, Stoffels P.***, Van Den Broeck R.***, Janssen P.AJ.***. *Johann Wolfgang Goethe Universitat, Frankfurt, **The Belgian Alpha-APA clinical trial group, ***Janssen Research Foundation. R 18893, the lead compound of the Alpha-APA series has proven in phase 1-2 studies to induce a borderline increase in CD4 count in asymptomatic HIV, patients. R89439 (loviride) is the most potent product of the alpha-APA series with a good oral bioavailability. Objective: The objectives of the present trial were to compare the anti-lIlVlI activity of R 18893, loviride and placebo in vivo and to assess the safety of loviride and R18893. Methods: 114 asymptomatic HIV,-positive patients with CD4>20% or>400 CELLS/mm' received either loviride 100 mg t.i.d., R18893 200 mg t.i.d. or placebo in this randomized, double blind trial. Results: Loviride induced a significant increase of CD4 count (p=0.005) and CD4%(p=0.035) versus placebo (see abstract De Brabander et al). Samples from 18 patients, treated for 3 months, were found negative for the Yl81C and VIO6A mutation (method: selective RNA PCR). The incidence of adverse events was not significantly different between the three treatment groups, except for rash (6/22) and pruritus (3/22), which was only reported in the R18893 group. Conclusion: A sustained increase of CD4 count above baseline was noticed during the 6 months treatment with loviride. Loviride was well tolerated in this trial. R18893 demonstrated a lower activity compared to loviride. Note: A complete report of the trial will be presented at the Conference. STASZEWSKI, Schlomo, Klinikum der Johann Wolfgang Goethe UniversitAt, Frankfurt, Germany Telephone (49)-69-6301 7688; Telefax (49)-69 6301 5712 512B PHASE II CLINICAL TRIALS OF DELAVIRDINE MESYLATE COMBINATION THERAPY, Freimuch, Bill, Davey* R. Batts D, Lane* C, Cox S, Wathen I., Peel B, Daenzer C, Morse G, Chaitt* D, & Herpin* B, Upjohn & NIH*, USA Objective: Evaluate the safety, tolerance, pharmacokinetics (PK) and surrogate marker response of the non-nucleoside reverse transcriptase inhibitor (RTI) Delavirdine Mesylate (DLV) in combination therapy with ZDV or ZDV plus ddl and as monotherapy in nucleoside RTI experienced HIV-l + patients with CD4=100-500/mm3. Methods: ITwo open-label, dose-escalation clinical trials. NIH trial (CD4=100-300): 400, 600, 800, 1,000 or 1,200 mg/day of DLV plus ZDV+ddl (32 pts) vs ZDV+ddl (15 pts), and 20 subjects receiving )DL.V(400 mg Tll)) alone or combined with ZI)V. Upjohn trial (CD4=200 -500): 34 patients receiving ZDV for at least 6 weeks given DLV doses of 400, 600, 800, 900 or 1,200 mg/day. Mean prior ZDV therapy >17 mo in both trials. Results: The most common medical events were maculopapular skin rash (-33%) and headache, which were not dose limiting. All DLV doses were well-tolerated with -95% subjects completing the 12/24 wk trials. PK was non-linear with high inter-subject variability, but low intra-subject variability (CV -20%). Mean steady state trough for 1,200 mg =16uM. The % of patients with a surrogate marker response at 2 consecutive timepoints (wks 1-24 for evaluable subjects): NIH; DLV+ZDV+ddl vs ZDV+ddl: CD4(50/25%T)= 59% vs 29%, ICDp24(>50%, )= 59% vs 0%, b-DNA(>5X1)= 44% vs 13%, and PLV(llog,)= 77% vs 25% and Upjohn; DLV+ZDV: CD4=50% and [ICDp24=23% and PBMC co-culture=100% at wk 6, 12, 16 or 241. Decreased viral susceptibility in the first 24 weeks to DLV (-100-fold, IC50=0.02uM-->3.0uM) in -70% subjects was associated with baselitie ZDV resistance and/or SI phenotype in the Upjohn trial. Conclusion: DLV is well-tolerated with anti-HIV-1 activity in combination therapy. William W. Freimuth, M.D., Ph.D. The Upjohn Company, 526 Jasper Street, Kalamazoo, MI 49007 7217-258-1, (616) 384-9365, fax (616) 384-9721 514B FINDING NEW INHIBITORS OF THE HIV-1 INTEGRASE: STRUCTURE-BASED AND EXPERIMENTAL STUDIES Weinstein. John N., Raghavan, K., Buolamwini, J., Zaharevitz, D., Paull, K.D., Mazumder, A., Fesen, M., Kohn, K.W., Pommier, Y. Devel. Therap. Progr., DCT, Natl Cancer Inst. (NCI), Belhesda, Md, USA. Objective; To identify new compounds active against the HIV-1 integrase using as a guide our hypothesis (Fesen, et al., Biochem. Pharmacol., in press) that'ortho-OH groups favor inhibition (of both the cleavage and integration steps). Fesen, et al. (PNAS 90: 2399, 1993) have identified inhibitors of this enzyme as potential therapeutic agents for AIDS. Me ds An assay using recombinant HIV-1 integrase and oligonucleotides identified 15 flavone analogues active against the enzyme. We then analyzed those molecules by 3 different quantitative structure-activity relationship (OSAR) methods: comparative molecular field analysis (CoMFA), electrotopological stale (E-state) analysis, and back-propagation neural networks. To identify new inhibitors, we also developed a set of pharmacophoric descriptors in Chem-X and searched the NCI database of 400,000 3D structures. Comlpounds predicted to be active are being tested in the integrase assay and in the NCI AIDS drug screen in CEM-SS cells. Results: CoMFA, E-state, and neural network analyses all support the ortho-OH hypothesis. OH substituents at positions C5, C6, C3', and/or C5 appear favorable. When a flavanone molecule without ortho hydroxyls was found to inhibit the integrase, it was built and analyzed in the Sybyl molecular modeling package. In one of its lowest-energy stales, the molecule folds on itself so that its two phenolic rings n-n stack with 2.78 A separation between OH centers -- in good agreement with the 2.79-A separation between ortho OH groups. Search of the NCI database for molecules similar to the flavone, to the flavanone, and to other inhibitors of the integrase yielded compounds that are currently being tested for activity. Conclusion: Analyses of molecular structure, 3D structural database searching, and experimental assays can be combined to identify new inhibitors of the HIV-1 integrase. John N. Weinstein, Natl. Cancer Inst., Bldq. 37, Room 5C25, 9000 Rockville Pike, Bethesda, MDn 200q? Tel: (301) 496-9571 FAX: (301) 402-0752 USA J1 0 44

Page  60 515B CHARACTERISATION OF "IN VIVO" SELECTED HIV-1 VARIANTS WITH REDUCED SENSITIVITY TO PROTEINASE INHIBITOR SAQUINAVIR. Mgus.Jan ('), Brun-Vezinet, F. ("), Duncan, I.B. ("'), HAitnggi, M.('), Jacobsen, H.('), Vella, S.("...) () F. Hoffmann-La Roche AG, PRTB, Basel, Switzerland; (") Laboratoire de Virologle, Hopital Bichat-Claude Bernard, Paris,.France; ("') Roche Products Ltd., Dept. Biology, Welwyn Garden City, U.K.; ("") Istituto Superiore di Sanita, Laboratory of Virology, Rome, Italy 516B IN VITRO INDUCTION OF HIV-1 WITH REDUCED SENSITIVITY TO HIV PROTEASE INHIBITORS, KNI-227 AND KNI-272. Anderson. Barry, Kageyama, S., Ueno, T., and Mitsuya, H. National Cancer Institute, Bethesda, Maryland, USA. OlIectlve: As reported previously, HIV-1 and HIV-2 resistance to Saquinavir (Ro 31-8959) can occur "in vitro" with extended, dose-escalating selection. The objective of this study was to investigate whether HIV virus with reduced sensitivity to this drug can be found in patients after treatment with Saquinavir. MeithQds: Plasma samples from patients treated with 600 mg Saquinavir tid alone or in combination with Zidovudine were cocultured with stimulated donor PBMC for drug susceptibility testing according to standard procedures. Genotypic analysis of HIV proteinase genes was performed on parallel samples using PCR amplification followed by DNA sequencing of subcloned genes. Results: Phenotypic analyses of virus Isolated from about 30 patients of the Phase I / II trial at baseline and 3/6 months of treatment with 600 mg Saquinavir were performed. From 2 patients virus could be isolated after 3-6 months that showed reduced sensitivity to the drug (more than 10-fold change in IC50) when compared to baseline values. Sequence analysis of a representative number of protelnase genes present in the viral samples revealed that a selection for specific genotypes had occured during treatment. Similar mutations as those found in n vitro selected HIV (G48V, L90M) could be identified but also some new, selected changes like G17R. Conclusion: Selection of HIV variants with reduced sensitivity to Saquinavir can occur in patients treated for prolonged periods of time but appears to be an Infrequent event at least after 3-6 months of treatment. Dr.J.Mous, F.Hoffmann-La Roche Ltd, CH-4002 Basel, Switzerland. Tel. 41-61-6886490, Fax. 41-61-6881448 517B/D CONFIDENTIAL SHARING - THE LINK FROM INDIVIDUAL TO COMMUNITY 517B/D _,,_,o_,,___..o COUNSELLING Campbell, lan D. Rader, A.D. The Salvation Army International Headquarters, London OBJECTIVE: A seropositive client, in the context of privacy may find it hard to integrate individual rights with public responsibility. The influences that help integration happen need to be explored. Counselling in some developing countries extends from the individual to the family and the community, and can be implemented as a means of facilitating change, as well as support. One capacity that links individual to community counselling for change is 'shared confidentiality'. METHODS: Information collected from field programme teams, communities and workshop participants shows that community counselling can be built on some key concepts such as community capacity for agreement, participation, accountability and confidential sharing. RESULTS: Experiences from India, Zambia and Brazil illustrate that the common link has been capacity for shared confidentiality. Examples illustrate the foundation of belonging that leads to participation and accountability, through confidential sharing, and show the strength of building on knowledge that exists within community, which if ignored can distract and disintegrate. CONCLUSION: Some communities have capacity to live with suffering and loss, yet find hope in deciding and implementing new safe community norms. This requires mutual accountability and respect for the privacy of individuals. Confidential sharing is a capacity that exists in communities in different cultures. An integrated care and prevention approach can develop from this, that adcelerates the capacity of communities to work together for positive change. ')r Tan n. Campbell, The Salvation Army, 101 1ueen Victoria Street, london '4P L 4: Tel 071 236 5222/'rax071 236 4981 Objective: KNI-227 and KNI-272 are transition-state mimetic tripeptide HIV protease inhibitors containing allophenylnorstatine with a hydroxymethylcarbonyl isostere which are highly potent against a wide spectrum of HIV strains in vitro. Clinical trials of KNI-272 are underway at the NCI. In this study we investigated the potential of the virus to develop resistance to these compounds. Methods: HIV-1LAI was serially passaged in the presence of increasing concentrations of the two compounds in a cell-free transmission system in which cell-free virions produced by previously de novo infected H9 cells were transmitted to a fresh H9 population every 7 days. Results: (i) Virus passaged with IC30o to IC60o (0.036 liM) concentrations of KNI-227 for 55 passages acquired a 3.3-fold decrease in drug susceptibility, while no protease-coding genetic changes were detectable. The drug concentration was increased with subsequent passages up to 0.27 iM when the viral population developed 1le84-Val and Val82-lle mutations. (ii) Virus cultured with an ICso concentration of KNI-272 (0.036 iM), which contains thioproline at the P1' site instead of the dimethylthioproline of KNI-227, demonstrated no decrease in sensitivity to KNI-272 by passage 30. Virus cultured in the presence of 0.5 iM KNI-272 (passage 42) contained Val32--lle and Ile84-Val mutations. Virus cultured in 1.0 giM KNI-272 (passage 45) demonstrated Val32-lle (10/10 clones), IleS4--Val (8/10), and Met46- lle mutations (3/10). Conclusions: The data suggest that the development of decreased virus sensitivity to the two drugs occurs and the virus may continue to replicate in the presence of KNI-227 and KNI-272 at concentrations 7.5- and 27-fold greater, respectively, than the IC o values previously inferred from in vitro data. The clinical significance of these in vitro findings requires further study. Barry Anderson, M.D., Ph.D., Experimental Retrovirology Section, Medicine Branch, National Cancer Institute, Bldg. 10, Rm 5A24, Bethesda, MD 20892, USA Tel: 301-496-9239, Fax: 301-402-0709. 518B/D HIV PREVENTION COUNSELING USING AN ON-SITE, RAPID HIV ASSAY Kassler, William J.'; Dillon B13'; Haley C'; Schenk T'; Hutcheson D'; Jones W'; Gerber R'; Reeker C'; Stark K' Holtgrave D'; Centers for Disease Control and Prevention, Atlanta. GA'; Dallas County Health Department. Dallas, TX2, US Background: Rapid, on-site HIV testing has the potential to improve delivery of prevention services at counseling and testing sites. This necessitates reconsideration of existing counseling protocols. Methods: We conducted formative research on: consent procedures, when ri conduct risk assessment and deliver prevention counseling, how to communicate the meaning of positive and negative results. We evaluated modified counseling by using the Single Use Diagnostic System (SUDS) HIV-I assay with Western blot confirmation of positives for three monthls at an STD clinic and anonymous test site (ATS). Diagnostic accuracy was determined by blinded, parallel testing before implementation of SUDS testing. We used focus groups of counselors and a survey of 200 clients to assess staff acceptance and client satisfaction. Results: Modified protocols included relevant consent language, deferral of those not ready to receive results, and risk assessment before the blood draw. SUDS negative and positive results were disclosed at the initial visit, and prevention counseling was conducted at that time. Counseling modifications were derived from the cognitive psychology literature and from the diagnostic accuracy of the test. To communicate the probability of beitg HIV infected given a SUDS positive result, in the ATS. with a positive predictive value (PPV) of 81%, we used the following phrases: "likely to be infected, good chance of being infected, usually infected." In the STD clinic, with a PPV of 88%, we used: "probably infected, very likely (or highly likely) infected, very good chance of beig infected." For SUDS positive clients, the importance of returnitg for cotfirmatory results, further counseling, partner notification, and referral, if necessary, was emphasized. Although initially reluctant, counselors found these protocols acceptable. 90% of clients liked getting their results the same day, and 86% of those who had a previous HIV test preferred the rapid test. Conclusions: The use of rapid, on-site HIV tests allows clients to receive results and results-specific prevention counseling at the initial visit. This eliminates the need for many clients to return for results. We present one model for counseling that appears to be well accepted by clients and counselors. William J. Kassler, Dfl, MPH (404) 639-8371 CDC (404) 639-8610 Fax 1600 Clifton Rd. MS E02 Atlanta, GA 30333 U' __L U' w J U' W

Page  61 519B/D Voluntary HIV counse lling and testing (c&t) in L.usaka, Zambia. SWhocomes, why and does it help. M.Kelly' R.Baggaley','t, M.Phiri', J.Dennetti,C.Chanda' 'Kara Counselling and Training Trust, Lusaka, **London School of Hygiene and Tropical Medicine, Objctives: 1. To establish why people wanted HIT CAT and why they felt at risk. 2. To explore people's understanding of lIlY and issues of HIV testing. 3. To assess hetthr llV C&f had helped people to make changes in their sexual behaviour. Methods: 500 consecutive people attending for voluntary confident ial HlIV C&T were interviewed by trained counsellors. The interviews were nondirective semi-structured last ing between 30-60 minutes. The interviews were carried out in the vernacular language or English as appropriate. The counsellors recorded their clients comments verbatim and made additional notes at the end of the interview where necessary. Further interviews were carried out at the time of post test counselling and at 3-6 months follow up.Rcsult... 700 of those attending were men. The most common reason for wanting to be tested was "to be able to make plans for the future"(23%) these included specific plans for marriage (10%) planning a pregnancy (1%) plans for overseas study (38) or entering holy orders (<1%). Current illness or worries about health, particularly amongst the men was also a common trigger factor for wanting to be tested. Men wanted to be tested because of their previous sexual behaviour "moving around with comen" whereas women frequently stated the illness or death of their husband as a reason for wanting a test. Knowledge about tilV was generally good with good understanding of the meaning of a positive and a negative test although there was much confusion over the window period. Many people felt that they knew what the result would be & in 69% their prediction was correct (p<0.001). Few people regretted being tested and many who were seropositive felt that it would help them cope with their health peroblems, however less than 50% felt able to share their result with their sexual partner. Most people stated that they intended to reduce the number of partners and use condoms but only 33'. of those with +ve results and 26%with -ve results bought them at the time of post test follow up. 50% returned for follow up counselling at 3-6 months. 50% of people were using condoms with their partner/s all or most of the time and 30% of those with re results and 3% of those with -ce results abstained from sex.Conclusions: 1. Some people had specific reasons for wanting an tilV test, men worried about their past sexual behaviour & women about their partner's behaviour. 2. HIV knowledge in this selected group was generally good. People felt that they would be able to cope with a positive result. At follow up few regretted laving had the test, even when found to be positive. 3. Various behaviourial strategies were adopted and these varied depending on the test result. Michael Kelly, Kara Counselling and Training Trust PO Box 37559 Lusaka Zambia Phone +260 1 229847 fax +260 1 22548 520B/D TRAINING TRADITIONAL HEALERS FOR AIDS AND STD PREVENTION AND COUNSELLING: A COMMUNITY EXPERIENCE IN UGANDA King R*t, Kyeyune Pf**, Kabatesi D*, Ssoli A*, Kitto D*, Kwamnya L*, Kaleeba N* Ilomsy J*; *TIIEWA, *TASO, tMSF-CII, Kampala, Uganda. Background: As first line conmmunity workers, traditional healers (TH) it Kampala have an intimate knowledge of their comumnity imelbers' disease perceptions, beliefs, attitudes and practices. lowever, healers have limited knowledge about IIIV/AIDS and STDs and have deep-rooted attitudes towards training. Objective: To train TI Is in providing I IIV/AIDSISTDs counselling and conununity education in Kampala. NMethods: Forty-eight healers were interviewed at their homes and practices, and 25 were invited to participate in workshops about AIDS and STDs. Initial training lasted 4 imonths; on average 4 days/month. Training included tfacts aboutt HIV/AIDS and STDs, cultural practices related to I IIV transmission, counselling, care antd support of PWAs, conununity education, and management of their clinics. Change in knowledge was evaluated tlhrotugh preaid post-tests Training activities provided a framework to develop an STD/AIDS cturricttltumt for and with Tlls in collaboration withls professional trainers. and an advisory group of 20 women representing various sectors of the oniUnility. Results: Seventeen healers participated in the majority of training sessions. Knowledge significantly increased alter 4 months of training. Group interaction anmid contacts with project trainers, through brainstorming, focus group disctssions, atnd lectures, facilitated a gradual clhange in healers' attitudes Seven of the participating TIlls have tiade their oWtt training materials and regularly provide unique and effective conununity education in IIV/AIDS and STDs using music, drama, and stories. Seven healers have started counselling their patients. Four of the 17 IlIs have had the occasion to demonstrate training skills while teaching other healers in miand outside Kampala Conclusions: TIlls have proven to be skilled counsellors and educators. Enthusiastic conununity responses to education by THls have empowered and tmotivated healers to disseminate katowledge through cotmmunity AIDS awareness workshops and to provide individual counselling for PWA clients. Therefore, it is essential to continue mobilizing, training, and supporting Tls as a critical complement to AIDS prevention and care in Uganda. Primrose Kyeyune, P.O. Box 5346 Kampala, UGANDA Tel: 256 41 530 619 Fax: 256 41 268 498 521B/D AIDS COUNSELLING IN RURAL MALAWI SLIEP YVONNE - MEIISA MEDICUS MUNDI - MALAWI 522B/D I)E.VU"I,)I'MI.,NT (O AN INTERVIEW MOL)IT, 1IV) (;VE REISULTS 1F lily' I'OSITIVI SEROI.O(IC TESTS. Casto P.', Guerra, A.* and Escalante A.' Casa de la Sal, A.C. Mexico, City', Banco Central de Sangre Centro Medico La Raza*. Objective: To formulate an approach in counselling that will enable the AIDS patient in a rural African community. Methods: The research was done in two phases. In phase 1 an in-depth investigation was conducted to elicit the experience of AIDS patients and the care givers of AIDS patients as well as the experience and opinions of AIDS counsellors. A purposive sampling method was used. In phase 2 a theory generating approach was used utilizing the results of phase 1 to generate a counselling model. Results: In phase 1 results indicated that resources and coping mechanisms of the patient in his/her socio-economic environment are inadequate to deal with the identified needs of the patient. In phase 2 enablement was identified as the central concept and associated concepts were described, and a model was designed. Conclusions: Proposals are made for national programme guidelines and policy regarding counselling of AIDS patients in a rural African commsunity. The model will be implemented during follow-up research. SLIEP, Yvonne, AIDS Programme, Diocese of South Malawi Telefax (265) 523058-33, PO Box 21, Chilema, Malawi Objective: 'To develop a model of interview that helps health professionals to give results of HIV positive serologic tests in a clear and specific way, so it provides medical information and psychological support. The focus of this interview is to promote, since the beginning, a positive attitude of the seropositive patients that helps them in their fltture mind/body self care. Method: We worked with 250 blood donors during a period of three years trying different styles of interviews. The proposed model is the restilt of a depurating process based in the reactions and needs of the patients. Results: After a phenomenological analysis we created an interview divided in three aspects: 1) Basic HIV/AIDS information; 2) Psycho-social analysis and support of the patient; 3) Contact with Institutions, according to their needs. We tried specific metaphors, language style, information content schemes and active listening techniques. Finally, we developed a written guide that explain step by step the process. Discussion and Conclusion: The best way to apply this model is by an interdisciplinary team, at least two, which could be a physician and an social worker or a psychologist. The training of the model consists in a careful lecture of the guide and group role playing sessions. Although we propose a semi-structured interview, creativity and adaptation skills are fundamental in the success of the technique. Pamela Castro, C6rdoba 76, Col. Roma Tel (525) 5140628 Fax (525) 2078042. N v)

Page  62 523A ANALYSIS OF HIV-1 LTR VARIANTS FROM MULTIPLE BODY SITES. Ait-Khaled M & Emery VC, Royal Free Hospital Med. Sch., London, UK. The differential and temporal expression of the HIV genome is regulated through the long terminal repeat (LTR). The' objectives of this study were to perform a cross-sectional and longitudinal analysis of HIV-LTR quasispecies in proviral HIV-1 DNA present in the LN and PBMC compartments of HIV-1-infected individuals. 10 to 20 clones of LTR amplimers were analysed using standard sequencing methods and their relationships determined using phylogenetic programmes available in the "Phylip" package. The data show that the LN harbours an HIV-1 proviral population that is more heterogeneous than that present in the PBMC for patients with an intact LN histological structure; but no significant difference in heterogeneity between these two compartments was observed for patients with disrupted LN structure. The phylogenetic trees revealed an independent clustering of LN and PBMC variants, indicating a compartmentalisation of the 2 quasispecies for patients with and intact lymph node; the analysis for the patients with disrupted LN did not show this site-specific compartmentalisation. The significance of these results with respect to the site of HIV-1 strain evolution will be discussed. A similar phylogenetic analysis has been carried out on LTR variants from multiple sites (spleen, lung, spinal cord, ganglion, lymph node), the results of which will be discussed with respect to tropism. M Ait-Khaled. Virology, Royal Free Hospital Med.Sch. Rowland Hill Street London NW3 2PF, UK. Tel: 71 794 0500 Fax: 71 830 2854 524A COMPARISON OF HIV-I ENV AND POL EVOLUTION N LYMPH NODE, PERIPHERAL BLOOD AND PLASMA. Robb Merlin L. Frey S, Polonis V*, Mayers DL, Ruderman J, White FA*,Lane J*, Michael NL, Turnicky R. Redfield RR. WRAIR and *SRA Technologies, Rockville, MD. OBJ: Genotype and phenotype of env and pol were determined to assess the relationships of HIV quasispecies in plasma, peripheral blood (PB) and lymph node (LN). METHOD: Samples were obtained from a pediatric patient on AZT undergoing evauation for possible malignancy. A context for the comparison was provided by analysis of 5 additional time points, three of which included plasma and viral stocks as well as provirus. A fragment of env encompassing VI, V2 and V3 was employed to evaluate env sequence diversity using phylogenetic tree analysis and consensus sequence comparisons. Pol genotype was characterized by determining codon 215 mutations with PCR typing. Phenotypic AZT resistance, syncytia phenotype and neutralization response are being determined. RESULTS: Over 300 env clones were obtained for analysis. Virtually no difference in env sequence (V I-V3) diversity was observed between LN and contemporaneous peripheral blood provirus. Viral isolate, cell-associated RNA and plasma sequence diversity were considerably more restricted than proviral diversity. Plasma env sequences did not predict subsequent proviral populations. The proviral populations were distinct over time and the diversity generally reflected sequence changes outside V3. Phylogenetic trees based upon V3 alone provided little basis for discriminating among samples or compartments compared to VI V2 or the entire env sequence available for study. Glycosylation site utilization also discriminated among samples from different time points and supported the conclusion that contemporaneously collected PBMC, plasma and LN were more similar to each other than to other time points. LN stocks were very similar to contemporaneous proviral and plasma populations but stocks derived from peripheral blood were as different from these species as clones from a different time point. Pol genotype and phenotype are under analysis. CONCLUSION: These data suggest that HIV positive cells circulating in the peripheral blood are representative of the LN population in respect to env defined quasispecies but cell-associated RNA, plasma and viral stocks are restricted subsets of this population which are not clearly linked. Merlin L. Robb, Walter Reed Army Institute of Research 13 Taft Ct., Silver Spring, MD. USA 20850 tel 301-762-0089; fax 301-762-4177 526A IMPAIRED CTL RECOGNITION DUE TO GENETIC VARIATIONS IN TIlE MAIN IMMUNOGENIC REGION OF TIlE IV-1 NEF PROTEINAO INTEY 1. COUILLIN, B. CULMANN-PENCIOLELLI, E. GOMARD, J. CIIOPPIN, J-P. LEVY, J-G. GUILLET and Sentob SARAGOSTI INSERM, ICOM, lospital COCHIN, 75674 Paris Cx 14, FRANCE Objective To understand the mechanism by which HIV persists during disease progression in spite of the strong and polymorphic immune response of the host. The high mutation rate of HIV could probably facilitate the selection of mutants which may avoid immunological detection. Methods To further understand this process, we have analyzed the variability of the main epitopic region of the HIV-1 NEF protein recognized by CTL in responding and non-responding donors from a geographically defined cohort. Results Titration experiments in a cellular assay allowed us to identifie mutations in epitopic regions which may prevent binding of peptide to HLA molecules, impair recognition of the HLA/peptide complex by the T-cell receptor (TCR) or disturb efficient antigen processing. Mutations in the dominant anchoring residues of particular HLA molecules, were identified in a simple binding assay. SARAGOSTI, Sentob, INSERM U363, I.C.G.M H6pital COCHIN, 75674 Paris, Cx 14, FRANCE Telephone: 33-1-, Telefax: 33-1- N N 0)1 525A PCR ANALYSIS OF HIV-1 ENV VARIABILITY IN LONG-TERM BENIGN INFECTIONS. Scolaro, Michael J.; Brucker, R.; Javaherbin, P.; Sevall, J.S. Objective: Characterization of (3) env regions by PCR analysis and correlation of sequence alterations with benign clinical course. Methods: Amplified DNA from a primer site in gag and 3 env regions was characterized by PCR analysis. DNA was obtained from fresh lymphocytes fram 2 long-term asymptmatics. Controls included BH10 and one wild-type symptcmatic strain. Results: All patients were positive for conserved regions of gag and env. In the env gene, an amplicon of 1200 bp was generated for the control strain (BHl0) and the symptomatic patient, but not for the 2 patients with long-term benign infection. A 500 bp target (5'-AAGGAAGCAACCACCACICT-3'/5'-ATGGGAATXGGCICAAAGGATA-3') was amplified with primers that include the 5'-end site of the 1200 bp amplified product (5' -AACCCCACCT XIT-3 '/5' -CCATAGGCICCGCGC-3'). Both the BHl0 and the wild-type strains showed this 500 bp amplified product. The patients with long-term untreated benign clinical course had complex banding patterns which imply a sequence modification [deletion(s) or clustered mutations] within this portion of the env gene. Conclusion: An env variant of HIV-l has been identified. DNA sequencing and additional clinical specimens will be examined for possible correlations with benign clinical course.

Page  63 527A SERA FROM HIV-1 INFECTED THAI SUBJECTS CROSS-NEUTRALIZE A VARIETY OF HIV-1 SEQUENCE SUBTYPES Kliks S,Ichimura H*,Visrutaratna S**,Dumitrescu Ovidui, Levy JA.Univ. of Calif.SanFrancisco CA USA,*KureNat.Hosp Kure Japan,**ChiangMaiPubl. Health Chiang Mai Thailand Objective: To evaluate cross-reactive neutralizing antibodies in sera of'Thai subjects infected by Thai clade E virus. Methods: Sera from twenty individuals in Thailand infected by HIV-1 clade E were tested by ELISA for their ability to bind to V3 peptides representative of the different HIV-1 clades. The sera were also evaluated for their ability to neutralize clinical isolates of HIV-1 from clade B from the U.S. and Thailand as well as lade A from Africa and lade F from Romania. Results: The sera from subjects infected with the HIV-1 clade E were strongly reactive with the gpl20 and a representative V3 peptide of a Thai clade E isolate. Cross-reactivity at lower titers was observed with some sera using V3 peptides from clades A, B and F. Neutralization of the clinical isolates, using PBMC assays, indicated that the majority (55%) of the Northern Thailand sera neutralized a representative Thailand isolate and a North American strain in lade B. All the sera also neutralized HIV-lsF70, an isolate from Rwanda belonging to lade A. Studies with HIV-I clade F strains are in progress Conclusions: V3 binding and varying cross-reactive neutralizing activities were detected among sera of individuals from Thailand infected by HIV-1 subtype E. This information has relevance to future vaccine development. DUMITRESCU, Ovidui, University of California, San Francisco CA USA 94143-0128 Telephone (415) 476-4071 Telefax (415) 476-8365 529A FUNCTIONAL ANALYSIS OF HIV-1 VIF AND VPU PROTEINS TOKUNAGA Kenzo, Ikuta K.*, Kawamura M., and Adachi A., Institute for Virus Research, Kyoto University, Kyoto, *Institute of Immunological Science, Hokkaido University, Sapporo, Japan Objective: We have already demonstrated that the requirement of Vif and Vpu proteins for HIV-1 replication is cell-type dependent. To determine the target cell-specificity of these two proteins, functionality of the mutant proteins was evaluated by a number of experimental system. Methods: Functionality of the Vif and Vpu proteins was monitored by single-round replication assays. In some cell lines, growth potentials of the mutant viruses were also determined. Results: The two viral proteins were not required for efficient virus replication in some particular cell lines out of many lines we have tested. The expression of CD4 gene had no effects on the functions of Vif and Vpu proteins. Conclusions: The Vif and Vpu proteins of HIV-1 act at the very late stage of virus life cycle in a cell-specific manner. These results may facilitate basic functional studies on non-essential genes in various types of target cells for HIV-1. TOKUNAGA Kenzo, Institute for Virus Research, Kyoto University, Kyoto, Japan Tel 075-751-4010 Fax 075-751-3995 2 A VARIATION OF VI AND V2 DOMAINS OF HIV-2 ENV GENE IN AEXPERIMENTALLY INFECTED MACAQUES. BBvon-Aubover Marie-Helene, Boussin F., Vogt G., Le Grand R., Vaslin B., and Dormont D. CRSSA/CEA, Fontenay-aux-Roses, France. Objective: We assessed the molecular variability of the env gene of HIV-2 SBL 6669/H5 in rhesus and cynomolgus macaques during the first two years of their infection. Animals: 10 rhesus macaques, preimmunized with recombinant vaccinia viruses expressing HIV-2 ROD genes, and 6 control monkeys (4 rhesus and 2 cynomolgus macaques) were experimentally infected by HIV-2 SBL6669/H5. Vaccinated monkeys were unprotected from heterologous infection. Methods: A 440 bp fragment encompassing V1 and V2 of HIV-2 env gene was sequentially analyzed during the first two years of the infection. Viral sequences were determined by direct sequencing of PCR products from both monkey PBMCs and sera. Results: Very few mutations were observed in the region studied in both monkey PBMCs and sera during the two years following monkey infection. Conclusion: Variability of V1 and V2 does not appear to be implicated in viral escape of the preimmunization in vaccinated monkeys. We have previously determined variations in the V3 to V4 region of HIV-2 env gene in macaques: significantly variation occurred less in V1 and V2 domains than in the V3-3' adjacent region. Furthermore, variation of V1 is lower in HIV-2 than in SIV in experimentally infected macaques. Bayon-Auboyer, Marie-Hel6ne, CEA CEN-FAR DSV/DPTE/SSA 60-68 Av. Division Leclerc, 92265 Fontenay-aux-Roses, FRANCE Tel.: (33-1) 46 54 76 74; Telefax: (33-1) 46 54 81 90 530A IIIV-I NEF DOWN-REGULATES CD4 RECEPTOR BY A BI-MODAL MECHANISM YVenkatesan, Sundararajan*,Hiller,.S.* Popov, S.,* Gratton, S.#, Chandrasekhar, S.*, and Sekaly, R. P. # Lab Mol Microbiolo, NIAID*, Bethesda,. MD 20892, and IRCM #, Montreal, Quebec, Canada H2WIR7. Objective: To elucidate the mechanisms of CD4 down-regulation induced by the HIV-1 Nef protein. Methods: HeLa cells were transfected with expression plasmids for CD4 and Nef, and CD4 surface expression was quantified by FACS and 125I labeled anti-CD4 antibody binding. The steady-state levels & kinetics of CD4 synthesis were measured by immunoblottin and pulse-chase labeling. Metabolic inhibitors of sub-cellular organelle function delineated some of the kinetic pathways. Recombinant vaccinia Nef vector was also used to infect various CD4 expressingT cell lines. Results: In transient transfection, the Nef induced reduction of surface CD4 expression paralleled the decrease in steady-state levels. Nef did not decrease the half-life of CD4 or interfere with its intracellular trafficking. CD4 reduction was not reversible by lysosomal inhibitors. Reduction of CD4 protein levels occurred during or immediately after translation and could be detected in an in vitro system. CD4 mutants with deletions in the extracellular domains were Nef responsive, while a deletion in the cytoplasmic domain had reduced Nef responsiveness. Protein kinase C target sites and Ick p56 bindig domain were not required for the Nef effect. In contrast, Nef reduced the surface expression of presynthesized CD4 by enhancing the receptor internalization. In both T cells and HeLa cells expressing CD4, Nef induced endosomal sequestration of of internalized CD4; in T cells, this process was reversed by lysosomotrophic agents, while in HeLa cells, it was not. This mechanism was mediated by the cytoplasmic tail of CD4, but did not require protein kinase C phosphorylation or Ick p56 interaction. Conclusions: Nef induced down-regulation of CD4 was mediated by two distinct mechanisms. Venkatesan, Sundararajan, LMM, NIAID, Bldg 10, Rm 6A05, National Inst. of Health, Bethesda, MD 20892, USA, Tel # (301)-496-6359, FAX # (301)-402-4122 (J1 0

Page  64 531AMECHANISM OF CELL SURFACE CD4 DOWN-REGULATION BY 53s1A______ __ NEF. Garcia, J. Victor, and Anderson, S. J. Dept. of VirologyiMol. Biol., St. Jude Children's Research Hospital, Memphis, TN 38101, USA. OBJECTIVE: To determine the mechanism of cell surface CD4 downregulation by Nef. METHODS: Retrovirus mediated gene transfer was used to introduce the nef gene of HIV into human cells expressing CD4 in the presence or absence of p561ck. Steady state levels of CD4 and p561ck were determined by western blot and half life studies by metabolic labeling and immunoprecipitation. RESULTS: A dramatic drop in the steady state levels of CD4 was observed in cells expressing Nef. This decrease was due to an increased rate of degradation of CD4. The levels of p561ck were not significantly affected in cells expressing Nef when compared to control samples. DISCUSSION AND CONCLUSIONS: Based on our results and those of others the mechanism of CD4 down-regulation by Nef involves an increased rate of internalization followed by degradation of CD4. The fact that the levels of p561ck are not significantly altered suggests a redistribution of this kinase in cells expressing Nef. Our understanding of Nef function forms the basis of ongoing evaluation of potential inhibitors of HIV replication; J. Victor Garcia, Dept. of Virology @ Mol. Biol. St. Jude Children's Research Hospital, 332 N Lauderdale, Memphis, TN 38101, USA. 901-531-2611, FAX 901-523-2622. 532A POSITIVE EFFECT OF NEF ON SIV REPLICATION IN MICROGLIA Kawahara T.*, Desrosiers R.C., and Kodama. Toshiaki*, *Oregon Regional Primate Research Center, Beaverton, OR, USA. Objective: We have isolated SIVmac155/BR variant from the encephalitic brain of a rhesus macaque following infection by cloned SlVmac239. In contrast-to the parental SIVmac239, the BR variant was competent to replicate in rhesus macaque peripheral blood-derived macrophages (Me) and replicated extremely well in microglial cells of rhesus macaque primary brain cell cultures. This study seeked to identify the viral determinants of the replicative potential of the BR variant for Me and microglial cells. Methods: Viral sequences encoding gpl120, gp41, and Net were derived from the BR variant by PCR and recombined into the corresponding regions of SIVmac239 by several combinations to construct a series of recombinants. The replicative ability of the recombinant viruses was analyzed on rhesus macaque Me, microglial cells, and lymphocytes, and on human T-cell lines in culture. Results: Genetic mapping revealed that specific viral sequences in the gp120 of the BR variant were essential for virus replication in both Mo and microglial cells; however, it conferred only a basal level of replicative potential for these cells. The gp4l and net gene of the BR variant on its own did not impart replicative competence to Me and microglial cells; however, they augumented replicative potential in these cells to the highest level when the gp120 of the BR variant was present. The gp41 and nef gene contribute equally to enhance virus replication in Me; however, only the nef gene was required for the high level replication in microglial cells. The nef gene did not enhance virus replication in rhesus macaque lymphocytes and human T-cell lines. Mutations in the nef gene not overlapping with U3 of 3'LTR were responsible for this activity; they were specifically present in the BR variant, but not in other SIVs with no or low replicative capacity for microglial cells. Concluslon:This study demonstates that mutations in not only gp120 but also in nef gene are important indetermining the replicative ability in microglial cells in culture, and suggests that specific net alleles may positively regulate virus replication in the affected brain in vivo. Kodama, Toshiaki, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006 USA Telephone (503) 690-5284; Telefax (508) 690-5563 534A VIF &VPU of HIV-1: CROSSTALK IN THE HIV GENOME? Elahe T. Dayton, ouis Cantaloupo & Andrew I. Dayton:LIR/ NIAID/NIH OBJECTIVES: The goal of this work is to examine possible interactions among VIF, VPU and other regions of the HIV genorme. RESULTS: Fragments were exchanged between the HXB-2 and the pNL43 provirus genomes. The effects of introducing VIF mutations were then monitored by measuring supernatant viral reverse transcriptase. Paradoxical effects were seen when the VIF mutations were introduced into chimeric strains consisting of HIVHXB2 with the "VPU + ENV" region replaced by that from pNL43. In these chimeras, vif mutation enhanced viral replication. If the VPU genes of these chimeras were deleted, the opposite result was seen: VIF mutation impaired replication. These results were only seen in Jurkat cells. Furthermore, VIF mutation of wild-type pNL43 severely impaired viral replication in Jurkat. This was also clearly different than VIF mutation of the HXB2-pNL43 chimeras. Currently we are initiating mechanistic studies on the effects of VIF mutation in the chimeras. We are also mapping the regions of pNL43 which appear to be responsible for the severe impairment effects of VIF mutations when the virus replicates in Jurkat cells. CONCLUSIONS: VIF and VPU may interact in a previously unrecognized fashion. These genes may be involved in crosstalk with other regions of the HIV genome. These other regiors of the HIV genome may represent target sequences for these genes. Elahe T. Dayton, 10/6A08 NIH/9000 Rockville Pike, Bethesda MD 20892 FAX:301-4024122 (J1 W~ uJ WA 533A Compliance With Prevention Guidelines: Congenital Syphilis Outcomes Among Urban Women in Atlanta, GA, 1990-1993. 'Fichtner RR RWarner DL. 'Rochat RW, & 'Conlon, Richard T.; 'CDC. Atlanta. GA; 2GA DHR, Atlanta, GA. Objectives: To evaluate compliance by prenatal service providers with congenital syphilis (CS) prevention guidelines by assessing CS outcomes among women who received prenatal care and were delivered of infants in Atlanta, Georgia, during 1990-1993, a period in which Atlanta had the highest incidence rates of infectious syphilis among women of any major U.S. city. Methods: In order to maximize the likelihood of idenctifying the totality of 1990-1993 incident cases of CS among infants who were delivered in an inner-city, publicly-funded hospital which reported approximately 40% of all Georgia cases and 95% of all Atlanta cases, we consolidated data from three sources. State report forms were matched against surveillance records at the Centers for Disease Control and Prevention (CDC), and comnputer linkages were made to Georgia's infant vital statistics system to ensure inclusion of stillbirths. Levels of provider comipliance with CDC prevention guidelines were quantified. Results: Of the 179 hospital cases of CS identified to have occurred during the three-year period. 94 (53%) received prenatal care with a median of four prenatal visits. Mothers of CS cases were black (93%), not married (84%), and 20-30 years of age (57%). Adverse outcomes among all cases of CS delivered included perinatal mortality (13%), low birth weight (84%), and prematurity (36%). Among CS cases who were live births, those born to mothers with prenatal care were of greater gestational age (37.8 mos. vs. 35.6 mos., X2=10.5, p<0.01) and had greater birth weight (2796 g vs. 2372 g, X2=9.8, p<0.01). Among mothers of CS cases who received prenatal care. 71% were not treated during pregnancy. But, among those treated, 81% received treatment inconsistent (i.e., late or non-penicillin) with CDC guidelines; 19% were reinfected after initial treatment during pregnancy. Conclusions: These findings suggest that compliance with CS prevention guidelines is inconsistent. Although continuity of prenatal care for many urban women cannot be always assured because of factors unrelated to care. more uniform adherence to guidelines, in addition to timely and aggressive outreach to womenwho have initiated care, are indicated. Richard T. Conlon, MPA; CDC, 1600 Clifton Road E-44, Atlanta, GA (404) 639-8300 FAX (404) 639-8622

Page  65 535B ANTI-HIV AND IMMUNOSTIMULANT EFFECTS OF GSPH-l ON HIV INFECTED PATIENTS M. Ben Amar, R. Morisset and G. Poirier, Dept. of Microbiology, Hotel-Dieu Hospital, Montreal, Canada. 536B EFFECTS OF THALIDOMIDE ON WASTING SYNDROME IN PATIENTS WITH AIDS. A RANDOMIZED, DOUBLE-BLIND, PLACEBO CONTROLLED CLINICAL TRIAL ReysTeren G, Sierra-Madero JG. Martinez del Cerro V, Mutioz-Trejo T, Armoyo-Figueroa H, Pasquetti AavTa JJ, Ruiz-Palacios GM. Instituto Nacional de la Nutrici6n, Mexico City, Mexico. Objectives: * Assess the efficacy and safety of GSPH-1 in a phase I study. * Compare GSPH-1 (an oral vegetal preparation) to AZT (the first-choice anti-HIV drug). Methods: Twenty HIV infected subjects with fewer than 500 CD4+ lymphocytes/mm3 were matched on the basis of equivalent CD4 cells count at onset of treatment and received either GSPH-1 (10 patients) or AZT (10 patients). Evaluation of responses to therapy included general status of patients, volume of lymph nodes, levels of beta-2-microglobulin, CD4( +), COD8(+), CD3( +)T cells, total lymphocytes and white cells counts. Results: All data were obtained after 10 months of treatment: In the GSPH-1 group, 7 patients improved their clinical status while the other 3 remained stable. GSPH-1 had anti-HIV properties illustrated by an important regression of adenopathies and a significant group reduction of beta-2-microglobulin. GSPH-1 also demonstrated immunostimulant activities resulting in the following mean variations of immunologic markers: GSPH-1 group AZT group CD4(+) T lymphocytes / 0.24% 1.33% CD8(+) T lymphocytes 2' 32.68% / 2.36% CD3(+) T lymphocytes /24.60% /' 2.71% Total lymphocytes / 23.16% / 2.72% White cells / 10.19% \ 15.58% No toxicity of GSPH-1 was observed at therapeutic doses. Discussion and Conclusion: GSPH-1 is a promising anti-HIV and immunomodulatory drug which could control HIV infection. BEN AMAR, Mohamed, Hotel-Dieu Hospital, Montreal, Quebec, Canada Telephone: (514) 843-2675, Telefax: (514) 849-8986 537B JOHREI IN THE TREATMENT OF HIV/AIDS Bihari, Bernard Foundation for Integrative Research. New York, New York USA Objective: To collect data in a small 12 week pilot study on the safety of Johrei, a method involving healing energy transfer developed in Japan, as a treatment for HIV/AIDS and to look for trends in immunologic function in order to determine whether a larger controlled study would be justified. Methods: Four HIV seropositive patients with CD4s of 200 -500 were enrolled in this 12 week open pilot study. Laboratory data included CBC and Differential, Chem Screen, T cell subsets, HIV viremia, NK activity and lymphocyte responses to various mitogens, all of which were repeated at 4, 8 and 12 weeks. Patients were administered Johrei 3 to 5 times a week by trained practitioners. Results: Mean CD4 numbers at baseline were 365, rising to a mean of 451 at 12 weeks, an increase of 24%. Mean CD4 percentage was 25.3% at baseline rising to 31.5% at 12 weeks, an increase of 25%. Symptom check lists and interviews indicated a general improvement in overall sense of well being in all 4 participants. Conclusion: Johrei may have some immune enhancing activity in people with HIV/AIDS. Only a much larger propetly controlled study would determine whether it in fact does, and what the clinical significance of such immune enhancement is. Bihari, Bernard Foundation for Integrative Research, 29 W. 15th Street, New York, New York 10011 (212) 229-9374 Background: Wasting syndrome (WS), a major feature of HIV disease, contributes to the mortality of AIDS patients. At present, there is no specific treatment for WS. Tumor necrosis factor a (TNFa) has been implicated in the pathogenesis of WS and it may play a role in the progression to AIDS. Objectives: To evaluate the efficacy of Thalidomide (Thal), a selective inhibitor of TNFa, in the treatment of WS in patients with AIDS, and to assess the effect of Thai on HIV viral burden in peripheral blood mononuclear cells (PBMC). Methods: Adult AIDS patients under antiretroviral therapy, without active opportunistic infection, and with > 10% of weight loss in the previous 6 months, were included. Patients were stratified by severity of weight loss and CD4 T cells count, and assigned in a random and double-blinded fashion to receive Thal (100mg PO qid) or placebo for 12 weeks. Weight and anthropometric data were recorded every fortnight. Clinical events were registered, and CD4' T cells/pL and HIV viral burden in PBMC by end-point dilution cultures were tested. Clinical outcomes of That use were weight gain or no progression of WS. Results: From 23 individuals (22 men and 1 woman) enrolled from January'93 to February'94, 18 have finished the treatment protocol. Both Thal (n=9) and placebo (n=9) groups were comparable in their baseline status. Stabilization or weight gain occurred in 8/9 patients from the Thal group and in 2/9 from the placebo group (OR=0.036, 95% CI: 0.003-0.48, p=0.008). Karnofsky index was significantly higher by the end of the study in the Thal group (Mann-Whitney U test: p=0.034). Mild and transient somnolence and erythematous macular lesions were only observed in the Thal group (8/9). Neither CD4' Tc count nor HIV viral burden showed changes in either group. Conclusions: Although the number of patients is small, these preliminary findings of an ongoing study demonstrate that That significantly reverted the natural history of WS in AIDS patients. Our results suggest that That, at the dosage used in this study, had no in vivo effect on HIV viral burden, and had no important toxicity. GUSTAVO REYES-TERAN, M.D. INSTITUTO NACIONAL DE LANUTRICION D DNUTRICION VASCO D QUIROGA 15 TIALPAN 14000, MEXICO.DF, MEXICO PHONE: (525)655-96-45 FAX: (525)6~55-96-75 538B PRELIMINARY STUDY ON TREATMENT OF HIV INFECTION WITH AIDS-NO.1 NI YA,ZHEJIANG ACADEMY OF MEDICAL SCIENCES Objective: According to the theory of Traditional Chinese Medical Science, AIDSNO.1 of Chinese Herbal Extracts(CHE) was selected to treat the hemoph -iliacs of HIV infection so that a kind of effective way treating AIDS was obtianed. Methods: The hemophiliacs showing strong positive results which were confirmed by ELISA, immunofluorescence test and Western blot were given oral AIDS-NO.1 0.5g three times a day for 10 months. The curative effects were affirmed by examing patient's weight, height, lymphocyte(OKT4, OKTB, OKT4/OKT8 etc.) and immunoassay. Results: The hemophiliacs administrating AIDS-NO.1I have improvement in physical strength, weight, height, OKT4, OKT8, OKT4/OKTg etc. All physiolo -gical parameters approached normal level. Discussions and Conlusions: It's a key that dialectics treat AIDS patients with CHE. Invigorative CHE was main and detoxificating CHE was auxiliary in order to protect immunization of patients against development of disease. The data indicated that cellular immunity was protected and humoral immunity was enhanced, thus HIV multiplication was inhibited. NI YA, Hangzhou, zhejiang, P. R. of China Telephone (86)-571-8076765, Telefax (86)-571-8075447 W 01 WA

Page  66 C 539B COLLABORATING WITH TRADITIONALHEALERSINAIDS RESEARCH, PREVENTION AND CARE IN UGANDA. Kabatesi D#, King R#t, Kwamya L#, Katabira E*, Kalibala S**, Kaleeba N*, Homsy J#t. #THETA\THIEWA.*TASO..MSF-CH, Kampala, Uganda, **WHO/GPA/IDS,Geneva. (?liecave: To explore the challenges of collaboration between traditional healers (TI) and medical personnel in AIDS research, care and prevention through a comparative clinical study of herbal treatments for AIDS symptoms and a project for training TII as AIDS counsellors and educators for their clients and communities. MAehods: Visits to TH and regular meetings were used to explain the projects. TH participated in AIDS awareness seminars, research methods sessions and focus group disussions. 111 and investigators developed guidelines for collaborative research which emphasized TIFs rights of ownership of research outcomes and guarantee of doctors' respect for their beliefs. The collaboration brought together TH of various backgrouids and practices. Results: First visits started in December 1991. Since then, 112 healers have been contacted in Kampala. 40 were selected for training and have remained in contact. Of these, 20 have been actively participating in the projects. Some THl have now started conducting community AIDS education seminars and support activities for their clients. Preliminary results of the clinical study have shown that some TH's preparations may be effective for chronic diarrhea and herpes zoster. TH feel this research is an entry into the medical community and are anxious for recognition and improvement of the quality of their treatments. THI's role in AIDS care and prevention has been recognised and supported by the Uganda AIDS Commission and other AIDS organisations. They have now started to organise themselves into a National Council of TH associations with the support of the Ministry of Health. During all this time, THI have not received any financial rewards in exchange for their contributions to the projects. Cunclusios: Collaboration with TH requires a considerable amount of time and patience. THI tend to fear and distrust researchers and scientists who they feel will not respect their rights and knowledge. However, TH are selfmotivated and given mutual commitment, openness, continuous communication and respect, they can substantially contribute to AIDS prevention and care efforts. Professionals need to recognise the role of traditional medicine in health care and AIDS care in particular. Governments should provide an institutional and regulatory framework that will preserve, promote and improve the practice of traditional medicine. Dr. Donna KABATESI, THEWA Project, P.O.Box 5346, Kampala. Uganda. Telephone: 256-41-530'619 - Fax: 256-41-268'498. 540B ALTERIJATIVE TREATIIENT AS COINTRIBUTION TO CARE OF pwHIV/AIDS I.WJolffers, S. de Moree, Dept. of Soc.Med. Vrije Universiteit, Arimsterdam O ective: Research was done to describe and understand health-seeking behaviour of pwAS/HIV, especially concerning alternative treatment. Methods: For quantitative data an questionnaire was designed that was sent to different HIV organizations in the Netherlands and that were left at regional AIDS-platforms. In-depth interviews with a random sanmple of those who returned their questionnaire. Results: It appears that 30.6% of pwHIV/AIDS used alternative treatment. Of these 24% already used alternative methods before diagnosis. Of those using alternative treatment 46% assumed that this treatment was effective, 66% thought that the alternative treatment would strengthen their resistance, 34% felt better because they had the feeling to be actively involved in treatment. Analysis of in-depth interviews with a sanmple of the research-group suggests that alternative treatment is seen by many as means to do something positive against the negative influences of the virus and the toxic effects of allopathic medicine (like AZT., co-trimoxazole). In addition, in the process of their HIV-infection pwHIV/AIDS seem to have little decisions left to take. Discussion and conclusions: Having the feeling to be in control and to be able to take decisions strongly adds to feelings of self-esteem and thus to the quality of life. Some of the decisions left to pwHlV/AIDS are whether to start AZT-treatment and when (at the moment CD4 counts are low enough), whether or not to follow alternative treatment, to decide about euthenasia. It seems that alternative medicine can play a positive role in care and improving of quality of life of pwHIV/AIDS. Prof. Jolffers, Van der Goeclnorststraat 7, A'dam. Tel. 31-20-5483366, Fax 31-20-6462228, no 1 542A I"TIikEST:F ANI-ItTLAI Orm DY I'ER IN SYMPTOMLESSINDIVIDUALS JOUBERT L, MARIOTTI M. REED D*, PETIT J.C. LEFRERE JEAN-JACOUES. INTS, H6pital St-Antoine, Paris, France. *Diagnostic Biotechnology, Geneve, Suisse. Objectives: 1) to determine whether anti-HTLV-I antibody titer is correlated with viral DNA load in symptomless infection. 2) to follow-up the titer of each antibody ZAB) over a 3 year period in this population Methods: 22 symptonmless HTLV-1 infected individuals were studied through quantitative DNA polymerase chain reaction to determine the proviral copy (PVC) number and through Western blot (WB) with end point dilution method to determine AB titer. The exact quantitation of each AB was performed through ScanBlot by determination of peak height of each band of WB. This latter procedure was applied every year over 3 year period. Results: Important variation in AB titer were observed according to the individuals studied. A significant positive correlation was observed between the PVC number and the titer of total antibodies (p=0.004). When the association between the peak height of each AB and the PVC was studied, a significant positive correlation was observed only with anti-p19 (p=O.02). No variation of every AB titer was observed over the 3 year period. Conclusions: The risk of developing a HTLV-I-related disease being linked to the viral load, antiHTLV- AB titer, by reflecting this load, could be used as a marker of disease progression. The determination of anti-HTLV-I titer through ScanBlot analysis appears as a reproducible and pratical tool for the management on a large scale of symptomless HTLV-I infected individuals. LEFRERE, Jean-Jacques, INTS, H6pital St-Antoine, Paris, France Tel (33)-1-43-44-46-83; Fax (33)-1-45-12-75-03 U' Wo CD w4 N C,' 541A CHRONOLOGICAL EXAMINATION OF HISTOPATHOLOGY AND mRNA EXPRESSION OF CYTOKINES AND NITRIC OXIDE SYNTHASE IN THE NERVOUS SYSTEM OF THE HAM RAT DISEASE Q 2ya, U. Tomaru., T. Kasai, I. Yamashita, C. Hanlon*, M. Abe, K. Seto, H. Ikeda, A. Wakisaka, H. Koprowski*, T. Yoshiki The First Dept. Pathol., Hokkaido Univ. Sch. Med., Sapporo, JAPAN, *Thomas Jefferson University, Philadelphia, PA, U.S.A. -- Objective: To investigate the pathogenesis of HTLV-I associated diseases, we tried to make rat models with HTLV-I infection, and succeeded in establishing rat model which shows HTLV-I-associated myelopathy/tropical spatic paraparesis (HAM/TSP)-like myelopathy, HAM rat disease. Here, we investigated the chronological changes of histopathology and mRNA expression of cytokines and nitric oxide synthase (NOS) in the nervous system of the HAM rat disease. Methods: Infection of rats with HTLV-I: New born WKAH rats were infected intraperitoneally with l0 of MT-2 (HTLV-I producting human cell line) injection. Histopathology: Spinal cords were stained with hematoxylin and eosin or Luxol fast blue and PAS for light microscopy. Detection of mRNA: By 3 month intervals, cerebrum, cerebellum, spinal cord and sciatic nerve were serially collected and mRNA of IL-lIa, J3, IL-2, IL-6, TNF-a, IFN-y and inducible NOS (iNOS) were detected with RT-PCR method. Results: In the HTLV-I infected rats, demyelination, vacuolation and macrophage infiltration were observed both in the spinal cord and peripheral nerve after 12 months of infection, and followed by gait disturbance and hind leg paraparesis in 4-10 months later. In these rats, mRNAs of IL-[l, TNF-a and iNOS were detected with the development of neurological changes. Conclusion: These data suggest that cytokines and nitric oxide may contribute to the pathogenesis of HAM rat disease, a rat model of HAM/TSP in humans. OHYA. Osamu, The First Department of Pathology, Hokkaido University School of Medicine, N-15, W-7, Kits-ku, Sapporo 060, JAPAN. Telephone (81)-11-758-4127; Telefax (81)-l1-758-4127

Page  67 543A PRENATAL AND POSTNATAL TRANSMISSION OF HTLV-I IN RATS. Mitsuo Hori. S. Kushida*, K. Uchida*, T. Abe and M. Miwa* Dept. Hematology Institute of Clinical Medicine. *Dept. Biochemistry, Institute of Basic Medical Sciences. University of Tsukuba, Tsukuba city, Ibaraki, Japan. 544A HTLV-I in Japan. Evidence for two molecular subtypes with a north south Gradient. A. Gessain*, A. Ureta Vidal*, R. Mahieux*, M. Yoshida**, K. Nishioka***, D. Tekaia*, L. Rosen*, G. de Th6* - *Institut Pasteur, Paris - **Tokyo University - ***Japanese Red Cross Society, Tokyo. Objective: To determine the route of mother-to-child transmission of HTLV- 1 in rats. Methods: A human T-cell line producing human T-cell leukemia virus type-1 (HTLV-1), MT-2 cell, was injected intravenously into female F344 dam rats before or after delivery. (1) To analyze the intra-uterine transmission. MT-2 cells were injected into cighl pregnant rats and Caesarean section was performnned at the 23rd day of pregnancy.(2) To analyze postnatal transmission, MT-2 cells were injected into a dam rat within 24 hours after delivery and six offspring were fostered by this dam. HTLV-1 provirus was analyzed by PCR. Results: (1) HITLV-1I provirus was detected from the DNA of the liver and the spleen taken also by Caesarean section from one of the eight fetuses. Seventy-one offspring were taken from the remaining seven dam and fostered by seven normal F344 rats. Four weeks after the Caesarean section. HTLV-1 provirus was detected in peripheral blood mononuclear cells (PBMCs) from two out of the seventy-one offspring. (2) HTLV- I provirus was detected from PBMCs of all six offspring at 4 weeks old. Conclusion: These results clearly indicate that the intra-uterine transmission of HTLV-1 occurs, and that postnatal transmission of HTLV-1 occurs with high frequency when the offspring do not have the maternal antibody. This model would be useful to control the mother-to-child transmission of retrovirus. MITSUO Hori, Department of Hematology, Institute of Clinical Medicine, University of Tsukuba, 1-I-1 Tennoudai, Tsukuba. Ibaraki 305, Japan. Tel. (81)-298-53-3293: Fax (81)-298-53-3039 67 ex vivo HTLV-I proviral DNAs from PBMCs of Japanese ATL, TSP/HAM and asymptomatic HTLV-I infected individuals were studied by RFLP on PCR amplified LTR. The results, together with those of 19 previously studied Japanese specimens, permitted to observe 2 molecular subtypes: the most frequent (observed in 78 % of the specimens) was named "Japanese subtype 3", exhibiting a diffuse geographical distribution within Japan. The second was the cosmopolitan subtype 2, present all around the world and seen in 22% of the Japanese specimens with a significant increasing North to South gradiant, representing 25 % of the specimens in Kyushyu and Okinawa islands. The sequencing of a fragment of 414 bp of the U3/R region (NT21 to 434 of the LTR) confirmed the existence of two molecular HTLV-I subtypes in Japan diverging in the LTR region by 1.6%. These data raise questions on the origin and dissemination of retroviruses in Asia. Guy de THE - Unite d'Epiddmiologie des Virus Oncogenes INSTITUT PASTEUR - 28, rue du Dr Roux, 75724 Paris Cedex 15. Tel. 33 1 45 68 89 30 - Fax 45 68 89 31 545A VIRAL AMPLIFICATION VIA CLONAL EXPANSION, RATHER THAN BY REVERSE TRANSCRIPTION, WOULD HELP EXPLAIN THE GENETIC STABILITY OF HTLV-1 EWa JP.Vartinian,S.Wain-Hoban.Servk desMaladiesduSang,Lile.Un deRoirobieMo l,airssutPaseur,Paris 546A RESTRICTED TCR Va AND VIlI USAGE BY HTLV-1 SPECIFIC CD8+ CTL FROM PERIPHERAL BLOOD OF PATIENTS WITH HAM/TSP. Elovaara, Irina, Utz, U., Smith, S., Lehky, T., Jacobson, S. Neuroimunology Branch, National Institutes of Health. Objecive: HITLV-! is characterised by a genetic stability. The second hallmark of IIlL-I is the high provirus load in most symptomless individuaLs.Taken together, these two characteristics of IITLV-1 would seem to be at odds with our knowledge of retroviruses replication which excludes the combination of an important replication with genetic stability. These data emphasize the question of IITLYV-1 replication status: ilow does the virus stay stable while replicating itself at a high rate? Our objective was to answer this question. Methods: -t') In order to assess the degree of genetic stability, a segment of two hundred and sixty-five base pairs spanning the hydrophobic envelope leader sequence was amplified from P19 and P20 DNA (two asymptomatic IITLV-I cariers from Guadeloupe) PCR products were cloned and one hundred molecular clones from each patient were sequenced. -2~) In order to assess the degree of clonality of cells harbouring HlTLV-I proviruses the 3 LTR and flanking cellular sequences were amplified by PCR. PCR products were cloned and more than 100 clones from each patient were sequenced. Results -I~) Sequencing of the envelope gene revealed only 3 point mutations, indicative of the remarkable genetic stability of IITLV-l. -2~) Among the 102 cellular flanking sequences established for P19, 9 clones accounted for 94 of the 102. The remaining 6 were unique. In P20 DNA, 50/104 cellular flanking sequences belonged to 15 clones, the remaining were unique. Ten other asymptomatic HTLV-1 carrier, 10 TSP/HAM patients and 4 ATLL were also studied: in all cases there were >10 discrete integration sites per sample. Discussion and conclusion These data showed that clonal expansion of HTLV-I bearing T cells was the norm for both symptomatic and asymptomatic carriers. Sequencing 100 molecular clones derived by PCR amplification of part of the envelope gene from the two asymptomatic carriers revealed almost no genetic variation. Viral amplification via clonal expansion, rather than by reverse transcription, would help explain this remarkable genetic stability. Oligoclonal expansion is therefore a way of life for HTLV. Our result would suggest that anti-reverse transcriptase drugs will be of little use in controling IITLV-I proliferation. WATTEL ERIC-Service des Maladies du Sang-i place de Verdun-590000 LILLE - Tel.: (33) -ax t,3) zo..",,.,o.04 Objective: To determine T cell receptor (TCR) V a and V B chain usage in HTLV-1 specific cytotoxic lymphocytes (CTL) obtained from peripheral blood of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: To characterize TCR repertoire, CD8+ lymphocytes from peripheral blood were cloned in limiting dilution, and the resulting wells were screened for HTLV-1-specific precursor CTL activity. The RNA was isolated from HLA-A2 and HLA-B14 restricted HTLV-1 tax 11-19 and tax 90-55 -specific CD8+ CTL lines, and cDNA was analyzed by PCR amplification using Va and V 8 chain family-specific oligonucleotide primers. Results: In patient with early HAM/TSP, HLA-A2 restricted Tax 11-19 specific CTL lines used preferentially Va 2 (9/9) and Va 3 (4/9). This was combined with preferential usage of V 813 9 (3/5). Among tax 11-19 specific CTL lines from 2 HLA-A2 patients with longer duration and severe paraparesis, the CTLs used preferentially Va 2 (6/9) and/or Va 12 (5/9) or Va 7 (2/4), respectively. Their TCR V 8 usage was preferentially restricted to V 131 or V1312. The TCR V a and VB usage of HLA-B14 HAM/TSP patient was limited but heterogeneous. Discussion and Conclusions: HTLV-1 tax 11-19 specific CD8+ CTL obtained from different HLA-A2 HAM/TSP patients express a restricted heterogeneity in TCR Va and V 813 chain usage with little or no overlap between individuals. HTLV-1 specific CTL using restricted TCR Va and V 813 repertoires may play a critical role in the development of HAM/TSP. The immunotherapeutic strategies that focus on eliminating these cells could be clinically significant. The information obtained from such therapy could be extended to other CNS diseases of retroviral origin. Dr.!rina Elovaara, Departiment of Neur-clogy, Tampere University Hospital, 33520 Tampere, Finland. Tel. 358-31-2476692, Fax: 358-31-247-4351 U' U') 44h 0)

Page  68 547D S T/HIV PREVENTION IN TURKEY: PLANNING A SEQUENCE OF INTERVENTIONS Aral. Seva O. * Fransen L.* * Koral S.*** Fincancioglu N.*** *C DC Atlanta, GA ** EC Brussels Belgium *** FPA and HRDF Turkey Objective: To rapidly assess which mix of early STD/HIV prevention interventions would potentially be effective, cost-effective and sustainable in Turkey; and to plan an intervention sequence to maximize synergy among the interventions. Methods: During rapid assessment we: 1) reviewed past issues of 3 leading newspapers; 2) collected infomation on TV coverage; 3) interviewed convenience samples of taxicab drivers, hotel employees, grocery store owners, academicians in public health and law, nvestigators of STD/HIVl and women's reproductive tract infections, and officials in the ministry of health; 4) reviewed available evidence on STD/HIV morbidity, sexual behavior patterns, migration patterns and same/opposite gender sex trade. Results: We found: 1. discrepancies between decision makers' perceptions and social realities with respect to the epidemiology of sexual behavior and STDs, and the state of public health programs; 2) discrepancies between sexual practices and public expression regarding sexual practices; 3) economic, demographic and political pressures, in Turkey and surrounding countries, towards the enlargement of prostitute supply; 4) a sexual double standard and gender specific migration patterns which sustain high demand for commercial sex; 5) patterns of health care seeking and STD clinmcal services which indicate other STDs may play a very important role in spread of HIV infection; 6) an important mass media role in opinion formation; 7) consensual denial of risk for the majority based on beliefs embedded in machismo, nationalism and religion and a resulting marginalization and externalization of STD/HIV risk; 8) high prevalence of syphilis among both Turkish and immigrant female prostitutes in Istanbul (early latent 8 and 13%; late latent 0 and 4%; previous history 9 and 22%) and 9) high rates of syphilis among male prostitutes (early latent 11%, late latent 21% and previous history 58%). Conclusion: We concluded interventions should initially include the following order: 1) awareness raising for decision makers and opinion leaders including members of parliament and mass medial 2) awareness raising for members of general population; 3) needs assessment and intervention development for sex workers; 4) training for medical personnel and 5) quality control for laboratory procedures. Sevgi O. Aral, Ph.D. Centers for Disease Control and Prevention 1600 Clifton Road, N.E., Mailstop E02 Atlanta, Georgia, 30333 Tel. (404) 639-8259 FAX (404) 639-8608 548D STD/HIV PREVENTION PROGRAM FOR WOMEN OF LOW SOCIOECONOMIC STATUS IN LIMA, PERU. Sanchez $jSixto, Woolcott D. Mejia A, Rouillon M, Blume E, Mazzotti G, SAnchez J. VIA LIBRE, Lima, Peru. Objective: To develop and evaluate educational material for women of low socioeconomic status (SES) organized in "clubs de madres" (mothers' clubs), community-based organizations settled in Lima. Penr. Methods: Eighteen focus groups among women from mothers' clubs, and 7 focus groups and 7 in-deep interviews among men of low SES were performed to obtain information at 2 levels: a) sexuality, machismo, STD/HIV, condom use; b) Information and communication network. A set of educational material (poster, brochure. manuals and flipcharts) was developed and pre-tested using in-deep interviews. Health promoters (120) from the community were trained to use the educational material; changes on knowledge and attitudes among the promoters were measured among the promoters. After pre-test, a sample of 600 women from mothers' clubs not previously interviewed entered into an educational intervention made by the trained promoters. A post-test will be performed after 6 month. Results: Educational materials were develop using messages to improve perception of risk for STD/HIV and health seeking behavior; to give information on STD/HIV prevention and family planning; to change attitudes and beliefs towards sexuality; and to empower women's decision making in health behavior. Among community health promoters, a significant improvement was found between pre and post-test regarding knowledge about STD/HIV and attitudes towards sexuality. In the pre-test evaluation, women from mothers' clubs were found to have low perception of risk for STD/HIV, poor health seeking behavior, low or no power in the family decisions, low or no power in contraceptive use decisions, and poor knowledge regarding STD/HIV prevention. Conclusions: This program develop educational materials for women of low SES using information from qualitative assessment and perform a successful intervention to encourage STD/HIV prevention. SAnchez Sixto. Calle Paraguay 478, Lima 1, Peri. Phone: (5114) 331396; FAX: (5114) 331396 (31 4 0 549D SlD PREVETION IN IM-RISK POOR WCM24E OF DELHFI - Dr. Senqupta Sushma, Dr. Mathur R., Dr. Yadav N., Ms Sarin E., Ms. Sahi R., Dr. Rao B,AIWC-Delhi,INDIA 550D KAP STUDY ABOUT STDS & FAMILY PLANNING AMONG RURAl YOUTH IN UGANDA. Sentuitse Simrn.Lyomoki S.W,Cujattude D.1 kerere thi.AIDS Ass. The study is based on 12 months work: OBJECTIVES - 1. To assess the prevalence rate of STDs: 2. To Demonstrate the need for providing STD services: 3. To provide counselling to waomen with STDs. 4. To make women aware. of STDS/HIV infection. METHODS-*Five STD centres established in collaboration with Family Welfare Progranmmes of the hospitals. *Individual counselling to STD+ive women. *IEC strategies. RESULTS - 8276 cases secreened, 521+ive for STDs,(prevalance rate 6.6%), 550 random sample tested for HIV none+ive, 4028 women and men contacted in interactive awareness programmes, 428 individual counsellings. CONCLUSIONS - *STD services should be supported by consistent awareness programmes on STDs/HIV and vice-versa, in the corresponding population. *The incidence of STDs/HIV infection is strongly linked with other problems resulting from over population, poverty, disease, illiteracy and gender bias. *Ma)ority of men and many women(housewive and girls) of these cormunities indulge in very very high risk behaviour. *Religion used as vehicle to spread the message may be more acceptable to these people. Dr. Sengupta Suslna, Medical Director,t9aen-in- AIDS ALL INDIA WOMEN'S COFERENCE, 6-BHA(MAN DASS ROAD, New Delhi-ll0 001,india tel:389680/314(R)3291550 (bjective: To determine the knowledge, attitude & practice of rural youths regarding STDs & family planning so as to generate data for planning future interventions. Mthod: Rural youth(n=118)of ages 15-25 yrs from 5 parishes were surveyed from a randomly selected sample of 371 households(36% population).Using a questiomnnaire administered by 40 interviewers,data gathered was analysed using EPIlFO statistical car puter package. Results: 69% reported to have had sex before, majority having started at age of 16years. 93% knew the existence of STDs.97% had heard about AlDS,58% gonorrhea,3L% syphilis.67% mentioned symptaoms like skin rash,genital sores & discharges & pain at micturition.33% did not krnow.13-66% mentioned several modes of transmission of STDs/HIV & 5% didn' t know.0nly 16% new clearly the concept & meaning of family plaming.Althourj many mentioned several methods of contraception,9% reported to the using condorms,86% abstaining & 20i% zero grazing as both means of contraception and avoiding SIDs. Discussion & Cmclusion: Knowledge about SIDs & family planning seems to be high, but so shallow & doesn't seem to be protective against STD/HIV or to promote safe parenthood. There is a need for in depth study of the dynaics of sexual behaviour among youths so as to come up with appropriate interventions. DR. INIdiE S11,P.O.ID( 12785 KfWPLA,TEL.256-OY41-241877, FAX 256-1-241877 LUGA. Iblaerere thiversi ty Students AIDS Control Association.

Page  69 551D AIDS and STD as told to children LAFOREST F.G.(0 / ALEXANDRE G.( I NDIMURUKUNDO N.( / NYABARAHIA L~ AGOtI J.~ / RUBOTI D. / MARORON E.) / ALEXANDRE Y.(Z) 552D Sexual Behaviour & revalence of STD & HIV Infection B. jsotimehin,L.Dare, in Nigerian Adolescents. 0 oengbeMed; University of Ibadan. College of Med; University of Ibadan. This anti-AIDS project was presented, as an idea and as a global campaigning strategy, in a prospectus at the conference on AIDS which was held in Africa, in Marrakech. We have now reached the last stage of the project, which has given rise to two handbooks aimed at: a) children and youngsters (popularizing tales increasing children's awareness of: AIDS, STD, sexual education, how HIV and STD are transmitted and preventive approaches). b) educators (parents and teachers), directions for a pedagogical exploitation of the elements laid out in the first handbook. These two handbooks originally written in French, have been adapted and translated into English, Spanish, Creole and Kirundi, but will be presented in French. (The authors wish to give two presentations: one oral and one through posters). AiMS: - To stop the spread of HIV-AIDS by creating, in time, with the children, a natural barrier against the transmission of the virus and of STD. - To palliate the lack of works of this type in the educational environment of children and youngsters. - To develop the appropriate tools for the primary and secundary level curriculums, and by so doing, institutionalize sexual education and the campaign against STD within the training provided to children and youngsters at school. - To offer a quality tool to parents and teachers in order to be able to respond to the legitimate curiosity of children about the issues of sexuality, STD and AIDS. - To raise funds for the benefit of campaigns against AIDS. METHOD: By using fairy tales and easily understandable symbols the message will be communicated to the children. By identifying with some heroes, the children will be put in situations where a safe choice can be made. PRODUCTS/RESULTS: Production of two pedagogical handbooks, tested and translated into several languages for an international use...Two other volumes in the progress. CONCLUSION: An original and appealing strategy against AIDS and STD, which will provide a first class weapon in the campaign against AIDS at a global level in the coming years. (DCRIPS - FRANCE. Tel: 33 (1), Fax: 33 (1) ~,)Universitd dHAITI fUniversitd du BURUNDI ~PNLS - BURUNDI 1Assemblde Nationale du CAMEROUN (OMS/PNLS - BURUNDI Adolescents in all cultures re probably the most sesually ractive and are the mest esposed to dangers of prevalent sexually transmitted disease given their behaviour. However, there is very little infonrmution about adolescent sessual behaviour and the prevalence of sexually transmitted diseases in this sector of the population in the Sub Saharun AfriHea even though the adult population has been shown to curry a large burden of disease wbich has greeat consequences in term of mortulity. This has becams more striking with the advent of the AIDS pandemic. This study set out to document (a) the sexsal behaviour in adolescent in the South West of Nigeria and (b) the prevalence of kay sexually transmitted diseases within the population. It is the hope that the output will be useful in (a) understanding the dynamics of infection and disease and (b) serve to inform meaningful inteentuion amongst 479 female adolescents aged between 17-24 with a mean age of 19.5 who have at lease high school education, an average of 2 seual partners. They possess high fertility ideals and only 9% regularly use barrier contraception. 1 heir knowledge of STI) is restricted to Gonococcal and HIV infections and 10% of them will admit to haing had a STD as evidenced by discomfort discbtge ulceration. Objectively 4% have T. varinulis iufection, 19% have candidu infection, 0.7'% has N. gonorrhea infection, 4% tested positive in an antigen ELISA for Chlamydia. None of the subjects had a positive VURL test nor were IIV positive. The relevance of these results to current efforts at control STDS in Sub-Sahara African will be discussed. Prof. Babatunde Osotimehin College of Med. University of Ibadan. 234-117135(Phone) Fax. 234-022-417135. 553B OCCULT 01'S IN HIV DISEASE Carroll. Eleanor. Salvato. P., Thompson. C.. Flores, R. Houston Immuno. Institute. Houston, TX USA. 554B BACILLUS CALMETTE-GUERIN INFECTION IN CHILDREN WITH AIDS-Sato HelenaK Ferrazoli L; Gonalves AMF:Pastorino AC:Jacob CMA;Marques HHS;Grumach AS Dept. Pedatrics, Univ Sbo Paulo & Inst.A.Lutz, Sbo Paulo - BRAZIL QBJECTIVE To describe the prevalence of occult 01's in HIV+ patients (pts.) METHODS: 175 HIV-positive gay males. age 23 to 47 provided data. Serum cryptococcal antigen and AFB blood and stool cultures were obtained, regardless of symptomatology, as part of the initial hospital work-up. Data were collected over an 8-month period. Number and Percentage Positive by CD4 Cell Grouping: Ocult s's < 50C- > 200 CD4 rypto(+) 32 (59) 18(33) 2 (4) 2(4) AFB (+) Blood 47 (632) 23 (311) 5 (72) 0 (02) AFB (+) Stool RESULTS: Of the 175 pts. cryptococcal antigen was positive in 54 (31%). 22 (41%) of these pts. denied headache and fever. Blood cultures were positive for MAIC in 75 (43%). 14 (20%) denied fever > 101 F or night sweats. Stool cultures were positive for MAIC in 38 (22%). 16 (42%) of these pts. denied chronic diarrhea (defined as diarrhea > 1 month with no known etiology) or weight loss. CONCLUSIONS: The majority of these OI's occurred at CD4 counts < 100: with 60% occurring with CD4 cells < 50. The asymptomatic presence of these life-threatening infections makes early diagnosis essential. Consequently, routine testing for cryptococcal antigen and MAIC is indicated for pts. with CD4 counts of 100 and below regardless of symptomatology. CARROLL, Eleanor, Houston Immunological Institute Houston, Texas USA TELEPHONE (713) 791-7520 (713) 797-1729 OBJECTIVE: The use of BCG vaccine in asymptomatic children HIV positive remains controversial. The goal of this report is to evaluate the adverse effects of BCG immunization in children with AIDS. METHODS: We reviewed the charts of 124 children with AIDS, admited to Instituto da Crianga during the last 8 years(1985/1993). They were Immunized with BCG(Moreau strain-Fundago Ataulfo de Palva-RJ-Brazil) within 2 months of birth by intradermal Injection. RESULTS: Among 57 children(perinatally infected) vaccinated previously to the diagnoses of HIV infection, only 3(5,2%) developed adverse effects. All of then had a severe cellular immunodeficiency at diagnosis. Two months after immunization one of children developed suppurative lifonode enlargement from wich BCG was isolated. Four monts after Isoniazid therapy it disappeared. The other two cases had disseminated form, 4 and 9 months after BCG, They developed fever, hepatomegaly, linfonode enlargement and one of them had a large local ulcer. One of these two children died with sepsis(not BCG strain) one month before diagnosis and the isolation of BCG in the bone marrow. In the last child BCG was Isolated from linfonode, liver and bone marrow. After Isoniazid, Ethambutol and Rifampicin treatment all the cultures became negatives. CONCLUSION: In Sio Paulo tuberculosis incidence is very high(50/105 inh) and BCG immunization is one of the most important control strategies. The occurrence of few cases of BCG dissemination pointed a big dilema for management of HIV infected chidren. The WHO recomendation must be considered and followed until further evaluations provide data for more precise risk-benefit of BCG immunization in AIDS era. Aquino,MZilda, R.Afonso Jose de Carvalho # 62, SSo Paulo CEP 05451-000 -BRAZIL. Phone:55-11-263.4996 FAX:55-11-853.2602 (31 (31 -I (1 (31 44h w

Page  70 O 555THE CANADIAN RANDOMIZED OPEN-LABEL TRIAL OF COMBINATION THERAPY FOR MAC BACTEREMIA: BLOOD CULTURE RESULTS. PHILLIPSI.P: Chomyc S; Talbot J; Gill J; Raboud J; Singer J; Shafran S and the Canadian MAC Study Group. Provincial Laboratory of N. Alberta, Canadian HIV Trials Network, Canada. Objective: A randomized open-label trial is comparing the efficacy and safety of ciprofloxacin 750 mg BID, ethambutol 15 mg/kg QD, rifampin 600 mg QD and clofazimine 100 mg QD versus clarithromycin 1000 mg BID, rifabutin 600 mg QD and ethambutol 15 mg/kg QD in HIV+ patients with Mycobacteriumn aviumn complex (MAC) bacteremia. This abstract describes the blood culture results observed in the combined cohort and examines predictors and correlates of culture negativity. Methoda: All patients are followed intensively to 32 weeks; survival is monitored beyond this period. The primary outcome is blood culture negativity defined by consecutive negative cultures at least two weeks apart within 16 weeks of randomization. Cultures were performed at a central laboratory using BACTEC and conventional methods. The Cox proportional hazards models was used to assess the effect of baseline variables which were considered to be of prognostic importance. Results: Of the 125 patients who were blood culture positive at local laboratories, 102 (81.6%) were confirmed at the central study laboratory. Of the 94 patients culture positive at baseline with at least 8 weeks of follow-up, 52% became blood culture negative; 28% by 2 weeks, 48% by 4 weeks and 52% by 8 weeks. Only 2 patients relapsed by 16 weeks; 8 others discontinued study medications before 16 weeks, 3 just before death, 3 for compliance, I for toxicity and I for illness. In a logistic regression analysis, there were no variables significantly related to clearing of bacteremia. A comparison of change in symptomrelated health status was made between those who cleared bacteremia by four weeks compared to those who did not. Patients who died were assigned the worst rank score. Differences in favour of the clearance group were seen at week 8 (p=.06), week 12 (p=.01) and week 16 (P<.001). Conclusion: Patients tended to clear early (.4 weeks) or not at all, and rarely relapsed within 16 weeks. Early clearing was correlated with a change in clinical condition as evidenced by changes on a MAC symptom-related health status questionnaire. Dr. Peter Phillips BC Centre for Excellence in HIV/AIDS 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z I Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. 557B MAC PROPHYLAXIS TREAT IND *F.M. GORDIN, P.M. SULLAM, B.R. SMITH, J. SCHOENFELDER, N. DEPIETRO, B. WYNNE. WASHINGTCN, DC-VAMC, VAMC-UCSF, SAN FRANCISCO, PACT, INC., ST. DAVIDS, PA., and PHARMACIA ADRIA, DUBLIN, OH. This program was designed to provide qualifying patients with HIV/AIDS access to rifabutin for the prevention of MAC bacteremia. A total of 2560 patients (302 were previously enrolled in 023/027 controlled studies) received rifabutin prophylaxis. Of these, 95.9% were male and 90.4% were caucasian. At baseline, patients had a mean age of 38.7 (12-78) years, CD4 count of 53.3 (0-270) cells/microliter and Karnofsky Performance status of 86.1 (30-100)%. The recommnended dose of rifabutin was 300rg/day. There was a mean interval of 160 (14-464) days between the baseline and final study visits. MAC bacteremia occurred after a negative baseline blood culture in 4.4% of the patients. The most frequently reported adverse events were: leukopenia, rash, nausea, diarrhea and abdominal pain. Within the design limitations of this study, rifabutin was shown to have an effectiveness and safety profile similar to those observed in controlled studies (023/027). Fred M. Gordin, M.D. 50 Irving St., NW, Washington, DC 20422 202-745-8301 556B THE CANADIAN RANDOMIZED OPEN-LABEL TRIAL OF COMBINATION THERAPY FOR MAC BACTEREMIA: QUALITY OF LIFE OUTCOMES. SINGER J: Fanning M; Thorne A; Turgeon F: Duperval R: Schlech W; Shafran S; and the Canadian MAC Study Group. Canadian HIV Trials Network, Vancouver, Canada Objective: A randomized open-label trial is comparing the efficacy and safety of ciprofloxacin 750 mg BID, ethambutol 15 mg/kg QD, rifampin 600 mg QD and clofazimine 750 mg BID versus clarithromycin 1000 mg BID, rifabutin 600 mg QD and ethambutol 15 mg/kg QD in HIV+ patients with mycobacterium avium complex (MAC). This abstract describes the results observed in the combined cohort in terms of changes in quality of life and how these may relate to laboratory outcomes. Methods: Karnofsky score, symptom questionnaire, bacteremia and the MOS-HIV instrument adapted for MAC were measured at baseline and then at 2 weeks, 4 weeks, and then every 4 weeks thereafter. RESuli 49 of 94 patients with at least 8 weeks of follow-up cultures have cleared bacteremia, 28% by 2 weeks, 48% at 4 weeks and 52% by 8 weeks. A comparison of change in symptoms and change in quality of life was made between those who cleared bacteremia by four weeks compared to those who did not. This comparison was conducted at 4, 8, 12 and 16 weeks. Patients who died were given the worst rank score. MAC symptom-related quality of life was superior in the cleared group at week 8 (p=.06), week 12 (p=.01) and week 16 (p<.001) but not at week 4 (p=.68). The proportion of patients with improvements at 2, 4 and 8 weeks; symptom score - 36%, 32% and 41%; symptom-related quality of life - 40%, 38%, and 49%; and Karnofsky 31%, 39% and 34%. Patients who withdrew because of death or concomitant illness were assumed to be worse while others lost to follow-up were considered as missing. Patients who indicated an improvement in overall symptom-related quality of life, a single question, were compared to those who did not; the symptom-related quality of life questions showed the largest effect size followed by the symptom questionnaire and the Karnofsky. There were significant differences on each of these outcomes for those who indicated improvement compared to those who did not. _Cngil lion Early clearing of bacteremia in this study was a good surrogate for improvement in clinical status although differences in clinical status lagged behind the clearing of bacteremia. The MAC symptom subscale of the adapted MOS-HIV showed greater effect size than a symptom questionnaire or the Karnofsky score. Dr. Joel Singer, BC Centre for Excellence in HIV/AIDS 606-1081 Burrard Street, Vancouver, BC, CANADA V6Z 1Y6. Telephone (604)-631-5305; Facsimile (604)-631-5464. U'7 UJ1 U'1 wJ U0 U' 558B CLARITHROMYCIN FOR THE PREVENTION OF M.AVIUM COMPLEX IN AIDS, Pierce. Mark*, Heifets L*, Crampton S*. Vanderbilt U., Nashville, TN'; Nat'I Jewish Center, Denver, CO*; Abbott Labs, Chicago, IL*. OBJECTIVE: The main objective of this prospective, randomized, double-blind, placebo controlled, multicenter trial is to determine if clarithromycin (clari) can prevent disseminated Mycobacterium-avium Complex (dMAC) infection in AIDS patients. METHODS: After informed consent was obtained, HIV-infected patients with CD4 < 100 cells/mm3 who met the inclusion criteria were randomized to receive clari 500 mg BID or placebo. Blood specimens for mycobacterial culture and sensitivity were obtained at screening, days 1 and 8, then every 4 weeks. Clinical and laboratory evaluations were periodically performed to assess clinical status, toxicity, compliance, and dMAC-related symptoms. RESULTS: This trial is ongoing and we report here the preliminary data and results of breakthrough isolates examined to date. World-wide enrollment since 11/92 totals 675 patients; 333 in the US and 342 in Europe. Data is available for 613 patients. Median followup as of 12/1/93 is 6 months; 4.6 months for the 420 patients remaining on study. 51 patients have experienced adverse events possibly related to study drug (clari or placebo). There have been 38 patients with positive mycobacterial blood cultures, 23 in the US and 15 in Europe. These occurred after a mean of 19.7 weeks on study (range 8 days - 36 weeks). Antimicrobial sensitivity data is available for 22/23 US isolates and reveal that 95% remain sensitive to clari (MIC < 2.0 ug/ml). One isolate was resistant (MIC > 512 ug/ml). CONCLUSIONS: This trial remains blinded, thus we cannot determine if Clari prevents dMAC in AIDS. However, prophylactic clari appears to have acceptable tolerability in this population and the high incidence of resistance seen when clari is used as monotherapy for active infection has not appeared. The latest efficacy and MIC data will be presented. Mark A. Pierce, M.D.; 1211 21st Avenue South, Ste 539, Nashville, TN, 37212; Ph (615) 936-1174; FAX (615) 936-1170

Page  71 559C THE BASICS BOX: A BOX FOR SAFER INJECTING AND THE PREVENTION OF NEEDLE STICK INJURIES. Doornlnck, M. van; and 'A. de Loor; NIAD; Advlesburo Drugs; The Netherlands. Objective: The development of a box for injecting equipment that; (1) supports drug users to prevent high risk situations (drugs available but no sterile equipment), (2) reduces the risk of needle stick injuries, (3) reinforces the Dutch syringe exchange programs, (4) fits in existing injecting practises and (5) is attractive for drug users. Methods: After studying the injecting practises a prototype was developed and pretested among 48 injecting drug users in 7 Dutch cities. The specifications for the box were determined and a first edition was produced. Results: Drug users find it important to be prepared for future injections but in practice only 31 of the 48 respondents always or almost always had syringes on them. Less than half of them had a spoon, a spare needle, alcohol swabs, filters, sterile water or ascorbic acid on them. The box was seen as supportive by more than two third of the respondents. Drug workers were satisfied with the special compartment for used syringes. Conclusion: The last version of the box (filled with two syringes, a spare needle, two bottles sterile water, ascorbic acid and some alcohol swabs) meets the wishes of most responoents and is found supportive and attractive. A special compartment for safe disposal of used syringes emphasises the difference between used and new syringes, reduces the risk for needle stick injuries and supports drug users to keep used needles for disposal at syringe exchange programs. DOORNINCK, Maarten van; NIAD, project 'AIDS and Drug Use' Postbox 725, 3500 AS Utrecht, The Netherlands. Telephone (31) 30 341 300; Telefax: (31) 30 316 362 561C DECLINING RISK FOR HIV AMONG IDUs IN KATHMANDU: IMPACT OF A HARM REDUCTION PROGRAMME: Shiba Hari Maharian*.Aaron Peak* ** Sujata Rana*. Nick Crofts**: *Lifesaving & Lifegiving Society, Kathmandu. Nepal.**Macfarlane Burnet Centre for Medical Research. Melbourne. Australia Objective: to measure the impact of a needle/syringe exchange programme on HIV prevalence and risk behaviors among IDUs in Kathmandu over a three year period. MAethods: All IDU clients of LALS were asked to participate in the studies if they had injected within the preceding 30 days. A questionnaire was completed and blood taken by the Community Health Outreach Workers who had previously establishcd a rclationship with the clicnt. All samples were tested for anti-HIV I & 2. HBsAg and VDRL. Results: 127 IDUs were enrolled in 1991. and 200 in 1993. There was no difference in demography between 1991 and 1993 samples: 90% cf94% male (ns); median age 25 years. age at first injection 22 years: mean monthly incomes 1211 Rs and 1318 Rs (ns). Significant decreases were observed in drug use, HIV risk behaviour: mean frequency of injecting fell from 24.4 per week (sd 10.8) to 17.0 (sd 9.5) (p<0.001). 1991 1993 Decrease p per week: unsafe injections per person 13.9 7.0 50% (0.00) persons shared with 3.9 3.1 21% (0.00) times shared with person 4.9 3.5 29% (0.00) Little change was seen in sexual risk behaviour, with the proportion reporting never or rarely using condoms with prostitutes: 84% (32/38) in 1991 and 91% (31/33) in 1993. HIV prevalence remained low over the years: 1.6% (2/127) in 1991. 1.0% (2/200) in 1993. Conclusions: Clients of this harm reduction programme in Kathmandu have significantly modified their HIV risk behavior in relation to drug use. from a starting point of high risk. The programme was instituted before HIV had spread. and seems to have achieved a sufficient level of behaviour change to have kept HIV prevalence low. Our indigenous model of HIV prevention tlhrough harm reduction in a developing country is proving effective, culturally appropriate and comparatively cost-effective. Mr. Shiba Hari Maharjan. Lifesaving and Lifegiving Society Post Office Box 3517. Kathmandu. Nepal Telephone: 977-1-413976. Fax: 977-1-417133 560C CHANGES IN HIGH-RISK BEHAVIORS AMONG HETEROSEXUALS FOLLOWING ALCOI IOL AND DRUG TREATMENT. Avins. Andrew L.. Woods WJ, Lindan CP, Boscarino JA,l Hudes ES, Clark W, Hulley SB; Center for AIDS Prevention Studies, University of California, San Francisco, USA OBJECTIVE: We have previously reported that urban heterosexual alcoholics and those who use noninjection drugs are at increased risk for HIV infection compared to individuals without these addictions. We now report the results of one-year follow-up of these subjects to examine changes in substance use and high-risk behaviors. METllODS: Consecutive clients entering 5 San Francisco alcohol treatment centers underwent a baseline interview and testing for HIV antibodies (response rate: 68%; overall seroprevalence, 5%). At baseline, 89% of subjects also reported using non-injection drugs within the prior year. We reinterviewed 687 (79%) heterosexual subjects approximately one year later. RESULIS: Of those subjects contacted, 58% completed their treatment program (outpatient: 21%, residential: 76%, p<.001); 182 (26%, 95% CI: 23%, 30%) remained completely abstinent from alcohol and 233 (35%, 95% CI: 32%, 39%) of subjects who had previously used drugs abstained from drug use during the follow-up period. Among subjects who reported the respective risk behavior at baseline, subjects who abstained from alcohol were less likely to continue having multiple sexual partners (OR=0.52, p=.02), having nonmonogamous partners (OR=0.44, p=.02), or to inject drugs (OR-=0.13, p<.001) compared to those who continued to drink alcohol. Among those with multiple partners who previously had inconsistent condom use, abstainers from alcohol were more likely to use condoms with all sexual encounters (OR=3.0, p<.01). Subjects who stopped drug use were also less likely to continue having multiple sexual partners (OR=0.46, p<.01) or non-monogamous partners (OR=0.34, p<.01) compared to those who continued to use drugs. CONCLUSIONS: Successful alcohol and drug treatment is associated with large reductions in high-risk behaviors. Providing adequate access to substance abuse treatment is essential for limiting the spread of HIIIV in heterosexual alcoholics and non-injection drug users. ANDREW L. AVINS, MD MPH 74 NEW MONTGOMERY ST., SUITE 502 SAN FRANCISCO, CA 94105 USA 415/597-9196 (P11); 415/597-9125 (FAX) 562C INTEGRATION OF HIV PRIMARY CARE IN METHADONE MAINTENANCE TREATMENT PROGRAMS(MMTP) IN HARLEM. L.Govia, M.Scimeca, W. El-Sadr, G.Patel, N.Nkanga, L.Ampy, Harlem Hospital Center N.Y.N.Y. OBJECTIVE: Four affiliated MMTP programs include a significant proportion of HIV-infected persons. Many have not received care. A program was initiated to integrate HIV care in the routine clinic procedures to ensure access and availability of these services.METHODS: The program has included:1)a mobile staff of a program manager, two internists, a dermatologist, a nurse and an administrative assistant 2) integration within the substance use treatment 4)linkage with on staff, health educators,social workers,addiction counselors 5)access to referral:peci.alties 6)incentives 7)access to clinical trials 8)promotion of non-judgmental atmosphere 9)linkage with other programs to complement services.RESULTS: In one year, the program has enrolled 295 HIV infected patients, 58%men 42%women of a total 1285 IDU's, conducting 1,000visits. Good compliance with follow-up rates of almost 100%. CONCLUSIONS: Provision of on site HIV care is an effective method of early interventions for HIV-infected IDU's. Development of programs that focus on health maintenance, provide needed services and are integrated in the drug treatment programs can have a powerful positive effect on the patients' health and well being. LESTER M. GOVIA, 506 LENOX AVENUE WP126 NEW YORK, NY 10037, TEL# (212) 939-3910 FAX/ (212) 939-2670 cyl ~n 0j1 ') 0

Page  72 563C TRAINING PEOPLE TO REACH INJECTING DRUG USERS IN IN.E.INDIA -,NAGALAN EXPERIENCF. *** Basu,AmitR;Ghosh,S;Zaveri,Y;Roy,T;Panghal,J.S. Objectives:Training in Outreach for intervention among injecting drug users (IDUs)in N.E.India is important as they account for half of the HIV positive persons there. A training team of Outreach & Counselling;IEC and Monitoring & Evaluation Consultants impart training to the NGOs which is specially designed to reach a hidden population like IDUs.Authors,in their attempt to assess the impact of this training also try to conceptualise training in outreach.Hethods:Mainly Experiential Training Methods - in the form of Group Work, Skills Building Exercise, Lecturettes and informal individual sessions. Evalua t ion is done through Post-Training questionnaires,Follow up group sessions and Field Supervisions. Out of 5, 1NGO from Nagaland is selected as there had been less exposure to any form of training compared to NGOs in Manipur. Results:Out of 9 participants 6 of them said they have gained skills and 3 said "don't know".All of them said to have gained knowledge/insight to their work.Within two months the trainees contacted 30 Male & 10 Female IDUs. Women Outreach Workers are helpful for contacting women IDUs.Conclusions:Delearning the Abstinence Model is an important area of training.Training philosophy plays a vital role in empowering trainees by exploring & sharpening their own potentials.Attitude & Values troini s the kp factor to reach the target audience when counselling sklls flelp to ma ita n the contact. BASU,Amit R. BE 318,Salt Lake,Calcutta 700 064, INDIA Telephone: (091)-033-349805 FAX: (091)-033-376290. 564C THE PUBLIC HEAL'ITH IMPACT OF NEEDLE EXCHANGE PROGRAMS (NEPS). Lurie Peter,1 Sorensen J,1 Lane S,l Kahn JG,1 Guydish J,l Foley J,l Chen D,1 Bowser B,1 Jones TS,2 Reingold AL.3 lUniversity of California (UC), San Francisco; 2Centers for Disease Control and Prevention; 3UC, Berkeley. Objective: To conduct a comprehensive assessment of the public health impact of NEPs in the US and abroad. The evaluation was funded by the US federal government. Methods: 1. A review of over 2,000 documents on NEPs including published articles, conference abstracts, unpublished materials and newspaper articles; 2. Site visits to 23 NEPs in four countries; 3. Mail survey of US NEPs not visited; and 4. Mathematical models permitting the assessment of the effectiveness and cost-effectiveness of NEPs in preventing HIV infection. Results: As of September 1993, there were 37 NEPs in the US, half of which were legal. They had distributed over 5.4 million syringes and had referred thousands of injection drug users (IDUs) to drug treatment programs. Based on our review we concluded: 1. The majority of studies of NEP clients demonstrate decreased rates of IIIV drug but not sex risk behavior, 2. Available data provide no evidence that NEPs increase the amount of drug use by NEP clients or change overall community levels of noninjection and injection drug use; 3. There is limited evidence that NEPs are associated with reductions in subcutaneous abscesses and hepatitis B; 4. Models developed by ourselves and others suggest that NEPs can prevent significant numbers of HIV infections, in almost all cases for less than the lifetime cost of treating an HIV-infected person; and 5. Although the need for large sample sizes and the inability to randomize IDUs to NEPs preclude direct demonstration of reduced IIIV incidence, findings 1, 3, and 4 above, combined with the plausibility of the intervention (removing potentially infected syringes from circulation, analogous to mosquito eradication for the prevention of malaria), suggest that it is likely that NEPs reduce HIV transmission to IDUs, their sex partners and their offspring. Discussion and Conclusions: We concluded: 1. The US government should repeal the ban on the use of its funds for NEP services and should commit substantial funds to NEP services and research; 2. Laws restricting the availability of clean syringes for IDUs should be revoked; and 3. The availability of sterile syringes can be further increased by the sale, distribution or exchange of syringes by pharmacists. PETER LURIE, MD, MPII 74 NEW MONTGOMERY ST., SUITE 600 SAN FRANCISCO, CA 94105 USA 415/597-9138 (PH); 415/597-9213 (FAX) U' CA) Un 0) 0) 565D DEVELOPMENT OF GENERIC - IEC PACKAGES FOR TARGET POPULATIONS IN INDIA Larivee C.*, Kashyap,Neera*, Prashar,A.,**, Panghaal, J.** * WHO,** National AIDS Control Organization, India 566D BRAZILIAN PLAYBOY AGAINST HIV: A PIONEERING APPROACH Lemes. Conceico *; Varella, D.**; Ilearst, N.***; Castilho,E. **** Editora Abril*, Unip**, CAPS***(San FranciscoUSA), Fiocruz****. Brazil. Objective: To provide NGOs and organizations working with target groups with standardized materials and messages: especially materials for a non-literate audience. Methods: Experienced communication professionals were placed with NGOs working with particular target audiences. Using interactive participatory techniques, the communication needs of the NGO and the target audience were examined and appropriate visual print materials developed. The resulting package was then tested with other NGOs in other states dealing with a similar target audience. The images and materials were adapted for more universal use and a manual on use and adaptation of the materials produced. Results: A series of packages using this approach have been developed for the following target audiences: commercial sex industry; injecting drug users; truck drivers; STD clinic attenders. Plans are underway to produce packages for target audiences as well. Packages are being distributed through NGO networks, private sector initiatives and state and local government. The packages generally contain education cards, leaflets, posters and a manual on use and adaptation. Discussion and Conclusions: NGOs and community organizations need communication tools to effectively support outreach, peer education and awareness raising efforts. Generic packages provide organizations with a basic format for consistent visual messages and a base from which specific materials can be developed according to felt need. Larivee Carol, Health Education Specialist, Global Programme on AIDS, World Health Organization, New Delhi, India Telephone (91)(11)(331 7519); Telefax (91(11)(331 8607) ObIjective, To clarify men's risk behaviors for HIVY infection and to arouse their awareness about the need to prevent infection,Brazilian Playboy (320,000 monthly circulation) developed a project that resulted in a 13-page special report, "AIDS -The Distance Between Intention and Action," published in January,1994. Methods: The magazine used three resources. First: it conducted a national survey to evaluate knowledge, attitudes, and sexual practices of the Brazilian nan related to AIDS to provide solid inforxmation on which to the base the report. This was dlone with the assistance of specialists from important scientific institutions. Second: it interviewed 25 fatuous Brazilian wotent and men, including P'et, the soccer star. Their opinions were used to endorse safer sex and desmystify prejudices related to condoms and AIDS. Third: it interviewed 37 specialists who work with AIDS to give scientific information. The intiforuation collected was presented in the article in a factual and sex-positive manner. Results:This combinationt resulted in an in-depth and pioneering approach for Brazilian media. Letters and phone calls [frouits readers have praised the project atnd the way it was presented, in iharmony with the magazine's editorial line, showing that prevention can be pleasant and erotic. Many said the report opened their eyes about the real risk of men being infected in heterosexual intercourse. The survey (results presented elsewhere at this conference) gives the best data yet available on the sexual behavior of Brazilian men and demonstrates that most are not protecting themselves against AIDS adequately. Playboy has shared these results with other Brazilian AIDS prevention organtizations.With this repxrt, the Brazilian edition has broken the international Playboy taboor of not discussing AIDS. Conclusions: This project supplies a nRalel of co peration between the mtedia anuld the scientific coniununity. Simitlar efforts, if conducted with ethics andl responsibility, can benefit everyone and help prevent IIIV infection. LEMES, ConceiVio, Revista Playboy,Editora Abril Ruta Geraldo Flausitto Gones, 61, Sio Paulo, SP, Brazil CEP 04573-90 Telephone 55 11 8525601: Telefax 55 11 8525601 or 55 11 5345396

Page  73 567D ARGE!JNTINA:USE OF THE PREVENTION },!DBOOK IN THE FORMATION OF HIIAN RESOUR SES BURGOS," ric; SIROTTI,C,.; RE"SIN,N., DIAIZ,A.; MORENO,S. -R.E.D. FOUNDATION PROBLEM: Argentina have not got a prevention official campaign and there are not actions for to prepare human resourses.Instead, from differents institutions (NGO's, schools, clubs, trade unions), are made in the whole country diffusion activities in direct contact with population. The actions have not got coordination and common axis, wich impede the development social resourses. HIPOTHESIS: Trough an education text by distance, is possible to form local staff of Communitary Prevention Agents (CPA), between proffesionals and non proffesionals members of institutions with social influence. This will make easy the homogeneity of activities in perspective of form a social prevention network AIDS. METHOD:Trough agreements with municipal governments, trade unions and social institutions, the handbooks have been distributed between teachers, social leaders, health agents, schools, etc. RESULTS: In 1992/ 1994, 8000 hadbooks have been distributed. Was reported the capacitation of 1,800 CPA. There are local and regional programs of formation of human resourses based on the handbook, with incidence in an area with 5 million people. More of 500 schools, 200 health unities, proffesional associations and provincialand municipal AIDS programmes have informed about the handbook utilization as basic text for the prevention AIDS planning. CONCLUSIONS: The use of a text based on the social prevention experiences, available for the use of people with or without proffesional formation, make possible the multiplication of the social and human resourses for the AIDS prevention and care. DIAZ, Arnoldo Fundacion R.E.D. Telefax: 54 1 962 4628 Anchorena 970 -l"A"- 1170 Buenos Aires, Argentina 569D ETHNIC YOUTH'S SOCIAL NETWORK STRUCTURE CAN INFORM HIV PREVENTION. Lovely. Richard H,. Liebow. EB. Battelle Seattle, Vera. M*. McGrady. GA**. Mueller. C, & Mann, E***. Klovdahl, AS****. *Univ. Puerto Rico, **Morehouse School of Med., ***Univ. Hawaii, ****Australia Nat. Univ. (Obiective: To determine if social network data can inform HIV primary prevention in different groups of minority youth at risk for HIV/AIDS. Mehius: Two populations of urban adolescents (12-15 years old), stratified by age and gender. were studied: African American youth in Atlanta Georgia and Puerto Rican youlh in San Juan. Puerto Rico. Initial respondents (n = 41) to 5(1) were a probability sample representative of the population of youth in the census tracts studied. They were administered a survey including probes for their HIV risk-related hehaviors and probes for the names of associates. in general. and associates who also engaged in HIV risk-related behaviors. Subsequent reslpondenls were suampled by randomly selecting a nune from the list of associates provided hy the initial respondents. They were then administered the same interview. Second. third and fourth node respondents were all sampled by this link-tracing "random walk" methodology. Resulis: Findings indicate that it is feasible to implement this unique sampling methodology so as to characterize the social networks of these youth. and to obtain valid data with regard to alcohol/drug use and sexual activity from these youth's and their associates. Socidal network properties of youth in Atlanta and San Juan youth were quite different. Reachahility (the probability that any two randomly sampled respondents can be connected by a series of links) was 1.01 in San Juan and approximately 11.5 in Atlanta. Conclusion: Based on these different social network properties (everyone in San Juan is connected to everyone else in the social network). different intervention strategies are implied bfor the two populations studied. In San Juan. changing peer group norms should have the effect of influencing all who are connected (the entire conmmunity): in Atlanta a more decentralized approach is required. 568D The Effect of Education Campaign for AIDS/HIV through Mass Medias from 1992 to 1994. Migiko Maeda, T.Inagaki, A.Ishii, M.Ashizawa, M.Minamltani; Tokyo Metropolitan Government Research Group of AIDS/HIV. Objective: To investigate the alteration of knowledge, attitude and behavior against AIDS of adolescents aged at twenties after the education campaigns through mass medias. Methods: We conducted 3 times of AIDS preventive education campaign in Tokyo. The 1st one was done in October 1992 by Tokyo Metropolitan Government (TMG), the 2nd was in February 1993 by Ministry of Health and Welfare and the 3rd was in November 1993 again by TMG. Before and after these campaigns, we had the chance of 4 times to investigate the people's knowledge, attitude and behavior against AIDS. The subjects were randomized sample of twenties of both sexes living in Tokyo. Results: TI7-Imost all subjects know that the Infection route of AIDS is sexual Intercourse. 2, From '92 to '94, it seems to be increased their amount of knowledge with AIDS, because most people know that AIDS dose not Infect through casual contact with other person. 3, In the same period, most people have become recognized exactly the facIlities where they can get the examination of IIIV antibody. 4, There seemed to be Increased the number of people who showed an acceptable attitude towards HIY positive person. 5, It seemed to be decreased the number of people who did not change their risky sexual behaviour. Discussion and Conclution: 1, However it is impossible to isolate specific advertising effects, the results indicate that the campaigns were successful in the alteration of people's knowledge, attitude and behavior against AIDS. 2, We have to continue the same education campaign, because the change of one's life style is not so easier compared development of one's knowledge which related serious health problem. Migiko Maeda, AIDS/HIV-Infection Control Office, Division of Medical Welfare, Bureau of Public Health,Tokyo Metropolitan Government,2-8-1Nishishijuku Shinjuku-ku,Tokyo 163-01 Japan, Tel 03(5320)4487, Fax 03(5388)1432 570D "AIDS RISK" AND "RISK POLICY" IN TH MEDIA: COPARIING FRANCE, GERNY N AN HE UK IN iHE 90'S Preda,Alexandru, Bielefeld University, Germany OBJECTIVE: The study compares print media representation strategies for risk and risk policy in three Western European countries with similar epidemic patterns. The years 1989-93 are covered. The main research question is if and how these media approaches are mutually consistent at internal and comparative levels. METHODS: Print media from France, Germany and the UK were selected according to: national audience, circulation level and impact on opinion and policy making. Reports and articles on AIDS were first quantitatively analyzed. The variables 'risk' and 'risk policy' were operationalized along the dimensions: level, seltrum, typc, localisation and then analyzed with semantic and rhetoric analysis techniques. RESULTS: Internal and mutual inconsistencies and paradoxes are shown and discussed for lggel, ~pgs nm and localisation of 'risk' and 'risk policy'. The implications for the public perception of risk are discussed on the ground of comparatively analyzing national differences for lcel and spectrum. DISCUSSION: The results are significant for understanding how the frames of the public perceiving AIDS risk and risk policy are constructed, as well as for prevention policy. How the public reads these media representations should be a further research theme. Alexandru Preda, Faculty of Sociology, Bielefeld Lhiversity, Germany, Tel: 0521-106-4613, Fax: 0521-106-5844 0 0) 0 v:

Page  74 571D THE RESPONSE OF MULTINATIONAL CORPORATIONS TO HIV/AIDS. Albers. Carolien: Tarantola, D; Bezmanilovic, B; Barese, P.; Francois-Xavier Bagnoud Center for Health and Human Rights, Harvard School of Public Health, Boston, MA, USA. Objective: To analyze the scope and magnitude of the response of major multinational corporations (MCs) to the HIV/AIDS pandemic in view of the forthcoming publication of AIDS in the World., Vol.2. Method: A survey of 50 MCs with headquarters in 17 countries, was conducted between 1/94 and 3/94. Questionnaires were designed in consultation with members of the US National AIDS Coalition and sent to MC's headquarters. Respondents were human resources managers, directors of health services or corporate HIV/AIDS program directors. The survey covered corporate HIV/AIDS related policies, training/prevention programs, attitudes, financial and organizational impact of HIV/AIDS. Results: As of 2/1994, preliminary analysis of 20 returns indicate that the response of MCs was motivated both by a concern for their staff and for possible liability. Two-third of the MCs have a training/prevention program established for employees working in domestic facilities, and half of the MCs also had a program for their employees in foreign offices. All MCs reported to provide mandatory HIV/AIDS training to their management. Although participation in training programs, for other staff had not been mandatory in most cases, two-third of all employees attended. Programs consist mainly of distribution of written materials, presentations, counseling and management training. They have often been started out of concern for corporation's employees, especially for those who travel. MCs with a policy regarding employees with HIV/AIDS, stated that no specific restriction was applied domestically to applicants or employees with HIV infection and that the same policies and services were applied to AIDS as to any other serious illness. Restrictive policies were, however, applied to employees to be assigned abroad. Companies reported that production or labor costs have not increased as a result of HIV/AIDS. Conclusion: MCs are responding to the HIV/AIDS pandemic by developing policies and prevention and training programs, especially for their domestic staff, those expected to travel and those assigned to foreign countries. The analysis performed on all retumrns will be presented in detail at the Conference. Carolien Albers, Francois-Xavier Bagnoud Center for Health and Human Rights, Harvard School of Public Health, 8 Story Street, Cambridge MA 02138, USA. Tel. (617) 496 4376- Fax (617) 496 4380 572D Peer education in the workplace by sembers of the positive and living squad In Luaks, sambia: Is it effective? R.Kagssley*.**. J.ukoosha***, D.Cbipanta***,W.Eulu***, J.Bennett*, P.Oodfrsy-Paussett**,**** K*Rare-Zmbart Project. Lusaka **London School of Hygiene and Tropical Medicine ***Kara Counselling and Training Trust, Lusaka, ****Zasbart Project. Department of Medicine, University Teaching Hospital, Lusaka Background: The "Poitive and Living Squad" (PALS) was established in August 1991. They are individuals who live openly and positively with HIV. Some members of the PALS have undergone training in HIV education end counselling and conduct outreach prograsmmes at local workplaces. During 1992-1993 the PALS visited over 850 workplaces and spoke to more than 65,000 people about HIV. Objectives: I. To collect baseline knowledge, attitudes and practices (KAP) information from those attending outreach sessions in a sample of workplaces. 2. To develop appropriate HIV education in this setting and to plan the focus of follow up outreach. 3. To see if HIV KAP changes following HIV outreach. Methods: Anonymous self-admlnistered, pre-tested questionnaires were distributed to a sample of the workers prior to the outreach sessions and collected in before the start of the sessions. 3-6 months later a second outreach session was carr ied out and the same questionnaire technique repeated. Results: We analysed 146 questionnaires from 5 companies in Lusaka prior to the introduction of an education programme and 187 questionnaires from the same companies 3-6 months following the education programme. All the questionnaires were returned. There were no significant differences in the demographic details of respondents in the pre and post education groups. Knowledge about HIV transmission was good at the start of the project but improved significantly. Therewas also improvement in attitudes to HIV. Prior to education 34% of people felt that those with HIV had lead Immoral lives, this fell to 16% following the education programse. 26K of people felt that those with HIV should be isolated and this fell to 1o following education. 66K of thought that people with HIV should have the equal opportunities, this rose to 86% poet education. Condom use increased from 46% to 64% but use with regular partner remained low. Prior to HIV education 39% thought that condoma reduced sexual pleasure and this attitude was unchanged. Conclusions: 1. Knowledge about HIV transmission Is high in the working population surveyed and improved with HIV education. 2. Attitudes to those with HIV improved significant following outreach programmes conducted by the PALS. 3. Although condos musage Increased many people continued to express negative attitudes towards condoms. David Chipanta, Kara Counselling and Training Trust Po Box 37559 Lusaka Zambia Phone +260 1 229847 Fax + 260 1 22984% u1 -4h v 573D AIDS EDUCATION AND INTERVENTION TRIALS AfONG YOUTH IN FACTORIES: A PILOT PROJECT. 574D EDUCATION PROGRAMME FOR TRANSPORT STAFF IN WORK PLACE-Dr.KRISHNAMURTHY PALANIAPPAN Abstract JS.IJTVlES: To provide knowledge on AIDS and motivate attitudes and preventive behaviours. To conduct rapid research and to design models for education and intervention among young factory workers. To pilot a study and intervention trials to recommend for expansion throughout, the country. lE72tiQ: After contacting and obtaining agreement from owners/managers of factories, a mini-survey was conducted with 300 workers to determine their level of knowledge and awareness of AIDS and high risk behaviour. A series of indepth interviews with 16 workers and group discussion with 8 groups were conducted to find out their possible motivation for prevention and their acceptance of interventions/media. The data was used as a baseline for evaluating change after interventions and to modify the intervention education strategies and content. BErllIS: The study showed that the groups of factory workers which were not involved in the AIDS prevention had a different level of knowledge, attitudes,and behaviour related to AIDS prevention than the groups which received the intervention and the methods used in the intervention acheived a level of success. The information we collected also showed that the best kinds of media for this purpose were videos and the informational cartoons, which were also of special interest to the study group. COCILUSI(US:The project aims to conduct a pilot study and intervention trials among the youth in a factory of Khon Kaen. With 2 rounds of trials and revisions, the project discovered that the best kinds of media for this purpose were video and informational cartoons. It is hoped that the models will be adopted by relevant government and non-government agencies to be used in factories throughout the country. CtsaaclaL Sakoodbavat Faculty of Medicine Khon Kaen University, Khon Kaen. Thailand 40002 Fax.043-243064 Until 1985 AIDS was a disease alien to India. During 1986 the first case with HIV/AIDS was reported. Some occupational groups are classified as high risk group based on the sexual behaviour pattern. The study is among a group of transport staff working in Public Undertaking. Present exercise is on a methodology of reaching people in their work spot in a working atmosphere. The results of KABP survey:96% are marriedage group 20-50 years- 35 X' believed AIDS can not be cured-40% believed condoms are only for contraception-OO% do not have an idea on the long incubation of HIV-58% never experienced sex before marriage - by and large inter personel discussion about sex among to male is prevalent. Intervention through group meeting and counseling in the depots where they are working is preformed. Educational programmes are done in a bus stationed in the depots creating a near working atmosphere. Follow up visits arranged. The results are encouraging and recommended for replication in all transport sectors. Dr.KRISHNAMURTHY PALANIAPPAN,JOINT DIRECTOR STATE AIDS PROJECT CELL, MADRAS.(INDIA)

Page  75 575D A MULTI-CENTER STUDY ON THE CHANGE OF KNOWLEDGE, IMAGINARY AND BEHAVIOURS IN A WORKING ENVIRONMENT THROUGH A PREVENTION PROGRAM. Agnotetto, Vittorio*; Ceserant, N.*; DoAlessandro, A.*; et at.; *L.I.L.A., MIlan, Italy. 576D AIDS POLICY AND EDUCATION AT WORKPLACE IN JAPAN: ONODA Kaori, FUKUWATARI Y, MUTO T (Juntendo Univ.), SAKURAI Y (National Medical Center), UMETADA Y (Sanwa Bank), NAGUMO A (Nippon Express) Objective: To compare the improvement in knowledge in groups of workers and trade union delegates of chemical and metal-mechanic industries and of the public adain. through a prevention program, with the verifiable changes in control groups not exposed at aimed preventive programs in the same period of time. Nethods: A sample of workers and trade union delegates, statistically significant, was picked out in Lombardy and Campania regions. The study identified, through stratified randomization: Sample groups (A), to whom a questionnaire and a symbolic representation test were aubmitted before and after the prevention programs. Two control groups (B and C) for each of the sample groups (A), to whom the questionnaire and the symbolic test were submitted in absence of any prevention programs. Results: In the sample groups (A), knowledge and imaginary improved after the prevention program; in the check groups (8 and C) knowledge -after one year- appeared In some cases to have worsened: e.g. %OX -> 2,86% believed that the virus can be transmitted through toilettes; 0% -> 2,99% believed in HIV air transmission; in the imaginary test groups B and C linked the word AIDS with the words mistery and fatality, white A groups with protection and silence;groups 8+C, differently from A, thought it necessary to tell the employer of HIV+ colleague. Discussion and conclusions: Direct info aimed at specific target improves knowledge and helps solidarity and rationality. Mass-media exposition and publictitery campaigns atone -aimed at the general population- may lead to a decrease of knowledge and develop anguish and discriminatory behaviours. Information and prevention strategies utilized up to know must be modified. Three-years project ('90-193) financed by Istituto Superiore Santti (State Health Department). Agnotetto Vittorio, L.I.L.A. V.le Tibaldi, 41, 20136 Milan, Italy, tel 02/58114980, fax 02/89400941 OBJECTIVE: To identify the current situation of HIV/AIDS policy and education at workplace in Japan. METHODS: Questionnaires were mailed to personnel managers of all companies listed on the Tokyo Stock Exchange in June 1993. Response rate was 37.5%(n=620). RESULTS: About 37% of the companies have taken some actions to deal with HIV/AIDS. Nearly 40%, however, were either undecided or had not thought of it. Of companies which are taking actions, 91.2% chose the education for HIV prevention, and 52.2% selected the education to reduce prejudice or discrimination against HIV/AIDS as their main activities (Multiple choice). The department of the personnel management(55.6%) and industrial physicians(44.0%) were chosen as main educators. DISCUSSION AND CONCLUSION: The results revealed that the importance of AIDS policy is not fully recognised by responded companies. It is suggested that Japanese companies should realise HIV/AIDS problems as real issues in the workplace. ONODA Kaori, Juntendo University, 2-1-1 Hongo, Tokyo, Japan. Tel 03-5802-1049; Fax 03-3814-0305 577A QUANTITATION OF HIV-1 RNA BY BRANCHED DNA ASSAY DURING LONG-TERM ZIDOVUDINE THERAPY Cotte Laurent, Trabaud MA, Bissuel F, Trepo C, INSERM U 271 - FRANCE 578A DIRECT DETECTION OF HIV-1 DNA OR RNA AT THE SINGLE CELL LEVEL BY IN-CELL PCR. GOROCHOV G.,PARIZOT C.,DEBRE P.,AUTRAN B.,ANGEVIN E. Lab.Immuno.URA CNRS 625 CHU Piti6-Salp6tribre, 75013 PARIS, Fr. Objective: To assess HIV-I RNA levels in plasma by branched DNA (bDNA) assay in patients receiving long-term Zidovudine (AZT) therapy and in a placebo group. Methods: 36 initially asymptomatic IIIV infected patients enrolled in a controlled trial receiving AZT Ig/day (n= 17) or placebo (n= 19), followed for 4 years. Quantification of IIV-1 RNA in plasma using bDNA signal amplification assay (Chiron) before treatment, and 0.5, 1, 2 and 3 years after initiation of the study. Results: HIV-1 RNA was detected at enrolment in 2/17 patients in the AZT group and in 1/19 patients in the placebo group. Less than 15% of the patients in AZT group had detectable HIV-1 RNA during the first 2 years, while this proportion regularly increased to 36% at 2 years in the placebo group. At 3 years, HIV-1 RNA was detected in 50% patients in both groups. Twelve patients (AZT group: 3; placebo group: 9) progressed to AIDS during follow-up and HIV-1 RNA increased in 10 of them. Twenty-four patients (AZT group: 14; placebo group: 10) remained asymptomatic or paucisymptomatic during the 4 years follow-up. Three of these 24 patients exhibited transiently low levels of HIV-I RNA, 4 patients turned HIV-1 RNA positive only at 3 years and 17 gave constantly negative results. Discussion and Conclusions: HIV-I RNA quantification using branched DNA technology correlates with clinical progression in HIV infected patients. AZT therapy delays positivity of the assay during the first 2 years, but this difference is no longer observed at 3 years. These results suggest that the transient benefit of AZT is limited by breakthrough after 2 years. Technical improvements may increase further the sensitivity of the assay. COTTE Laurent, Hepatogastroenterology and AIDS unit, HlOpital de 1-l8telDieu, 1 place de lll68pital, 69 288 Lyon - FRANCE Tel: (33) 72 41 30 79 Fax: (33) 72 41 31 40 We have developed a fluorescent labelling method for the direct detection of HIV mRNA and DNA in cells in suspension. This avoids the need for in situ hybridisation, and retains cell structure. The HIV DNA or cDNA is amplified either using fluorescent primers, or fluorescent nucleotide triphosphates. The presence of fluorecently labelled PCR products is then detected inside the cells by flow cytometry analysis and/or confocal microscopy. A single copy of proviral DNA could be detected in U1 cells. In control experiments, the fluorescent labelling was highly specific for the infected cells diluted in non infected cells. Small subsets of cells bearing HIV transcripts were detected with a low threshold (0.1%). Samples from 28 HIV infected donors, ranging from stages II to IV-D were studied. Intracellular HIV DNA or RNA can be detected at all stages, and more readily from lymphoid tissue (6/6 donors positive, mean of positive cells: 2.8% for RNA, range 0.13-5.80% and 4.23% for DNA, range 0 52 -6.5%) than blood (17/28 positive; mean: 0.8% for RNA, range 0-2.7% and 1% for DNA, range 0-6.4%). Lymphoid organs could indeed represent a major viral reservoir. However we could not correlate the clinical stage of the disease and the level of infection. Nevertheless, our results strongly support the emerging view that the level of infection was first underestimated. We recently extended the investigative potential of the technique by coupling in-cell PCR with antibody surface staining, for dual colour analysis of defined cell subsets. Dr Guy GOROCHOV, Lab.d'Immunologie cellulaire et tissulaire, URA CNRS 625, H6p. Piti4-Salpetriere, 83 Bvd de I'hopital, 75013 PARIS, FRANCE. Tel; 1 42177492, FAX; 1 42177490. 0 -,1 00

Page  76 579ANHANCED DETECTION RATE OF HIV DNA AND RNA BY PCR IN SALIVA FROM HIV SEROPOSITIVE IDUs. Hewlett Indira K.*, Joshi, B.*, Lee, S.F., Epstein, J. and Qureshi, M.N.**, *CBER, FDA, MD 20852, USA, **Beth Israel Hospital, N.Y Objective: Studies on saliva have widely varying results for HIV isolation making it difficult to evaluate HIV transmission via contaminated saliva. In this report, we describe studies on the detection of proviral DNA and virion-associated RNA in saliva from 75 IDUs. Methods: Saliva and paired plasma samples from these individuals were studied blinded with regard to serostatus. Saliva was ultracentrifuged and nucleic acid extracted from the pellet was used for subsequent DNA and RNA amplification assays. 90ul of salivary pellet was mixed with O10ul of 0.5% NP40 and vortexed prior to heating at 75 C for 10mn. Supernatant was used for DNA-PCR with gag primers SK 431/ SKI145. Amplified products detected by liquid hybridization with radiolabelled SK102 probe. Results: 18/52 (34.6%) samples harbored between 50-100 copies of HIV-1 DNA. 2/24 (8.3%) seronegative IDUs were also found to be HIV positive in our study. For RT-PCR studies, RNA was extracted from 250ul of salivary pellet by the guanidinum isothiocyanate/phenol/chloroform method. RT-PCR studies revealed the presence of HIV RNA in saliva from 39/51 (76.5%) seropositive IDUs. Paired plasma samples from the same individuals are also being analyzed for the presence of HIV. Discussion: Our data suggest that HIV RNA and DNA are present in saliva of HIV seropositive individuals. The results obtained in our study show a high prevalence of HIV-1 in saliva relative to previous reports. We are currently investigating the infectivity of HIV for salivary epithelial cells. Indira K. Hewlett, 1401, Rockville Pike, Rockville, MD 20853-1448, USA. Ph: (301)-594-6727: Fax:594-6989 580A QUANTITATION OF HIV VIRAL LOAD IN PBMCS AND PLASMA USING BRANCHED DNA (bDNA) SIGNAL AMPLIFICATION. Dailey P., Wilber J., Neuwald Paul Chiron Corporation, Emeryville, CA, USA Objective: To evaluate the feasibility of quantitating HIV RNA in Peripheral Blood Mononuclear Cells (PBMCs), and examine the relationship between HIV viral load in these cells with viral load in plasma using a bDNA signal amplification assay. Methods: Plasma and cell samples were obtained from 30 HIV seropositive patients attending an AIDS outpatient clinic. PBMCs were purified by Leukoprep density gradient separation and frozen at -80~C as a dry pellet. Samples were homogenized in 8M guanidine HCI and 0.5% sarcosyl, and RNA was selectively precipitated with ethanol. The precipitate was resolubilized in the bDNA assay sample buffer containing Proteinase K and detergent and added directly to a 96-well microplate for quantitation of HIV RNA (QuantiplexTM HIV-RNA, Chiron Corporation) in the same manner as plasma samples. HIV RNA levels were expressed as HIV RNA Eq/ml in plasma and HIV RNA Eq/10' cells in PBMCs. Results: HIV RNA was detected and quantitated in 89% and 83% of 30 paired plasma and PBMC specimens, respectively. There was a significant correlation between the plasma and PBMC HIV RNA levels (r=.54, p=.003). The mean ratio of plasma/PBMC HIV RNA levels for all patients was 13.1 (range: 0.1-115.0). This ratio increased with progression of disease. The plasma/PBMC ratio increased from 6.1 to 14.02 and then to 23.8 in samples from patients in CDC clinical categories A (asymptomatic), B (symptomatic), and C (AIDS), respectively. Discussion and Conclusions: The bDNA assay is a simple and convenient method which can be used to quantitate HIV viral load in PBMCs as well as plasma after a simple processing step. A significant correlation was observed between plasma and PBMC viral load. The plasma/PBMC HIV RNA ratio increased with disease progression. Evaluation of HIV viral load in both compartments of the peripheral blood may prove useful in evaluating antiviral therapy and studying the natural history and clinical progression of AIDS. NEUWALD, Paul D.; Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 USA; Telephone 510/601-3044; Telefax 510/655-7733 U' m~J CD U' N) 581A GENETIC CHARACTERIZATION OF INFECTIOUS HIV-1 POPULATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS KATOL Shingo, Hiraishi, Y., Sugita, T., and Takano, T. Department of Microbiology, Keio University School of Medicine, Tokyo, Japan 582A AN APPROACH TO MONITOR VIABLE HIV-1: [I] POLY A LINKED NON-ISOTOPIC MICROTITER PLATE ASSAY FOR REVERSE TRANSCRIPTASE OF RETROVIRUSES. Suzuki.Kazuo*, Saito,T**, Takahashi,T*, Kano,K*, Watanabe,S**, Imai,M** *Olympus Co., Tokyo, Japan. **Kanagawa Prefectural Institute of Health, Kanagawa, Japan. Objective: To examine whether genetic diversity of the infectious HIV-1 population in peripheral blood mononuclear cells (PBMC) of infected individuals differ from those of the total population involving defective viruses. Methods: We have developed a plaque hybridization assay for HIV-1 which enable us to quantitate and clone biologically active viruses in PBMC simultaneously. Nucleotide sequences in the env and pol genes of the infectious viruses isolated by this method were determined and compared with those of the unselected proviruses. Results: The total HIV-1 population in the env gene was genetically more diverse than the infectious population. The major variants of these two populations were much different. The amino acid markers in the pol gene specific for drug resistance were observed in infectious viruses but at a different frequency from the unselected population. Discussion and Conclusions: Infectious HIV-1 strains in vivo isolated by our method provided different genetic aspects from the total proviruses obtained from PCR-amplifiled HIV-1 DNA and thus it will provide important informations for disease prognosis and detection of drugresistant viruses. KATO, Shingo, Department of Microbiology, Kelo University School of Medicine, Tokyo, Japan Telephone (81)-3-3353-1211; Telefax (81)-3-3353-0145 Objectives: An assay for detection of reverse transcriptase of HIV (RT assay) was developed using poly A linked to microtiter plate. In this study we tried to increase the sensitivity and improve quantitative analysis of retroviruses, HIV-1, HIV-2, HTLV-1 and FIV. Methods: Non-isotopic RT assay was performed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction step, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. This incubation time of RT was varied from 15 minutes to 15 hours for the comparison of the sensitivity of this assay. Results: The sensitivity of this RT assay was increased with the reaction time of the RT. After 15 hours incubation, the sensitivity was increased 50 times more than that of 15 minutes incubation of RT reaction. This RT assay detected other kinds of RT activity of retrovirus, HIV-2, HTLV-1, and FIV. Quantitative analysis also became possible. Discussion and Conclusion: Our RT assay was able to detect active retroviruses, such as HIV-1, HIV-2, HTLV-1, and FIV, depending on the activity of their reverse transcriptase with high sensitivity. This assay could be used for quantification of active retroviruses. Suzuki, Kazuo, Olympus Co., Tokyo, Japan. Telephone (81)-426-91-7119, Fax (81)-426-91-6239.

Page  77 POSTER ABSTRACTS Wednesday 10 AUGUST & Thursday 11 AUGUST

Page  78 " PA0191 PCR METHOD FOR DIRECT SEQUENCING OF THE HIV-1 PROTEASE 0 GENE. Yamaguchi K, Groopman JE, BYRN, Randal A. New England Deaconess Hospital, Boston, MA, USA Objective: To develop a rapid method for sequencing the predominant genetic species of HIV-1 protease gene in tissue culture samples. Methods: The complete protease gene was amplified from cellular DNA extracts by PCR and standard external primers. Single strand DNA was prepared by asymmetric PCR using the same primers. Dideoxy sequencing reactions were performed using internal fluorescent primers and PCR. The samples were analyzed using the Fluorescent A.L.F. DNA Sequencer (Pharmacia) nonradioactive system. Results: The A.L.F. sequencing system displays the relative intensity of each possible nucleotide present at each position. Because HIV accumulates variants during replication this system provides a simple method to observe major population shifts between related samples. Small differences, averaging 3 amino acid substitutions, were observed between primary isolates and the prototype HXB2 sequence. Comparison of sites of sequence variation with structure/function models of the protease suggest functional regions of the gene are highly conserved. Repeated sequencing of a single sample produced reliable results. Discussion and conclusions: This method is suitable for determining the major species present in tissue culture samples. It works well for primary isolates of HIV-1 propagated in PBMCs, monocytotropic strains of HIV-1 grown in macrophages, and lab strains of HIV grown in lymphoid cell lines. This method should be useful for analysis of HIV populations grown under the selective pressure of anti-viral agents, either in vitro or in vivo. PA0192 USE OF PCR FOR DETECTION OF HIV INFECTION IN INFANTS IN TANZANIA Nkya W.M.M.M', Nyombi B', Bergsjo P', Shao J' 1. KCMC Research Centre, 2. Tanzanian Norwegian AIDS Project (MUTAN), 3. Muhimbili Medical Centre OBJECTIVE: To establish PCR in Tanzania and evaluate it with respect to the detection of HIV infection in infants. METHODOLOGY: Venous blood sample were obtained from suspected patients and mothers while cord blood was used for infants. The samples were analysed by ELISA, W/Blot and PCR. RESULTS: 63 blood samples were analysed. 35 blood samples (56%) were HIV positive while 24 blood samples (38%) were HIV negative by all tests. 4 blood samples (6%) were HIV positive by both ELISA and W/Blot but negative by PCR. These are being Investigated further. Out of 8 blood sample pairs (mother and infant) screened by ELISA, and W/Blot 6 pairs were seropositive while 2 pairs were seronegative. When PCR was done on the 6 seropositive pairs all samples from the mothers were PCR positive while all the samples from the infants were PCR negative. The 2 pairs that were seronegative were also PCR negative. CONCLUSION: 1. The PCR results were in general agreement (94%) with ELISA and W/Blot results. 2. PCR technique seems to discriminate HIV negative from HIV positive infants. "o O "0 -b BYRN, Randal A., Division of Hematology/Oncology, New England Deaconess Hospital, 185 Pilgrim Road, Boston, MA 02215, USA Tel. 617-632-0161, FAX 617-424-6237 PA0193 GENE-LEVEL MONITORING OF HIV INFECTION BY POR WITH TWO INTERNAL STANDARDS A.A. Yolov. A.V. Kozlova and E.V. Karamov D. I. Ivanovsky Inst.of Virolocry, MoscowRu:ssia Liec!ti. The estimation of HIV genomes per host cell,eonomes has been carried out for slumples of patients' blood and cell culture:s. In such a way the study of the influence ct drugs and other conditions by the gene-level monitor i ne c t' innotect:on seems to be possible. Me thods. We propose the quantification by PCR with two respective internal standards of both HIV,enomes and host cell,enomes for such moni torin to be carried out for any crude sample-. The both standards were constructed by PFCRmediated joinin of DNA fraements. Results. Estimation of HIV DNA per HLA-DQa cenes (these markers were re arded as two copies per cell enome) indicate:. that for- IV-infected persons the number of viral DNA enomes varies in the range 2-500 per 1U000 lymphocytes. This value was found to change in the course of treatment. In contra:t. the respective manitude estimnated for the cell cultures was up to 5000. The broad applicability of POR with an internal standard for gene quantification was also supported in this study. wtQh cIusiL. The observed DNA-level decree of infection with immlunological and other data (their comparison reveals corell ations at least in some cases) seem to represent the versatile monitoring of retroviral infection. Andrei A. Yolov. Moscow. 123098. Russia. D. I. Ivanovsk.ki Institute of Virology; Telephone: (095) 1903062; Fax: (095) 1902867 PA0194 DETECTION OF HIV-RELATED GENES IN VIRUS-INFECTED CELLS BY ELECTRON MICROSCOPICAL IN SITU HYBRIDIZATION GOTO Toshivuki. FUKUI,T., NAKANO,T, HIRAI,K and NAKAI,M. Depertment of Microbiology, Osaka Medical College, Takatsuki, Osaka JAPAN and *Medical Research Institute, Tokyo Medical and Dental University, Tokyo JAPAN Objective: In situ hybridization (ISH) is a useful method for detection of viral RNA or integrated DNA sequences in virus-infected cell. Here we report optimized procedures for ISH at electron microscopic (EM) level in HIV-infected cell lines. Methods: Oligonucleotides for HIV LTR regions were synthesized. The HIV-infected cells and non-infected cells were fixed with 2% paraformaldehyde and 0.04% glutaraldehyde for embedding in Lowicryl K4M. The samples and the biotinylated DNA probes (in 50% formamide) were denatured by heating at 72 ~C just before hybridization Hybridization was performed for over 16 hours at 37 ~C in a moist chamber. Samples were then incubated in goat anti-biotin antibody coupled to lnm gold particles for 2-4 hours at room temperature. Silver enhancement of the gold signal was performed for 4-7 min. The sections were stained with uranyl acetate and lead citrate. Results: The oligonucleotides at LTR region in HIV were reacted mainly in the nuleous and scatteredly in the cytoplasm of HIV-infected cells. But there were no reactions on the extracellular virus particles and uninfected cells (Molt4) for negative control. Discussion and Conclusions: These observations suggest that the procedure used here is applicable to EM-ISH with oligonucleotide probes showing the viral or integrated DNA sequences in HIV-infected cells. GOTO,Toshiyuki, Depertment of Microbiology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569 JAPAN Telephone(8 I )-726-83-1221 (ext2647);Telefax (81)-726-84-6517 nI

Page  79 PA0195 ARE CLINICAL ISOLATES MORPHOLOGICALLY DIFFERENT FROM LABORATORY STRAINS OF HIV? Gelderblom. HR, Asjo, B*, Moore, JP**, Ho, DD**, Sandfort, J and Pauli, G Robert Koch-Institut Berlin, *National Centre for Research in Virology, University of Bergen, ** The Aaron Diamond AIDS Research Center, New York Objective: To study by thin section electron microscopy three different kinds of specimens: HIV from plasma of infected individuals, first passage clinical isolates of HIV, slow/low and rapid/high phenotype isolates with regard to virus morphology and anchorage stability of the glycoprotein knobs. There is growing evidence that the established laboratory strains of HIV are biologically different from clinical isolates and/or from the virus in the infected individual. Methods: (1) Virus was sedimented (100.000g, 1 h) from the plasma of p24 antigen positive individuals and processed for electron microscopy. ( 2) HIV was isolated in PBMC and first passage virus was studied and (3) CD4+cells infected with non-passaged or low passage of the slow/low or rapid high phenotype variants were investigated by electron microscopy. Pellets and virus infected cells were Epon embedded using tannic acid for contrast enhancement and studied by thin section electron microscopy. Results: First attempts to detect HIV directly in the sediments of 3 ml of p24 antigen positive individuals (200 -250 pg/ml) failed. Using polystyrene beads as concentration marker a detection limit of 100.000 particles per 3ml was established. When plama HIV was used to infect PBMC, first passage virus was detected in about 20 % of cells. Virus particles in 2 such cultures were morphologically mature and showed loss of envelope glykoprotein knobs. Discussion and Conclusion: In sediments from p24 positive plasma, the sensitivity of detection was too low. Nevertheless direct evaluation seems to be a promising approach to study HIV in its natural behaviour. First passage HIV in PBMC did not differ in morphogenesis and anchorage stablility of the glycoprotein knobs from established laboratory strains. H.R. Gelderblom, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Tel.: 4547 2337, FAX: 4547 2605 PA0196 ISOLATION AND CHARACTERIZATION OF HIV-1 CLINICAL ISOLATES IN JAPAN WATANABE Kuhomi.1Kitamura K.,1 Nakasone T.,1Shiosaki K.,2Yamada K.,3 Moritsugu Y.,l Yamazaki S.,1 and Honda M.11)AIDS Res.Cent., NIH., Tokyo, 2)Chemo-Sero-Therapeutic Res. Institute. Kumamoto, 4)Institute of Med.Scie., St.Marianna Univ., Kawasaki, Japan. Objective: In order to study the characteristics of clinical HIV isolates prevalent in Japan, we analyzed the genotype, phenotype and immunotype of the virus. Method: We have been isolated HIV strains from blood specimens of HIV infected individuals in Japan from 1988. Further, we analyzed C2-V3 region from 150 HIV infected individuals. We, then, characterized 15 clinical isolates from mainly hemophiliacs seropositive for HIV, employing sequencing methods of C2-V3 region, HIV-V3 slot blot analysis and CEM neutralization assay using both serum and their peripheral blood mononuclear cells. Results: The number of clinical specimens studied reached more than 1,000. More than 300 of HIV were successfully isolated from the samples. The isolation rate of HIV has decreased to 30 percent in 1993 from 40 percent in 1992. These results suggested that treatment with drugs such as AZT and/or ddl may be effective to those HIV-infected individuals. The viral and genomic sequence of HIV belongs to clade B virus of HIV-l. By polyclonal analysis, PND-sequences of long survived individuals with HIV infection showed less diversity. In contrast, rapidly progressing individuals showed diverse viral sequences. When progressed to disease, GPGIR core sequence changed to GPGG and hydrophilic amino acid changed to hydrophobic amino acid, correlating the loss of binding activity of anti-MN neutralizing antibody to the mutated viruses. Glycosilation sites were relatively conserved in both N-tenninal and Cterminal of V3 region. Conclusion: These results suggest that the characterizing the viruses may be a useful tool to estimate the clinical states of HIV infection and HIV-induced diseases. WATANABE, Kuhomi, AIDS Research Center, Tokyo, Japan Telephone (81)-3-5285-I 11 l(Ext.2737); Telefax (81)-3-5285-1 150 PA 0197 IILITI,| 1i1 UratStsI t eElAEC TtCltiTuL P Ifat-I ITAIII1 55 Ciii. Lobsies, iLeor; losa, 1; levee, L; l ed, U; ilatttbi,.l; lattlue, I; list,, i.l1 a1llleCl Lilelit1la. li11li. ciI. OBJICTIPI: Biological characterization of 1IV-1 isolates from patients at different disease stage. IITIOD: Clinical epidemiological and virological aspects of 30 infected people were studied. Isolate Ill-I strains were characterized and classified in relation with replication kinetics and cellular tropism as well as syncytium inducing (SI), and non syncytium inducing (BSI) capacity. The biological phenotype of viral isolates were correlated with each patient disease clinical stage. IISLTS: HIV was isolated from all AIDS patients, but not in all asyntomatic seropositive carriers. 20 out of 21 strains of AIDS patients were classified Rapid High and SI, however 3 out of 5 strains of asyntomatic seropositive carriers were considered Slow-Low and SI. COICLISIOI: The percentage of recovered viral isolates agrees with the patient's clinical stage and it was demonstrated that there is an association between the isolate biological phenotype and the patient's clinical evolution. LIIIIIl, Leaser. Laberstertle de Ivestilgacluee del I11. ptdea a 131Istisaac 14, C. Dabs eeba TeleS. I4 isti. tar. ti $6216t. PA0198 PHARMACO-MECHANICAL APPLICATIONS TO HIV INFECTION Mark W.i ter,Ft. LauderdaleFL, USA Objective: A method by which to harvest IHluman Immunodeficiency Virus (IIV) negative aul.ologous CD4+ and CD8+ lymphocytes and their precursor (primitive) cells; to prepare cultures for multiple growth and genetic alterations by removing autologous lymphocytes from a donor; to determine HIV status; to discard HIV positive lymphocytes and/or return genetically, chemically, and/or pharmacologically altered HIIV negative lymphocytes directly or indirectly to the donor. Method A mechanical device employed with apheresis and flow cellU technology collects and separates HIV negative and HIV positive autologous lymphocytes. HIV positive lymphocytes exit the system via a discard chamber. HIV negative lymphocytes are routed to one of several chambers. In a chamber, lymphocytes undergo genetic, chemical, and/or pharmacological alterations and returned directly to the donor or collected for future culture. Results: This procedure and mechanical device is currently being studied by the scientific and manufacturing industry. Currently, grants have been made available for stem cell reconstitution, genetic applications, and developments in harvesting viable cell lines from culture. Data gathered from these programs show great promise. Conclusions: Genetically altered lymphocytes would prohibit viral destruction to cellular DNA. The use of Culture Derived Lymphocytes (CDL) would aid in the reconstitution of the immune system. Mark W. Lister 1729 E. Commercial Blvd. Suite 239 Fort Lauderdale,FL 33334 USA phone/fax(305)491-1167 0 0 __D

Page  80 O PA0199 HIV: A BLACK TULIP IN THE BOUQUET OF HEMOTRANSMISSIVE O DISEASES. E.Zhiburt, N.Belgesov, A.Leschev, I.Voltchek. Medical Military Academy, "Avalanche Ltd". St.Petersburg, Russia. Human immunodeficiency virus I/II, hepatitis B and C viruses, treponema pallidum are the agents of most significant hemotransmissive infections. Markers of these infections are usually analysed in blood donor screening. It is determined that among young (18-30 years) donors in St.Petersburg (Russia) prevalence of HBsAg is about 1,9%, anti-HCV- 1,3%, anti-TP - 0,1%. Quantity of anti-HIV I/Il serepositive donor's blood is depended on kind of ELISA kit and is from 0,2% to 2,8%. More than 15000 donors have been investigated. Confirmatory tests have given no HIV-positive results. With Russian donors' blood screening high specific (lizate) test-systems must be used. In our opinion test-system (Avicenna), is acceptable. In spite of low level of HIV-infection in Russia anti- HIV blood donor screening must be powerful preventive barrier. Other hemotransmissive diseases can to be used as model with organization of antiepidemic measures. E.B. ZHIBURT, MD. The Center of Blood and Tissues, Medical Military Academy, 47, Zagorodnyi, St.Petersburg, 198013, Russia. Fax: (812) 311-8549. PAO200 HIV VIRAL LOAD IN LYMPHOID TISSUES USING BRANCHED DNA (bDNA) SIGNAL AMPLIFICATION. Dailey, P., Wilber, J., and Urdea, Mickey S. Chiron Corporation, Emeryville, CA. Objective: To evaluate the feasibility of quantitating HIV viral load in lymphoid tissues in a simple, rapid and reproducible manner. Methods: Lymphoid tissues including lymph node, tonsil and spleen specimens were frozen at - 80"C immediately after collection. Samples were homogenized in 8M guanidine HCI and 0.5% sarcosyl, and RNA was selectively precipitated with ethanol. The precipitate was resolubilized in the bDNA assay sample buffer containing Proteinase K and detergent and added directly to a 96-well microplate for quantitation of HIV RNA (QuantiplexT' HIV-RNA, Chiron Corporation). Results: HIV RNA was quantitated from lymphoid tissues obtained from HIV seropositive patients. Viral load in these samples ranged from 9.31 x 106 to 9.28 x 10' HIV Eq/gram. Separate samples of left and right tonsil from the same patient were virtually identical in viral load (9.13 x 10' vs 9.28 x 10' HIV Eq/gram). Similar results were observed with spleen samples. Lymph node tissue obtained from three patients was divided into multiple samples. The difference in quantitation between samples from the same lymph node ranged from 1.4 to 3-fold. HIV RNA in small samples (ca. I mg) of lymphoid tissues was detectable by the bDNA assay. Discussion and Conclusions: The bDNA assay can be used to quantitate HIV viral load in lymphoid tissues. The results obtained suggest that HIV RNA in a single sample is representative of viral load elsewhere in that tissue, and perhaps of lymphoid tissue elsewhere in the patient. Further study of this issue will be required. This assay should prove useful in the study of the pathogenesis of AIDS and the efficacy of antiviral agents. URDEA, Mickey S., Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 USA; Phone 510/601-3040; Fax 510/655-7733 "v a 0 CDO "0 0 N 0 N PA0201 ENHANCED HIV-1 ACTIVITY AFTER REACTIVATION CRYOSTORAGE INFECTED CELLS. Selinva Ludmila*; UryvaevL*; YurinO#; GuschinaE*; IrovaT#; PokrovskyV. #; LvovD*. Ivanovsky Institute of Virology*;Specialized Research Lab. for AIDS Epidemiology & Prevention#,lMoscow, Russia Objective: To understanding different mechanisms and factors that determine regulation HIV-1 expression in cell lines. Methods:Virus specific activity were evaluated in Jurkattat cells by electron microscopy, fluorescent antibody and immunoblott methods. Resalts: Recent HIV-1 isolates (MS-1974,NS-1978; Vopr. virus. 1991,3,239) infected only Jurkat-tat cells and exhibit low raplicate activity without cytopathicity. There were the increase in these capacities after reactivation frosen cells persistently infected with both isolates. Isolates show some differences in the spreed virus in cells. Discussion and Conclusions:Low virus isolates were recovered from seropositive children with the appearence symptoms of AIDS and immunodeficiency status before HIV infection. So our results suggest presence in children's PBMIC all HIV-1 genome elements required for productive viral expression and give new approach in the recognition of HIV-1 pathogenesis. SELIJMOVA,Ludmila,c/o Uryvaev Leonid,The D. I. Ivanovsky Institute of Virology,Gamaleya st,16 123098, Moscow, Russia, T095-193-54-93, F095-190-74-85 PA0202 SEQUENCE IN V2 AND V3 REGIONS OF SYNCYTIUM INDUCING HIV-I MIYAMOTO Kanji,*TOMITA N., **NISHIZAWA T. Sch. Health Sci. Okayama Univ. *Okayama Red. C. **Sumitomo Metal Indu. Japan Objective: To determine which V2 and V3 sequences are associated with syncytium formation of T cell trophic HIV-I. Methods: HIV-I was isolated from the peripheral mononuclear cells of five anti-HIV-I antibody positive hemophilia patients. Isolated HIV-I strains were adapted to a T cell line, Molt-4 -clone 8, to examine syncytium-inducing (SI) ability. The V2 and V3 regions of the SI HIV-I strain were sequenced. Results: In one of the five HIV-I strains, formation of numerous large multinucleated syncytial cells was induced in Molt-4 -clone 8, but in the other four strains, only a few multinucleated syncytial cells were observed. The hypervariable V2 sequences of the SI HIV-I strain were almost same as those in the reported data but the V3 sequences differed from those in the reported data. Discussion and conclusions: The Molt-4-clone 8 T cell line appears to be useful for the examination of SI ability. This unique SI HIV-I strain may contribute to a better understanding of the pathogenesis of HIV-I infection. MIYAMOTO, Kanji, Sch. Health Sci. Okayama Univ. 5-1 Shikata 2-chome, Okayama, Japan Tel. 086-223-7151; Telefax 086-222-3717

Page  81 PA0203 AFFINITY ISOLATION OF HIV-1 EPITOPE-SPECIFIC ANTIBODIES DenisoivaS.,Meshcheryakova D., Andreev S., Khaitov R., Tarasova S., Institute of Immunology, Moscow, Russia. OBJECTIVE: The isolation of human antibodies directed to virusneutralizing epitopes of HIV-1 envelope protein by affinity purification and investigatioin their serological properties. METHODS: Peptides from V3 loop (301-323 aa, MN strain) and gpl20 carboxyl end region (495-516, BRU) were coupled to AH-Sepharose 4B by means of N-ma eimidopropionic acid linker via peptide N-terminal Cys. The cross-reactivity of anti-peptide Abs isolated from sera of HIV-1i infected patients was investigated with synthetic fragments from different HIV-1 gp120/41 regions by ELISA, and with HIV-1 proteins by Western blot. The Abs concentrations and purity was measured by spectrophotometry and gel-electrophoreze. RESULTS:The peptde-S epharose columns showed high selectivity for HIV-1 Abspecific adsorbtion. Practically all amounts of specific Abs from HIV sera were capable to bind with these sorbents. Calculated conc of specific Abs in human sera were 148 and 152 ug/ml for anti-V3 and anti-C-120 respectivly, that are consistent with earler reported data, and it is likely corresponds to their true content in the HIV-1sera. Both anti-V3 and anti-C-120 Abs showed specific reactivity with corresponding peptides. Anti-V3 Abs cross-reacted with only V3 peptides from BRU strain, while anti-C-120 Abs reacted to V3 peptides of MN and BRU strains. Data of Western blot indicated that the Abs recognized only gpi20 and 160 proteins, and the electrophoreze showed the presence of gG chains and only minor amount of human serum albumin. CONCLUSION: We have developed convenient method of preparative isolation of natural epitope-specific HIV-1 antibodies based on peptide-Sepharose column chromatography. It is important, that suggested method of conjugation peptides to Sepharose does not result in damage of the epitope structure, and completely retains biological activity of purified antibodies. Spphia Tarasova, Institute of Immunology Rashirskoye shosse 24-2, 115478 Moscow Russia, fax: (095) I17 1027, tel: II?7 8177 PA0204 EPITOPE ANALYSIS OF HIV-1 p17 Akemi OLa, Liu Xiao-Liang, Nobuo Sakato, Hajime Fujio, and Shigeharu Ueda Res. Inst. Microb. Diseases, Osaka Univ. Objective: Examination of immunological role of HIV-lpl7 gag protein is important for the design of AIDS vaccine. We established 20 anti-pl7 monoclonal antibodies (MoAbs) and analyzed epitopes of pl7 by using 6 synthetic peptides. Methods: MoAbs were obtained by immunizing Balb/c mice with a recombinant p17 of pNL432. Six peptides (P1-12, P12-29, P30-52, P53-87, P87-115, P118-132) were synthesized as antigens according to hydrophilicity and flexibility analysis of the amino acid sequence of HIV-1 p17. Twenty MoAbs were analyzed by a binding assay in liquid phase and ELISA. Localization of p17 or its derivatives on the surface of HIV-1-infected MT-4 cells was examined by immunofluorescent staining tests. Results: There were 6 MoAbs recognizing P12-29, one recognizing P30-52, three recognizing P53-87, and nine recognizing P87-115. No MoAbs recognized P1-12 and P118-132. One MoAb did not reacted with any of these synthetic peptides, but reacted recombinant p17. The highest affinity constant of them was 1.4 X 10' (M') and the lowest was 2 X IO' (M'). Immunofluorescent examination revealed that these MoAbs reacted with native p17 or its derivatives in and on HIV-1 (JMH-1 strain)-infected MT-4 cells. Discussion and Conclusions: Using 20 MoAbs against recombinant p17, we detected 4 epitopes in p17 of HIV-1. One MoAb revealed a comformational epitope. Immunofluorescent tests suggest that these epitopes are present on the cell surface of the HIV-1-infected cells. OTA, Akemi, The Research Institute for Microbial Diseases, Osaka, Japan Telephone (06)-875-1170, Telefax (06)-875-1170 PA0205 SIMULATION OF HIV-1 CULTURE PASSAGE: EFFECTS ON V3 LOOP PHENOTYPE- GalvanVeronica;Rabinovich R;Gutierrez P;Gomez Carrillo M;Marquina S;Ubonatti O- Nati.Centre of Ref. for AIDS, Argentina Objectives: Design a simulation model to predict potential phenotypical changes affecting the V3 loop in a HIV-1 infected U937-2 cell line culture system. Methods: Assuming that proviral genomes that replicate integrated with cellular one suffer no changes, the model generates a distribution of the number of culture passages during which the viral genome replicates via reverse transcriptase. Random punctual mutations are generated on the starting sequence, according to published frequencies that consider the G->A hypermutation and the position effect for the GpA dinucleotide. The model then translates the result into amino acids Indicating the distribution of charge and viability for each sequence. It also shows the number of phenotypes identical to the original one. This procedure is iterated as many times as the distribution Indicates. This model was applied to 28 V3 loop proviral sequences corresponding to an equal number of local patients. Results: For all sequence studied, 7 culture passages were simulated with a starting concentration of 0.001% Infected cells. The probability of obtaining an phenotype Identical to the original one ranged between 50 and 73.8%. The highest percentages of identity corresponded to sequences with high A contents. When the simulation was started with a higher proportion of Infected cells, the sequences recovered showed less divergence. Sequences with high G contents showed a tendency to generate defective genomes. In all cases, sequences with more polar and positively charged amino acids appeared. Conclusions and discussion: This model serves to estimate the significance of the phenotypes obtained from isolates derived from the circulating viral populations. Galvan Veronica- CNR para SIDA, Dept. Microbiologla, Fac. de Medicina, UBA - Paraguay 2155 - Piso 11 (1121) - Buenos Aires, Argentina - TE: 541 961-8124 Fax: 541 962-5404 PA02 06 8IIV-1 SPECIFIC ACC IS Of(RfOILED BY CD55 AND CD59. 0Sch6itz*,**, J6rn, Zier*, J.P., Kluxen*, B., Aries*, S., Schlitz*, H. *Dept. of Virology, Bernhard-Hocht-Institute, Hamburg, Germany; **Dept. of Immunology, N.E. PRIIATE RES CTR, Harvard Medical School, Boston, MA, USA Objective: An increased complement sensitivity of PBNC from HIV-1 infected patients has been directly correlated to a decreased expression of the complement restriction factors CD55 and CD59. We studied the effect of antibody-dependent complement-mediated cytotoxicity (ACC) on the lysis of BIV-1 infected cell lines, differing in the expression of CD55 and CD59. Methods: The HIV-1 virus strain IIIb was replicated in the human permanent cell lines H9 and KE37. At the peak of the infection the cells were incubated with HIV-1 positive sera and complement. The specific lysis was measured by propidiun iodide uptake or chromium release. Results: When the HIV-1 infected target cell line KE37, which do not functionally express CD55 and CD59, was used, a highly significant cytotoxic effect was detected in the presence of complement and HIV-1 positive sera (n=50), while negative sera (n=50) were unable to induce cytolysis. All sera of HIV-1 infected patients contained antibodies, which mediated a specific lysis. Furthermore, when blocking antibodies against CD55 and CD59 were used, which abrogate the function of these molecules, the complement resistant cell line H9 showed a HIV-i specific lysis as well. Discussion and Conclusions: We conclude that complement and antibody-mediated lysis of CD55 and CD59 deficient lymphocytes should be considered in the pathogenesis of the progressive loss of CD4 lymphocytes in the course of the infection. In earlier studies, ACC was not observered in human HIV-1 positive sera. However, in these studies the complement resistance of target cells was not taken into account. To further elucidate the role of ACC, different cell subsets from HIV-1 infected patients will be investigated. SCHIITI, J6rn Department of Virology, Bernard-Nocht-Institute, Bernhard llocht Str. 74, 20359 Bamburg, German; Tel: (49)-40-31182-455; Fax: (49)-40-31182-400 0 O 0 O -o 0 0 0

Page  82 PAO207 INFECTION OF VAGINAL AND COLONIC EPITHELIAL CELLS BY HIV-I IS NEUTRALIZED BY ANTIBODIES RAISED AGAINST CONSERVED EPITOPES IN THE ENVELOPE GLYCOPROTEIN GP120 Furuta Yasushi, Eriksson K.*, Svennerholm B., Fredman P.", Horal P., Vahlne A., Czerkinsky C.* Dept.of Clin. Virol., *Med. Microbiol. and Immunol., and I Neurochem., University of GSteborg, G6teborg, Sweden. Objective: The rectal and genital tract mucosae are considered to be major sites of entry for HIV-1 during sexual contact. We have attempted to develop HIV-1 infection in human vaginal epithelial cells and to identify the receptor for HIV-l invaginal epithelial cells. Furthermore, we examined sites on gpl20 involved in antibody-mediated neutralization of HIV-1 in vaginal and colonic epithelial cells in vitro. Methods: Two vaginal epithelial cell lines, Hs 760.T and Hs 769.Vg, were infected with HTLV-IIIB and infection was analyzed by cocultivation with T lymphocytes and by PCR. The binding capacity of lipid extracts from the vaginal cells to gpl20 were tested by thinlayer-chromatography-ELISA. The HIV-1 neutralizing activity of monkey hyperimmune sera raised against a series of 40 overlapping synthetic peptides covering the entire sequence of HIV-I gpl20 was evaluated in the vaginal and colonic (HT-29) epithelial cells. Results: The vaginal epithelial cells could be infected by HIV-1I via a mechanism involving initial interaction of sulfated lactosylceramide with gpl20. Antisera raised against five synthetic peptides, corresponding to three relatively conserved regions and to the V3 loop, efficiently neutralized HIV-1 infection of the vaginal and colonic epithelial cells in vitro. Conclusions: These results have obvious implications for the design of mucosal subunit vaccines against sexually transmitted HIV-1 infections. FURUTA, Yasushi, Dept. of Clinical Virology, Goteborg University, Guldhedsgatan 10, S-41346 Goteborg, Sweden Telephone(46)-31-60100 Telefax(46)-31-604960 PA0208 V SYA GT ASSOCATD wlRANSUDATIgOFSERUM-BORNE lgG TO HIV, EXPRESSIN D, ERNTA AVIDITY TO GPI60, IN HIV-1N IED WIEN. L. B6ec, Carol T6viB6nissan, J. Pillot. Unit6 d'immunologie Microbienne, Institut Pasteur, France. Objective. To determine the origin of specific IgG and IgA antibodies (Ab) against HIV in cervico-vaginal secretions (VS) from HIV-1-infected women. Method. IgG and IgA (after immunoprecipitation of IgG) Ab to a whole lysate of HIV-I, albumin (HSA) and lactoferrin (LF) were evaluated in paired serum (S) and VS from 30 HIV- I-infected women, and secretory IgA (S-IgA) to gpl60 in their VS. Avidities of IgG and IgA to gp 60 were compared d in paired S and VS. Results. IgG Ab to HIV-1 titers in S [median: 2 106, range: 105-8 106] were 100-fold more elevated than IgG Ab to HIV-1 titers in VS [median: 2 104, range: 104l-8 104], whereas IgA Ab to HIV-l titers in S [median: 104, range: 102-105] were 10-fold more elevated than IgA Ab to HIV-1 titers in VS [median: 2 103, range: 102-2 104]. Specific activity of IgG Ab to HIV (OD at 1:1000/[IgG]) was 50-fold higher in VS than in S. S-IgA to gpl60 in VS were detected in all cases. Mean ratio HSA in S/HSA in VS (14.8; nl ratio#90) and mean LF level in VS (95 g/l; nl=187 g/I) were significantly decreased. IgG and IgA Ab titers in S and VS highly correlated. Relative avidities of IgG to gpl60 were similar in S and VS, whereas avidities of IgA to gpl60 in VS were higher than those of IgA to gpl60 in S. Conclusions. (i) IgG, IgA and S-IgA Ab to HIV are always detected in VS from HIV-linfected women. (ii) Humoral cervico-vaginal immunity against HIV is characterized by local synthesis of S-IgA and IgG Ab to HIV associated with transudation of serum-borne IgG Ab to HIV. (iii) High avidity of IgA Ab to the surface glycoprotein gpl60 in VS suggests that IgA Ab to HIV could be particularly efficient for immune exclusion of HIV particles in VS. (iv) Increased HSA in VS is related to alteration of cervico-vaginal mucosa due to local opportunistic infections and/or HIV replication. (v) Decreased LF indicates disturbance of mucosal functions. (vi) Finally, IgG and IgA Ab to HIV in VS could play a major role in limiting the virus infectivity on cervico-vaginal mucosa. Dr. L. B61ec Unite d'Immunologie Microbienne Institut Pasteur, 25 rue du Docteur Roux 75 015 Paris France; Telephone: (33.1) 45688254; Telefax: (33.1.) 43958055 O 0 0 "0 0 0 PA0209 Bongertz Vera, Costa CI, Morgado MG & dos Santos VGV HIV-1 NEUTRALIZATION IN BRAZIL. Dept Immunol, Evandro Chagas Hosp, IOC-FIOCRUZ, Rio de Janeiro, RJ, Brazil Objectives: Determination of the susceptibility of brazilian HIV-1 isolates to neutralization by brazilian sera. Methods: The neutralization assay in PHA activated PBMC (Albert et al. AIDS 4:107, 1990) evaluated with the DuPont HIV antigen assay was used. Neutralization titre was defined as the reciprocal of the highest dilution without antigen detection. Results: The TCID50 of brazilian HIV-1 isolates (CDC stages II, III, IV, Rio de Janeiro, RJ) was measured, with results varying from 75 to 6250. Isolates with titres above 100 were selected for evaluation of their susceptibility towards autologous and heterologous neutralization. None of 6 isolates tested were neutralized by their autologous sera, while heterologous neutralization varied. All isolates were neutralized by the control pool of brazilian plasma. Discussion and Conclusions: Autologous HIV-1 neutralization in Brazil appears to be as rare as in other countries, indicating that vaccines capable of inducing HIV-1 neutralizing antibodies would be indicated also in Brazil. Vera Bogertz, Dept. Iiaunol. IOC FIOCRUZ, P0 Box 926, 21045-900 Rio de Janeiro, RJ, Brazil. Tel 55-21-2801486 Fax 55-21-2801589 or 5903545 PAO21O NEUTRALIZING MONOCLONAL ANTIBODY AGAINST AN PA0210 EXTERNAL ENVELOPE PROTEIN (GPl20) OF SIVmac251 MATSUMI.Shintaro, MATSUSHITA,S., YOSHIMURA,K., JAVAHERIAN,K.*, TAKATSUKI,K. Kumamoto Univ. School of Med., *Repligen Corporation Objective The purpose of this study was to identify a neutralizing epitope of SIVmac251 by raising neutralizing monoclonal antibodies. Methods Monoclonal antibodies (mAbs) were obtained by immunizing mice with SIVmac251 recombinant gp 110. Binding activities of the antibodies were analyzed by enzyme linked immunosorbent assay (ELISA), flowcytometry (FACS), and Western blot analysis. Neutralization of SIVmac251 infection was determined by fusion inhibition and indirect immunofluorescence assays. Neutralizing epitope was identified by pepscan analysis. Results After screening hybrid clones by ELISA, three mAbs (M318T, M56S, M815) were obtained. Strong reactivity of M318T against SIVmac251 infected cells was detected by FACS analysis. M318T crossreacted with ROD and GHI strains of HIV-2. M56S and M815 also reacted with the surface of SIVmac251 infected cells,but no crossreaction was noted for these two clones. Complete neutralization against SIVmac251 was observed in the presence of M318T, but was not in the presence of M56S and M815. By pepscan analysis, the neutralizing epitope recognized by M318T was localized between amino acid 178 and 185 in the V2 resion of gpl20. Discussion and Conclusion The availability of this neutralizing mAb will allow further investigations to elucidate the role of neutralizing antibody in an animal model system of HIV-1. MATSUMI, Shintaro, Kumamoto University School of Medicine, Kumamoto, Japan Telephone (81)-96-344-2[11; Telefax (81)-96-363-5265

Page  83 PA0211 BROADLY NEUTRALIZING ANTIBODIES TO HIV-1 OCCURING IN AN AUTOIMMUNE PARADIGM. Douvas, Angeline and Takehana, Y. University of Southern ali ornia, school of Med. Los Angeles, CA USA Objective: The objective was to determine if autoimmune antibodies occurring the disorder mixed connective tissue disease (MCTD) neutralize HIV-1. There is extensive homology between a RNAsplicing protein 70K, which is the natural target of the autoantibodies, and gpl20/41. A key homology occurs between a dominant neutralizing epitope in the V3 loop, having the conserved amino acid configuration G-AF-T, and the RNA-binding site of 70K. Methods: We screened 49 anti-70K sera by HIV-ELISA and HIV-western blot. All 49 were then screened by ELISA for reactivity to gpl20, gp41, and V3 (MN and 1111B stains). These reactivities were compared to 12 HIV-infected sera, to a pool of IgG from infected sera (trademark HIVIG) and to suitable controls. Conversely, HIV-infected sera were tested for reactivity to 70K by western blot. Nine of the anti-70K sera were tested for neutralization of both a primary HIV-1 isolate and the MN strain, using a p24 based neutralization assay. Results: False-positive HIV-ELISAs were recorded in I1 of 49 anti-70K sera. The mean reactivity of the 49 sera to V3 was higher than that of HIV-infected sera and HIVIG. Moreover, anti-70K sera showed only a 33% difference in reactivity to MN vs 11113B strains by ELISA, vs a 3.7-fold difference in HIV antibodies. Of 9 anti-70K sera tested for neutralization, 3 were >80% neutralizing. Moreover, the titer for 50% neutralization was >2-fold higher for the anti-70K antibodies, as compared to HIVIG. Neutralization correlated with a V3/gp41 ratio by ELISA of > 1. All HIV-infected sera gave a positive reaction to 70K by western blot. Conclusions: The mutual cross-reactivity between antibodies in HIV-infection and in MCTD is based on homology between a neutralizing epitope in the V3 loop, and the RNA-binding site of 70K. The homologous sequence is both a B and T cell epitope in both diseases. Moreover, antibody production in MCTD is a T cell-dependent process. These findings have profound implications for modeling immunoprotective strategies against HIV-infection based on the autoimmune paradigm. DOUVAS, Angeline, University of Southern California School of Medicine, Los Angeles, CA, USA Tel:(213)342-1951, Fax:(213)342-2874 PA0213 TWO DISTANT ANTIGENIC LINEAR SEQUENCES OF HIV-1 P24 (aa 181 -185 and 316-319) MIGHT BE PARTS OF A CONFORMATIONAL EPITOPE. JANVIER Blandine, BAILLOU A. HOEBECKE J, LASARTE JJ", SAROBE P', BARIN F., URA CNRS 1334, Tours, France, and *Univ. de Navarra, Spain. Objective: To identify B-cell epitopes of the major core protein p24 recognized during natural HIV1 infection. Summary: Reactivity of 20 HIV1 infected patients and 8 HIV1 negative patients were analyzed using enzyme immunoassays developed with 45 pentadecapeplides, spanning amino acids (aa) 133 to 363 of the HIV1 p55 gag precursor. Two peptides covering aa 178-192 and 288-302 were recognized by 40% and 45% of HIV1 positive human samples. The C-terminal moeity of p24 (aa 278-322) seemed particularly antigenic since it was recognized by 7 of 10 murine monoclonal antibodies (mu MoAb). A peptide covering aa 272-322 of p55 was synthetized and recognized by most of human sera (85%). However, inhibition studies indicated that this sequence was not an immunodominant domain of p24. Using the sequences alignments proposed by Argos (EMBO J 1989, 8: 779), molecular modelisation based on the 3D structure of the Mengovirus VP2 by insertion of Ihe two p24 linear epilopes showed that both regions could be part of a conformational epitope encompassing aa sequences 181-185 and 316-319. The modelisation also explains why human sera reacting only to sequence 173-188 by indirect peptide EIAs can inhibit binding of native p24 by a mu MoAb specific for sequence 285-304. Recognition of the former could lead to steric hindrance inhibiting recognition of the latter sequence. JANVIER Blandine, Laboratoire de Virologie, CHRU Bretonneau, 37044 TOURS Cedex, France. tel. (33) 47 47 47 47 ext. 4086; fax. (33) 47 47 36 10. PA0212 OUANIIIAIIVE[ ANALYSIS OF TIlE HUNORAL IMtUNE RESPONSES IN HIV-I SEROPOSIIIVE PATIENTS. Efremova Elena V., Papuashvili N.N., thaitov R.H. Inst.of Inmunology, Noscow, Russia. )bjec.tive: Quantitative monitoring of the HIV-1 antibodies to HIV-1 antigens in seropositive patients during disease development. Methods: Quantitation of antibody responses to HIV-1 antigens was accomplished in dinamics during 6 years in 30 patients at different stages of disease by ELISA prepared from recombinant proteins and by densitometry of immunoblot (IB) pattern. Results: In these studies the decline of p24 antibodies titers and increase in gp4l/p24 antibodies ratio were correlated to the clinical evolution. Moreover, quantitative IB exhibited the presence of low p24 antibodies reactivity when zero titers were obtained in ELISA. I)iscussion and Conclusion: Thus quantitative IB appears to refine prognostic assessments of HIIV infection and could also be used in monitoring of response to antiviral therapy. EFREROVA Elena, Institute of Immonology, Raoscor, Russia Telephone (095) 117-81-00; Fa (095) 117-10-21 PA0214 SLOWER CLINICAL DETERIORATION OF HIV SEROPOSITIVE INDIVIDUALS WHO ARE ANTI-P24 HIGH RESPONDERS. BARIN Francis, BESNIER JM, ROUILLARD E, GUEROIS C, CHOUTET P, GOUDEAU A., CHRU Bretonneau and CHRU Trousseau, Tours, France. Objective: To evaluate the prognostic value of antibody titer to p24 in HIV1 infected Caucasian patients. Methods: One hundred and twenty five HIV1 seropositive individuals classified as CDC2 (n=65) or CDC3 (n=60) stage at entry in the study. Mean follow-up was 59.6 ~+ 20.8 months. P24 antigen (p24 Ag) and antibody to p24 were quantified at entry using the HIVAG-1 and HIVAB p24 assays from Abbott. Results: Seventy seven patients (61.6%) evolved to a CDC4 stage. Sixty three (58.3%) of those who were p24 Ag negative at entry and 14 (82.4%) of those,20 who were p24 Ag positive evolved to CDC4 stage. Only,~ 34.9% of high responders to p24 (titer > 1000) evolved -o>,, to CDC4 stage compared to 77.2% and 72.0% of those 0o with titers below 100 and comprised 100-999, respecti- ~ vely. Kaplan-Meier estimates of CDC4 stage-free time for o the different categories are shown in the figure. 20 Conclusion: 0 20 0o 0o s0 1oo High response to p24 is a slrong predictor of a slower AIDS development. BARIN Francis, Laboratoire de Virologie, CHRU Bretonneau, 37044 TOURS Cedex, France. tel. (33) 47 47 47 47 ext. 4086: fax. (33) 47 47 36 10. -o 0 N 0 N._L i 4"h

Page  84 PA0215 ASSAY FOR AVIDITY OF P24 SPECIFIC IMMUOGLOBULIN G IN SERA OF IIIV-1 INFECTED PATIENTS IKEGAMI, Nobuko, AKATANI, K., IMAI, M., YOSHIOKA, K. Clin. Res. Inst. Osaka National Hospital, Osaka, Japan. Objective: Anti-p24 antibody (IgG) levels and avidity of IgG specific to p24 antigen were examined to characterize the nature of p24 specific IgG produced in patients persistently infected with HIV-1. Methods: Antip24 antibody (IgG) levels in sera were examined by ELISA using microplates coated with purified p24 antigen and sera of IIIV-1 infected hemophiliac patients. Avidity of p24 specific IgG was assayed by avidity-ELISA using purified p24 antigen and IgG fractionated from individual sera. Avidity of p24 specific IgG was evaluated by avidity index. Results: The data obtained from the assays of sequential serum samples from asymptomatic HIIV-1 carrier (AC) show that increase in p24 specific antibody levels correlate to increase in avidity index with time after infection in majority of AC patients. The data also show that avidity index of p24 specific IgG derived from p24 antigen positive sera is low, but the anti-p24 IgG antibody levels are not low. Conclusion: Avidity of p24 specific IgG may be an useful marker for evaluating humoral immune response in HIV-infected patients. PA0216 STUDY OF THE ANTIGENICITY OF SWIV GP140 WITH MONOCLONAL ANTIBODIES TI.aba, R. Legrand2, L Montagnler3 and EBahraoui. 1. CNRS URA1455, Marselle, 2. CEA, Fontenay aux roses, 3.lnstitut Pasteur, Paris, France. Objective: To study the antigenicity and immunogenicity of gpl40 of SIVmac251 and to determine epitopes of neutralization. M h..ds: Mice immunized with soluble recombinant gpl40 of SIV were used to produce monoclonal antibodies (Mab). Anti-gpl40 Mabs were tested for their affinity for gpl40, for their ability to inhibit gp-CD4 interaction, and for their capacity to neutralize SIV infection in-vitro. Re.sults: Four Mab were obtained. After epitope mapping against synthetic peptides, 3 Mab (1B9, 6C11, 8C5) recognized the region 411-430 which is deleted in gpl4O of HIV-2. The fourth Mab (3C8) seems to be directed against a conformational epitope. Interestingly, preincubation of SIV gpl40 with 3C8 Mab blocked its subsequent binding to CD4+ cells. All these Mab will be tested for their capacity to inhibit SIV replication and syncytium induction in-vitro. 0 0 N~ -J IKEGAMI,Nobuko, Osaka National Hospital, Osaka, Japan Telephone(06)-942-1331; Telefax(06)-943-6467 PA0217 RABBITS IMMUNIZED WITH RECOMBINANT VACCINIA VIRUSES EXPRESSING TRUNCATED FORMS OF THE ENVELOPE GLYCOPROTEIN GP160 OF HIV-2 DEVELOPED NEUTRALIZING ANTIBODIES Schaffner Jra, Otteken A., Jurklewicz E., Voss G., Dittmer U., Bodemer W. and Hunsmann G.; German Primate Centre, G6ttingen, Germany Objective: The cellular and humoral Immune response of rabbits immunized with gp 160 of HIV-2 was examined. Methods: Recombinant vaccinia viruses (rVV) expressing the entire HIV-2EN envsgene or its Cterminally truncated derivates were prepared. Rabbits were Immunized wit hthese specific rW and their immune response was assessed. The antigen-specific proliferation of peripheral blood mononuclear cells restimulated with HIV-2E was determined. Serum samples were analyzed by radioimmunoprecipitation (RIP) and an IA. Neutralizing antibodies (nab) were assayed on MT4 cells. Resuls: Sera of all immunized animals precipitated ENV from HIV-2BEN Infected cells. The size of the inserted env gene sequences did not determine the extent of antigen specific blood cell proliferation. Ukewise ELISA antibody titres against HIV-2 did not correlate to the size of the expressed rgp. Serum samples from rabbits Immunized with W-env constructs comprising only 198 and 332 N-terminal aminoacids contained the highest nab titers. Discussion and conclusion: At least one neutralizing domain resides within the 198 N-terminal aminoacids of the env of HIV-2. It is hard to explain why the larger env fragments did not efficiently induce nab. Perhaps nab-inducing epitopes are hidden by sugar residues. Such epitopes can only be recognized by B-cells after the expression of shortened env molecules. J6rg Schaffner, German Primate Centre, Dept. of Virology and Immunology, Kelinerweg 4, D-37077 G6ttingen, Germany phone +49-5513851153, fax +49-551-3851184 PA0218 CHARACTERIZATION OF THE COMPLEMENTARITY DETERMINING REGIONS IN IMMUNOGLOBULIN BY THE 6-MERS WHICH ARE MOST VARIABLE IN THE HIV-1 GENOME TAKIGUCHI. Kvoko, and Doi, H., Fujitsu Labs. Ltd., Chiba, Japan. Objective: To estimate the characteristics of complementarity determining regions (CDRs) in immunoglobulin (Ig) gene which are essential to interact with variable epitopes of HIV-1 proteins. Methods: Using the data base DDBJ we analyzed CDRs in human and rodent Ig genes by the purine (u)/pyrimidine (y) 6-mers [uuyuuy], which are most variable in the HIV-1 genome (Doi, H., Proc. Nail. Acad. Sci. USA, 1991, 88, 9282-9286), and their complementary 6-mers [uyyuyy]. Results: Source Chain Frequency of uuyu and/or [uyyu % Source Chai CDR1 CDR2 CDR3 human H 72.2 29.6 87.7 L 63.2 68.9 50.8 H 30.9 37.0 50.6 rodent L 50.0 81.3 50.8 The 6-mers [uuyuuy] and/or [uyyuyy] characteristically and differently appeared between in human and rodent Ig genes or in each CDRs of H-chain and L-chain. They most frequently appeared (87.7%) in CDR3 of human H-chain, but infrequently in CDR2 of human H-chain (29.6%). In contrast, they frequently appeared in CDR2 of rodent L-chain (81.3%), and infrequently in CDRI of rodent H-chain (30.9%). Codon frames in the 6-mers also differed between the two species. Discussion and Conclusions: The characteristics of CDRs are different between in human and in rodents Ig. The results suggest that chimeric anti-HIV antibodies having CDRs from rodents would not effectively operate or would cause any side effect in human because of idiotype network. TAKIGUCHI, Kyoko, Fujitsu Labs. Ltd., Chiba, Japan. Telephone (81)-43-299-3211; Telefax (81)-43-299-3075

Page  85 PA219 INFECTION ENHANCEMENT AND COMPLEMENT ACTIVATION BY HUMAN GP41 ANTIBODIES E sLGorg, Prohidzka Z, Hidvigi T, Ujhelyi E., T6th D.F1, Thielens N2 NIHBTI.,Budapest, 'Inst.Microbiol.,Debrecen,Hungary, 2Inst.Biol.Struct., Grenoble, France, Objective: LiUttle is known about the mechanism of complement-dependent enhancement of HIV infection(CDEI). Antibodies responsible for CDEI may play a significant role in the progression of HIV disease according to our recent studies (F0st et al., AIDS, in press). In the present work, a possible correlation between CDEI and complement activation was studied by using 3 human anti-gp41 monodonals (a kind gift of Dr.S.Zolla-Pazner, New York) and a polyclonal human anti-gp41 preparation. Methods: Complement activation by the antibodies was tested in gp41-coated EULISA plates. Binding of Clq, C3b, C4b and IgG to the plates incubated with mixtures of different amounts of antibodies and normal human serum (NHS) was measured. CDEI was determined as described previously (D.T6th et a., AIDS, 1991) using mixtures of different dilutions of the antibody preparations and 1:4 or 1:40 final NHS dilution; HTLVIIIb, and CR2 canrrying MT-4 target cells. Growth of HIV in the cultures was monitored by RT assay. Results: All 3 monodonals (181-D, 240-D, 246-D) increased the C1q binding ability of solid phase gp41, the effect of 246-D was the highest. C3b and C4b binding from NHS was increased only by the mAb 246-D and the polyclonal antibody. A significant decrease in antibody binding to gp41 was observed with each antibody preparation in the presence of both NHS and purified Clq,similarly to our earlier results (Hidvegi et al. 1993) - All 3 monoclonals had a marked CDEI effect. The peak RT activity in the cultures was significantly higher at the higher NHS concentration. The extent of CDEI was dependent on the antibody concentration as well. Discussion and Conclusions: Similarly to our previous results obtained with sera of HIV patients, human antigp41 monocdonals had a marked CDEI effect, especially at near-physiological concentrations of complement. The extent of CDEI, however, did not correlate with the ability of the antibodies to increase complement activation by gp41. Further studies are necessary to explain the mechanism of complement-dependent HIV infection enhancement. PA0220 TR-FIA TEST SYSTEM FOR HIV-I ANTIBODY IN HUMAN SERA. Khristova Marina, Kushch A., Busse T., Stachanova V. Institute of Virology, Moscow, Russia. Objective: A competitive time-resolved fluoroimmunoassay (Tr-Fia) for detection of antibodies to p 24 of HIV-1i was developed. Methods: In this assay the solid phase coated with recombinant anti-gens of HIV-I and Eu-helate labeled monoclonal antibody (Mab) to p 24 were used. Results: The developed test system was used ror examination of sera from HIVinfected persons.It was shown that the sensitivity of Tr-Fia test system was compared to that of reference HIV antibody detection EIA, however the time of analysils was 2-3 times less. Conclusions: The developing of analogous Tr-Fia test system with Eu- labeled Nab to both HIV env and gar proteins could be very perspective for rapid AIDS diagnosis. Dr George FOst, Nat.Inst.Haematol.Blood Transfimmunol., Dar6czi Ot 24, P.O.Box 44, H-1502, Budapest, Hungary, phone/fax: 361-2092310 KHRISTOVA M.L. Address: 123098 Moscow, Gamaleya str., 1B. The D. I.Ivanovsky Institute of Virology Tel.: (095) 190-30-54. Fax:(095)190-28-67 PA0221 "PERSPECTIVE NEW METHOD FOR ESTIMATION OF AIDS PATIENTS IMMUNE STATUS" Emelianov B.A., Pokrovsky V.V.,Sooolov I.A.,Semenov V.A., Buravtsova E. Institute of epidemiology - AIDS Center and Anti-Doping Center, Moscow, Russia Method: The ratio is evaluated of the amount of antibodies and immunoglobulines absorbed on blood cells to their amount in blood plasma. Results: Balance of irununoglobulines between absorbed and liquid phases is moved distinctly to the absorbed phase when latent AIDS patients converse to active disease. Linear correlation is essential between such disbalance and parameters of cells immunity. Discussion: Estimations of immunoglobulines balance between blood cells and plasma are rather laborous but this ratio fluctuates in accordance with enviroment and can possibly help to monitor drugs therapy of AIDS patients. Conclusion: Distribution of antibodies and immunoglobulines between blood plasma and cells is related to the immune state of living beings and this ratio is perhaps the integral function of the whole immune status. Emelianov B.A. Moscow AIDS Center Russia 105275 Ulitsa Socolina Gori b2, 15, 8-la (anti-doping) ph.365-30-09 Telex 411677 AIDS SU PA0222 Effect of slalic acid removal on the antibody response to V3 of HIV-1 envelope glycoprotein. Abdelaziz Benjouad1', K. Mabrouk2, J.C Gluckman1, and E.Fenouilletl. (1) CNRS URA 1463, H6pital Piti6-Salpetrire, Paris; (2) CNRS URA 1455, Facult6 de M6decine Nord, Marseille; France. Objective: HIV-1 Env and its principal neutralizing domain, the V3 loop, are essential components of current vaccine trials. Because glycans may Influence gp160 immunogenicity (A.B. et al., J. Virol. 1992,66:2473; A.B. et al., J. Virol. 1993,67:1693), here we characterized anti-V3 antibodies from rabbits immunized by desialylated recombinant HIV-1(LAI) gpl60 (rgp160). Methods: Native or neuraminidase-treated rgp160 was Injected to rabbits. The titers and avidity of the resulting sera were determined by ELISA using V3 peptides from divergent isolates (MN, SF2, CDC4, WMJ2, NY5, HXB2, SC, RF, Z2, Z6, Eli). Their ability to inhibit syncytium formation was studied. Results: Amino acid residues flanking the GPGR tip of V3 were necessary for binding of anti-V3 antibodies elicited by either native or desialylated rgp160. Both types of antibodies reacted to V3(MN) and (SF2) and to the North American/European V3 consensus peptide, and both impaired HIV(LAI)-induced syncytia. Anti-desialylated rgpl60 antibodies reacted in addition to V3(CDC4), (WMJ2) and (NY5). V3 peptides did not differ in their secondary structure, as determined by circular dichroism. The titers and avidity for V3(MN) of antidesialylated rgpl60 antibodies were significantly lower than those of anti-native gp160, which likely accounts for the inability of anti-desialylated rgpl160 sera to neutralize HIV1(MN)-induced syncytia. Conclusion: The immunogenicity of V3 may be influenced by changes in gp160 glycosylation pattern, which may then represent an alternative to elicit broadly reactive antibodies. E.Fenouillet: CNRS URA 1463, 83 Bd de I'H6pital, H6pital Piti6 -Salp~triere, 75651 Paris Cedex13; France. Tel: 33 1 42 17 74 50; Fax: 33 1 42 17 74 41 0 I% -o 0 __) "N) N) @ J'

Page  86 co PA0223 VIROLYSIS AND IN VITRO NEUTRALIZATION OF HIV BY HUMANISED MONOCLONAL ANTIBODY hNM-01. Nakamura, M,I Armour, K,2 Terada, M,3 Carr, FJ,2 Ohno, T, 1 & Harris. WJ.2 IJikei University School of Medicine, Minato-ku Tokyo, Japan, 2Scotgen Biopharmnnaceuticals Inc, Scotland, UK and 3NMBI, Boston, USA. Objective: Murine monoclonal antibody NM-01 has been shown previously to neutralize a broad range of isolates of HIV in vitro and also to induce complement dependent lysis of HIV particles. We have humanised NM01 with the aim of retaining full biological activity within a human IgG Iin preparation for clinical studies. Methods: The heavy and light variable immunoglobulin genes from NMOI were cloned and sequenced and the CDR's transferred into human IgGI immunoglobulin constructs and transfected into mouse NSO cells for expression. Strategic changes were made in human frameworks to restore antigen binding affinity. In vitro neutralization assays were carried out by measuring both reverse transcriptase and p24 levels and syncytium formation. Virolysis was confirmed by electron microscopy and flow cytometry analysis. Results and Discussion: Humanised hNM01 was at least as effective as the original murine antibody in neutralizing MN and IIIB strains of HIV by all assays studied and was several fold more potent at neutralization in the presence of human complement. Neutralization studies of clinical isolates are in progress and will be reported. Presenting Author WJ Harris, Scotgen Biopharmnnaceuticals Inc. Kettock Lodge, Campus 2, Aberdeen Science and Technology Park, Balgownie Drive, Bridge of Don, Aberdeen, AB22 8GU, Scotland, UK. Tel:( 224 ) 822822 Fax:( 224 ) 822966 PA0224 HUMAN ANTIBODY TO MATRIX PROTEIN (P17) NEUTRALIZES HIV INVITRO Kageyama, Seiji, Ismail, S., Katsumoto, T. t, Hossain, M., Gao, M., Taniguchi, K.', Sasao, F., Toya, M.1, Wakamiya, N., Tsuchie, H., and Kurimura, T. Dept. Pathol., Res. Inst. for Microbial Dis., Osaka Univ., Suita; *Eiken Chemical, Ohtawara; tTottori Univ., School of Med., Yonago; ISawai Pharmaceutical, Osaka, Japan. Objective: Because of its conservative amino acid sequence and the consistency of antip17 level in vivo with the deterioration of clinical status, it is of interest to investigate the role of anti-p17 in a natural course of HIV infection. In this study, neutralizing activity of human anti-p 17 purified from HIV carriers' plasma was determined. Materials and Methods: Affinity column chromatography was performed to detect human anti-p17. Recombinant pl17 (kindly provided by Dr. Shinagawa) and glycine-HCI (pH2.5) buffer were used for a ligand and an elution buffer, respectively. Anti-p17 was challenged to HIVJH3 (20 or 200 TCID50so/well) isolated from a Japanese HIV carrier to determine a neutralizing activity. Peripheral blood mononuclear cells from HIV seronegative donors were used for target cells. HIV replication levels in the additional culture were assessed by p24 gag protein production. Results and Discussion: HIV infectivity was impaired after exposure to each antip17, which was apparently observed when human anti-p17 was challenged to decreased HIV dose (20 TCID50so). p24 protein production was significantly suppressed to less than 10 % as compared to control after an exposure to anti-p17. Neutralizing activity of human anti-p1 7 observed in vitro may imply its actual influence on HIV infection in vivo. This result is encouraging the further careful investigation of anti-p17. KAGEYAMA, Seiji, Research Institute for Microbial Diseases, Osaka University, Sulta, Osaka, Japan. Telephone: (81) 06-875-2128. Telefax: (81) 06-875-3894 -o 0 0 3 N "N PA0225 HIV RESISTANCE? Rowland-lones S. Sutton J, Aryoshi K,* Dong T, Gotch F, McAdam S, Sabally S,*Whittle, H,* McMichael A, Inst. Mol. Medicine, Oxford, *MRC, The Gambia. Objective: to determine whether HIV-seronegative commercial sex workers (CSW's) repeatedly exposed to HIV have HIV-specific cytotoxic T lymphocytes (CTL). Methods: 20 seronegative Gambian CSW's, who had ben working at least 5 years, were studied. The peripheral blood mononuclear cells (PBMC's) of those found to have HLA B35 or B53 were stimulated in vitro with previously determined epitope peptides from HIV-1 and 2 restricted by these common Gambian HLA molecules. Assays for HIVspecific CTL activity were performed on days 7 and 14, and where it was detected, evidence of occult HIV infection was sought by viral culture and nested PCR. A control group with B35 at low risk of HIV infection was also studied. Results: 3/6 women with B35 had CTL activity against one or more of the B35 -restricted peptides, which are cross-reactive between HIV-1 and 2. Two of these had detectable CTL three months later None had evidence of persistent HIV infection by viral culture or PCR. Conclusions: The finding of HIV-specific CTL in seronegative and apparently uninfected women who have been highly exposed to HIV-1 and 2 may represent protective immunity to HIV. Dr Sarah Rowland-Jones Molecular Immunology Group, Institute of Molecular Medicine John Radcliffe Hospital, Oxford, OX3 9DU, UK PA0226 LYMPHOPROLIFERATIVE RESPONSES TO MICROBIAL AND HIV-1 SPECIFIC ANTIGENS IN HIV POSITIVE THAI INDIVIDUALS. Supapongse, Thippawan, et al. AFRIMS, Bangkok, Thailand. Objective: To determine the ability of HIV-infected Thai individuals to generate lymphoproliferative responses to HIV-1 envelope antigens from the 2 genotypes present in Thailand and to microbial antigens. Methods: PBMCs from 5 ELISA seronegative and 12 HIV-infected persons were tested for their ability to proliferate after exposure to rgp160 IIIB(genotype B),rgp160 Chiang Mai(genotype E),PPD,tetanus and Candida. The HIV-specific antigens were produced in a Baculovirus expression system and partially purified. A response was scored as positive if the stimulation index was >3. Using PCR with genotype-specific primers, it was determined that both genotype B and E were represented. Results: Observed frequency of lymphoproliferative responses Microbial Antigens HIV-Specific Antigens HIV-Infection PPD Tetanus Candida gpl60IIIB gpl60CM Negative (n=5) 5/5 5/5 4/5 3/5 0/5 Positive (n=12) 11/12 9/12 7/12 2/12 1/12 Lymphocytes from Thai HIV-infected persons exhibit recall responses to microbial antigens nearly comparable to those of uninfected subjects. Responses to HIV gp160 were poor, even when using antigens representative of the HIV-1 genotypes in Thailand. Responses to gp160 cannot be distinguished from a background of apparent cross-reactivity with insect proteins in HIV-negative subjects. Conclusions: The good response to microbial antigens and the relatively poor response to HIV-1 gpl60 observed in other locations are also observed in HIV-positive Thai subjects where a highly distinct genotype of HIV-1 is prevalent. MAJ Thippawan Supapongse AFRIMS, 315/6 Rajvithi Road Bangkok 10400 Thailand Telephone: 66-2-246-0071; Facsimile: 66-2-247-6030

Page  87 PA0227 SPECIFIC CELLULAR IMMUNE RESPONSES IN HEALTH CARE WORKERS AFTER HIV EXPOSURE. Kessler. Harold*: Sullivan, J.*; Pinto, L.**; et al. *Rush Med College, Chicago, IL, **NCI, Bethesda, MD, USA Objective: To evaluate HIV specific T-helper and cytotoxic T-lymphocyte responses in seronegative health care workers (HCW) after occupational exposure to HIV-infected blood/body fluids (B/BF). Method: Peripheral blood mononuclear cells (PBMC) were obtained serially from HCW following occupational B/BF exposures (1-6 phlebotomies per subject at 0 to 52 weeks). PBMC (3x103) were cultured in the presence of influenza, alloantigen, PHA and synthetic HIV envelope peptides. HIV specific T-helper responses were measured by IL-2 production from these cultures. A stimulation index >4 was defined as a positive response. HIV-1 env-stimulated PBMC from HCW were assayed for cytotoxic T-lymphocyte (CTL) activity against env- or medium-pulsed autologous EBV transformed cell lines using a standard 6 hour s'Cr release assay. Evidence of transmission of HIV infection to exposed HCW was evaluated by HIV-Ab and PCR tests. Baultl: Nineteen HCW exposed to HIV-infected B/BF, 20 control HCW exposed to HIV-negative B/BF and 10 controls without occupational histories of B/BF exposure were evaluated. All subjects to date have remained negative for HIV infection. T-helper responders/ CTL responders/ Exposure group HCW evaluated HCW evaluated HIV+ B/BF 14/19 (74%) 3/7 (57%) HIV-- B/BF 4 / 20 (20%) 0 / 5 ( 0%) No exposures 0 / 10 ( 0%) 0 / 5 ( 0%) Conclusion: HIV-specific T-helper responses occur frequently following occupational exposure to HIVcontaminated B/BF in the absence of infection. Cytotoxic T-lymphocyte activity also develops following such exposures and suggests these cellular responses are specific for HIV envelope antigens. KESSLER, Harold A., Rush Medical College, Chicago, IL 60612, USA Telephone 312-942-5865; Telefax 312-942-2184 PA 22 INCREASED EXPRESSION OF CD38 MARKER ON CD8+ T CELLS AND 24 ANTIGEN LEVEL IN HIV DISEASE PROGRESSION. PA 2NGCH SA G I C M I:A EDP A UNIVERSITY OF PAVIA ITALY The authors studied uy f'low-cytometry the usefulness of combined evaluation of immunologic changes in CD8+ subset (CD38 marker of lymphocytes activation and differentiation) and p24 antigen level in HIV disease progression,confronting two study groups of seropositive subjects. The patients were randomized into 2 groups on the basis of CDh absolute count: A) group - CD4 < 200 cells/mm7 (68 subjects) B) group - CD > 200 cells/mm3 (100 subjects) Statistical analysis revealed significant differences among the two groups for both of the markers (CD38+/CD8+ subset and p24 antigen concentration) (p<O,001). They demonstrated a correlationship between increased percentage of CD38+/CD8+ cells and p24 Ag higher level in patient with absolute count of CD4+ < 200 cells/mm. Our results suggest that the monitoring of CD38 on CD8+ cells, in HIV positive patients, associated with the most commonly screened parameters, may facilitate the early diagnosis of AIDS. CAMPISI D. CL. INF. DIS. IRCCS POL. S. MAT E VIA VIALE TARAMELLI 5 ITALY 0382/502675 FAX 032 i320 PA0228 LACK OF CTL RESPONSE ASSOCIATED WITH LOW VIRAL LOAD IN TWO HEALTHY O SYMPTOJIATIC IIV, INDIVIDULS WITl UGI, STABL4 CD4+ CELL COUNTS..M. Dalod, O. Dufresne, 3.0. Guillt, D. Salmon, D. Sicurd, and. Oonmar4 INSERM U 152, Intitut Cochin de GOd tique Moleulaire, Hapital Cochin, 75014 Paris, France. &Service de Mddecine Interne et d'Hmatologie, Hpital Cochin, 75014 Paris, France. Most HIV-infected individuals develop a strong CTL response against mutiple HIV antigens during clinical phases 2 and 3, whereas this response disappears with evolution to AIDS. Although some of the negative influence of CTLs on the loss of CD4+ cells is still unclear, they are believed to play a major role in delaying the emergence of the disease. We therefore studied the anti-HIV CTL response of two atypic asymptomatic patients with high, stable CD4+ cells count (>700/mm3) who have been HIV+ for 3-7 years. No CTL response was found using autologous irradiated blasts for in vitro restimulation. As it was impossible to isolate virus from these patients by classical coculture protocols even after CD8 depletion, we looked for the virus in PBMC by PCR. We used two different pairs of primers, one for GAG, one for POL, internal probes, and beta-globin primers and probe as DNA quality control. Although the resulting signals were weak unlike those from normal asymptomatic controls, all the samples from the first patient were positive for the two genes. By contrast, the samples from the second patient varied widely. Some were repeatedly negative, others positive for only one of the two genes, but none were positive for both. This suggests a low viral load, and we are now doing further quantitation. We are also studying other patients with a similar high, stable CD4+ cell counts. We are looking both for CTL response using different in vitro restimulation protocols, and for CD4+ proliferative responses to PPD, mitogens, and HIV antigens. D. SICARD, CHU COCHIN, 27 rue du Fg Saint Jacques 76754 PARIS CEDEX 14. TEL 42 34 13 43 FAX: 42 34 13 40 PA0230 ESTABLISHMENT OF CD8+ CELL LINES FROM SEROPOSITIVES Tateno Masatoshi*, Takahashi T.*, Sato H.*, Tateno Y.* *, Togashi T.* *, Yoshiki T.** * *Dept. of Pathology, Sapporo City General Hospital, Sapporo, JAPAN, **Dept. of Pediatrics and ***Pathology, Hokkaido University, Sapporo, JAPAN Objectives: HIV-1 seropositive individuals are known to have CD8+ cells which suppress the viral growth. These anti-viral effects are thought to be mediated by CTL and/or unknown soluble factor(s) secreted by them. To investigate the factor(s), we tried to establish CD8+ cell lines from seropositives. Methods: CD8+ cells were isolated by using anti-OKT8 monoclonal antibody and panning plate. After stimulation with PHA for three days, they were cocultured with mitomycin-C treated MT2 cells (HTLV-I immortalized cell line). Results: We obtained three CD8+ cell lines (240,245 and 259). Unexpectedly we could not establish CD8+ cell lines from normal subjects. All of them were HIV-1 negative and positive for HTLV-I. When they were added to acutely infected cultures, they suppressed the viral replication dose-dependently. These suppressive effects were supposed to be mediated by soluble factors secreted in the culture supernatant. Blocking experiments showed that the these factor were different from TNF or INF. Conclusion: The CD8+ cell lines might provide the useful tools for the analysis of antiviral effects of CD8+ cells TATENO, Masatoshi, Dept. of Pathology, Sapporo City General Hospital, Kita 1,Nishi 9, Chuoku, Sapporo 060.JAPAN Tel(81)-11-261-2281; Fax(81)-11-251-7593 O O 0 0 co

Page  88 c0 Go PA0231 IDENTIFICATION OF HIV-I T CELL EPITOPES BY REVERSE IMMUNOGENETICS. Shiga. Haiime, Shioda, T., Takamiya, Y., Oka, S., Kimura, S., Miwa, K.* and Takiguchi, M.: Institute of Medical Science, University of Tokyo and *Ajinomoto Central Research Laboratories. Japan Object: HIV-1 specific CTL is thought to play an crucial role in the clearance of HIV-1. Identification of CTL epitopes is very important for generation of the vaccine preventing the HIV-1 infection. We try to identify multiple T cell epitopes of HIV-1 using "reverse immunogenetics". Methods: 1. Amino acid sequences of HIV-1 were searched for 8 to l1-mer patterns carrying the anchor residues of the HLA-B*3501 binding self-peptide motif. Sixty-four candidates of HLA-B*3501 binding HTLV-1 peptide were synthesized and quantitatively analyzed in a peptide binding assay by an indirect immunofluorescence of their affinity to HLA-B*3501 molecules using RMA-S cells expressing HLA-B*3501 molecules. 2. PBL of two HIV-1 patients carrying HLA-B35 were stimulated in vitro with HLA-B*3501 binding peptides and CTL activity of stimulated PBL were tested after several stimulations. Results: Of sixty-four peptides, twenty seven bound to HLA-B*3501 molecules. Thirteen of twentyseven peptides could induce CTL in both or either HIV- 1 patients carrying HLA-B35. Discussion and Conclusion: We identified numerous CTL epitopes presented by HLA-B*3501 molecules using reverse immunogentics. This strategy should be used to identify CTL epitopes presented by other HLA class I molecules SHIGA, Hajime, Dept. Tumor Biology, Inst. Med. Sci., Univ. Tokyo Tokyo, Japan. Tel:03-3443-81 11, FAX:03-3443-6319 PA0232 PENTOXIFYLLINE REDUCES CYTOKINE RELEASES AND THE ABILITY TO STIMULATE CELL SURFACE ANTIGEN AND HIV-1 EXPRESSION BY CD8+ CYTOTOXIC T LYMPHOCYTES (CTL) Helnkelein, Martin; Schneider-Schaulles, J.; Walker, B.D.; Jassoy, C.; Institute for Virology and Immunobiology, University of Wilrzburg, WUlrzburg, Germany and Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, U.S.A.. -jegtyie To examine whether the phosphodiesterase inhibitor pentoxifylline (POX) downregulates cytokine production by CD8+ CTL and affects biologic functions mediated by CTL-derived cytokines. Mthods POX-treated and untreated HIV-l-specific CTL were incubated with B-lymphoblasts presenting either the cognate epitope or a control peptide and supematants harvested after 24 hours. Concentrations of cytokines in the supematants were determined by ELISA. In addition, U-937 and IMR-32 cells were incubated with these supernatants for 24-48 hours and HLA class I and ICAM-1 expression determined by flow cytometry analyses. Stimulation of HIV-1 expression was determined using the chronically infected monocytoid cell line U1. Results: POX inhibited IFN-y, TNF-a, and GM-CSF releases by antigen-stimulated CTL at the transcriptional level. Similarily, the cAMP analog dibutyryl cAMP inhibited release of these cytokines indicating that the effect of POX on cytokine releases by the CD8+ CTL is mediated by increased intracellular cAMP levels. Target cell lysis by the CTL remained unaffected. The ability of supemrnatants from POX-treated antigen-stimulated CTL to induce HLA class I and ICAM-1 expression and to stimulate HIV-1 replication was significantly reduced. Conclusions: Production of IFN-y, TNF-ax, and GM-CSF by HIV-specific CD8+ CTL is downregulated by POX resulting in restricted biologic activity of these cells. This observation suggests that it might be important to determine the role of cytokines released by these CTL in infected individuals when considering treatment of HIV-1-infected individuals with POX. Martin Heinkelein, Institute for Virology and Immunobiology, Versbacher Strasse 7, Wtirzbutg, Germany. Phone: (931) 201-5954; FAX: (931) 201-3934 O 0 O 0 N W,, "4b PA0233 EXPERIMENTAL STUDY OF TRADITIONAL CHINESE MEDICINAL HERBS-GLYKE ON TREATMENT OF AIDS GUAN Chongfen et al,China Academy of TCM Object: The Traditional Chinese Medicine-Glyke revealed some effects in 112 HIV/AIDS patients treated in Tanzania. Experimental study was made in laboratory. The Immunomodulatory and antivlral effect of Glyke was observed. Hethod: Antiviral effect was investigated by means of SIV and HIV. Immune functional tests was observed with PFC,SRFC,MLR,IL-I,IL-2 AND T cell subset and so on. Result: The experimental results indicated that Glyke escalated the function of plaque forming cells, specific rosette forming cells and the delayed hypersensitivity, strengthen the mixed lymphocyte reaction which showed that it promoted immunological reaction induced by homogenous alloantigen. It also promoted the biological activity of IL-1 and IL-2, especially it could increase the number of T helper cells and recover TH/TS ratio. Results indicated that Glyke could also inhibit the HIV-I, and SIY, whose Inhibitory rate reached 78.817. Conclusion, The experimental results suggest that Glyke not only incresses the immunity of organism but also inhibits the proliferation of HIV. It has no toxic and side effect. Glyke is a safe and effective medicine in treating the HIV/AIDS. Guan Chongfen. 18 Belxinceang, Beijing 100700 China. Tle:4014411-2556. Fax 00861.4013896 PA0234 ANTIGENIC PROFILE OF EARLY HIV CTL RESPONSES IN A SERIES OF SEROCONVERTER PATIENTS - HADIDA F.*, DEBRE P.*,OKSENHENDLER E.**,KATLAMA C.*,SELIGMAN M.**, AUTRAN B.*-* CH Piti6-Salp6tri6re;**CH Saint-Louis;PARIS, France Objective: The aim of our study was: 1/to determine whether the specificities of the early CTL responses observed during the first months after seroconversion might differ from those observed later on in the same donors. 2/ To compare early CTL responses to the CTL profile of stable HIVinfected individuals or in patients progressing towards AIDS. 3/ To analyze the early phenotype changes after seroconversion. Methods: Using recombinant vaccinia viruses expressing HIV-1 LAI genes, HLA class I restricted CTL lines specific for gag, env, pol, nef, rev, tat and vif proteins were analyzed in three groups of patients: a) A group of 9 donors with symptomatic HIV-1 primo infection observed from 1 to 24 months after seroconversion; b) Two groups of HIV-infected individuals with a seropositivity known for more than 2 months, either asymptomatic with stable CD4 counts above 500/mm3 or progressing towards AIDS with mean counts of 200/mm3. Results: Our data indicate that CD8+ cells detected in HIV seroconverters were activated and differentiated in vivo into specific CTL. Very early after seroconversion, CTL responses could be detected against the majority of VIH proteins but mainly directed against the structural proteins. The striking point is that the percentage of CTL responses directed against nef evolves in the same manner as the structural proteins. Finally, the frequencies of CTL responses directed against the early regulatory proteins rev, tat and vif were more prominent after the period of seroconversion compared to the asymptomatic patients with stable CD4 counts. Fabienne HADIDA, Lab. Immunologie Cellulaire et Tissulaire, URA CNRS 625, CH Piti-Salpdtriere, 83 bd de l'h6pital, 75013 PARIS, France. Tel., FAX.

Page  89 PA0235 ANTIBODY RESPONSE TO PNEUMOCOCCAL VACCINATION IN HIV-1 INFECTED PATIENTS. Cimoch, Paul J; Loss SD, Houghton RA, Reiter WM, Keller RH, Vorce DE, Nemechek PM, Berger DS, Piperado JR, Tomaka FL. Center for Special Immunology, Ft. Lauderdale, Florida, USA. OBJECTIVE: To assess the response in HIV-1 infected patients to pneumococcal vaccination and correlate this response with other immune system parameters. METHODS: Polyvalent pneumococcal vaccine, 0.5cbc sc, (Pneumovax 23, MSD) was administered to HIV-1 infected patients. Paired pre- and post-vaccination IgG titers measured to pneumococcal antigens types 03, 08, 12 and 14 (ELISA, Microbiology Reference Laboratory, Cypress, California) were obtained. Absolute CD4+ cell count and T-cel activation index to mitogen stimulation (patients with >200 CD4+ cells/mm3) performed at time of vaccination (+/- 3 months) were compared with quantitative changes in titers of pneumococcal antibodies. Patients having a three-fold increase in antibody titer to one of the two major antigens (03 or 08) were considered responders. RESULTS: A total of 102 patients were studied withpaired pre- and post-vaccination pneumococcal titers performed on the average. rane of 4.5 weeks apart (range 4-7 weeks). Patients were stratified by CD4+ cell range, with numbers of responders in each range as follows: >500 cells/mrm3 (n=29), 8 (28%); 200-500 cells/mm3 (n=28), 6 (21%); <200 cells/mm3 (n=45), 11 (24%). There was no correlation between CD4+ cell counts and response to pneumococcal vaccination (p=0.86 Chi-square). In the cohort of patients above 200 cells/mm3, responders had a higher mean T-cell activation index than non-responders (33.9%, SD=16.0 versus 20.2%, SD=18.4; p=0.028 independent t-test). CONCLUSIONS: An antibody response to polyvalent pneumococcal vaccination was observed in 24% of HIV-1 infected patients studied. Presence or absence of a response did not correlate with numbers of CD4+ cells. In patients who have above 200 cells/mm3, an antibody response did significantly correlate with better T-cell function as measured by a greater T-cell activation index. Response to pneumococcal vaccination in HIV infected individuals, as a measure of immune system function, may serve as a useful assay for guiding clinical therapeutic or prophylactic decisions. PA0236 ANTI-LACTOFERRIN ANTIBODIES IN HIV-1 INFECTION M.CDEPERIJ.-Y.FOLLI:.OU2,O. PICARD3 ANDCDAMAIS1 1U313 INSERM, Cordelicrs. F-75006 Paris; 2H6pital de la Pitid Salpdtritre F.75013 Pais; 3H6pitnl Saint-Antoine 11-75012 Paris. Antineutrophil cytoplasmic antibodies (ANCA) have been described in several forms of vasculitis and recently in patients with AIDS. Auto-antigen targets recognized by ANCA are mainly protcins found in azurophilic granules of neutrophils or lysosomes of monocytes whereas the specificity of ANCA in HIV-infected patients was still unidentified. Here we proposed that these ANCA found in HIV-positive patients recognized lactoferrin (LI), a protein found in the specific granules of neutroplls and involved in non-specific immunity. Sera from 85 HIV-infected patients have been tested for the presence of antilactoferrin antibodies (anti-LF Ab). 58 sera were found positive, this include sera from asymptomatie (18/28, 64.3 %) and AIDS patients (39/57, 68.4 %). In the control group, only one out of 26 normal donors show any reactivity. The auto-antibodies found were IgG, neither IgA nor IgE isotype was detected. None of the tested patients positive for anti-LF had clinical evidence of vasculitis, the previous reported anti-LF-associated pathology and the level of anti-LF Ab was not directly correlated either with the stage of the disease or the hypergammaglobulineamia. Moreover, no correlation was found between the levels of the auto-antibodies, the lactoferrin itself and the neutrophil counts. The clinical significance of the autoantibodies anti-LF in AIDS remains to be elucidated and will be discussed. Acknowledgments: we express our gratitude to Pr. D. Zagury for his cooperative attitude In providing serum of the HIV+ patient. Follzou J.-Y., Service de Radiothdrapic, Hpital de laI Pitid Salp6tribre, 25, Bid de I'HOpital F-75013 Paris (Prance). TdI: (33) Fax # (33) Paul J. Cimoch, MD, FACP 515 East Las Olas Blvd, Suite 1600 Ft. Lauderdale, Florida 33301, USA 305-767-4715 (F) 305-766-2552 (T) Note: If you are submitting an ebstract, you must rbgister slmultaneously. P A023 7 EVIDENCE OF POLYCLONAL B CELL ACTIVATION IN IV INFECTION W.K. Chia, F. Wright, X. Li, J. Freedman, S.E. Read., University of Toronto, Canada. Objective: To examine the presence of autoreactive and cross-reactive antibodies in HIV-infected individuals. Methodr: Magnetic beads were used for CD4' and CD8' cell isolation. The lymphocytes were then stained with FITI'C-conjugated goat anti-human IgG. Serum and plasma specimens from HIV' individuals were also examined for immunofluorescent (IF) antibodies and neutralizing antibodies against vaccinia virus (vSC-8). Results: 20% of lymphocytes from early stage HIV* patients were found coated with automreactive antibodies. Lymphocytes coated with these autoreactive antibodies increased to 50% in patient with AIDS. An unusually high proportion HIV patients also have IF antibodies against vaccinia virus (vSC-8). In paired serum samples from HIV-infected individuals collected before and after HIV seroconversion, anti-vaccinia IF antibody was detected only in postseroconversion samples. However, this was a non-neutralizing anti-vaccinia in that it did not protect cell culture (CV-1) from vaccinia virus infection. Discussion and Conclusion: We have demonstrated auto-reactive antibodies against T-lymphocytes, and also non-protective antibody reactive to vaccinia virus associated with IlIV infection. Based on these observations, we propose that the presence of these antibodies might be due to polyclonal B lymphocyte activation related to IIlIV infection. Mr. Wah Kiam Chia, Dept. of Microbiology, University of Toronto 150 College St., Toronto, Ont., Canada, MSS IA8, Tel: 416-864-5184 Fax: 416-864-5693 PA0238 SERUM IgG AND IgA SUBCLASS CHANGES OVER THREE YEARS IN HEMOPHILIAC CHILDREN WITH HIV INFECTION. YOSHIHIRO TAKAHASHI, DEPARTMENT OF PEDIATRICS, ODATE MUNICIPAL HOSPITAL, ODATE, AKITA, JAPAN. Objective: Children with HIV infection have various dysfunctions in humoral and cellular immunity. We have investigated serum IgG subclass, IgA subclass and peripheral blood lymphocyte subset(CD4,CD8) in 7 HIV infected hemophiliac children over three years, 1990 to 1992. Methods: IgG subclass were measured by a radioimmunoassay using monoclonal antibodies to IgGl-4, IgA subclass were also measured by enzyme immunoassay using monoclonal antibodies to IgAl-2. Results: Three of 7 cases have progressed from AC to ARC, and the 4 other cases have remained AC over three years. IgGl level of the 5 cases have been stationary or increased as total IgG level. The IgG2 and IgG4 levels of all 7 cases, and IgG3 level of 6 cases have decreased in these 3 years. Above all, IgG2 levels of 3 cases are below the normal range in 1992. We have also observed the correlated decreasing between IgG2,IgG3 and CD4 cells count. The IgAl and IgA2 levels of 5 cases have increased as total IgA level, and those levels of the other 2 cases have decreased. Discussion and Conclusions: These data indicate that IgG and IgA subclass abnormalities are found in most HIV-infected hemophiliac children as progression from AC to ARC, and quantitative deficiencies of IgG subclass, particulaly IgG2 and IgG3, might explain the high frequency of recurrent bacterial respiratory infections occurring in these progressed patients as acquired humoral immunodeficiency due to HIV infection. These data also suggest that the prophylactic use of intravenous immune globulin for IgG subclass supplemetation may be effective in preventing serious bacterial infections in symptomatic HIV-infected children. TAKAHASHI, Yoshihiro, Department of Pediatrics,Odate Municipal Hospital, 3-1, Yutakacho, Odate, Japan,017 Telephone 0186-42-5370; Telefax 0186-42-2055 -v 0 N 0

Page  90 e PA0239 CHANGES IN AUTOREACTIVITY DURING HIV rgpl60 VACCINATION. Lundholm. Peter, Wahren, M., Sandstrom, E., Volvovitz, F. & Wahren, B. Dept. of Clin. Virology, Karolinska Institute, Stockholm, Sweden. PA0240 MOLECULAR MIMICRY OF HIV gpl20 PROTEIN TO VOLTAGEDEPENDENT Ca++ CHANNEL RECEPTOR Blinov, V.M., Resenchuk. S.M., Chirikova, G.B., Denisov, S.I., Zverev V.V. Objective: The HIV-1 envelope protein contains several regions with amino acid homology to HLA class I and class II molecules. Occurrence and changes of autoimmune reactivity to these molecules were studied in asymptomatic HIV-infected patients and during vaccination with HIV envelope gp160 glycoprotein. Methods: Forty asymptomatic I IV-infected patients with CD4 cell counts above 400 were vaccinated 6 times during 6 months with rgpl60 of HIV-l, produced in a baculo virus vector. Microcytotoxicity and enzyme-linked immunosorbent assay to HLA class I and II IIIV homologous peptides and II1V related synthetic peptides were performed with sera taken over a 1-year period for each individual. Results: 13/21 ILA A2 typed and HIV-I infected patients exhibited cytotoxic lymphocyte antibodies to CD8+, HLA A2 cells. This reactivity decreased in all patients during vaccination with rgpl60. Three regained cytolytic activity after immunizations were discontinued. 25-65% of I IIV-I infected individuals had antibodies reactive with peptides representing HLA class I DR or HLA class I A2 regions. HlLA-specific antibodies also decreased during immunization while HIV specific antibodies increased. Conclusions: Autoantibodies directed to bistocompatibility antigens occur in HIV-I infected patients. It appears feasible to redirect the humoral response from autoimmune to HIV-specific antibody responses by repeated immunization with HIV rgpl60. LUNDIHOLM, Peter, Department of Clinical Virology, Karolinska Institute and Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Sweden. Telephone: +46-8-735 13 00; Telefax: +46-8-27 22 31 PA0241 SEROLOGICAL ASPECTS OF AUTOIIMUNITY IN HIVINFECTED PATIENTS. Stanekova Danica, Habekova M., Mokras M., Bratislava, Slovakia Objective: ie(.i ).1. (i, a.cs. I at.itoiIur, i t.y phe'rt - trc cfte n disu( n"c, t in IltV in frection.re itndjried in HV',' pu:si tive pa tir:-.r t.. Methods: 18 HI V-rp:.itive r:patii.nto (stage 01-. A.: 15:ca--.. C2: 1 t: -: cane) were eva I.t ted. An ti- - S rd.. E and arint.i. -I, arnt.ibodie were m..acurrid by EL IMF; ii i, ur-.i fiied n..cti ger. CIt were gut..n ti ied by tI urbi - cli m(.t.-.. ing rni:romooetod with E)G aend '32, 4i and Bf t.b,,ct1E by radial. immunodif fusion. Results: m ti - UI-. antibodies wter-e i"ut i outrnd in I. p itive pat ien te. Anti --ds--Lt tA antibodies weri?: c t.i tated inr 2 of 1.1 ( 18.2%) patienrts, (stage:/A). No..iific:nt diff(-_rences in) C3,C4, Bf.nd CIK::.t:..-els wer..-- r n.ci d b t-w.:nrt hi:e. groutP. of HIV. - in rfer ted and hea 1tI, irt vi d t i 1 Disscusions and Conclusions. The o)rigait of autoimmrnune:. (mtirt ai it:rin:t s 11 of. irtfecti.(~n n is rctmple acud lob.cr:t.tre. It'-ro.tct, ti ct o)f autt.ar'ti. bodies is only a. ien- er;al.i zed in - (lit..o otcf imitnto r'l.a ti tin t.lisor e-.r., Stanekova, Danica, Inst. of Preventive and Clinical Medicine, Limbova 14, 83301 Bratis lava, Slovakia, tel: 42-7-374980, F: 373906 Two-thirds of AIDS patients suffer from dementia and a host of other neurological disorders caused mainly by the damage of patient's neurons. HIV envelope glycoprotein gp120 is the probable culprit. Structural features of the alphal subunit of L-type voltage-sensitive calcium channels includes four homologous repeats (I-IV) containing six membranespanning regions (S1-S6). S3 is a putative transmembrane helix with considerable negative charge that forms the salt bridges with positive charges on S4 (where the voltage-sensitive mechanism is thought to reside). Structural features of HIV gp160, being the precursor of gp120, are similar to Ca++ channel receptor. gp160 contains six membrane-spanning regions (H1-H8). H2 and H6 segments are putative transmembrane helixes, with negative charges that compete across the salt bridges with positive charges on S4 of Ca++ channel. The competitive interaction between H2 segment of gp120 and S4 segment of brain Ca++ channel may enhance rbC receptor mediated vulnerability by promotoring the essentially irreversible increase in Ca++ ions with subsequent cell death. Thus, mimicking of gpl20 to the Ca++ channel receptor are the possible cause of HIV neurotoxicity. RESENCHUK, Sergey, Institute of Molecular Biology, Koltsovo, Novosibirsk, Russia. Tel. (007)-3832-643-942 0 N) "D W 0 N) 44h N PA0242 HEPATITIS C VIRUS (HCV) RNA AND HUMAN IMMUNE-DEFICIENCY VIRUS (HIV) P24 ANTIGEN IN THE CRYOGLOBULIN OF HEMOPHILIACS WITH HIV AND OR/ HCV INFECTION. GOHCHI Kengo, Matsuda J, Gotoh M, Tsukamoto M and Saitoh N Department of Medicine, Teikyo University School of Medicine, Tokyo, Japan Objective: Cryoglobulin (cryo) as a form of immune complex (IC) frequently occurs in association with infectious pathogens, including hepatitis C virus (HCV). It is well known that circulating IC is present in patients with HIV infection, We investigated whether HIV is associated with the formation of cryo in hemophiliacs (H) who are infected with HIV. Methods: We studied 52 H with HCV antibody and HCV RNA (30 were also positive for HIV), 5 male homosexuals (Ho) positive for HIV without HCV infection, and 20 healthy controls (C). Cryoglobulin was measured by the modified method of Brouet et al. Results and Conclusion: cryowas detected in 48 of 52 H. The 4 remaining H without cryo were HIV positive. HCV RNA was detected in the cryo of 85% H. p24 antigen was detected in 46% of HIV positive H with cryo. However p24 antigen was negative in the cryo of these patients or in that of the remaining patients without p24. Ho and C were all negative for cryo. We treated two HIVpositive patients with HCV-infection with interferon. In one patient' s case, cryo was still positive, however, HCV RNA was no longer detected in cryo. These facts suggest that a certain virus(es) including HIV other than HCV and/or an immunologic abnormality(ies) might be associated with the formation of cryoglobulin. GOHCHI Kengo, Teikyo University School of Medicine,2-11-1 Kaga Itabashiku Tokyo, Japan Tel: 03-3964-1211, Fax: 03-5375-0272

Page  91 PA0243 THEORETICAL STUDY OF THE MECHANISM BY WHICH HIV INDUCES AIDS Shiosawa.Masasumi, Kashimori.Y, and Kambara.T The University of Electro-Communications PA0244 DIMINISHED SURFACE EXPRESSION OF IgA Fc RECEPTORS (FcaR) CtON BLOOD NEUTROPHILS AND MONOCYTES AND THEIR SATURATION BY ENDOGENOUSIgA IN AIDS PATIENTS. GROSSEIETE, B., VIARD J.P., BACH. J.F., LEHUEN, A., MONTEIRO. R:; H6pital Necker, PARIS, FRANCE. Objective: There are various length of the period between HIV infection and AIDS. It has not been clarified yet what determines the length. In order to clarify the mechanism by which HIV induces AIDS, we investigate the response of the immune system to HIV infection using a theoretical model of the system. Methods: We construct a model of the immune system where the variations in the number of many kinds of antigens, helper T cells and killer T cells and of HIV are described by the differential equations. we consider several kinds of mechanisms for the development of AIDS and investigate how the immune system is destroyed through each mechanism by using computer simulation. Results: We have investigated the effect of five possible mechanisms: (1)Increase of the ability that HIV avoids the attack of immune system by mutation of HIV, (2)Destruction of lymph nodes by HIV, (3)Reduction of function of thymus induced by HIV, (4)Transformation of HIV to a super antigen by HIV mutation, (5)Coinfections of some pathogens and HIV. All of the mechanisms may induce AIDS if we choose suitably the values of the parameters included in the model. However, the variation process in the state of the immune system from the HIV infection to AIDS is qualitatively different for each mechanism. Discussion: It could not be determined from the present simulation which mechanism is most plausible. We need much more microscopic experimental information about the action of HIV in human body. Shiozawa.M, Kashimori.Y, and Kambara.T Department of Applied Physics and Chemistry, The University of Electro-Communications, Chouf, Tokyo, 182, Japan Telephone (+81)-0424-83-2161; Telefax (+81)-0424-89-9748 PA0245 COMPUTER SIMULATION OF HIV INDUCED DEFORMATION OF IMMUNE NETWORK SYSTEM Hirai Masahito, Kashimori Y. and Kambara T. The University of Electro-Communications Objective: A functional idiotypic network is a useful model of the immune system. In order to clarify how HIV destroys the immune system, we investigate how and through what mechanism the idiotypic network is deformed by HIV infection. Methods: We have developed a model for the immune network system that is easily simulated on a computer. An antibody is represented as a pair of the paratope and the epitope strings. The connections between antibodies and between antibodies and antigens are made by complementary matches between the strings. Results: Before HIV infection the network stays in a stationary state where many idiotopes may stably coexist. However, the network is deformed by the HIV infection such that the numbers of many idiotopes change temporarily. If we treat HIV as a super antigen, the network is completely destroyed. Discussion and Conclusions: Although the results are based on a theoretical model of the immune system, it is a possible hypothesis for the cause of AIDS that HIV becomes a super antigen after many times of its mutation. M.Hirai, Y.Kashimori and T.Kambara Kambara lab., Dept. of Applied Phys. and Chem., Univ. of Electro-Communications,chofu,Tokyo 182, Japan Telephone +81-424-83-2161; Telefax +81-424-89-9748 Objective: Elevated IgA plasma levels have been detected in patients with AIDS. Since hyper IgA plasma level could be due to an aberrant IgA catabolism, we analysed FaR expression and saturation by endogenous IgA in blood phagocytic cells from patients with AIDS. Methods: This was realized by using a quantitative flow cytometry method in which whole blood cells were stained either with anti-FeaR mAbs recognizing epitopes outside the IgA-binding site, or with F(ab')2 fragments of anti-IgA antibodies. Results: The expression of FeaR was decreased on neutrophils and monocytes in patients (n=27), as comparedto controls (n=21) (mean fluorescence intensities: neutrophils: 33.5~16 versus 41.6~12, p=0.05; monocytes: 53.9~16 vs 77.7+21, p<0.001). In AIDS patients, however, neutrophils and monocytes had higher levels of receptor-bound IgA than controls (mean fluorescence intensities: neutrophils: 3+2 vs 0.92~0.4, p<0.001; monocytes: 3.4+2.7 vs 0.8+0.5, p<0.001). Conclusion: These results suggest that diminished expression of FeaR on blood phagocytes could contribute to the formation of hyper IgA levels in AIDS. VIARD, Jean-Paul, Service d'Immunologie Clinique, H3pital Necker, PARIS, FRANCE. Tel: (33) 1 44 49 54 25; Fax: (33) 1 43 06 23 88 PA0246 DISTINCT T CELL DISTURBANCES IN HIV2 INFECTION Jaleco A.,Cov s MJ., Pintq LL.., Vi orino, Rui M.M aculdad e edicna de Lis -o sa anta Maria Medicina 2 / Imunologia Clinica. Lisboa-Portugal Objective: As progression of HIV2 associated disease appears to be slower than in HIV1, we investigated whether there were distinct alterations in T cell subsets ("memory" and "naive" T cells) as well as in T cell death and apoptosis. Methods: Lymphocyte subsets were quantitated in the peripheral blood mononuclear cells from HIV2 (n=43) and HIV1 (n=46) infected subjects by flow cytometry using monoclonal antibodies to CD3, CD4, CD8, CD45RO and CD45RA. For T cell death and apoptosis studies, PBMC were cultured for 48 hours alone or in the presence of PHA, PWM or staphilococal enterotoxin. Cell death was measured by trypan blue exclusion and apoptosis by measuring the % of hypodiploid nuclei corresponding to fragmented DNA, by flow cytometry. Results: There were no differences in the degree of reduction of CD4+ CD45RO+ cells in asymptomatic HIV1 (8.9%~3.5 SD) versus asymptomatic HIV2 (10.0%~6.8 SD) patients. However, in HIV1 AIDS patients (3.4%~2.6 SD) the reduction in the percentage of this subset was more pronounced than in HIV2 Infection (6.3%t4.5 SD). CD8+ CD45RO+ cells were significantly increased both in asymptomatic (8.3%~5.8 SD) and in AIDS (18.5%~12.4 SD) HIV1 patients as compared to normal subjects (3.7%~4.0 SD). HIV2 asymptomatic patients had normal levels of these cells (5.2%~4.0 SD) and HIV2 AIDS patients had increases (9.1%~7.9 SD) which were much less pronounced than the ones observed in HIV1 AIDS patients. Cell death (HIV1 n=27, HIV2 n=30) and apoptosis (HIV1 n=17 and HIV2 n=15) percentages were increased in both HIV1 and HIV2 infected subjects particularly in AIDS patients. However, asymptomatic HIV2 patients show less pronounced increases in cell death and apoptosis when compared to normal subjects than HIV1 asymptomatic subjects. Conclusion: 1) The depletion of "memory" CD4+ cells and the expansion of CD8+ CD45RO+ cytotoxic subset are less pronounced in HIV2 infection. 2) Increased T cell death and apoptosis also occurs in HIV2 infection although asymptomatic HIV2 patients exhibit lesser increases. Victorino R.M.Hft. - Faculdade de ledicina de Lisboa3-. floniz 5100Li oa-Portugai Phone:3a1-i-7 lonFax: 351-1-30 1445 0 N 4b 0 0)

Page  92 c PAO247 THE EFFECT OF HIV-INFECTION ON EARLY AGING OF THE IMMUNE?'J SYSTEM & THE POSSIBLE ROLE OF MODIFICATION OF REVERSED CD4/CD8 RATIO IN HIV-INFECTION TREATMENT. Raoufi, Mohammad; Iran's University of Medical Sciences, Tehran, Iran Objective: To evaluate the effects of the rate & duration of CD8+ cell count alternations & CD4/CD8 ratio reversion on immunodeficiency occured In HIV-infection. Method and Results: The emergence of idiopathic CD4+ T lymphocytopenia (ICL) & its related severe immunodeficiency has served a similar condition to HIV Infection through which the direct HIV-related effects on the immune system has omitted & the more accurate assessment of Immune system's abnormalities, being common between ICL & HIV-infectlon has been provided. In addition, according to close similarities between the immune system of HIV-infected individuals & aged persons, the following speculations could be considered: 1) Like CD4+ T lymphocytes, the CD8+ T lymphocytes may play an important role In induction of immunodeficiency & in prediction of HIV-infection progression; 2) Neither the CD4+ T cell counts nor its declining rate could sufficiently predict the HIV-infection progression; the CD8+ cell counts (whether constant or increased) & the severity of CD4/CD8 ratio reversion should also be considered; 3) It seems that the rate & duratrion of simultaneous alteration of CD4+ & CD8+ T cell counts are the most important factors affecting the HIV-infection progression. Indeed, a persistent low CD4 cell count & reversed CD4/CD8 ratio (> 1.0) is likely not to be associated with an Impending severe immunodeficiency. In contrast, a progressive & long-term CD4+ T cell depletion and CD4/CD8 < 1 could strongly alarm a more rapid progression of HIV infeciton. The active chronicity of CD4/CD8 ratio reversion may result in Irreversible immunodeficiency; 4) The modulation of reversed CD4/CD8 ratio by using of available antiretroviral drugs, being able to increase the CD4+ T cell counts even transiently, and by suppression of CD8+ T cell counts to a normal limit may contribute to injured immune system to reconstruct itself & conqure the HIV-infection & its progression more properly. M(,M][MAD RAOXJFI 21,Hafez Shirazi Alley,Vali-e-Asr St.,Tehran,19679,IRAN PA0248 CD4 AND CD8 LYMPHOCYTES IN llIV INFECTED AND NON-INFECTED BABIES BORN TO IIIV-1 SEROPOSITIVE MOTHERS. BANGKOK. THAILAND. LikanonsakuLSirirat;' Wasi C;' Thepthai C;' Sutthent R;' Louisirirotchanakul S;' Chearskul S;' Vanprapar N;' Department of Microbiology', Immunology', Pediatrics'. Faculty of Medicine Siriraj Hospital. Bangkok, Thailand. Objeclive: To determine the numbers and percentages of CD4 and CDs T-lymphocytes in I lIV infected and non-infected babies born to HIV seropositive mothers at various ages. Method: From April 1992 to December 1993, a total of 108 babies born to I-V seropositive mothers were studied. The 43 babies were 13 asymptomatic and.o symptomatic I IIV infected halies, classified as l'I and '2, respectively. The ns babies showed seroreversion and assumed to be the non-infected cases (C). The CD4 and CDs bearing lymphocytes were determined by flow cytometry. Approximately, 3 ml of IDTA blood were collected for CBC, T-cell subpopulaion (SimutestM CD4/CDs) and HIV testings (ant-I IIV, immune compcx dissociation 1'24 Ag and proviral DNA) Result: A total of 275 subsequent follow- up blood samples from 108 subjects were analysed in this study. The result of T-cell subpopulation in each group were shown in Table. Proviral DNA wsas detected in all infected babies. P24 Ag was puositive in 9n%. T-cell subsets 0-3 too. >3-6 me. >6-12 mo. >12-42 mo. (x 1 SD) C Pt P2 C PI P2 C Pt P2 C Pt P2 "/CD4 4317 33~9 171~9 40~7 27.9 2619 36_5 23~7 1418 3415 18~6 1216 %CD8 24~7 2717 39012 21~7 29~6 33~16 24~1 37~14 46~12 27~7 43111 5411 CD4/CD8 ratio 2.0~0.7 1.3~0.6 0.5~0.3 2.2~0.9 1.0~0.6 1.2~1.0 1.5~0.6 0.7~0.4 0.410.4 1.310.4I 0.4~0.2 0.210.2 N 48 13 6 42 10o 5 47 9 8 14 40 11 31t Conclusion: The numbers and percentages of CDI and CDs lymphocytes in asymptomnatic and symptomatic I IV infected babies were significantly difference from the control cases (p<oos) as well as CD4/CD8 ratio. Siriral t.ikanonsaku l)c. Department of Microbiology, Faculty of Medicine Siriraj I lospital, Mahidol University, Bang'.ok Thailand. (Tel e062-4t13111; Fax 662-4110201) 0 N 4.b 0 0 PA0249 CIIARACTERIZATION OF TIME V82 T CELL SUBSET SPECIFICALLY DELETED DURING IlYV-INFTCt]ON F.Poccia, S.Boullier, M.Cochet and M.L.Gougeon Unit6 doncologie Virale, Institut Pasteur, Paris, France Qkllcsijyj: As we have previously observed that the V52 T cell subset was strongly reduced in the course of HIV-infection, we characterized the repertoire and the function of this subset in healthy donors and patients. Methods: Peripheral yS T cell subsets were investigated for the expression of the VS chains using specific mAbs and FACScan analysis.The V82 T cell gene rearrangements were analysed by PCR using specific V52 and CS oligonucleotides.TCRs rearrangements and CDR3 size distribution were analysed on an Automatic DNA Sequencer using fluorescent JS's oligonucleotides. In addition influence of various stimuli such as anti-y8 TcR or anti-CD3 mAbs, MTB, Daudi etc.. was studied on the fitction of this T cell subset. Phenotypic analysis of V52 T cells was performed using specific mAbs (CD45RO, bcl2, Fas, etc...) and FACS analysis. Results: Repertoire analysis of the V52-C8 transcripts indicated no major difference in the CDR3 size profile between normal donors and HIV-infected patients. Functional analysis of V52 T cells in infected vs. healthy donors indicated a normal response to anti-yS TcR mAbs, Daudi and MTB stimulations. Since V82 T cells are mainly CD45RO+, express surface Fas antigen correlated with low levels of intracytoplasmic Bcl-2, they could be more sensitive to activation-associated cell death. Indeed these cells showed a selective inability to respond to ionomycin, PHA or anti-CD3 mAbs. Interestingly enough patients' V82 T cells were in addition defective in their response to IL-2. In order to investigate the influence of HIV-1 infection on V52 T cell decresase and dysfunction, normal lymphocytes were productively infected with HIV in vitro. We observed under these conditions an IL-2 dependent selective expansion of V82 T cells. Since noe of Ithe HIV recombinant proteins were able to induce this activation, it suggests that HIV may play an indirect role in this phenomenon through cytokine production. oncluslon: The reduction of the V82 T cell subset during HIV-infection is not correlated with a specific antigenic stimulation. This selective deletion of the primed/memory V52 Fas expressing subset may be the consequence of the in vivo activation of this subset by cytokines chronically produced in patients. POCCIA Fabrizio,Unitd d'oncologie Virale, Institut Pasteur. Paris, France Tel. (33) 1 45688914; Fax (33) 1 45688885 'ONPATT250,o.NGOF C I.CD CD38+ LYPHOCYTE COUNTS IN PA0250 tns i tgC r P~B' PA^ "'=U FBCT!Vz FOLLOW-u} STUDY Teertevadue TngLr UhavdLa, N., Mebonia, 1. Geo6tain AIDS Research Centor, Tbilisi, Georgia. Obj sotive s Prospective follow-up study of CD6+ and ODO+CD38 lymphocyte counts and evaluation of their interrelationship in the course of disease in HIV infected patients. Methods a Four year prospective follow-up study of 14 adult paLiwents with II1 stage of IIV nfeotion (by CDC) haa boon uarried out. CD6+ (Activated Helper T Cell.) and CDS+ lymphocytes expressing CD38 (activation markers), as well as other immunologic indices were determined every three months. During the follow-up period 4 patients developed AIDS. Results= Increasea of CD6+ lymphocytes number was observed in 4 of 14 patients with II stage, in 6 of 9 patients with III stage, in 2 of 3 patients with IV A and IV c2 stages and in all 4 patients with AIzDS. Inc.rease of CD8+CD38+ lymphocyte count was found in 5, 7, 3, and 3 patient. respectively. Simultaneous stable increase of CD6+ and CD8+CD38+ lymphocyte counto was observed in 6 patients with II/III stage of disease. Subsequently 4 of them showed decrease of CD4+ lymphocytes amount and developed AIDS. Dlsoussio mnad Conolueious, DInelo elevation of CDS+ and CD8+CD38+ lymphocyte levels in most patients proceeds decrease of CD4 lymphocytee amount and is the earliest immonologic sign of HIV infection progress. fsZtSVADAk;eh~i "oG diOh AIDS'Research center, Tbilisi, Georgia. Telephone (8832) 39-80-18 Telex: 21200 eaoDS CaU, 212223 Lha3b 1u

Page  93 PA0251 HGH CD ANTIGEN DENSITY ON T CELLS IN ADVANCED HIV DISEASE g,1 Bdnhegyi D2Horvath A,3 Tarjan V,1 F0st G,1 1NIHBTI; St LAszl6 Hosp.; inst.Dermat.Semmelweis Univ.Budapest, Hungary Objeclie: The abscute number of CD4+ cells is continuously decreasing in the blood of patients with HIV infection. At the same time an elevation in the number of CD8+ cells can be observed. When the CD4+ cell count drops beyond a critical level - usually considered to be 200/ul - AIDS very frequently develops. Some AIDS patients, however who have CD4+ cell count of less than 50 cells/ul iare in relatively good condition and can survive for months or years. The purpose of the present work was to study the CD4 antigen density on T lymphocytes of these patients (n=11; group C) as compared to asymptomatic HIV seropositive (n=8; group B) and HIV seronegative (n=8; group A) persons. Methods: Absolute number of CD3+CD4+ cells and CD3+CD8+ cells were determined by flow cytometry. The amounts of CD4 and CD8 antigens on CD2+ T lymphocytes were measured by the CAPCELLIA test (Sanofi Pasteur) in parallel. Using these two parameters, the cellular density of CD4 and CD8 antigens on T lymphocytes of different persons could be calculated. Results: The mean cellular density of the CD4 antigenson T lymphocytes was found to be 0.15~0.08, 0.25~0.04 (p<0.05) and 1.12~11.56 (p<0.01) pmolx107/T cell respectively, in the groups A, B, and C. By contrast, no significant differences in CD8 cellular density were found between the three groups. Discussion and Conclusions: To detect the CD4 receptors, mAb F101-69 was used which recognise an epitope near to the gpl20 binding site of CD4. Therefore we have measured concentration of only the gp120-free CD4 antigens on the T lymphocytes. The paradoxical increase in the CD4 cellular density in advanced HIV disease can be due to the neoantigen formation induced by gp120 CD4 interaction. The selection of the CD4+ lymphocytes with high CD4 receptor density can be responsible for the surprisingly good clinical condition of some patients with extremely low CD4+ cell counts. Eszter Ujhelyi, Budapest POB 44 H-1502 Hungary 361-1652-059, 361-1667-020 PA0D252 NEUTROPHIL FUNCTION AS A PROGNOSTIC MARKER IN AIDS. P 0VShepeleva Galina, Serebrovskaya LV., Gabrilovich D.I., Ivanova LA., Kravchenko A.V., Poknwsky V.V. Russia AIDS Research Centre, Moscow, Russia Objective: To identify the role of changes in HIV infection of polymorphonuclear neutrophils (PMNs) in prognosis of HIV infection. Subjects and methods: Functional activity of PMN (chemiluminescence (PMN CL),phagocytosis, intracellular killing, NBT-test, activity of intracellular enzymes) was tested in 63 HIV-1 infected patients and 20 healthy HIV seronegative donors. In addition, the numbers of T-cell subsets,level of nonspecific circulating complexes(CIC),IgG,IgA,IgM and fourth component of complement in sera were measured. Results and Conclusion: PMN CL, cytotoxic activity of PMN and activity of intracellular enzymes were decreased in patients at all stages of infection, more pronounced in patients with AIDS. A lower level of PMN activity in patients with infection rapidly progressing towards AIDS was found than in patients with relatively stable course of infection. The initial low level of PMN CL correlate with development of symptomatic HIV infection during the first year after testing, upper level had strong correlation with stable course of HIV infection: CLmv- 865~ 155 (control, n-20),134~ 116 (patients with development of symptomatic HIV infection during of one year period, n-8), 368~146 (patients with stable course of HIV infection during five years, n=14,p,05). The unfavourable prognosis was associated both with low level and very high level of NBT-test (5-6 time more than normal). SHEPELEVA, GALINA, Russia AIDS Research Centre, Moscow, Russia. Telephone: (095)365-30-09. Fax: (095)365-46-80 PAO253 SJOGREN'S SYNDROME: HIV IgE-LIKE BINDING PROTEINS CAUSE THE AUTOIMMUNE DISEASE Blinov, V.M., Grinev, AA., Zverev, V.V. PA0254 SUPPRESSIVE EFFECT OF V3 LOOP ON IL-2 INDUCED T CELL GROWTH: Sakaida HitgoshiMurakami T,Kawase S,Hattori T and Uchiyama T, Inst. for Virus Res, Kyoto Univ, Kyoto, Japan Symptoms similar to autoimmune disease Sjogren's syndrome and glandular pathology are observed in certain HIV-infected persons, hence a search was initiated for a possible retroviral etiology in this syndrome. Surprising homology on the protein level of immunoglobulin E-binding factor (IgE-BF), playing significant role in autoimmune diseases, with coding sequences of the GAG and POL genes from human retroviruses (HIV1, HIV2, HTLVI, HTLVII) was revealed. These data suggest that pathogenic retroviruses may produce viral proteins similar to cellular IgE-binding proteins causing an autoimmune diseases. Models of HIV1 mRNAs precursors secondary structure were elaborated. According to these models GAG-POL mRNA have alternative splicing sites to produce IgE-binding proteins. The revealed similarities suggest that the major GAG-POL protein is likely to be important not only in viral reproduction, but in affecting the B-cellular immunity as well. We propose that the monoclonal antibodies against the HIV-produced IgE-like binding proteins may be used for treatment of SS and AIDS patients. GRINEV, Andrian, Institute for Viral Preparation, Moscow, Russia. Telephone (007)-095-274-56-05 Objective: The immunodeficiency caused by HIV is primarily due to a progressive decline in the number and function of T4 cells. We investigated the effect of the third variable loop (V3 loop) of gpl20 of HIV which has various biological activities on the proliferation of T cells. Methods: V3 loop of HTLV-III BH10 strain as well as linear V3 peptides (CTR36,IRI(F/G), IRI12) was synthesized. The cells used for the proliferation study (3H-TdR uptake) and cell cycle analysis (FACScan analysis) were IL-2 dependent cell lines (Kit225,ED40515(+),KT3), IL-2 independent cell lines (Hut102, Jurkat) and PHA-stimulated PBMC. Results: V3 loop markedly suppressed (>95%) IL-2-Induced proliferation of IL-2 dependent cell lines but not IL-2 independent cell lines at 10 M, whereas other used V3 peptides did not show any suppressive effect. Cell cycle analysis after 24 hr culture with V3 loop revealed G1 arrest of Kit225 cells. IL-2- and IL-4- but not IL-7- driven cell growth of PHA- stimulated PBMC was Inhibited by V3 loop. Discussion and Conclusions: The cell growth through the IL-2/IL-2R system was strongly inhibited by V3 loop, and the loop structure was Important for Its biological activities. The suppressive effect of V3 loop may play an Important role In the development of immunodeficiency in HIV- infected people. SAKAIDA, Hitoshi, Institute for Virus Research, Kyoto University, Kyoto, Japan. Telephone (81)-75-751-4035;Fax (81)-75-761-5626 "O 0 N (J -o 0 N)

Page  94 SPA0255 SYNOVIAL CELL APOPTOSIS BY HIV-1 INFECTION; Tomoko Hasunuma b Yoshio Koyanagi*, Hiroyuki Aono, Naoki Yamamoto*, Kusuki Nishioka Institute of Medical Science, St. Marianna University *Department of Microbiology,Tokyo Medical and Dental University Objective: Several cases have been reported in which the symptoms of rheumatoid arthritis (RA) resolve in parallel with the development of symptoms of AIDS. This phenomenon is thought to occur secondary to reduction of CD4 positive cells in the peripheral blood. We investigated whether any changes could be detected in synovial cells infected with HIV-1. Method: Synovial tissues were obtained from RA patients during surgery for total knee replacement or synovectomy. Synovial cells were separated by digestion with collagenase and cultured. HIV-1 NL4-3 and IIIB strains were added into the medium of synovial cells and cultured for 3 to 5 days. Cells were assessed by immunostaining, nick-end labeling and DNA gel electrophoresis. Results: Synovial cells infected with HIV-1 NL4-3 showed apoptotic change such as shrinkage and cell death, whereas non infected cells and cells infected with IIIB strain did not. By nick-end labeling method, these NL4-3 infected cells showed positive reaction in their nucleus. Moreover, DNA extracted from these cells showed ladder formation by electrophoresis. Discussion and Conclusion: From these data, it seems that was induced programmed cell death in synovial cells by infection with HIV-1 NL4-3. Our findings suggest that remission of RA symptoms after the development of AIDS may be caused by synovial cell apoptosis. HASUNUMA,Tomoko, Institute of Medical Science, St. Marianna University, Kawasaki, Japan Telephone (81)-44-977-8111 ext.4109; Fax (81)-44-976-9319 PAC256 INFLUENCE OF 1IV IN TIlE INDIUCTION OF THE VB8-SPECIFIC ANERGY IN T CELLS FROM 1IIV-INFlECTED INDIVIDUALS. Dadaglio Gilles. Garcia S., Montagnier L., Gougeon M.L. Viral Oncology Unit. Institut Pasteur, Paris, France. Objective: We have previously shown the existence of a specific VS8 anergy in peripheral T cells from IIIVinfected individuals. This V88 unresponsiveness affected both CD4+ and CD8+ T cells and was frequent since it affected 56% of the patients tested. Since in murine models a VB-specific anergy is known to be the consequence of a previous in vivo superantigenic activation, and because comparison of the clinical status of the anergic versus responder patients did not show any correlation with previous bacterial or viral infections, we postulated that this superantigen might be encoded or tightly associated with HIV (G. Dadaglio, S. Garcia, L. Montagnier, M.L. Gougeon, J. Exp. Med. 1994,179: 413-424). The present study was aimed at defining the role of HIV in this phenomenon. Methods: The superantigen erythrogenic toxin A of Streptococcus (ETA) which stimulates specifically CD4+ and CD8+ T cells expressing the VB8 or VBI2 elements of the TcR was used to stimulate either PBLs or lymph nodes (LN) cells from healthy donors or WIIIV-infected individuals at CDC stage I to stage IV of the disease. After 7 days of stimulation in the presence of IL-2, VS expression was analysed on activated CD4+ and CD8+ subsets by dual staining with corresponding mAbs and FACScan analysis. Phenotypic characterization of the VB subsets was also determined by FACScan analysis Results and discussion: If the superantigen responsible for the VB8 anergy is closely related to HIV, we might expect that due to its more important representation in the LN, the main site of HIV replication, a VB8 specific expansion of this subset might be observed in this organ. Therefore we have compared in a series of patients the ex-vivo VB repertoire as well as the activation markers expressed on the VB subsets in LN vs peripheral T cells. Preliminary experiments performed on LN and peripheral T cells from a few very recent seroconverters did not allow to detect any significative differences in the VB repertoire. However none of these patients was found VB8 anergic. Interestingly enough, we found one patient who showed a VB8 specific anergy in peripheral T cells whereas his LN T cells appeared normal responders to VB8 specific stimuli. These experiments will be extended to other patients. This observation suggests that in this patient the VB8 anergy in blood may be the consequence a VB8-specific stimulation in the lymph nodes induced by HIV. In parallel, in vitro experiments are in progress in order to define whether HIV or iV-infected cells show a selective V8 s uperantigenic activity. DADAGLIO Gilles, Institut Pasteur, 28 rue du Docteur ROUX, 75015 Paris, France Telephone (1); Telefax (1) -o 0 U' U' D -o 0 UN No PA0257 SCREENING FOR HIV-Ab IN SALIVA. A MULTICENTER STUDY. BURKE M,VARDINON N,BENTWICH Z,BURSTEIN R,MAAYAN S,BEN PORAT E,POLLACK S,YUST I,MORAG A. ISRAEL TASK FORCE Objective: HIV antibodies (Ab) may appear in saliva, a readily available body fluid which may be stored without refrigeration. A multicenter study was designed to determine the feasibility of using HIV Ab detection in saliva as a diagnostic tool as compared to serum. Methods: A total of 1224 subjects were examined in four participating AIDS centers (Rambam, Hadassa, Kaplan and Ichilov Hospitals). The study was double-blind and the results were compared using a numbered code for each subject. Testing for HIV Ab was performed on saliva specimens and examined by Immunocomb (Orgenics, Israel) HIV 1/HIV 2 Bi-spot kit. Confirmation was carried out on sera using Recombinant HIV 1/HIV 2, 3rd generation (Abbott, USA) kit. Results: All 99 HIV-seropositive samples were detected by Immunocomb saliva kit (Sensitivity 100%). Of the remaining 1125 HIV-seronegative subjects, 21 specimens of saliva were positive and 1101 were negative (Specificity 98.2%). Demographic and risk factor analysis suggested that certain factors may affect false positive results, such as age (p=0.025) and country of origin outside Israel (p=0.05). Discussion and Conclusion: This multicenter study shows the Immunocomb saliva kit to be a useful, highly sensitive and specific technique for testing large populations with a Negative Predictive Value of 100%. Saliva testing could become a valuable method for HIV Ab screening. BURKE Michael, Sourasky Tel Aviv Medical Center Tel Aviv,ISRAEL,Tel 97236974587,Fax 97235469580 PA0258 Enhanced immunodeficiency in hemophiliacs co-infected with HIV1 and HTLV-1 K.USUKU, S.YASHIKI, T.FUJIYOSHI, M.OSAME and S. SONODA Dept. of Medicine and Dept. of Virology, Kagoshima Univ. Objective: Since the south-west part of Japan is an endemic area of HTLV-I, co-infection of HIV1 and HTLV-I may occur in hemophiliacs. To evaluate an impact of double infection, we examined clinical course and immunological state of these patients. Methods: We investigated 33 hemophiliacs for production of antibodies to HIV-1 and HTLV-l by ELISA, PA, IF and WB, and cellular immunity by examining changes of CD4 +T cell s, response to PHA and autologous proliferative response (APR) observed in a HTLV-1 infected individual. Effect of HIV-1 infection on HTLV-I specific CTL clones were examined in vitro. Results: Of 33 hemophiliacs, 16 and 9 were infected only with HIV-1 and HTLV-I, respectively. Three were doubly infected. While HIV-1 single infected patients remained ARC, double infected patients developed AIDS. Production of antibodies to gp41 and p24 of HIV-1, CD4+T cells, response to PHA and APR were declined in double-infected patients. Cytopathic effect of CTL clones was decreased after HIV-1 infection. Discussion and Conclusions: Our result clearly showed that double infection is a potent risk factor to develop AIDS. APR is shown to consist of growth of HTLV-l infected cells and proliferation of T cells against HTLV-I infected cells. Since in vitro CTL response was reduced after HIV-1 infection, decreased APR in double infected patients may be due to impaired T-cell proliferation to HTLV-I infected cells. These observations suggest that augmentation of CTL response in vivo is essential to preserve remaining immune function in HIV-1 infected individuals. KOICHIRO USUKU M.D. Illrd Department of Internal Medicine Kagoshima University 8-35-1 Sakuragaoka Kagoshima 890 JAPAN TEL (81)-992-75-5332; FAX (81)-992-65-7164

Page  95 PA0259 IMPAIRMENT OF T CELL PROLIFERATION T INCREASED INTRA CELLULAR CAMP DOES NOT PREVENT INDUCTION OF CYTOKINE MRNA. B Hofmann, A Sorensen, JES Hansen, JO Nielsen. Dept. of Infectious Diseases, Hvidovre University Hospital, Copenhagen Objective: To investigate how changes in cAMP influence T cell proliferation and the capability of T cells to produce cytokines. The cytokine-driven immune activation in HIV infection may seem contradictory to the decreasing cellular immune competence. We have earlier shown that part of the T cell anergy is mediated by an increase in intracellular cAMP possibly caused by viral proteins (Hofmann et al Proc. Nat. Acad. Sci. USA 1993 and Hofmann et al. AIDS 1993). Method: PIIA driven lymphocyte proliferative assay and a semiquantitative cytokine mRNA PCR for IL-2, INF-gamma, IL-4, IL-10 and beta-actin. Results: T cell proliferation in response to PHA was reduced by 50% by increasing intra cellular cAMP levels in normal T cells by addition of Br-cAMP. In contrast, an increase in intracellular camp levels did not affect the T cells ability to express mRNA for IL-2 and INF-gamma after stimulation with PHA. Br-cAMP also failed to affect mRNA levels for IL-2 and INF-gamma when mRNA for these cytokines were already presentls in PHA blasts. mRNA for IL-4 and IL-10 was not/only slightly induced by stimulation with PHA for 2 hours or 20 hours. Addition of HIV proteins, which have earlier been shown to increase intracellular cAMP did not in preliminary experiments induce cytokine mRNA. Discussion and conclusion: Increased intracellular levels of cAMP affect T cell functions such as cell proliferation but without interfering with induction or regulation of cytokine mRNA levels for the Thl associated cytokines 11-2 and INF-gamma. This may explain the simultaneous immune activation and functional T cell impairment in HIV infection. Bo Hofmann, MD., D.Sc., Dept. Infectious Diseases, Hvidovre University Hospital Kettegaards alle 30, DK-2625 Phone +45 31 35 53 58 Fax +45 31 47 49 79 PA0260 HEAT-INACTIVEATED HIV-I INDUCES PRODUCTION OF BOTH TUMOR NECROSIS FACTOR ALPHA AND ITS SOLUBLE RECEPTOR (TNF-SR75) BY HUMAN MONOCYTES. AC RIMANIOL*. F BOUSSINt, A IIERBELIN', D DE GROOTE *, D DORMONTt., JF BACH*, B DESCAMPS-LATSCHA" and EL~ JA A *INSERM U25, Necker Hospital, Paris, tLaboratoire de Neumpathologie exp rimentale, Fontenay-aux- Roses, Fance ** Medgenix Researh GOmup Fleum, Belgium. Obectiy: TNFa appears to play a major role in HIV expression. Elevated levels of TNF soluble receptors (TNF-sR) were recently demonstrated in the serum of HIV patients at the asymptomatic stage of infection and TNF-sR were shown to be markers of HIV progression. This study investigates whether circulating monocytes could be a source of TNF-sR75 production upon interaction with HIV-1. Metholds: Monocytes from normal donors were incubated with heat-inaclivated HIV-I LAI containing culture supernatants from infected or uninfected (mock supemrnatant) human cord blood. At various times, TNFsR75 was measured by ELISA and TNFa by both ELISA and cylotoxicity towards L929 cells. Results: Following a 20 h incubation of monocytes with 3.12 ng/ml p24 equivalent HIV-1 TNFa production was significantly higher than in controls (4229~1524 vs 208~148 pg/ml, p<0.001) and a four fold increase of TNF-sR75 production was observed (376~47 vs 85~25 pg/ml, p<0.001). Dose response study revealed a plateau of TNFa and TNF-sR75 responses at 2 ng/ml of HIV-I and half maximal response at 1 ng/ml and 0.25 ng/ml, respectively. Kinetics study showed that TNF-sR75 response occurs after 5 h and reaches its plateau at day 2, while TNFax response is more rapid and transient (peak between 12 and 20 h). Finally, a specific monoclonal antibody to TNFzc (I ftg/ml) that totally neutralized TNFo inhibited only 30% of TNF-sR75 production, suggesting that at least 2/3 of the monocyte production of TNF-sR75 is directly induced by HIV-1 and is not the result of autocrine stimulation by TNFa. Discussion and Conclusions: These resulls demonstrate for the first time that heat-inactivated HIV-1 induces production of both TNF and its naturally occurring specific inhibitor, TNF-sR75, by normal human monocytes. Although the mechanisms of TNF-sR75 production (active synthesis and/or protease induced shedding) remain to be elucidated, these in vitro data could explain: i) in vivo observations suggesting that TNF-sR75 could be a marker of HIV progression and ii) monocyte desensitization in the latency period of HIV infection. ZAVALA, Flora, INSERM U25, H8pital Necker, 161 rue de Sevres, 75743 Paris Cedex 15, France. Telephone (33)-1-44-49-53-78; Telefax (33)-1-43-06-23-88. PA0261 INHIBITION OF TNF PRODUCTION BY LENTINAN AND CURDLAN SULFATE. Masihi. K, Noel, Chihara, G.*, Kaneko, Y.** Robert Koch Inst., Berlin, Germany; *Biotech. Res. Ctr., Teikyo Univ., Japan; **Ajinomoto Co., Tokyo, Japan Objective: Tumor necrosis factor (TNF) can exacerbate several microbial infections including HIV and plays an important role in bacterial septic shock and adult respiratory distress syndrome. Development of novel strategies to block the production of TNF therefore have a potential therapeutic relevance. Methods: TNF activity was determined by a sensitive L929 fibroblast cytotoxicity assay using recombinant TNF standard. The functional activity of macrophages was assayed by luminol-dependent zymosan-stimulated chemiluminescence. HIV activity was determined using p24 ELISA. Results: Significant production of TNF and high oxidative respiratory burst of phagocytic cells in BCG-rimed animals following LPS treatment could almost be completely inhibited by either lentinan or curdlan sulfate. The magnitude of the inhibition was similar to that achieved with pentoxifylline, a known inhibitor of TNF. Lentinan and curdlan sulfate also modulated the production of TNF in human monocytoid U937 and THP-1 cell lines following interferon-gamma treatment or after HIV-1 infection. Discussion and Conclusions: Since lentinan already is being used in cancer patients, these results suggest the feasibility of using lentinan and curdlan sulfate as inhibitors of TNF production in clinically relevant conditions including AIDS. MASIHI, K. Noel, Robert Koch Inst., Nordufer 20, 13353 Berlin, Germany Tel.(49)-30-4547 2453; Fax (49)-30-4547 2328 PA0262 Downregulation of ADF/human thioredoxin as a possible mediator of HIVinduced apoptosis. N. Sato, S. Iwata, H. Masutani, K. Nakamura, T. Hori and J. Yodoi Dept. of Biological Responses, Inst. or virus Research, Kyoto Univ., Kyoto, Japan and Dept. of Anesthesia, Kyoto Univ. Hospital, Kyoto, Japan. Objective: Recent evidence suggests that oxidative stress may mediates HIV-induced apoptosis of lymphocytes. We have previously shown that ADF/human thioredoxin (hTrx), an endogenous antioxidant, was downregulated by HIV infection. To study the possible involvement of ADF/thioredoxin in apoptotic process, we have analyzed the effect of thiol modification on a human T lymphoid cell line. Methods: A human T lymphoid cell line, Jurkat, were treated with either diamide (a sulfhydryl-specific oxidant) or buthionine sulfoximine (BSO; an inhibitor of glutathione synthesis). After drug treatment, we have analyzed 1) intracellular GSH content by calorimetric assay, 2) oxidation state of ADF/hTrx by native-PAGE/immunoblotting, 3) DNA content after detergent extraction by propidium iodide staining/flowcytometry. Results: When cells were treated with diamide, decrease of GSI content, oxidation of ADF/hTrx and induction of apoptosis were observed, whereas the treatment with 13SO did not induce apoptosis or oxidation of ADF/hTrx upto 48 hours in spite of marked decrease of GSH content. Discussion and Conclusions: Our results implies that oxidation or inactivation of ADF/hTrx may lead to apoptotic cell death. The downregulation of ADF/hTrx may have pathogenic or facilitatory roles in HIV-induced apoptosis. Whether replenishment of ADF/hTrx blocks HIV-induced apoptosis remains to be determined to establish the therapeulic potential of ADF/hTrx for HIV infection. Supported by a Grant-in-Aid for Scientific Research on Japan. SATO, Norihito, Institute for Virus Research, Kyoto Japan Telephone (81)-75-751-4924; Telefax (81 )-75-761-5766 a -1 Co 0 *0 N 0) N

Page  96 PA0263 REGULATION OF TNF-a RELEASE BY DIFFERENT NEOPLASTIC B CELLS. C. Simonelli*", S. Coral*. A. Gasparollo*. U. Tirelli~. M. Altomonte* and M. Maio*. *Ilmmunotherapeutics Unit and "Medical Oncology-AIDS INRCCS-CRO, Aviano. 33081, Italy. PAO264 DECREASED SPONTANEOUS PRODUCTION OF IL-1 AND TNFa IN HIV SEROPOSITIVE INDIVIDUALS TREATED WITH AZT. SADEGIII H., WEISS L., LEDUR A., KAZATCHKINE M.D. and HAEFFNERCAVAILLON N., INSERM U 28, Unitd d'Immunopathologie, Universitd Pierre et Marie Curie, H6pital Broussais, Paris, France Tumor Necrosis Faclor-a (TNF-a) has been demonstraled to be involved in the upregulation of HIV replication. In addition, resting or SAC/IL2 -stimulated B cells fronm HIV-positive individuals release TNF-a. The mechanism(s) underlying this phenomenon remains largely unknown. We have recently shown Ihat crosslinking of HLA class II antigens strongly upregulales the release of TNF-a by the EBV-B lymphoblasloid cell line JY (Altomonte M. et al., J. Immunol., 151:5115-5122, 1993). In the present study we investigaled whether crosslinking of HLA-DR molecules by mAb L243 could affecl TNF-a release by the B cell lines Daudi and Raji (Burkitt lymphoma), Ril, Cil and Sol (NHL lymphoma) and NALM-6 (pre-B ALL). Triggering of HLA-DR molecules consistently upregulated, in a time- and dose-dependent manner, TNF-a release by Daudi, Raji and ScI cells. These data demonstrate that different B cell types mighl be induced to secrete TNFa through crosslinking of HLA class II antigens. B cell lymphomas are likely to develop in the course of HIV-infection. Therefore, our data might suggest that neoplastic B cells, in addition to normal B lymphocytes, might sustain Ihe high levels of circulating TNF-a and, possibly, viral replication in HIV-infected patients. Cecilia Simonelli, M.D. Immunotherapeutics Unit and Medical Oncology-AIDS, INRCCS-CRO, Via Pedemontana Occ.le, Aviano, 33081, (PN) Italy - Tel: 39-434-659342 - Fax: 39-434-659428 Individuals infected with HIV produce constitutively high levels of pro-inflammatory cytokines such as IL-1 and TNFa. In the present study we have investigated if antiretroviral therapy could modify the constitutive dysregulation of cytokine production by monocytes from patients treated with AZT or AZTIDDI for 8 months. Serum levels of IL- Ia, IL-11, IL-Ira and TNFa were quantitated by ELISA in samples obtained from patients seropositive for -HIV before treatment with AZT (n=15) or AZT/DDI (n=9) (To) and I month (T1), 3 months (T3) and 6-8 months (T6-8) after. Production of cytokines was also measured in cell lysates from isolated uncultured monocytes and from monocytes cultured in the absence of any cytokine inducer for 24h. Production of biologicaly active IL-1 was assayed using the C3HIHeJ cell comitogenic assay in the presence of ConA. Production of IL-la, IL-I.0, IL-Ira and TNFa antigens was determined by ELISA. The results from samples obtained at To indicate that serum levels of TNFa was higher in patients infected with IIIV (mean values (m): 525 pg/ml) than in seronegative individuals (n=12; m-O pg/ml). TNFa levels decreased upon time to undetectable levels in almost all patients. IL-1 ra levels were similar in both HIV-infected patients and control (323pg/ml vs 312pg/ml) and remained unchanged following treatment. No IL-la nor IL-l. were detected in serum samples. However, monocytes isolated at To from the 24 seropositive individuals and cultured for 24h produced IL-i a and IL-1 iP spontaneously. Upon treatment there was a decrease in IL-1 production by cells from all patients as assessed both in functional and antigenic assays. Similarly, the mean of spontaneous TNFa production by cells isolated at To was of m=870pg/ml and decreased to m=133pg/ml by cells obtained at T6-8. Taken together, these results demonstrate that AZT or a combination of AZT/DDI therapy reduces the production of pro-inflammatory cytokines which are involved in viral replication. WEISS Laurence - H8pital Broussais 96, rue Didot - 75014 PARIS - FRANCE Tel.: 19 (33).1.43 95 94 19 - FAX Number: 19 (33) 1.43 95 94 24 -o 0 0 PAO265 EFFECT OF NF- AND IFN-y ON HIV-1 REPLICATION IN VITRO PA0265 EFETOTN-ANIN Han X. Becker K, Degen H, Jablonowski H*, Strohmeyer G*, Jungwirth C Inst.of Virology, Univ. Wurzburg/Dep.of Medicine, Univ. Dusseldorf*, FRG bjZectuve: To investigate the effects of TNF-a and IFTN-y alone and in combination on HIV-1 replication in vitro. Methods: Replication of HIV-1 was measured via determination of reverse transcriptase activity in the chronically HIV-1-infected human macrophage cell line U1 being treated with TNF-a and/or IFN-y. RNA-analysis was performed by Northern blot and viral p24 antigen was detected by Western blotting. Cell death by apoptosis was examined by Southern analysis of cellular DNA fragmentation. Resultst: TNF-a dose-dependently stimulates HIV-1 replication in chronically infected Ul cells. IFN-y alone only marginally enhances reverse transcriptase activity and accumulation of HIV-RNA, but it significantly potentiates HIV-stimulation induced by TNF-a. The amount of p24 antigen in infected U1 cells is elevated slightly after treatment with IFN-y, markedly after treatment with TNF-a, and even further after simultaneous addition of both cytokines. Combined treatment with TNT-a and IFN-y also significantly increased programmed cell death. Conclusions: Previously it was reported that TNF-a exerts antiviral effects on HIV-1 which were synergistically increased by IFN-y. In contrast, we found that synergistic action of both cytokines in vitro significantly increased BIV-1 replication and programmed cell death. This study was supported by the Heinz-Anamann-Stiftung, DP(aseldorf, FRG PAO266 TNF-alpha IN AIDS ASSOCIATED TOXOPLAMOSIS S.Singh, N. Singh, JK Maniar, DG Sapte, Trupati Seth; A.I.I.M.S., NEW DELHI & GT Hospital, BOMBAY (India). INTRODUCTION:Tumour Necrosis Factor (TNF)-alpha is cytokine which has been associated with beneficial effects to the host in some diseases, white in other diseases it has been incriminated with bad prognosis. METHODS: We compared the serum tevels of human-TNF-a in 18 patients of Toxoplasmosis with AIDS and 12 HIV negative Toxoplasmosls patients, using the ready-to-use EIA kit from Boeringer Mannheim 8iochemica. RESULTS: Of the 18 patients 14 were males and 4 females; 13 had HIV-1 and 5 HIV-1+2 infection. 8 died and 10 are living. Our results showed that the anti-toxoplasma IgG antibody (EU) values were more in AIDS patients (420.4 SD 324.6) than non-NIV infected toxoplasmosis patients (210.2 SD 49.5). The TNF-a values in AIDS patients were found highlty raised (p<.001) than in non-HIV infected anti-toxoptasma antibody and age matched patients (114 SD 213 Vs 67.6 SD 81.2 pico grams per ml). CONCLUSIONS: The raised TNF values in toxoplasmosis are in concurrence with others reports, but, very high concentrations of human-TNF-alpha in AIDS associated toxoplasmosis in contrast to the non-HIV infected patients of toxoptasmosis are being reported first time. But whether these raised values of this cytokine were in response to toswoplasma or HIV or it was an augmented effect of both the infectious agents. Dr. med. Klaus Becker, Medizinische Universitltsklinik Dtisseldorf, Ableilung fur Gastroenterologie, Moorenstr. 5, 40225 Dusseldorf, FRG Tel. 0211/311-7795, Fax 021119348076 Dr. Sarman Singh, F/35 Ansari Nagar, New Delhi-29 Tel-091-011-652039; FAX-091-011-6863153, 6868316

Page  97 PAT267TUMOR NECROSIS FACTOR PRODUCTION IN HIV-INFECTED PATIENTS.UTILITY AS A SURROGATE AND RELATIONSHIP WITH t 4 Ae:ngual,M.Sasal,M.Cervantes y F.Segura OBJECTIVES 1.- To determine plasma TNF-alpha levels in HIV-infected patients at different clinical stages.2.- To study the correlation of plasma TNF levels with surrogate markers (CD4), karnofsky index, triglycerides, LDH, hematocrit and lymphocyte count.3.- To compare plasma TNF-alpha levels in four groups of infected-patients: assymptomatic (CD4)500), PCP, cerebral toxoplasmosis and CMV retinitis and to assess if high TNF-alpha lead to worse disease outcome. RESULTS Fifty four samples were analyzed, sixteen of assymptomatic patients, nine PCP, nine cerebral toxoplasmosis and fifteen CHV retinitis. The mean value of TNF-alpha was 14 in assymptomatics, 17 in PCP, 31 in toxoplasmosis and 26 in CMV group. Mean value of TNF in healthy controls was 6. TNF was statistically significative (p(O.002) comparing assymptomatic group with whole AIDS group. The comparation for TNF values between toxoplasma-assymptomatic and CMV-assymptomatic, was also significative (p<O.002).TNF values had a good statistic correlation with CD4, Ht, lymphocyte count and Karnofsky. There was no correlation between TNF values and LDH and triglycerides. DISCUSSION Our results suggest that TNF-alpha levels are higher in patients with advanced disease except in PC pneumonia. We observe very similar TNF levels in PCP and assymptomatic patients suggestivy that the elevation of TNF in PCP may occur only locally in the lung. High levels of TNFalpha demonstrate activation of macrophages as the predominant mechanism during toxoplasma infection CMV induction of TNF-alpha may play a critical role in CMV-induced inflammation.Toxoplasma gondii and CMV may be both AIDS-enhancig cofactors because TNF production increases HIV-1 activation. Longitudinal studies are in progress to avaluate if TNF levels can change the course of HIV- I infection.. CERVANTES.- HOSPITAL DE SABADELL, Parc Taulf,s/n. SABADELL Barcelona.-Spain 93/723.10.10 FAX 93/716.06.46 PA0268 TRANSCRIPTIONAL ALTERATION OF TNF-ALPHA AND IL-6 IN AN HIV-INFECTED MONOCYTE/MACROPHAGE LINEAGE CELL LINES TERUNUMA, Hiroshi, OHIARA, Y., MIZOBUCHI, M., TSUNODA, I., and IWASAKI. Y.; epartment of Neurological Sciences, Tohoku University School of Medicine, Sendai, Japan Objective: To study the effect of human immunodeficiency virus (HIV) infection on the cytokine production in monocyte/macrophage lineage cells, we measured the levels of TNF-alpha and IL-6 mRNA in a human monocyte/macrophage lineage cell line chronically infected HIV (U937/HIV) and its uninfected parental line (U937) with or without phorbol 12-myristate 13-acetate (TPA) stimulation. Methods: Total RNA was extracted by acid guanidinium thiocyanate method from non-stimulated cells and those stimulated with TPA (80 nM) for 3 days. cDNA was synthesized from 1 mg of the extracted RNAs, and the levels of TNF-alpha and IL-6 were quantitated by the PCR using serial dilutions of the cDNAs. Results: 1) Both TNF-alpha and IL-6 mRNA were constitutively expressed in U937/HIV but were hardly found in parental U937. 2) The TPA stimulation induced IL-6 production in U937 and it also upregulated that in U937/HIV. 3) The TPA stimulation also induced TNF-alpha production in U937 but down-regulated that in U937/HIV. Discussion and Conclusions: In HIV encephalitis, it has been proposed that HIV-infected macrophages and microglia may play a role in the etiology of HIV-related neurologic dysfunction because the predominant infected cells in the brain are macrophages and microglia. Our results demonstrate that HIV infection of monocyte/macrophage lineage cells causes the transcriptional alteration of cytokines, suggesting that HIV-infected monocyte/macrophage lineage cells could induce the development of CNS diseases through the alteration of cytokine production. TERUNUMA, Hiroshi, Department of Neurological Sciences, tohoku University School of Medicine, 2-1 Seiryo-Machi, Sendai, Japan; Telephone (81)-22-273-9105; Telefax (81)-22-274-3847 PA0269 TUMOR NECROSIS FACTOR-a, INTERLEUKIN- 8lB AND IL-6 CONCENTRATIONS ASSOCIATED WITH LIPID ABNORMALITIES IN THE SERUM OF HIV-1 INFECTED PATIENTS. DUPOIliche. SION A, IRON A. PATY MC, CASSAIGNE A. LACUT JY. Pellegrin Hospital. Bordeaux, France. Objectlve.Patientswith the acquired immunodeficiency syndrome exhibit lipid disturbances in relation with circulating cytokine levels. This study characterized serum cholesterol (CT) and triglyceride (TG) levels concurrently with circulating levels of tumor necrosis factor-alpha (TNF-ae), interleukin-113 (IL-li3), and interleukin-6 (IL-16) in HIV infected patients. Mlethods.Serum samples from 45 patients were analyzed.15 were HIV seronegative subjects (group A), 15 were asymptomatic HIV seropositive patients (HIV+) with CD4 T-lymphocytes )200/plI (group B), and, 15 were symptomatic HIV+ with with CD4 200/Ipl (group C). Cytokine levels were assayed by Cistron TNF-az ELISA kits and bylmmunotech TNF-a ELISA kit, by Cistron IL-1i ELISA kit, by Eurogenetics IL-6 ELISA kit and CT and TG levels were assayed by an automatic enzymatic analyser. Results. mean serum levels (S.D.) group A group B group C p (A vs B) p (A vs C) p (B vs C) CT (mmol/1) 5,1 (0.8) 4.2 (0.8) 4.2 (1.7) <0,05 <0,05 N.S. T6 (mmol/L) 1.1 (0.7) 1.3 (0.5) 3.1 (2.3) N.S. <0.01 <0.01 TNF-a Cistron kit <5 <5 <5 N.S. N.S. N.S. (ng/mI) Immunotech kit <5 12.7 (10.4) 18.4 (9.7) 0,05 0,05 0,05 IL-13 (ng/ml) <10 <10 10 N.S. N.S. N.S. IL-6 (ng/ml) <2 2.5 (1) 54 (76) N.S. <0.05 <0.05 Discussion. As others, we found raised serum levels of TG (especially in group C) and decreased serum levels of CT in HIV+, raised levels of IL-6 in symptomatic HIV+ (group C) and no change in IL- 113 between control and HIV+. We found discrepancies between the two TNF-a ELISA kits: only one assessed high serum levels for HIV+, especially for group C. DUPON Michel, Hipital Pellegrin, Tripode, CHRU, place Amblie Raba Leon, 33076 BORDEAUX CEDEX, FRANCE. Telephone: (33)-56-79-55-66. Telefax (33)-56-79-60-36. PA0270 TRIGLYCERIDES (TG) AND TUMOR NECROSIS FACTOR-a (TNF-a) IN IIV INFECTION. LLobet P, Muga R, Abad E, Gonzalez JA, Ruzafa A, Rey-Joly C. Internal Medicine Unit. Hospital Germans Trias i Pujol. Barcelona Objective. To determinate the TNF-c serum levels in HIV infected patients in order to Know the relationship with TG serum levels, CD4 count and clinical diagnosis. Methods. Seventy five HIV infected patients (58 M, 20 F) in different HIV stages, mean age 32 years, were estudied and analyzed for TNF-a (TNF-oa IRMA, Medgenix, Belgium), TG (enzimatic method), CD4 count (cytometry flow method) and clinical diagnosis at the moment of the study. Results. CD4 < 200 201-400 > 400 TOTAL N 51 17 7 75 TNF-a x~SD pg/ml 31.5~44.8 13.1~13.2 13.6~5.8 25.5~38.1 TO xjSD mg/dl 235~132 136.5~73.3 130.5~52.5 202~123.7 TG>180 ag/dl 55(%) 23.5% 16.7% 44.2% TNF>15 pg/ml 41(%) 23.5% 14.3% 34.2% The TNF-ac levels according to the clinical diagnosis were: protozoa (n= 12) 38.5~36.3; bacteria (n= 12) 38.3 ~46.2; mycobateria (n=4) 105.4~91.8 (p=0.001); assymptomatic patients (n=32) 13.2~ 15.5 There was no differences in TNF-a levels according to CD4 count (p=0.16). The correlation coefficient between TNF-a and TG was R=10.34. We found differences in TG levels according to CD4 count (p =0.005). Conclusions. Both TNF-a and TG enhance HIV infection with CD4 count <200 /mm' but there is no relationship between them. Enhanced TNF-a is highest in mycobacterial disease, and it could be relevant marker of opportunistic infections. Llobet P. Infectious Diseases Unit. Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain Tel. 34.3.4651200; Fax. 34.3.3954206 a -o 0 N) O N 0

Page  98 CD P A0 7 1 DETECTION OF LOCAL PRODUCTION OF INTERLEUKIN-DO IN KAPOSI'S SARCOMA PAO271 "'eo oo ecvn. 0,memaa oe scn ASSOCIATED WITH AIDS.,f tsoi.Lia Leik.o;Akridge,R.E.2&Reed,S.0.2.3. llnst.Infect.Emilio Ribas- S.Paulo, Brazil;?Infect. Dis. Research Inst.,Seattle,WA,USA, 3Cornell Univ.Aed.College,NY,NY,USA Objectives: The aim of this study is to determine "in vivo" local expression of Interleukin-10 (IL10) using mRNA-PCR and immunohistological techniques, in Kaposi's sarcoma associated AIDS lesions and the correspondent lymph node tissue. Methods: Skin lesions of KS and correspondent lymph nodes biopsies were performed as well as in normal skin from HIV positive individuals. All of them were young homosexual/bissexual men registered at the Institute of Infectology "Emilio Ribas". They did not have any previous treatment at the time of biopsies. In all patients the samples were examined by mRnA-PCR and microscopic examination of the tissue samples using a highly specific biotinylated anti-Hu IL-10 monoclonal antibody, followed by alkaline phosphatase /Fast red. In order to assure that the labelling system was functioning properly, a biotinylated antibody (anti-Ho IgO) was ran in parallel in another sample. Resolts: The IL-10 was evident in all lymph node tissue examined, was absent in normal skin biopsies and was detected in the Kaposi's lesion of one individual who had disseminated cutaneo-mucosae Kaposi' s sarcoma.These results suggest that IL-10 functions as a critical component in viral replication possibly by inactivating certain macrophage functions known to up regulate HIV replication. Microcospic examination resulted in diffuse labelling with no specific cells being more intensely stained than another. However the staining pattern was restricted to specific regions of the lymph node: primarily the lumen and the walls of the sinuses. All controls had minimal binding of the probe indicating the specificity of the antibody. conclusions: The observation that IL-O10 is present in lymph nodes from AIDS patients with Kaposi's sarcoma and in a skin biopsy from a disseminated Kaposi's sarcoma patient suggests that IL-DO may be involved in the alteration of the immune system and the pathology associated with this disease. Luiza Keiko K. lyafuso Institito de Infectologia Emilio Rihas Av. Dr. Arnaldo, 165, So Paulo, SP, Brazil CEP-01246 Tel: (311)852 8966 Fax: (55 l) 2803954 PAO272 DECREASED CONCENTRATIONS OF IL-10 IN PATIENTS WITH HIV-INFECTION Kucharzik, Torsten, Stoll R., LOgering N. and Domschke W. Department of Medicine B, University of MOnster, Germany Objective: Altered cytokine production with elevated mRNA levels of IFNy, IL-6 and TNFa have been reported in patients with HIV-infection. Interleukin-10 (IL10) inhibits the production of different cytokines like IFNy, TNFa and IL-6 through inhibitory effects on Thil-cells and macrophages and is thought to be a suppressor type cytokine. Some authors found increased IL-10 mRNA expression in patients with early HIV-infection. In the present study we determined serum concentration of IL-10 in patients with late HIV-infection. Methods: IL-10 was measured with our own newly established ELISA-system using DIANOVA-antibodies. Serum concentrations of IL-10 were assessed in 60 patients with HIV-infection CDC IV and in 60 healthy controls. Results: IL-10 levels were significantly decreased in patients with HIV-infection (mean + SEM, 508 + 90 pg/ml) compared with healthy controls (mean + SEM, 1189 ~+ 130 pg/ml; p<0.0001). Using 800 pg/ml as discrimination level, 84 % of the patients with HIV-infection but only 35 % of the control population presented IL-10 levels below that limit. Discussion: We assume that IL-10 production in late HIV-infection is markedly reduced presumable associated with diminished CD4 cell count. Our data suggest that IL-10 plays an important role in the altered immune response in HIV-infected patients. T. Kucharzik; Department of Medicine B, University of MOnster, Albert-Schweitzer Str. 33, D-48129 Monster, Tel.: 0251/83-6230; Fax: 0251/83-6211 PA0274 USEFULNESS OF CYTOKINES FOR ADVANCE)D IIV INFECTION DIAGNOSIS. Portilla J, Sanchez J, Boix V, Munoz C, Sen ML,Priego M, P6rez-Mateo Miguel. Hospital General Universitario de Alicante. Spain. OBJECTIVE: To investigate the usefulness of cytokines: tumor necrosis factor alfa (TNF), interferon (IFN) and soluble Interleukin-2 receptor (I1-2 r) for diagnosis of advanced HIV disease. METIlODS: In 100 HIV patients (IVDU 76%), 79 men and 21 women, we determined CD4 cells by flow cytometry, Agp24 by EIA and cytokines by enzyme amplified sensitivity inmmunoassay (EASIA; MEDGENIX diagnostics). The patients were classificated according to the CDC staging system (1987): stage A (11.0), stage B (IVA,IVC2), stage C (case definition for AIDS, CDC 1987) Statistical analysis: analisis of variance, Students t-test and Pearson's correlation test. (results in meansas ~ @) RESULTS: Sitage A Stage B Stage C p cases 56 36 36 TNF (pcg/ml) 15~12 22~121 30~22 0,01 IFN (Umil) 0.3~0.3 0.7+1.0 0.9~1.3 0,1 IL-2r (pcg/ml) 5300~3440 6259~3864 6326~3202 0,3 Agp24 (pcg/ml) 14~38 134~228 120~155 0,01 CD4 (cells/nunm3) 669~392 246~195 114~125 0.01 There was no correlation of cytokines with antigenemia and with CD4 counts. There were no significant differences among cytokines, Ag p24 and CD4 counts for sex, age and risk group. CONCLUSIONS:I)TNF increases in advanced HIV disease and is the only cytokine used in this study with prognostic value. 2)TNF, Ag p24 and CD4 cell counts are useful independents markers of AIDS.3) Immunologic. cellular and serologic parameters sarc independent surro.aste markers for 14HIV advanced disease. 4) The elevation of TNF could exrtain some of the y)wmtoms of advanced HIV disease. PEREZ-MATEO, Miguel. Medicina Interns. 8 plants. Hospital General Universitario de Alicante. Maestro Alonso 106. Alicante. Spain.Telephone and Telefax 96-5908355 0 0 4 PA027 3 SOLUBLE IL-6R IS GENERATED THROUGH AN ALTERNATIVE SPICING Horiuchi,Sankichi, Koyanagi,Y., Shoou, E., Tanaka,Y., Waki,M., Matsumoto,A., Yamamoto,M. and Yamamoto,N. Tokyo Med. and Dent. Univ. Objective: To solve the mechanism of generation of slL-6R, we investigated whether or not the IL-6R molecule lacking the transmembrane (TM) domain could be detected in mRNA isolated from cell lines or PBMC of HIV-l-infected individuals or ATLpatients. Methods: We employed PCR method using primer pairs selected on the basis of the IL-6R sequence, encompassing the TM domain, and examined to detect slL-6R protein in culture supernatants of cell lines by radioimmunoprecipitaion using specific antibodies against IL-6R. Results:We detected two main DNA molecules in the PCR products: one with a large size molecule for conventional IL-6R, and the other with a smaller size molecule. The sequencing analysis of both DNA molecules showed that a 94 bp-region coding for the TM domain of IL-6R was detected in the smaller size molecule generated by alternative splicing. We detected slL-6R protein with molecular weight of 45 k-Da in culture supernatants of cell lines. Discussion: Our results indicate that deletion of the TM through alternative splicing of mRNA is one of the major mechanisms for release of the slL-6R in the serum of the patients. IIORIUCHI, Sankichi, Department of Microbiology. Tokyo Medical and Dental University. School of Medicine. Tokyo Japan Tel (81)-3-3813-6111; Fax (81)-3-3818-7175

Page  99 PA0275 INTERFERON-a AUGMENTS INTERLEUKIN-8 RELEASE FROM LATENTLY HIV--INFECTED MONOCYTIC CELL LINE, U937 CELLS OHASHI, Kunihiro, Akazawa, R. and Kurimoto, M., Fujisaki Inst. Hayashibara Biochem. Labs., Inc., Okayama, Japan Objective: The effect of human interferon-a (IFN-a) on the release of antimicrobial interleukin-8 (IL-8) from latently HIV-l-infected U937 cells (U937/HIV-l(L)) studied in vitro to evaluate the potential of IFN-a in the management of AIDS-associated opportunistic diseases. Methods: The U937/HIV-I(L) cells were incubated with IFN-a at a concentration of 1 to 10,000 IU/ml for 24hr and IL-8 in the culture supernatant obtained was quantitated by ELISA. An involvement of IL-1 in the IL-8 release from U937/HIV-1(L) cells was evaluated by the neutralization test with anti-IL-1 antibodies. Results: The U937/HIV-1(L) cells showed a marked reduction of IL-8 secretion as compared to productively HIV-l-infected or uninfected U937 cells. IFN-a restored partially a reduced IL-8 release from U937/HIV-1(L) cells in a dose-dependent manner. Any significant inhibition of IFN-a-augmented IL-8 secretion by anti-IL-la or antiIL-113 antibody was not observed,indicating that the enhanced IL-8 secretion occurred without any augmentation of IL-I secretion. Discussion and Conclusions: These findings suggest that IFN-a, in addition to its antiviral activity, may have a beneficial potential to treat some bacterial or fungal infection frequently seen in patients with progressive stages of HIV-1 infection. OHASHI, Kunihiro, Fujisaki Institute, Hayashibara Biochem. Labs., Inc., Okayama, Japan Telephone (81)-86-276-3141; Telefax (81)-86-276-6885 PA0276 TNF AND IL-6 PRODUCTION IN LPS-STIMULATED BLOOD OF HIV-INFECTED PERSONS. Heyligenberg R, Romijn JA. Godfried MH, Endert E, Sauerwein HP; Academic Medical Center, Amsterdam, the Netherlands. Introduction: Cytokines have been implicated as pathogenic factors in HIV-infection. However, no consistent differences in plasma cytokine levels between AIDS and controls have been demonstrated. As cytokines are paracrine mediators, ex vivo lipopolysaccharide (LPS) induced cytokine production in whole blood could be a better indicator of cytokine production than plasma levels. In this study the effect of different concentrations of LPS on ex vivo TNF and IL-6 production in blood of AIDS-patients and controls is evaluated. Methods: 50 ml blood was taken from 10 AIDS patients (CDC-IV, no active opportunistic infection or neoplasm ooooo p<o.o other than Kaposis Sarcoma) and 10 healthy controls. Heparinized sterile aliquots were stimulated with 0, 1 10 0 ~ 10, 100 or 1000 ngLPS/ml for 8 hours at 37"C. TNF and C IL-6 concentrations were measured by ELISA. Eooo Results: Basal plasma concentrations of IL6 and TNF c were not different between both groups. LPS dosages of. 0 v - healthy volunteers a 10 ng/ml induced maximal production of IL6 in both ~, o - AIDS patlents groups. Submaximal stimulation (1 ngLPS/ml), resulted _t-10 o in a significantly higher IL-6 production in the AIDS- o samples (see figure). LPS stimulated TNF production 'o ie, was dose dependent and not different between patients L nmt and controls. LP ng Conclusion: IL-6 production, but not TNF production, is increased in AIDS without opportunistic infection when compared to healthy controls. The increase in IL6 production is only apparent after submaximal stimulation with LPS. HEYLIGENBERG, R. Academic Medical Center, Amsterdam, The Netherlands Telephone 20-5666071; Telefax 20- 566444 PA0277 CYTOKINES IN THE URINE OF HIV+ AND AIDS PATIENTS. TP. Stein, B. Koerner, M.D. Schluter and D. Condoluci, Univ. of Med. and Dent. of NJ-School of Osteo. Med., Stratford, NJ, USA. PA0278 CYTOKINES IN ACUTE HIV1 AND EBV INFECTIONS A. Biglino, B. Forno, A. Pollono, A. Sinicco Inst. Infect. Diseases, Univ. of Turin. Objectives: Measurement of cytokines and related factors in the urine offers the possibility of obtaining integrated 24 hr. estimates of cytokine activity. To investigate whether increased cytokine secretion could be detected in the urine we assayed the urine of controls and HIV+ve subjects for ILI-ra, TNF, IL-la, IL-lf3 and IL-6 by ELISA's. Methgd1: A ten hour pooled urine collection was collected from controls (C), asymtomatic (HIV+ve, AS), wasting syndrome (HIV+ve, WS) and AIDS. Results are shown below. Data are mean (SEM). n C 12 AS 5 WS 5 AIDS 24 CD4 IL6 ILl-ar ILl-13 ILl-ra TNF-ct 0.74(0.31) 0.13(0.12) 0.03(0.01) 1.71(0.28) 0.04(0.01) 415(86) 4.66(3.58) 0.80(0.71) 0.01(0.01) 2.86(0.98) 0.17(0.08) 27(11) 8.0(2.5) 0.09(0.09) 1.13(1.11) 2.98(1.08) 0.75(0.61) 61(15) 3.90(0.87) 0.54(0.40) 0.70(0.44) 4.26(0.53) 1.06(0.60) Serum cytokine profiles and T-cell subsets were evaluated in 15 patients with acute primary HIV-1 infection, and compared with those obtained from 18 patients with primary Epstein-Barr Virus (EBV) infection and from 18 control subjects, in order to elucidate possible defects of immune response to HIV in early phases of virus-host interaction. CD4+ and CD8+ cell counts, serum concentrations of 5'neopterin, 02-microglobulin, Interleukin (IL)-2, IL-4, soluble IL-2 receptor (sIL-2R), Tumor Necrosis Factor (TNF)-a and soluble CD8 antigen (sCD8) were significantly lower in acute HIV-1 infection if compared to EBV infection. By contrast, HIV-1 infection showed higher Interferon (IFN)-t concentrations if compared to either EBV infection or control subjects. In acute primary HIV-1 infection, impaired release of both macrophage- and Thelper derived cytokines could be responsible for an early defect of cytotoxic response, leading to viral persistence with continuing CD4+ cell loss. Conclusions: (i) AIDS patients have elevated IL-6 (92%, p<0.05), IL-Ira (79%, (p<0.05) and TNF-at (71%, p<0.05) in the urine. (ii) IL6 is also elevated in patients with wasting syndrome (100%, p<0.05). The elevated urine IL-6 levels in the WS patients supports our previous findings of increased acute phase protein synthesis (fibrinogen) in this group. (iii) In contrast to using a spot blood sample for monitoring cytokines, the use of urine is non-invasive and gives an integrated value over time. 0 N 0 N) A. Biglino, Inst. Inf. Dis. Univ. Turin, Cso Svizzera 164, 10149 Osp. Amedeo di Savoia, Torino, Italy. 39-11-4393859.

Page  100 PA0279 SUPPRESSION OF HIV-REPLICATION IN MACROPHAGES INDUCED BY GM-CSF M ATSUDA Shunji. Akagawa,K.Honda,M,Yokota.Y.Takebe,Y,Takemori,T. AIDS Research Center Dept.of lmmunology, NIH. Tokyo, JAPAN Objective: The mechanisms of HIV-infection and replication in different kinds of macrophages have not been clarified. M-CSF and GM-CSF have been reported to differentiate monocytes to macrophages having different characteristics. Using these macrophages,HIV-replication was examined. Methods: Monocytes isolated from normal peripheral blood were stimulated with either M-CSF or GM-CSF for one week. The monocyte derived-macrophages were examined on characteristics and HIV-replication. Results: The macrophages induced by either M-CSF or GM-CSF showed different characteristics. Macrophages indused by M-CSF replicated HIV but the replication was not detected in macrophages induced by GM-CSF. However. CD4 molecules were expressed on the surface of the macrophages and a small amount of provirus was detected by PCR. Discussion: M-CSF and GM-CSF differentiated monocytes to macrophages having different characteristics. In the macrophages induced by GM-CSF, certain steps of HIVreplication, possibly the late stages of replication, were suggested to be inhibited. The mechanisms may be important for the latency of HIV-infection in macrophages. M ATSUDA. Shunji. Dept of Clinical Research, National Kure Hospital, Kure, Hiroshima, Japan. Tel:0823-22-31 1 1I, Fax:0823-21-0478 PA0280 DIFFERENCE IN LATENCY BETWEEN TWO CELL LINES CHRONICALLY INFECTED WITH HIV OwatariSatsuki,Zhang,J.,Gao,M.,Hossain,M., Tanabe-Tochikura,A.,Kageyama,S.,Wakamiya,N., Tsuchie,H.and Kurimura,T. Dept.of Pathology,Research Institute for Microbial Diseases,Osaka University Cells latently infected with HIV are observed in peripheral blood and lymph nodes of HIV carriers. Activation of latently infected HIV is one of the most important events for the onset of clinical diseases.To analyze the latency of HIV, ACH-2 and U1 were used in this study. Provirus in Ul could be easily activated in the presence of murine resident peritoneal macrophages or human peripheral blood mononuclear cells(PBMC). Neither purified human CD4+ lymphocytes nor CD8+ lymphocytes could activate the provirus in Ul. Activation of the virus occurred without direct contact of Ul with activating cells separated by filter membrane of pore size of 0.45 pm. Provirus in ACH-2 was not activated under the conditions stated above. These findings are important for the analyses of natural history of HIV infection especially so-called latent period of 10 years. Studies on the active substance from murine macrophages or human PBMC is now in progress. OWATARI,Satsuki,Research Institute for Microbial Diseases,Osaka University,Suita,Osaka,Japan Telephone:(81)06-875-2128.Telefax:(81)06-875-3896. 0 c0 0 PA0281 Role of cytokines on herpes simplex infection in HIV disease.Smriti Kundu,T.C. Merigan.Stanford Univ. Med. Center Objectives: In chronic stage of infection, Th2 like immune responses eg. IL-4, IL-10 production predominates which downregulate possible protective Thl (IL-2, IFN gamma) responses, thus helping the organism persists in the host. During chronic stage of HIV disease, mucocutaneous ulcers due to HSV with duration of > 1 month is an AIDS defining illness. We propose to study the role ofThl and Th2 like immune responses on HSV infection in HIV disease. Materials & methods: 40 HIV seropositive, asymptomatic individuals with CD4+ T cells <50>500/cu.mm. and 10 HIV seronegative individuals were included in this study. Peripheral blood mononuclear cells (PBMC) were stimulated with PHA, tetanus toxoid and HSV-l antigen. Supernatants were collected at 48 hours to assay for IFN gamma and IL-4 concentrations by ELISA and also tested for their anti-HSV-I effect by plaque inhibition assay. Results: PHA, tetanus toxoid and HSV stimulation produced significantly higher concentration of IFN-gamma in controls compared to HIV infected individuals (p<.05, Willcoxon test). IL-4 production was significantly less in controls compared to HIV infected individuals (p <.05, Willcoxon test). Inhibition of plaque formation was significantly higher in controls compared to HIV seropositive group (p<.05, Willcoxon test). At least four fold changes in plaque counts were considered significant. Anti-IFN gamma and anti-IL-4 antibodies reversed the actions of IFN-gamma and IL-4 respectively by changing plaque numbers. There was significant correlation between IFNgamma and IL-4 concentrations with inhibition of plaque formation by HSV (p<.05, Willcoxon test). Discussion & Conclusion: IFN-gamma has antiviral effect. We observed here decreased level of IFNgamma and increased level of its downregulatory IL-4 in the HIV infected individuals. This pattern of cytokine production provides an explanation for persistent HSV infection in HIV disease. These observations may help in defining appropriate therapeutic regimens. Center for AIDS Research, Stanford University, 300 Pasteur Dr. Room S-156, Stanford, CA 94305, USA PAO282 ENHANCEMENT OF IN VITRO REPLICATION OF CYTOMEGALOVIRUS (CMV) BY IL-8. T. MURAYAMA, N. MUKAIDA & K. MATSUSIIIMA. Kanazawa Med. Univ. & Cancer Res.lnst., Kanazawa Univ, Kanazawa, Japan. Objectives) Reactivation of persistent or latent CMV infection is presumed to cause opportunistic infection amonng AIDS pateins. Plasma 11.-8 level increased among IllIV-infected patients, reaching above 1 ng/ml. The obsevation that IL-8 acts also on fibroblasts prompted us to examine the effects of IL-8 on CMV replication in human lung fibrobalsts. Methods) Hluman embryonic lung fibroblasts were infected with CMV in the presence or the absence of IL-8. The produced virions and DNA in the culture supernatants were determined by a plaque assay and Southern blotting analysis, respectively. Phophoprotein 71 RNA transcript and late cytoplasmic antigen protein were detected by in situ hybridization and immunohistochemistry, respectively. Results) IL-8-induced increase of CMV virion production was observed at 0.1 ng/ml, reaching maximum at 10 ng/ml. The effect was inhibited by a neutralizing antibody against IL-8 but not by a control one, indicating that the effect was attributed to IL-8. Ahead of increasing the replication of CMV DNA, IL-8 enhanced the transcription of phophoprotein 71 and the protein synthesis of late cytoplasmic antigen, suggesting that IL-8 has effects on all the stage of CMV replication. Discussions and conclusions) Considering elevated plasma IL-8 levels in lItV-infected patients, these results would imply that IL-8 may reactivate latent CMV infection in AIDS patients. MUKAIDA, Naofumi Dept. Pharmacol. Cancer Res.Inst., Kanazawa Univ., Kanazawa, 920,Japan Tel: 011-81-762-62-8151, Telefax: 011-81-762-60-7704

Page  101 PA0283 ONCOSTATIN M INHIBITS HIV-TAT MEDIATED TRANSACTIVATION IN HELA-TAT CELLS - Jost A. Est6 M. Witvrouw, J. Tu, R. Pauwels, E. De Clercq and A.-M. Vandamme, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Oncostatin M, a polypeptide cytokine, stimulates Kaposi's Sarcoma (KS) cell proliferation in vivo and in vitro. Related cytokines (leukemia inhibitory factor and interleukin 6), have been studied for their capacity to induce KS, as well as to enhance HIV replication. However, the influence of Oncostatin M on the replication of HIV has not been studied yet. Oncostatin M was unable to influence HIV-1 replication in acutely infected MT4 or CEM cells or in acutely infected peripheral blood lymphocytes (PBL). In persistently infected HUT-78 cells, Oncostatin M failed to demonstrate any activity at subtoxic concentrations. However, using a quantitative bioassay based on transactivation of a chimeric gene comprising the HIV-1 long terminal repeat (LTR), fused to the LacZ gene of E.coli and transfected in a HeLa cell line expressing TAT, Oncostatin M was found to inhibit HIV LTR-driven LacZ gene expression at a 50% inhibitory concentration (IC50) of 1.25 ng/ml. Oncostatin M did not inhibit the activation of HIV LTR by TNF in Molt pNAZ cells or TAT-mediated transactivation in Hep-2, COS 7, and Jurkat TAT cells. The specificity of Oncostatin M on HeLa Tat cells may be to an indirect action through the signal transduction pathway which can differ depending on the cell line or it could also be related to the particular assay and cell conditions. We have previously shown that the activity of Ro5-3335, a compound postulated to act as an inhibitor of TAT-mediated trans-activation is also highly dependent on the nature of the host cells. HIV-1 transcription has been studied in a wide variety of biological systems. Different transient transfection assays have shown internally consistent answers that are not necessarily in accordance with the results obtained in other assays. Our findings on the behavior of Oncostatin M support a basic rule of thumb to be considered when extrapolating data from any particular assay system to the in vivo situation that is, care must be taken so that the systems chosen emulate HIV infection as closely as possible. Jos6 A. Este, Rega Institute for Medical Research, K.U.Leuven, B-3000 Leuven, Belgium Telephone (32)-16-33.21.68; Telefax (32)-16-33.73.40 PA0284 THE EXPRESSION OF CELL ADHESION MOLECULES IS MODULATED BY THE GP20-INDUCED CYTOKINES. POSSIBLE IMPLICATIONS IN HIV-1 INFECTION.DianzaniF.,Capobianchi M.R., Castilletti C., Fais S., Ameglio F.*, Cordiali Fei P.*, Mercuri F., Ficociello B. Institute of Virology, University "La Sapienza" and * Institute San Gallicano, Rome, Italy. Objective:Cell adhesion receptors are involved in immuno-related functions, and are selectively included in HIV-1 virions, possibly playing a role in the virus absorption and spread. Since their expression is modulated by several factors, such as the HIV-I induced cytokines, we investigated whether cytokine treatment of host cells could influence also the envelope of HIV- I progeny with respect to the inclusion of adhesion molecules.Methods:PBMC cultures were established in the presence of recombinant gpl20. Biological assays as well as ELISA were used to measure the cytokines in the supemrnatants. FACS analysis, immunocytostaining and ELISA on cellular lysates were used to study the expression of cellular adhesion molecules. ELISA and the capture by immobilized MAbs was used to measure the cytokine-induced modifications of the adhesion molecules included in HIV-I virions. Susceptible CD4 positive and negative cells were used to investigate the replication ability of HIV-1 progeny from cytokine-treated cells. Neutralization with MAbs to cellular molecules was used to establish the role of such molecules in virus infection.Results:gpl20 induces IFN alpha and gamma, TNF alpha, IL6, ILl alpha and beta, as well as IL10. The cellular expression of adhesion molecules, namely ICAM-1, is up regulated by some of the cytokines induced by gpl20, such as IFN gamma and TNF alpha. The virus progeny from cytokine-treated cells also shows a corresponding increase of adhesion molecules, and has a different ability to infect either CD4 positive or negative cells, as compared to control virus preparations. Conclusion and discussion:Cell adhesion molecules incorporated in infectious HIV-1 virions could play a role in the early steps of infection. The modulation of the cellular expression of these molecules by cytokines could lead to alterations in the envelope of the virus progeny, with consequent possible alterations of the attachment ability of such virions. Since the pattern of gpl20 -induced cytokines is similar to that observed in vivo during HIV-I infection, our results could have pathogenetic implications. CAPOBIANCHI, Maria R., The Institute of Virology, Viale di Porta Tiburtina 28, 00185 Rome, Italy. Tel 39,.6.4452846. Fax 39.6.4469024 PA0285 PENTOXIFYLLINE(PTX) AS A TNF INHIBITOR IN HIV + INDIVIDUALS Sonnabend, Joseph*, Buimovici-Klein, E., Freeman, K., Smith, B., Mohan, V., Harrington, M. *CRIA, New York Objective: To determine if PTX affects spontaneous TNF production by lymphocytes from HIV infected patients. Methods: A randomized, double blind, placebo controlled 12 week study of PTX as a TNF antagonist was conducted by the Community Research Initiative on AIDS (CRIA) in HIV infected patients with under 300 CD4 lymphocytes/mm3, whose lymphocytes ex vivo produced TNF at levels greater than 15pg/ml over 24 hours, on two tests taken at an interval of 2 wks. Patients were randomized to receive placebo or PTX (400mg) 3 times a day. HIV levels were measured by quantitative microcultures of peripheral blood mononuclear cells (PBMC). TNF levels in the supernatants of cultured PBMC were determined by an ELISA method. Regular clinical evaluations were performed as well as blood counts and blood chemistries including triglyceride levels. Results: 15 patients on placebo and 19 patients on PTX completed 12 weeks on study. PTX was well tolerated and only one patient on PTX discontinued the study medication because of GI discomfort. TNF production by lymphocytes was generally lower on evaluations subsequent to baseline. The level in the PTX group was significantly lower than placebo at 8 weeks. There were no differences in HIV levels between the groups. Discussion and Conclusions: PTX, which was well tolerated, was associated with a lowered level of spontaneously produced TNF at 8 weeks compared to placebo. TNF production in the placebo group also fell probably reflecting fluctuations in TNF production over time, patients having been selected for high TNF production at baseline. Long term studies on levels of TNF production should clarify this issue which will have implications on sample sizes required to study modulation of cytokine production. PA0286 INDUcTION OFT IFN ODUCTION BY O, GONA CERTAN PAIDOIC SEQUENCE. TIOSliKO YAMAMOTO, YAMAMOTO, S., KATAOKA T., KOIASE, M., KOMURO, K. AND TOKUNAGA, T. NATL. INST. OF IIEALTIl, TOKYO. JAPAN. Objective: Interferon (IFN) plays an important role in the HIV disease. Recent studies show some effects of IFN in the treatment of early HIV-infection. Cell culture studies also support these effects. HIV infection of CD4 positive cells is blocked by IFN administered at the time of viral challenge. Here we present a paper that the ability of the synthetic single-stranded oligodeoxyribonucleotides (oligo DNAs) possessing particular hexamer palindromic sequences to induce IFN production of human peripheral blood lymphocytes (PBL). Results: When PBL were cultued with a 30-mer oligo DNA with the palindrome of AACGTT, IFN was detected in the culture fluid 8 hr later and the titer of IFN reached 10 -10 units/ml at the maximal level 18 hr later. Type of IFN induced with oligo DNAs was dominantly IFN-alpha. Discussion and Conclusions: It is suggested that certain palindoromic structures in the synthetic oligo DNAs are essential to induce IFN production of PBL. YAMAMOT Toshiko, National Institute of Health, Musashimurayama, Tokyo, Japan. Telephone 0425-61-0775; telefax 0425-61-7173 0 0 01 Joseph A. Sonnabend Community Research Initiat ive on AIDS 275 7th Ave., NYC, NY, USA Tel: (212) 924-3934 Fax: (212) 924-3936

Page  102 PA0287 Evaluative Investigation of psicosocial factors in 20 HIV more survival Peresonl Relatlonehlpe, Creatilty, Coemunilatone S oial Partploetleon. Repsrentaliatlon, In the field of health In 20 HIV Infected patients, during sore than 5 years. Authors Gonales Netorl M1G In Huesan Resovree lManogement, Phyoholosy Counsell.. Vice-President of Amieos do I Vide Foundation. Objleive, Evasluate th oyehololoa! rnle experi lenced by 20.V Inlfeoted Patients and Its effects In their health. Methodoloyl Thisto I study made with evaluative and Interpretative ourposes, represented by seens of the recorda sIntervies obtained fromee 20 HIV Infeeted ay-sent this Interview soehlved to ohk ua pall the reaotlone experlenced by these patiento feeing different events trough of the every day life during a period of tie of S vears free the very dialnosis beslining. Conolusins, the res ulto uggest that the devselooent or Interference of the fator oretlivity, gomuniloetien, solial pertplostion, end roeparentalloetlo process have an Imertant relatloneshl with t he state of health of the IV eoOplne so. I conelud that all these faotors are Juot the result of develepment thes lf-ste of Infeeted people and the well handelng in the diffsret eontexte. Results, At the end of five years period, from the very esent of receiving the results HIV. 76 5S of patients 116 peoplel died. and durinl g Its rocess they experienoed several types of behaviors suh ass poor or lack of oreativity to faoe problems of everytday life, an Inaoertive communloatlon, very unfreeoquent socialp erticipatlen, and strong strainsead eonflieteo ith parents- not worked or solved. All these behaviours were aesoclated to the Inoro.n.leg er of the patients until reasehlng death. The realinin 25% of patients showed a hlh rate of ereatlvlty to face problems of over-day life, a very as*rtive eoeunleatlen, frequent social partielpation and the resolution of Important conflicts with their parents. Nestor Gonzalez. Telep. 582-8612824 Fax 582-817213 PA0 288D CTIAL EXPRESSIONAND PROVIRAL INFECIONVECTORS FOR DIRECT CLONING OF PCR-GENERATED WILD-TYPE AND MUTANT (DUGFRESISTANT) HIV-1 PROTEASE GENES.B. Maschera, E. Furfine & f.D. Blair, Wellcome Research Labs, Beckenham BR3 3BS, UK and Burroughs-Wellcome Co., Research Triangle Park, NC 27709, USA. Objective; Viruses with altered sensitivities to at least ten different protease inhibitors have been generated by drug selection in tissue culture, and also, in at least one case, in the clinic. These variant viruses have one or more altered amino acids in the protease enzyme. To study the effects of individual and multiple mutations on enzyme function and on virus growth, we produced an E. coli expression vector and an infectious HIV-1 proviral clone which are matched so that both accept, by direct ligation, the same PCR-generated restriction enzyme frog ments. Nothodas, The bacterial expression vector was produced by attaching the 139-amino acid open reading frame for human 7-interferon, which is expressed at high levels in E. cell, to the HIV-I protease reading frame at aa -12 (i.e., 12 amino acids N-terminal of the Phe-Pro autocatalysis site) through a poly-glycine "hinge'. At the C-terminus of the protease ORF, a stop codon was introduced at aa.9. The "hinge" is encoded by a Nl= restriction enzyme site, and the stop codon is encoded by tandem Aol i stU sites. The HXBc2 proviral clone was modified by removal of about 6 kilobase pairs of flanking human sequence from the 5' and 3 end and then the unique ioLI and AgI restriction sites were introduced by site directed mutagenesis. These sites change three amino acids in the gaga-il polyprotein (A-13G, A-11R, P+9S relative to PRO 1 to 99) and three amino acids in p6 (Q28A, E29A, P30A). Rosulter Wild type enzyme was expressed at high levels in E. coli but only after induction with IPTGPI, and had a toxic effect on bacterial growth. We have purified about 1-2mg from 500ml of culture. Full levels of expression were determined using a PCR mutagenised PRO gene containing the mutation D25N; coomassie stained levels of the 7-IFN-PRO fusion were visible after lh of induction. D25N did not process any precursor, whereas wild-type enzyme processed more than 90% of precursor. The matched recombinant wild-type virus containing the lNotLtLAsDI sites, grew as well as unmodified HXBc2 in MT4, C8166 and H9 T-cells as measured by virus yield and p24 assays. The D25N mutant proviral recombinant yielded no virus and p24 levels were only detecteable at 2 days post-electroporation, falling to background by 10 days post-electroporation. Discussion We have developed a system for rapid mutagenesis and cloning which permits the matching of mutant protease activities, both in crude assays and also with purified enzyme (Km, k K), with virus growth and drug sensitivity (ICS). If we can relate the Ki value to the match virus IC, then we have the potential, after cryftallisation of purified mutant enzymes with appropriate inlbitors, to determine the interactions at the atomic level which ultimately affect HIV-1 drug sensitivity. Furthermore, the unique nature of using restriction sites spanning only 360 bp of the HIV genome has allowed us to prepare a library of infectious clones, with random mutations in the protease gene, which can be used to establish quickly the array of mutations that arise in viruses during drug selection. DR EDWARD D. BLAIR, WELLCOME RESEARCH LABORATORIES, BBCKENHAM, KENT, UK. 081 639 5508(T); 081 663 3532(F) v o 0 N -o 0 (0 PA0289 IGE SYNTHESIS DRIVEN BY IL-4 PRODUCING CD8+ CELLS IN AIDS Scala Enrico*, Paganelli R*, Ansotegui I.J.*, Ausiello C.M.**, Sirianni M.C.*, Halapi E.***, D'Offizi G.*, Mezzaroma I.*, Fanales-Belasio E.*, Cassone A.**, Aiuti F.*- *Department of Clinical Immunology & Allergy, University "La Sapienza", Rome, Italy; **Laboratory of Bacteriology and Medical Micology, I.S.S., Rome, Italy; ***Department of Immunology, Karolinska Institute, Stockholm, Sweden Objective: Increased serum levels of IgE are found in Aids patients even after CD4+ cells, the physiological inducers of IgE synthesis, have virtually disappeared. Therefore we investigated other cells for helper activity. Methods: T cell clones from three patients with AIDS and high IgE serum levels were grown in rInterleukin (IL)-2. Phenotypic analysis was performed by cytofluorimetry. Production of cytokines was assessed by both ELISA and reverse PCR for mRNA. Results: Most CD8+ T cell clones from these patients produce IL-4, IL-5 and GM-CSF, express a CD40 ligand on the surface, and thus functionally mimick TH-2 CD4+ cells. However Interferon-y is also produced by 40% of the clones, and TNF-z by 80%. The supernatants of these clones stimulate the synthesis of IgE by anti-CD40 activated B lymphocytes of normal individuals. CD8+ clones also have weak natural killer activity. About one third express CDI6 or CD56 molecules on the surface. One CDI6+ clone showed weak redirected killer activity by anti-CDI6. Only two clones have a y/3+ CD8+ phenotype. Discussion: These findings indicate that IgE production in human immunodeficiency virus (HIV) infection is efficiently driven by CD8+ lymphocytes. The finding of IL-4 producer CD8+ cells may be related to the TH-IITH-2 switch, which represents a crucial event in Aids pathogenesis. SCALA Enrico, Dept. of Clinical Immunology & Allergy Viale dell'Universita 37, 00185 Rome, Italy Tel.(39)-6-4454941 Fax (39)-6-4463328 PA0290 Oxidative stress & apoptosis in HIV infection. A role for synergistic antioxidant metabolites from plants. H. Greenspan, LGD Biomed.Grp,; 0. Aruoma, Univ. London Apoptosis is recognized as a pathway of immune cell loss in patients with HIV/AIDS. The cascade of events that results from "oxidative stress" seen in HIV/AIDS, a result of overabundance of reactive oxygen forms combined with a multilevel deficiency in metabolic sources of antioxidants, is markedly similar to that which can initiate apoptosis. This includes oxidation of cellular membranes, alteration of metabolic pathways, disruption of electron transort systems, depletions of cellular ATP production, loss of Ca homeostasis, endonuclease activation and DNA/chromatin fragmentation. Primary and secondary metabolites found in plants, act as synergistic antioxidants, and can protect plants from oxidation induced cell death. Experiments have shown that some of these same metabolites can inhibit cell killing by HIV. Can these compounds be useful in inhibiting viral activation and the apoptosis that immune cells are thought to undergo in HIV/AIDS through their synergistic antioxidant properties? A review of OS in HIV/AIDS is presented and the basis for OS initiating apoptosis is explored. The functional antioxidant activities of plant metabolites are illustrated and the use of these broad spectrum antioxidants from plants are proposed as a mechanism by which cell killing in HIV infection can be inhibited. Dr. Howard C. Greenspan, 23 Spring Hill Rd., Annandale, NJ, 08801 USA 908-730-8717(phone) 908-735-6842(fax)

Page  103 P A02891 B IRM.-CARBOHIDRATE OF LOU HOLECULAR WEIGHT ECA 10-142 CONTROLS AIDS P M u Cevallos A. Edwin-Dc.Chief of Oncology and Radiotherapy services of Metropolitan Hospital Quito,Ecuador. Objective: The BIRM ECA 10-142 in vitro, reports non-cytotoxic even at the highest concentration tested, and moderated anti.HIV activity with X reduction in syncitia-formation of 59%;(Southern Research Institute,Birmingham Alabama,U.S.A.;July 1991) In vivo doubte-blind reported an elevation in the number of CD4 cells. The chemical analysis report in Nutclear Magnetic Resonance stablished the empirical formula C3H502, or a multiple thereof. methods: Twenty patients in state IV of CDC, were chosen, and survived from january 1989 to april 1993 (Figure No 2). In every patient the CD4 cells were elevated notoriously. For example: from 149 CD4 cells to 480 CD4 cells,in 3 months of treatment with BIRM. Results: The results were evaluated based on the number of CD4,CD8 and quotient CD4/CD8; clinical evolution of the patient in his signs and sympthoms.Patients with CD4 so Low Like 1%, survived for 15 months, and in the second group the patients are still alive after 22 months of treatment. Discussion and Conctussions: The BIRM ECA 10-142 has demostrated been efective in all the states of HIV/AIDS, BIRM has been evaluated "in-vitro"and"in-vivo"; and its composition has demostrate to be an carbohydrate of Low molecular weight, wich performs at the level of celutlar adhesion and reduces in 59% the syncitia formation. The elevation in the number of CD4 cells indefinetty in all the patients treated, shows that we are in front of a modulater and stimulater substance of the Inmune System, a substance of enormous aplication in this kind of patients. CEVALLOS ARELLANO EDWIN Av.Eloy Atfaro 516 y Alemania,Quito,Ecuador Telephone (593)2 526898;Fax (593)2 569493 PA0292 CYSTEAMINE AND CYSTAMINE INHIBIT HIV-1 REPLICATION IN CELLS OF P A0 2 MONOCYTEIMACROPHAGE AND T CELL LINEAGES. Ho. Wen-Zhe.. Zhu, XH., Song, L., Cutili, J., Douglas, S.D. The Children's Hospital of Philadelphia, University of Pennsylvania Medical School, Philadelphia, PA, USA. The effects of cysteamine (2-aminoethanethiol, MEA) and its disulfide, cystamine (a drug known to be effective In treatment of nephropathic cystinosis)on the human immunodeficency virus (HIV) expression in chronically infected cells of promonocytic line (U1) and T cell line (ACH-2), as well as, In human peripheral blpod monocyte-derived macrophages were Investigated. U1 and ACH-2 cells constitutively express low levels of virus, which is Increased in the presence of tumor necrosis factor a (TNF-), Interleukin 6 (IL-6), and other Inducers. Cysteamine and cystamine supressed the induction of HIV-1 expression mediated by TNF-o, IL-6, or GM-CSF in U1 cells in a dose dependent fashion in the absence of cytotoxic or cytostatic effects. HIV-1 reverse transcriptase (RT) activity, induced by these mediators, was decreased by 50-90% after treatment with cysteamine or cystamine. Inhibitory effects of cysteamine or cystamine were also observed In the TNF-a-stimulated chronically infected ACH-2 line cultures. In addition, HIV-1 Infection was blocked or substantially reduced in the cysteamine or cystamine pre-treated primary monocytelmacrophage cultures compared with untreated cultures Infected in vitro as determined by RT activity. In the primary monocyte/macrophage cultures treated after Infection was established, RT activities were also suppressed 50-90% that of untreated controls. Untreated cultures showed giant syncytia formation after HIV-1 infection, whereas most of the treated cells remained free of the giant syncytia. Cysteamine or cystamine may have the potential to limit HIV-1 replication in monocytes/macrophages In vivo and may be a new class of agents with antiretroviral potential. Dr. Wen-Zhe Ho, Division of Allergy, Immunology and Infectious Diseases, Childen's Hospital of Philadelphia 34th Street and Civic center, Philadelphia, PAl9104 PA0293 INHIBITION OF HIV-1 REPLICATION BY SDZ NIM 811, A NONIMMUNOSUPPRESSIVE CYCLOSPORIN A ANALOG. Objective: To determine whether NIM 811 has a potential as an anti-HIV agent. Results: (Me-Ile-4) Cyclosporin (SDZ NIM 811) is a 4-substituted Cyclosporin devoid of immunosuppressive activity, but retaining full binding capacity to cyclophilin. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line and in HeLa T4 cells; its antiviral activity was demonstrated against laboratory strains and against clinical isolates in primary T4 lymphocytes and in primary monocytes. The compound shows antiviral synergy in vitro when combined with AZT, DDI or with HIV-protease inhibitors. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, neither free nor bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virusinfected and uninfected cells. SDZ NIM 811, however, blocks formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs and monkeys resulted in blood levels considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than Cyclosporin A. Conclusion: The properties of SDZ NIM 811 suggest a potential for antiviral therapy. Brigitte Rosenwirth, Sandoz Research Institute Brunner Strasse 59, A-1235 Vienna, Austria Phone (43)-1-86634-380, Fax (43)-1-86634-681 PA0294 Pentoxifylline enhances the antiviral activity of ddl against HIV-1 in cultured mononuclear cells. Zhang L, Novick W, Crumpacker CS. Beth Israel Hospital Boston, MA and Hoechst-Roussel Pharm., NJ. Obiectivg: To determine if Pentoxifylline (PTX) enhances the ability of ddl to inhibit HIV replication in PBMC cultures. M ld: PBMC were activated with OKT3 antibody and acutely infected with HIVum in the presence of increasing concentration of PTX, ddl or combinations of PTX and ddl. PBMC's had a media change at day 3 and at day 6 supernatant was assayed for the presence of HIV P24. Cell viability was measured by the M7T assay. Results: ddl alone was observed to inhibit HIV p24 expression by 50% (ID50) at a concentration of 0.11 iM. ID50 for PTX alone is 90.5 iM. When the two drugs were added together the ID50 dose occurred at.006 tM of ddl and 7.73 M PTX. The combination index (CI) of 0.151 indicated synergy with these two compounds on inhibition of HIV p24 antigen expression. TD5o (50% toxic dose) of PTX is 414.3 tM. When PTX and ddl were added together, TD50 is 472.8 iM. ddl alone was not inhibiting for cells up to 10.0 pM.These results were calibrated by the median-effect equation (Chou, 1976) and generalized multiple drug effect equations used for computer simulation (Chou and Talalay, 1983, 1984). Conclusions: In OKT3 antibody activated PBMC, the addition of PTX to ddl is synergistic for inhibition of HIV p24 antigen production with a CI value 0.151. This is accomplished with no added inhibitory effect on cell viability in the presence of the two drugs. This suggests that the effect of PTX to decrease levels of TNF-ot following OKT3 activation can be associated with enhanced antiviral activity of ddl. Lin Zhang, M.D. Beth Israel Hospital, Dana 617 330 Brookline Avenue, Boston, HA 02215, USA Tel: 617-735-3661; FAX: 617-735-5541 0 <O c0 44h

Page  104 PA0295 SEXUAL TRANSMISSION OF ZIDOVUDINE-RESISTANT HIV-1 Im rle.Ai l son,C1;Carr,A.1;Zheng,N.1;Roggensack,M.1;Duncombe,C.2; Finteyson,R.2;Vizzard,J.;Keatdor,J.3;Penny,R.1; endCooper,D.A.3;end the PHI Study Group.l.The Centre for Immunology, St Vincent's Hospital; 2.Private practice, Derlinghurst,;3.NCHECR,UNSW,Sydney,Austrella. bect ve To assess the prevalence of zidovudlne-reslstant HIV-1 in homosexual men who seroconverted at least 6 months after zidovudine therapy became available in Australia.Methods: HIV was Isolated from 28 seronegative men who presented with symptomatic primary HIV infection,and from 5 sexual partners. ZDV sensitivity was assessed by a PBL assay; selected Isolates were tested for mutations at codon 215 of the viral RT, by selective PCR. Results:The mean ZDV ICs0 for the 28 seroconverters was 0.301uM, range (0.001-6.7)uM, and geometric mean 0.018uM: ZDV C uMl_#subc215PR <0.1 20 ND 0.1-1.0 7 WT >1.0 (6.7) 1 mutant IC50 values for 4 of the 5 virus donor Isolates ranged from 0.001-0.113uM; all were WT at codon 215. The remaining Isolate, from a patient with symptomatic HIV disease who had been on ZDV therapy for 2 years, demonstrated a resistant phenotype (ZDV IC50 2.7uM) and genotype (mutation at codon 215). This patient was the sexual partner of the seroconverter with the resistant Isolate. Summary and conclusion; We have Identified a case of sexual transmisslon of zldovudine resistant HIV-1, from a homosexual man who had been on zidovudlne therapy for 2 years, to his seroconvertlng sexual partner. Despite widespread uptake of zidovudine therapy for all stages of HIV disease amomgst homosexual men in Sydney, transmission of zldovudlne resistant HIV-1 remains Infrequent. Allison Imrle Centre for Immunology, St Vincent's Hospital, Sydney, Australia tel(02)3617700 fax(02)3612391 PA0297 CELLULAR RESISTANCE INDUCED BY IN VITRO AZT-TREATMENT OF CEM CELLS. Riva E'., Turriziani O'., Simeoni E'., Di Marco P'., Bellarosa D'., Romagnoli G**., Cianfriglia M**., Antonelli G*.,Dianzani F'..Institute of Virology, University "La Sapienza", Rome. **ISS, Rome. *Deparnent of Biomedicine, University of Pisa, Pisa. It has been recently addressed the question whether HIV-1 resistance to AZT might depend also on cellular mechanisms of acquired drug-resistance other than on viral mutations. Specifically experiments were planned to address the question whether AZT itself was able to induce cellular resistance to the drug. Results of these experiments show that this possibility does exist. In fact, CEM cells propagated in the presence of increasing concentrations of AZT (CEMAZT) become resistant to antigrowth and antiviral activity of AZT. Specifically the concentration of AZT able to inhibit 50% of cellular growth (TC50) is 30 fold higher in CEMAZT as compared to the parental cell line. More importantly the concentration of the drug able to inhibith 50% of virus yield (ID50) is 3000 fold higher in CEMAZT as compared to parental CEM line. Further experiments using other compounds, such as vinblastine, vincristine, ddl and ddC revealed that the acquired AZT-induced resistance is specific for AZT. It was also possible, by RIA test and by radioactive AZT, to demonstrate that AZT concentration inside the cells is significantly diminished in CEMAZT as compared to CEM. The use of radioactive AZT, allow us also to rule out the possibility that the lack of the inhibitory activity in CEMAZT is due to the sequestration of the drug in specific organelles leading to the low availability of the drug in their site of action. These cells, although fully resistant to antiviral and antigrowth activity of AZT, when cultured for a long period in the presence of AZT fail to show detectable level of P-170, as measured by evaluation of specific mRNA and by FACS analysis. In conclusion, although some questions remain open, these findings strongly suggest that the resistance to AZT may be mediated by a specific mechanism of uptake inhibition. Work supported by Ministry of Health-ISS: AIDS Research Project. Antonelli Guido, Department of Biomedicine, Universi ty of Pisa, Pisa. tel. 39-50-555333 fax: 39-50-555477 PA0296 INHIBITION OF HIV-1 PROVIRAL DNA INTEGRATION IN CELL CULTURES BY AZT TREATMENT. *BRUNO R.,DEBIAGGI M.,CARLEVARI M.,ROMERO E.,'FILICE G. 'Institute of Infectious Diseases and Institute of Microbiology, Universly of Pavia- Pavia -Italy OBJECTIVE. Although studies conducted in animal models indicate the AZT prophylaxis activity on murine or feline retroviral infectious, few date are known about AZT prophylaxis of HIV-1 infection in human. In this study we evaluate "in vitro" the pre and early postexposure prophylaxis on HIV-1 infection. METHODS. At various times before and after HTLV-IIIB infection H9 cell cultures were treated with different AZT concentrations. Cultures were tested for p24 antigen production and proviral DNA integration at 7 and 14 days after infection. RESULTS. In our experimental conditions the pre and early post treatment with AZT (from 10 to 1 pg/ml) can prevent HIV.1 DNA integration in H9 cell cultures. DISCUSSION AND CONCLUSIONS. Results obtained indicate that AZT can effectively inhibit "in vitro" HIV-1 proviral DNA integration. BRUNO Raffaele - Institute of Infectiuos Diseases, University of Pavia, via Brambilla 74-27100 PAVIA - ITALY - 39-0382-526335 fax 525886. PA0298 ANALYSIS OF THE PRESENCE OF AZT-RESISTANT HIV IN CARRIERS Sakamoto, Takashi1'2 Tochikura.A,' Sasso.F,' Kageyama.S,t Wakamiya.N,' Tsuchie.H,' Uemura.H2 and Kurimura.T'.' Dept. of Pathology, Research Institute for Microbial Diseases, Osaka Univ., Suita, Japan 2 Foso Pharmacentical Industries., Osaka Japan. The appearance of AZT-resistant virus after treatment of HIV carriers with the drug is well-known. Selection of clinical specimens, proviral DNA, free virions or stocks of isolated virus, should give us different results by PCR in terms of drug resistance. To clarify this problem, we selected and followed 2 HIV carirriers before and on AZT treatment. Viral nucleic acid samples in I'PBMC, free virions in plasma and isolated virus stocks were amplified with nested PCR and clotted employing pUC18. At least 5 clones from each sample were sequenced and attalysed for mutations responsible for AZT-resistance. The appearance of AZT-resistant mutations varied among sample species taken at the same time point. Sometimes AZT-resistant mutations appear as early as 1 month after treatment. Mutants with stronger resistance to AZT appear with longer period of treatment. These observations indicate that, for the search for clinical resistance to AZT, choice of specimens for PCR is important. SAKAMOTO, TAKASHI Research Institute for Microbial Diseases, Osaka Univ., Osaka, Japan. Telephone (81)6-875-2128; Telefax (81)6-875-3894 0 I' N 0 tD 00

Page  105 PA0299 KYNOSTATIN (KNI)-272: AN ORALLY BIOAVAILABLE TRIPEPTIDE HIV PROTEASE INHIBITOR CONTAINING ALLOPHENYLNORSTATINE Kiso, Yoshiakil), Mimoto, T.1'2), Enomoto, H.0), Kisanuki, S.2), Moriwaki, H.1), Kimura, T.1), Hattori, N.2), Hayashi, H.2), Takada, K.'), and Akaji, K.1) 1) Kyoto Pharmaceutical University, Kyoto, Japan, and 2) Bioscience Research Laboratories, Japan Energy Co., Todashi, Saitama, Japan. The HIV protease, an aspartic protease, can recognize Phe-Pro and Tyr-Pro sequences as the virus specific cleavage sites. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine (Apns). Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and super-potent HIV protease inhibitor (Ki=0.0055nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. The practical efficient synthetic method was established. Because of the proximity to the virus specific substrate transition state, KNI-272 satisfies the requirements for anti-AIDS drugs: 1. in vitro enzyme inhibition (potency, mechanism, selectivity and stability); 2.antiviral activity in cells (potency, mechanism, cytotoxicity, stability and permeability); 3. in vivo animal tests (toxicity, stability and oral bioavailability). KISO, Yoshiaki, Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan Telephone (81)-75-595-4635; Telefax (81)75-591-9900 PAO301 T22 PEPTIDE INHIBITS MEMBRANE MIXING OF CELL FUSION Murakani,Tsut mu',Koyanagi,Y'.,Minoguchi,S'.,Shida,Hl.,Waki,M'..Matsumoto, A' and Yamamoto,N'. Tokyo Med. Dent. Univ.', Kyoto Univ.2, Seikagaku Co'. Objective: W e have already reported that T22 ([Tyr'," Lys']-polyphemusin II) strongly inhibits both cell-free and cell to cell HIV infection in vitro. Site of anti-HIV action of this compound was estimated to be at an early stage of HIV infecton. In the present study, we investigated the mechanism of T22 in more detail. Methods: Lipid (membrane) mixing and content mixing were measured by coculture between R18-loaded HeLa S3 cells which was infected with HIV-1 env-encoded vaccinia virus and HeLa cells infected with CD4-encoded vaccinia virus using fluorescence microscope. The reactivity of anti-gpl20 monoclonal antiobody (0.513) to MOLT-4/HIV cells was measured by flow cytometer. Results and Discussion: More than 0.lpg/ml T22 strongly inhibited lipid mixing between HeLa S3 and HeLa cells. Pretreatment of MOLT-4/HIV, but not MOLT-4cells with T22inhibited the lipid mixing. The reactivity of anti-gpl20 monoclonal antibody (0.513) to MOLT-4/HIV cells was enhanced by pretreatment of the cells with T22, which was also observed in pretreatment of the cells with soluble CD4. These results strongly suggest that T22 inhibits the early stage of cell-cell fusion by acting on HIV-1 env protein. MURAKAMI, Tsutomu, Department of Microbiology. Tokyo Medical and Dental University. School of Medicine. Tokyo Japan Tel (81)-3-3813-6111; Fax (81)-3-3818-7175 PAO300 STRUCTURE-ACTIVITY RELATIONSHIPS OF A NOVEL ANTI-HIV SYNTHETIC PEPTIDE, T22, AND ITS DERIVATIVES 0 H. NAKASHIMA', M. ITOY, T MURAKAMI. Y. TERAKAWA' M. MASUDA, H. TAMAMURA'. N. FUJII', M. WAKI, A. MATSUMOTO', N.YAMAMOTO'; 'Dept. Micobiol., Yamanashi Med. Univ., Yamanashi, 'Dept. Microbiol., Tokyo Med. Dent. Univ., Tokyo 'Fact. Pharmaceut. Sci., Kyoto Univ., Kyoto, 'Seikagaku Co. Tokyo, Japan. Objective: We synthesized more than 60 peptides which were associated with tachyplesin and its isopeptide (polyphemusin) and estimated the anti-HIV activity of them in vitro. Among them, we found that a novel compound, which we designated as T22, ([Tyr "' ',Lys ']-polyphemusin II), strongly and selectively inhibited the HI-V-induced cytopathicity and viral antigen expression. In the present study, we investigated the more detailed structure-activity relationships of those synthetic peptides. Methods: The anti-HIV activity was evaluated as the protection against HIV-induced cytopathic effects in MT-4 cells. The cell viability was measured by the MTT method. Results and Discussion: Through analysis of the anti-HIIV activity of T22 and its relative peptides, the importance of the Nterminal Arg residue and amino acids composition at the position of 5-7 and 14-16 which exist between two S-S bonds was suggestedThe solution structure of T22 was investigated using NMR. and its secondary structure was confirmed to be similar to that of tachyplesin I. The antiparallel 1-sheet structure and the several amino acid side chains on the (3-sheet of T22 are thought to be associated with the expression of anti-HIV activity. NAKASHIMA, Hideki, Department of Microbiology, Yamanashi Medical University, Yamanashi, Japan Telephone (81)-552-73-1111; Telefax (81)-552-73-6728 PA0302 Molecular Parameter for Anti-Human Immnunodeficiency Virus Activity of T22 (ITyr5,12, Lys7. polyphenmusin II) Nobutaka Fujii, Y. Terakawa, A. Otaka, -I. Tamamura, M. Masuda, T. Koide, K. Matsuzaki, T. Murakami', I-. Nakashima~, M. Waki^, K. Miyajima, and N. Yamamoto. Facul. l'harma. Sci., Kyoto Univ. fDcpt. Microbiot., Tokyo Mcd. Dent. Univ.,.Dcpart. Microbiol., Yamanashi Mcd. Univ., and ASEIKAGAKU Corp. Objective: T22 (jTyr5,12, Lys71-polyphemusin It) was found to have the strong anti-human immunodeficiency virus (HIV) activity and exert its effect on a virus-cell fusion procxess. In the present study, the all-D enantiomer of T22 and its related compounds were synthesized to examine the molecular parameters required for the interaction of T22 with membrane components of cell or virus to exert anti-HIIV activity. Methods: All peptides were synthesized by Fmoc-based solid phase method. The anti-HIV activity was evaluated as the protection against HIV-induced cytopathic effects in MT-4 cells. The cell viability was measured by the MTT method. Anti-HIV activity of peptides was investigated in comparison with membrane permeability against large unilamellar vesicles. Results and Discussion: The all-D enantiomer of T22 exhibited twenty times lower anti-HIV activity than T22, whereas both of them showed the same potency for membrane penneability. No positive correlation between anti-HIV activity and membrane permeability was observed. These results suggest that the anti-HIV activity of T22 is mediated through the interaction with chiral component(s) of cell or virus. FUJII, Nobutaka, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto Japan Tel (81)-75-753-455 1; Fax (81)-75 -753-4570 0 (ID CD 0 CA) 0 N

Page  106 - PA0303 THE STUDY OF CHITOZAN SULPHATE ANTI-HIV ACT ION MECHANISM OmI G. V. Korn i layeva. S.M. Andreev, A. I. Gamzazade and E. V. Karamov D. I. Ivanovsky.v Inst.of Virology. Moscow. Russia 0 b j e c t i v e: To investigate the action mechanism of chi tczan sulplhates ( tS) posses- i n high anti-HIV acti vity. M e t h o d A set of peptide-; repres_entine all the hi ch positive cItar'e density rei ions of ep120 and Q p41 was S:.ynthesized and used to determine whether electrostati c i nteract ions occur between neat i ve lyv c harged CS and positively charted lIV envelope frasmert:_. The inhibitory etfect of CS and an influence of synthetic peptides on it was determined in,:yncyt i um formation assayv by cocul ti vat i ng H19 cells with chronically HIV-infected H9/RF cells at a ratio 12. Se. u 1 t: Addition of synthetic peptides from PND.301-323) of two strains (BRU and MN) at concn of 250 lk/ml completely abolished the inhibitory properties of CS us.ed at the concn 2.5 /mIl (100 inhibition of syncvt i umf formation). The Rp120 C-terminus peptides showed 10-1 % activity in these experiments. Other s'p120 and p41 peptide= were not active. C o r c 1 u s i o n s: We concluded that sulphated polysaccharides probably inhibit:syvncy ti umn formatiorn by electrostatic bindine with ep120 PND. Galina V. Kornilayeva. Moscow, Russia, D. I. Ivanovskv Institute of Virolovy; Telephone: (095)1903062: Fax: (095)1902867; PA0304 ANTI-HIV ACTIVITY OF SULFATED ALKYLOLIGOSACCHARIDES IN A QUASI-IN-VIVO ASSAY SYSTEM AS AN INTERMEDIARY STEP TO CLINICAL TRIALS OK. INAZAWA', H. NAKASIllMA', M. ITO', N.YAMAMOTO", N.IKUSHIMA', TSHOJI', K.KATSURAYA. T. URYU; 'Dept.Microbiol., Yamanashi Med.Univ., SDept.Microbiol., Tokyo Med.Dent.Univ., ' Dainippon Ink, 'inst. Industrial Sci., Univ. of Tokyo. Objective: Previously, we reported that synthetic sulfated alkyl oligosaccharides have potent antiHIV activity (Biochem. Pharmacol., 43; 2385-2392, 1992). In the present study, we investigated if sulfated alkyl oligosaccharides were able to exist in their effective form against lllV and how long they could persist in the bloodstream of experimental animals which were administrated with compounds. Methods: Synthetic sulfated alkyl laminarioligosaccharides (DL-1 10) or maltooligosaccharides (MAL- I1) were injected to mice and whole blood was bled at serial times after injection. Blood was centrifuged and then anti-tllIV activity in each serum was determined by MTT assay based on protection of HIV- induced cytopathic effects in MT-4 cells. Similar experiments were carried out in rabbits or dogs. Results: When DL- 10 was injected to mice, the reciprocal number of the mouse serum which obtained 1 hr after injection and achieved 50% or 90% inhibition from HIV-induced CPE was 960 and 870, respectively, and those serum 12hrs after injection was 97 and 83, respectively. At 24 hrs after injection in rabbits and dogs, the reciprocal number of dilution which achieved 50% inhibition against HIV-induced CPE was 400 and 200, respectively. Discussion and Conclusions: The present study shows that even the culture medium containing only 0.25% or 0.5% of experimental animal sera significantly inhibits HlIV replication. These results suggest that sulfated alkyl laminarioligosaccharide (DL-110) may be a good candidate to be further pursued for the therapy and prophylaxis of IIIV infections. INAZAWA, Kazuhiko, Department of Microbiology, Yamanashi Medical University, Yamanashi, Japan Telephone (81)-552-73-1111; Telefax (81)-552-73-6728 O 0 CA) 0 0 0 0 #.4 PA0305 EFFECTS OF SULFATED DERIVATIVES OF PLANT ORIGIN ON HUMAN IMMUNODEFICIENCY VIRUS INFECTION Ogawara Hiroshi,Hirayama, F.2, Mizuno, T.3, Uchino, K.3, Saito, T.2, Nakashima, H.4, and Yamamoto, N.s Meiji College of Pharmacy, Tokyo, 2Yamanouchi Pharmaceutical Co., Tokyo, 3Nippon Flour Mills Co., Atsugi, 4Yamanashi Medical University, Yamanashi, 'Tokyo Medical and Dental Univeristy, Tokyo. Objective: The objective of this research is to get new, low toxic and clinically useful drugs against HIV infection. Methods and Results: Using inhibitory cffcct on iIlV-induced cytopathic effect in MT4 cells as a primary screening system, sulfated derivatives of plant origin were found to be more effective than the other known sulfated compounds. Among them, a derivative of sulfated tannic acid (Y3) was the most effective. Y3 showed EC value of 0.003 tg/ml in this system. On the other hand, for the HIV-non-infected MT-4 cells, it had a 50 % cytotoxic concentration of 1.3 jtg/ml. Moreover, Y3 inhibited HIV-induced multinucleated giant cell formation, and HIV-specific antigen expression. Y3 showed no significant increase of the clotting time in prothrombin-treated blood and of orthothromboplastin-added blood at less than 0.1 to 0.01 pg/ml, especially in the former system. To determine the effect of blood on the stability, the anti-HIV activity was measured of the samples which were obtained appropriate times after the injection to mice. When 10 mg/kg was injected to mice, the effective amounts of Y3 remained in the bloods after 30 minutes of injection. When 100 mg/kg was injected, the serums kept the effective amounts for more than 120 minutes. The side-effect was also examined. Conclusion: Considering the effectiveness and the side-effect, Y3 may become a promising compound against HIV-infection. Meiji College of Pharmacy Hiroshi Ogawara, 35-23, Nozawa-l, Setagaya-ku Tokyo 154, Japan. 03-3424-1001,03-3424-5040 ( telefax).. PA0306 DESIGN OF ANTI-HIV AGENTS BASED ON CGLYCYRRHIZIC ACID PlyasunovaO., Pol<rovsky A., Bal tina L., Mur inov Y., KTolstikov G. Institute of Molecular Biology, Koltsovo, *lnstitute of Organic Chemistry, Ufa, Russia Objective: In search for new highly effective drugs to control HIV-infection we performed some chemical modifications of glycyrrhizic acid (GA) and evaluated anti-HIV activity of these GA derivat i ves. Anti-HIV effect of combinations of GA or monoanTnonium salt of GA with azidothymidine (AZT) was examined in T-helper cells (MT-4) primary infected by HIV-1 as well as in monocyte cell line (U-937) chronically infected by HIV-1 and containing provirus. Methods: The level of HIV reproduction in cell cultures was examined by measuring of reverse transcriptase activity, viral antigen by ernnunoenzyme method. There was also apprec i ated the viabi l i ty of cel l s. Results: It. was established that the selective indexes (IS) for various GA derivatives were different. So, tested amides of GA were more effective HIV-i inhibitors (IS 29-40) than tested glycopept ides of GA and GA (IS 3-6), IS was 53 and 57 for monoarrmonium salt of GA and for penta-o-nicotinate of GA, respectively. It was founded that. glycyrrhetinic acid was not effective HIV inhibitor. We estimated the drug combinat. i on indexes of GA der i vat i ves combinat ions with AZT. It was established that tested compounds were not antagonistic for AZT. Discussion and Conclusion: The search of GA derivatives shown that most active HIV inhibitors were amide of GA with 5-amino-uracil, monoarrmon ium salt of GA, penta-o-n i cot inat.e of GA. For effect i ve cur i ng of HIV-infection a combination therapy may be advised, so, simultaneouse use of AZT with GA derivatives seemed to be most. promising. Plyasunova, O.A. Institute of Molecular Biology, NPO "Vector", Koltsovo, Novosibirsk Region, 633159,Russia Telephone: 8-383-2-64-70-21, FAX: 8-383-2-64-29-74

Page  107 PA0307 VESNARINONE, A QUINOLINONE DERIVATIVE, INHIBITS THE REPLICATION OF HIV-1 IN CULTURED CELLS.: Ikuro Maruyama', T.Nakajima', I.Kitajima', M.Osame', J.Q.Zhao, I.S.Y.Chen*, K.Aihara$. S.Nakais, M.Ikeda$, M.Adachi ('Dep. of Clin. Med. and 3rd Dep. of Intern. Med., School of Med.,Kagoshima Univ.,Kagoshima,Japan, UCLA School of Med., LA, California, sOtsuka Pharm.Co.,Ltd., lbkushima, Japan) PA0308 A SERIES OF SULFONATED AND PHOSPONATED DERIVATIVES OF DISTAMYCIN WHICH INHIBIT TIlE REPLICATION OF HIV. Clanton, David J.*, Rice, W.G*., Buckheit, R.W.t, Mongelli, N.@, Borgia, A.L.@, and Bader, J.P. * Program Resources, Inc., Frederick, MD., tFrederick Research Center, Frederick, MD, @Farmitalia Carlo Erba, Milan, Italy, *National Cancer Institute, Bethesda, MD. QObjectlive: Vesnarinone, a quinolinone derivative, is a synthetic oral positive inotropic agent that has been used for treatment of patients with congestive heart failure. We investigated the effects of vesnarinone on the replication of HIV-1 in human peripheral blood mononucleated cells, in human T cell lines, and in macrophage cell lines. Miethods: Anti-HIV effects of vesnarinone was determined using CEM-SS, MT-2, peripheral blood lymphocytes and macrophages infected with HIV-lms, HIV-IRF, HIVIsKi, HIV-2ROD, HIV-2Ms and clinically isolated viruses. The effects on chronically infected models were examined using U937 cells chronically infected with HIV-lm. Replication of virus was determined by measurement of CPE, p24, RT and infectious particles. Restna: In CEM-SS cells infected with HIVIe, vesnarinone reduced CPE of HIV-I at concentrations from 3.2 to 32pg/ml. The ICs0 was achieved at 6.34pg/ml. Vesnarinone decreased the amount of p24 and infectious particles in supernatant, demonstrating that anti-CPE activity of vesnarinone is related to reduction in viral production. Vesnarinone inhibited virus production of tested HIV-1 strains including clinically isolated strains in acutely infected cells. Vesnarinone also reduced replication of chronically infected HIV-lm in U937 by 30% or more at greater than 10pg/ml. Vesnarinone did not show cytotoxicity at concentrations up to 100 tg/ml. Vesnarinone had no anti-viral activity to HIV-2 strains. Discussions: Anti-HIV-1 effects of vesnarinone were observed at clinically achieved concentrations, suggesting that vesnarinone may be therapeutically useful in patients infected with HIV- I. MARUYAMA Ikuro, Dep. of Clinical Medicine, School of Medicine, Kagoshima Univ., Kagoshima, Japan. Telephone (0992)-64-221 1; Telefax (0992)-75-2629 Objective: To analyze a new series of sulfonated and phosphonated compounds which are active against I IIV with favorable clinical potential. Methods: 20 sulfonic and phosphonic derivatives of distamycin were evaluated for their efficacy in the National Cancer Institute's AIDS Drug Screen. Activity was assessed against several cell and viral isolates, including clinical isolates in PBMC's. PCR analysis of the LTR/gag region was used to determine the manner by which these compounds inhibit HIV. Results: The most potent of these derivatives, NSC 651015, had an ECso of 3.5pM in the anti-HIV assay. This compound was equally effective against the AZT resistant and pyridinone resistant mutants of HIV, as well as the macrophage-tropic strain HIV-l,.L. There was no effect on chronically infected cell lines. Virus was not directly inactivated by the drug, nor did pretreatment of cells with drug reduce HIV replication. Binding of virus to cells was shown to be inhibited, as well as cell to cell fusion. PCR analysis confirmed that 651015 acts early in the replication cycle of HIV. Conclusion: 651015 is a potent inhibitor of HIV replication and has a number of favorable characteristics as a therapeutic agent. NCI is currently considering this compound for clinical trials. Dr. David J. Clanton NCI-FCRDC Program Resources, Inc. fax 301 846-5304 P.O.Box B, Frederick, Md 21701 USA 301 846-5300 PA0309 MODIFIED CYCLODEXTRIN SULFATE (mCDS71); PROTEIN BINDING AND ANTI-HIV MECHANISM *OTAKE, Toru, *MORI,H., tMORIYA,T., *YAMASAKI,K., fYAMAKITA,H., *MORIMOTO,M.,tMATSUMOTO,K. and *UEBA,N. *Osaka Prefectural Institute of Public Health, t Tanabe Seiyaku Co.Ltd., Osaka, Japan Modified cyclodextrin sulfate(mCDS71), a new polyanionic anti-HIV agent, binds to human plasma proteins and is still effective in human sera. The binding of mCDS71 to the proteins was 98-99% and the major portions of the bound protein was albumin. The complex of mCDS71 with human surum proteins showed a high anti-HIV activity in vitro. These results suggest the efficacy of mCDS71 in vivo. Intense block of the syncytium formation in MOLT-4HTLV-lB/MOLT-4 cells and time course analysis of the anti-HIV-1 activity, have suggested an early stage inhibition of HIV entry into cells as the mode of action of mCDS71. However, the exact sites of action of the compound during virus-cell interactions remain undisclosed., mnCDS71 failed to suppress the binding of anti-Leu3a to MT-4 (CD4+) cells, and unlike dextran sulfate, mCDS71 did not suppress the binding of the anti-HIV-1 gpl20 monoclonal antibody 0.53 to MOLT-4/HTLV-IIIB cells. Futhermore, mCDS71 only slightly inhibited dipeptidyl peptidase IV (CD26), which is proposed as a cofactor of viral entry; and scarcely suppressed binding of a specific monoclonal antibody(Tal) to CD26 on the cells. Further studies of the sites of action are in progress. Toru OTAKE, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, 537, Osaka, Japan, Telphone 06-972-1321, Telefax 06-972-2393 PAO310 NP06: A NOVEL ANTI-HIV OLIGOPEPTIDE PRODUCED BY ACTINOMYCES Machida, Makoto*; Date, T.*; Thyota, K.*; Ishida, S.*; Watanabe, K.*; Ohta, K.*; Yoshioka, H.*; Chokekijchai, S.**; Kojima, E.**; Roller, P***; Mitsuya, H.** and Murakami, K*, *Research Laboratory of Bioscience, Nippon Paper Industries, Co., Ltd, Yamaguchi, Japan and **Medicine Branch and ***Laboratory of Medicinal Chemistry, National Cancer Institute, NIH, Bethesda, USA. Objectives: To identify compounds active against HIV-I from products of microorganisms and to define the mechanism of their antiviral activity. Methods: Extracts from more than 7,200 different bacterial colonies were fractionated by preparative thin layer chromatography and high performance liquid chromatography. Each fraction was tested for anti-HIV- I activity in various target cells by using cytopathic effect inhibition or MTT score as an endpoint. Antiviral mechanism wvas also investigated. Results: NP06, purified from extracts of an Actinomyces colony isolated from a soil of Yamaguchi, was identified to have an activity against HIV-I in vitro. The 50% inhibitory concentration (ICso) values of NP06 against HIV-I were 0.6 and 1/t g/ml as tested in PHA-PBM and MT-2 cells, respectively The IC50 value against HIV-2 was 15 Itg/mil as tested in ATH8 cells. The 50% cytotoxic concentration/ICso ratio for NP06 was 49 when assessed in MT-2 cells. 'H,"C-NMR and FAB mass spectrometric analyses indicate that NP06 is a hydrophobic 21-mer oligopeptide N-terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2164.5. Antiviral activity of NP06 appears to be due to its inhibition of the virus-cell binding. NP06 shows no activity against viral reverse transcriptase or protease. Conclusions: NP06, isolated from Actinomyces, exerts a potent and selective anti-HIV-I activity in vitro and may serve as a lead compound for the search andior chemical modifications for more effective compounds. Machida Makoto*, Research Laboratory of Bioscience, Nippon Paper Industries Co., Ltd, Iwakuni, Yamaguchi, Japan Tblephone: (81)-827-24-6405; Telephax: (81)-827-24-6407 0 CA) 0 V 0 0

Page  108 r. oD PAO311 PHARMACOKINETICS OF CENTRAL NERVOUS SYSTEM (CNS) - TARGETING 6-HALO-DIDEOXYGUANOSINES IN MICE AND MONKEYS Watanabe, Kazuhiro*; Fujii, Y.*; Kojima, E.*; Balls, F.**; Hawkins, M.**; Shirasaka, T.**; Machida, M.*; Yoshioka, H.*; Mitsuya, H.**; Murakami, K.* * Research Lab of Bioscience, Nippon Paper Industries Co., Lid, Yamaguchi, Japan, ** National Cancer Institute, National Institutes of Health, Bethesda, USA. Objectives: To develop CNS-targeting dideoxynucleosides, we studied the pharmacokinetics of a series of 6-Halo-2',3'-dideoxyguanosines (6-halo-ddGs) in mice and monkeys. Methods: Four 6-Halo-ddGs (F-ddG, Cl-ddG, Br-ddG, l-ddG) and ddG were administered to BALBic mice orally at an 80agmole/kg, intravenously and subcutaneously at a 200tamoleikg, or to rhesus monkeys intravenously at a dose of 25mg/kg. Blood, cerebrospinal fluid (CSF) and brain tissue were analyzed for the content of each 6-halo-ddG and ddG (dehalogenated product catalyzed by adenosine deaminase) by high performance liquid chromatography. Results: When orally administered to mice, the mean area under curve (AUC; tM. min) values were 320, 294, 222, 122 and 128 for F-ddG, Cl-ddG, Br-ddG, I-ddG and ddG, respectively. When F-ddG and Cl-ddG were intravenously administered to mice, brain levels of ddG (as compared with AUC) were 3.6 - fold and 2.8 - fold higher than the parent drug, ddG, was administered. Subcutaneous administration of Cl-ddG into mice achieved 4 - fold higher ddG concentration in blood (as compared with AUC) and ddG persisted S3 - fold longer in brain than intravenous administration. CSF to plasma ddG concentration ratios following injection of CI-ddG, l-ddG and ddG in monkeys were 23%, 17% and 6.5%, respectively. Conclusions: 6-halo-ddGs, especially Cl-ddG, represent a new class of dideoxynucleoside analogs which possess a potential for favorable penetration into CNS and may prove to be agents for therapy of HIVinduced neurologic disorders. WATANABE, Kazuhiro, Research Laboratory of Bioscience, Nippon Paper Industries Co., Ltd, Iwakuni, Yamaguchi, Japan Telephone: (81)-827-24-6405; Telefax:(81)-827-24-6407 PA0312 NOVEL HIGHLY PROMISING HEFT ANALOGUE: MKC-442 Ubasawa Masaru*; Takashima, H.*; Sekiya, K.*; Yuasa, S.*; Niwa, T.*; Yamamoto, M.*; Tsutsui, N.*; Tanaka, H.**; Miyasaka, T.**; Baba, M.*** * Mitsubishi Kasei Corporation, Yokohama, Japan; **Showa University, Tokyo, Japan, ***Fukushima Medical College, Fukushima, Japan. Objective: Anti-HIV-1 activities of some HEPT (1-[(2-hydroxyethoxy)methyl]-6 -(phenylthio)thymine) analogues (in MT-4 cells) diminished in the presence of human serum (-IS). The decreasing activity appeared to result from HS protein binding to the compounds. New HEPT analogues that could be less affected by HS were synthesized and evaluated in the present study. Method: The observed decrease in anti-HIV-1 activity was found to correlate with lipophilicity of the compounds. A series of HEPT analogues in which 6-phenylthio group was replaced with a less lipophilic substituent were synthesized Results: Benzyl group was found to be the most appropriate substituent on the 6 position. Among 6-benzyl derivatives, MKC-442 (6-benzyl-l-ethoxymethyl-5 -isopropyluracil) was selected as a promising analogue. Its inhibitory value against HIV-1 (ECso 15 nM) was not much affected by HS (ECso 63 nM in the presence of 50% HS). Furthermore, MKC-442 has several notable features: it is active against AZT-resistant mutants; it does not inhibit murine bone marrow cell growth; it shows synergistic inhibition of HIV-1 replication with AZT, DDI or DDC; it penetrates into the brain; its effective concentration in plasma is kept for a rather long period. Discussion and Conclusions: MKC-442 apparently possesses several characteristics which are required for clinical studies of AIDS chemotherapy. UBASAWA, Masaru, Mitsubishi Kasei Corp., Yokohama, Japan. Telephone (81)-45-963-3371; Telefax (81)-45-963-3977 0 CA) 0 W.) I 44 PA0313 MODULATION OF HIV PRODUCTION BY SALICYLATES Kotler DP*, Bulmovici-Klein E, Mohan V, Maitra U, Sonnabend J. St. Luke's/Roosevelt Hosp Ctr, NY, NY. Objective: To determine if salicylates can inhibit HIV production in vitro and in vivo. Methods: The ability of salicylated (5-aminosalicylic acid [5-ASA] and acetylsalicylic acid [ASA]) to inhibit HIV production in vitro was tested in 3 systems. Organ cultures (from intestinal biopsies of HIV- patients) which spontaneously produce HIV p24 antigen on incubation and chronically infected T-cell line (ACH-2) were incubated with and without 5-ASA or ASA respectively, and spontaneous and TNF induced HIV p24 antigen production was measured in both systems. Lymphocytes from HIV infected patients were cocultivated with donor lymphocytes with or without ASA and p24 antigen was measured. (HIV Infected patients were treated with 5 ASA (6G daily) for 2 months and tissue p24 contents (in rectal biopsies) determined. A randomized double-blind 8 week study of ASA versus placebo was commenced in February, 1994, and HIV levels are being tested by measurement of plasma viremia, quantitative microculture, acid dissociated p24 antigen and the branched DNA technique. Results: 5 ASA (2mM) and ASA (2mM) inhibited, by approximately 50%, HIV p24 antigen production, both spontaneous and TNF induced, by organ cultures (rectal biopsies) and ACH-2 cells respectively. ASA (2mM) also inhibited HIV production by lymphocytes from infected patients. Nine individuals receiving 5-ASA (6gm daily) for 2 months showed a reduction in p24 content in rectal biopsies. Discussion and Conclusions: Salicylates appear to Inhibit spontaneous and TNF induced HIV production in vitro. This anti-HIV effect was seen in the rectal tissues of 9 patients treated with 5-ASA and is being studied in randomized placebo controlled trial of ASA in 50 HIV infected patients. Preliminary results of the trial will be presented. PA0314 ANTIVIRAL EFFECT OF CONVERGENT VS. DIVERGENT DRUG COMBINATION ON HIV-1 SPREAD IN VITRO. K.T. Chong and P.J. Pagano. Upjohn Laboratories, Kalamazoo, MI, USA. Objective: We investigated the role of Delavirdine mesylate (DLV), a non-nucleoside reverse transcriptase (RT) inhibitor, in three-drug combination against HIV-1 spread in human PBMC culture. We also compared the efficacy of combining drugs which attack similar target sites or "convergent approach" (e.g., AZT, ddl and DLV as inhibitors of HIV-1 RT) to combination targeting dissimilar sites or "divergent" approach (with AZT, protease inhibitor U-75875 and CD4-PE40 or human alpha interferon). Methods: A viral spread assay was established by mixing infected human PBMC with uninfected PBMC at 1:100 ratio. We monitored drug effects on HIV-1 spread and cell survival during treatment and on viral recovery when drugs were removed from culture. Results: When AZT, ddl and DLV were combined at IC5o concentrations, we observed 99.9% viral inhibition by day 6 of drug treatment. When drug was removed on day 7 of treatment, there was still 73.4% viral growth inhibition on day 12 of infection. In contrast, AZT at three times IC50so concentration, produced 85.1% inhibition by day 6 of infection; with drug removed on day 7, there was complete loss of viral inhibition on day 8 of infection. Combination of AZT, ddI and DLV also produced greater cell survival than with AZT monotherapy. Discussion and Conclusion: Divergent therapy is as effective as convergent therapy during treatment but was less effective on viral recovery. This potential disadvantage may reflect more the weakness of individual non-RT inhibitors used than that of the ineffectiveness of the divergent approach. CHONG, K.T., Upjohn Laboratories, Kalamazoo, MI, USA Telephone (616) 385-7007; FAX (616) 385-6492 Donald P. Kotler, St. Luke's/Roosevelt Hosp Ctr, 421 West 113th St., S & R 1301, NY, NY 10025. Tel: (212) 523-3670 Fax: (212) 523-3678

Page  109 PAO315 Lactoferrin prevents infection of HIV in vitro. TANAKA SHIGEAKI, Tokai University, School of Medicine. Objective: In order to evaluate the anti-HIV activity of lactoferrin (LF), we investigated preventive activity of LF of HIV infection in vitro. Methods: HTLV Ill, was infected to MT-4 cells in the presence or absence of LF. Effect of LF was assayed by MTT method, by binding inhibition (IF). and by fusion inhibition. Several peptides were synthesized chemically based on sequences of N-terminal lobe of LF. Results: Human lactoferrin (hLF) as well as bovine lactoferrin (bLF) from milk inhibited the infection of HIV to tissue culture cells. The inhibition was also observed in virus adsorption to cells (Binding assay) and syncytium formation. In addition, inhibition of HIV infection to MT-4 cells was reconstituted with synthetic peptide derived from N-terminal lobe of LF. Particuraly, a peptide which is correspond to amino acid residues of 39-52 of N lobe showed significant inhibition of HIV infection in vitro. Discussion and Conclusions: LF prevents HIV induced cytopathic effects and viral antigen expression. These results suggest that LF possesses potent anti-HIV activity in its protein moiety and may be useful in preventing HIV infection in humans. TANAKA StIIG(;EAKI Molecular Life Science. Tokai Univ. School of Med. Bosei dai Isheharashi. Japan. 04163-93-1121. 0463-94-9058 PA0317 NOVEL NON-NUCLEOSIDE COMPOUND WITH IN VITRO ANTI-HIV-1 ACTIVITY De la Fuente, Jesus A.; Cross, S.; Marugin, J.J. PharmaMar S.A., Madrid, SPAIN Objective: Evaluation of a new non-nucleoside type compound (PM-92131 (~)) as a potent in vitro anti HIV-1I inhibitor. Methods: A new type of compounds, degraded system related with the limonoid family, has been identified as a potent inhibitor of HIV-1. Three different assays have been used to evaluate this in vitro activity: XTT cytoprotection, syncitial-forming and p24 ELISA assays, in which CEM-SS cells and HIV-1 RF strain virus were used. Results: PM-92131 (~) was identified as a potent inhibitor of HIV-1 infection in all three assays. The results from the cyto-protection assay were ECso= 0.2 pM, ICso= 20.0 pM with a ratio of SI= 100. We have isolated each enantiomer form the racemic mixture initially assayed by a chemical procedure, resulting to be the (+) enantiomer the active compound against HIV-1 infection. Conclusions: PM-92131 is a potent inhibitor of HIV-1 on the in vitro assays. This type of compound, with potential anti HIV in vivo activity, is a new and very interesting contribution to the development of additional antiviral agents for the prevention and treatment of the AIDS. De la Fuente, Jesus A., PharmaMar S.A., C/ Calera,3, Tres Cantos 28760, Madrid, SPAIN, Telf. 34-1-8032000, FAX# 34-1-8031143 PA0316 STRUCTURE-ACTIVITY RELATIONSHIPS OF NOVEL AMIDES OF BETULINIC ACID BLOCKING HIV-1 ENTRY Dereu N., Bousseau A., Poujade C., Evers M., Htnin Y., Mayaux J.F. Rh6ne-Poulenc Rorer S.A.Central Research Vitry sur Seine France Objective Betulinic acid amide RPR 103611 and related compounds are potent, non peptidic, inhibitors of HIV-1 entry by blocking a post-binding step necessary for virus-membrane fusion. Preliminary studies have shown the importance of the nature of the lateral chain attached to the amide moiety. The effect of chain variations on anti-HIV-1 activity was investigated by synthesizing a series of new betulinic derivatives. Methods Compounds were tested in CEM4 cells for their ability to inhibit the cytopathic effect induced by HIV-1 infection. Results The inversion of the amide moiety within the chain was found to only slightly modulate activity. Chain rigidification by introducing phenylene moieties near the triterpene proved detrimental for activity. A phenyl substituent or a phenylene near the terminal carboxylic acid led to very potent derivatives (EC50<50nM). Discussion and Conclusions: RPR 103611 and some related molecules are very potent anti-HIV-1 agents acting by a novel mode of action. These molecules represent an entirely new approach for the development of new anti-HIV drugs. Norbert DEREU, Centre de Recherche de Vitry-Alfortville, 13, quai J. Guesde, F-94403 Vitry sur Seine FRANCE Fax 33-1- 45738014 PA0318 ANTI-HIV-1 ACTIVITY OF THE EXTRACT OF FUSCOPORIA OBLIQUA MORI, Haruyo*, OTAKE, T.*, YAMASAKI, K.*, NISHIO, M.t, MORIMOTO, M.* and UEBA, N.* *Osaka Prefectural Institute of Public Health tTanabe Seiyaku Co. Ltd.,Osaka, Japan A water extract of Fuscoporia obliqua, which belongs to Polyporaceae, was evaluated for anti-HIV activity in vitro. The extract completely inhibited HIV-1-induced cytopathicity in MT-4 cells at concentrations as low as 15.6pg/ml, and suppressed giant cell formation in co-cultures of MOLT-4 /MOLT-4HIV-1 or HIV-2 cells. Inhibition was also observed against the replication of JR-FL, the macrophage tropic HIV-1 strain, in peripheral blood mononuclear cells. Althouth pretreatment of MT-4 cells with the extract prior to viral exposure prevented HIV-1 infection weakly, preincubation of HIV-1 particles with the extract before infection dramatically decreased infectivity of the virus. Flow cytometric analysis revealed that the extract selectively blocked the binding of monoclonal antibody(mAb) specific for CD26, which was estimated as a cofactor for entry of HIV in CD4+ cells, and OKT4A/Leu3a mAb to several T4+ cells, and 0.51 mAb to MOLT-4HTLV-IIIB cells. These observations suggest that the extract affects both virions and cells to exhibit the anti-HIV activity. MORI, Haruyo, Osaka Prefectural Institute of Public Health, Osaka, Japan Telephone 06-972-1321; Telefax 06-972-2393 0 CA) 0 CA) -J

Page  110 PA0319 SCREENING OF,ANTI-Hl, ACTIVITY OF KOREAN AND CHINESE TRADITIONAL MEDICINES. LEE. JOO-SHIL LEE, fl. R., NAM, J. G., LEE, Y. J., KANG, C., SHIN, Y. O0 Ojective: To evaluate anti-HIV activities in vitro cell culture of natural products used in Korean and Chinese traditional medicines. Methods: I IIV infected MT-4 cells were cultured with methanol or boiling water extract of each natural product used in traditional medicine and then its anti-HIV activity was screened by MTT assay. Confirmatory assay was performed by IFA, p24 antigen detection, and MTT assay using various cell lines such as MT-2, MT-4, or 119 cells. Methanol extracts showing anti-HIV activity were fractionated and evaluated. Results: Among the screened 92 methanol and 116 boiling water extracts from 202 species, 19 species showed anti-IV activity without cytotoxicity though the inhibitory effects of the species varied depending on cell lines and evaluation methods used. As representative species, EDso of water fractions of KN046 and KN049 were 34.8 ug/ml and 170.6 ug/ml respectively. Discussion and Conclusions: Although some species showed high cytotoxicity (SI:5-10), this syudy suggests that the traditional medicines could be useful candidates for drug development LEE, JOO-SHIT, Center for AIDS Research, NITH 5 Nokbun-Dong, Eunpyung-Gu, Seoul 122-020, Korea TEL(82)-02-380-1695 FAX(82)-02-382-4892 PA0320 ENCYCLAN DERIVATIVES AS ANTI-HIV DRUGS. Pokrovsky A. Plyasunova O.,Fedyuk N.,Kiselev O.,*Ponizovsky M.,*Charushin V.,*Chupahin O. Inst.Molec.Biol.,*Ural Politech.Inst.,Russia Objective: D)esing of anti-HIV agentsbased on amphypatic compound - encyclane woe performed. Anti-HIV activity of derivatives bee rig various hydrophobic and hydrophyl i c groups has beetn i nves tiga ted. Methods: Anti-HIV activity was examined by measuring of cell s viability, reverse transcriptase activity, p24 viral antigen by emmunroenzyme and flow cytofluorometry methods on HIV chronically and acute infected cells. Results: It was found the number of encyclan derivatives wi th different anti-HIV effects and cytotoxicity. The values of ID50 of active derivatives was determinated in the range from 0,005 t.o 2 pM. Some of these compounds provide the survive of HIV--infected cells and other are effective in the killing of HIV-infected cells. It was determirated additionally the index of drug effectivity as the ratio of the cytotoxicity for noninfected cells to the cytotoxicity for HIV-- infected cells. Discussion and Conclusion: It is supported the approach for screening of high effective anti-HIV drugs based on the simultaneously determination of the cells viability and inhibition of viral antigene expression. Pokrovsky A.G. Institute Molecular Biology. loltsovo, Novosibirsk Region. 633159, Russia Phone:8 3832-64-70- 21, FAX: 8-3832-32-88-31 0 C_) (0 J, 0 CA) PA0321 Inhibition of HIV Activity in vitro and in vivo by MDI-P. Robert E. Morrow, M.D., Medical Discoveries, Inc., and William n. Wlch Ph.D., WMCL. Objective: The objective of this report is to demonstrate the in vitro and in vivo noncytotoxic anti-HIV activity of MDI-P. Methods: MDI-P is comprised of HOCl", OC', Cl1, and 03 generated by the use of a sterile, patented electrolysis chamber utilizing inert nobel metal electrodes. Free Cl, OCl1 /HOC1, concentrations of MDI-P were quantitated using ion specific chlorine electrodes. 03 was quantitated using the indigo trisulfonate/malonate acid method. The chronically HIV infected HB2cell line with p24 antigen measurement was used to assess in vitro anti-HIV activity of MDI-P. Balb/c mice infected with a Friend leukemia virus and measured by Mean Plasma Virus Titer (MPV). Results: MDI-P chlorine concentrations of 5ppm and O3levels of 40ug/ml resulted in a 5 log reduction in HIV activity (i.e. no p24 antigen dected in concentrations of HB2cells from 107 to 102)after a one minute incubation with MDI-P (P<0.001 as compared to saline controls). Lower concentrations of chlorine, 0.Sppm and 0.05ppm (O3levels of 0.4 and O.04ug/ml) showed a three and two fold reductions in HIV activity respectively (P<0.001). Less than 5% cell death (via trypan blue) was observed at these concentrations and incubation periods with MDI-P. MPV in the Balb/c mice model following a five day treatment of MDI-P (5ppm chlorine and O3levels of 40ug/ml) showed a 20% decrease vs controls. Discussion and Conclusions: Previous studies have documented significant microbicidal activity of Cl', OHC1', OC1l,and 03 solutions up to 410 times greater than solutions containing chlorine compounds only ( Sci. Tot. Enviroment, 63:191, 1987) and anti-HIV activity by 03 alone (AntiViral Res., 16:281,1991). The present results suggest that MDI-P has significant anti-HIV activity without adverse cytotoxicity. William D. Welch, Ph.D., WMCL, INC., 6010 Sadring Avenue, Woodland Hills, CA 91367 USA, (818) 348-4866, Telefax (818) 348-7658. PA0322 CONFORMATIONAL STUDIES ON ANTI-AIDS DRUGS AND AGENTS Saran Arnil, T.I.F.R., Bombay, India Acquired Immuno Deficiency Syndrome (AIDS) is a fatal and infectious disease which is caused by a retrovirus known as human immuno deficiency virus (HIV). The rapid spread of AIDS has caused world wide concern and efforts are directed towards finding an effective drug against HIV. Azidotymidine (AZT) has been, so far, the most effective drug for clinical use in AIDS patients. Recently, 2',3'-dideoxynucleosides, namely ddl, ddA, ddG ddC and ddT...etc have also been shown to be active against HIV. Conformational studies on AZT and the above mentioned dideoxynucleosides have been carried out by using quantum-mechanical PCILO method. This presentation will review the results and their biological significance highlighting an excellent correlation which has been obtained between conformation and the biological activity of these anti-AIDS drugs and agents. SARAN, Anil, Chemical Physics Group, Tata Institute of Fundamental Research, Bombay 400 005, India Telephone (091) 22-2152971; Telefax (091)22-215210

Page  111 PA0323 HIV-1 REVERSE TRANSCRIPTASE (RT) BINDING AND BOUND RT ASSAYS WITHOUT USING RADIOISOTOPE. Sano, Kouichi, Nakamura, T., Nakano, T., Odawara, F.', Morimatsu, S., Misaki, H.', and Nakai, M., Department of Microbiology, Osaka Medical College. Takatsuki-shi, Osaka, Japan. ' Diagnostic Division, Asahi Chemical Industry Co., Tagata-gun, Shizuoka, Japan. Objective: We developed RT binding and bound RT assays for the analysis of template-primer binding site of RT and HIV-1 RT inhibitor. Methods: Non-radioisotopic RT assay using template-primer immobilized microplate (Nakano, et al. 1994) was modified and examined the influence of non-organic salt solutions and solution varied pH. For RT binding assay, RT was mixed with a solution and allowed to bind to template prior to washing. RT was bound to template prior to washing, and a solution was reacted to the RT for bound RT assay. After these reaction, the RT activity in the microplate was non-radioisotopically measured. Results: The binding was template specific and the activity of RT bound on the plate was quantitatively measurerable. High concentration of nonorganic salt solutions and pH were influenced to the assays. Antibody from HIV-l-infected men inhibited RT binding and bound RT in both asaays. Conclusion: We develolped RT binding and bound RT assay. We detected antibodies that inhibited RT binding and polymerization in sera from HIV1-infected men using the assays under the comparison with control sera. These non-RI assays will useful in the studies of epitope mapping of RT and of RT inhibitor. SANO, Kouichi, Department of Microbiology, Osaka Medical College, Takatsuki-shi, Osaka, 569 Japan Telephone (81)-726-83-1221; Telefax (81)-726-84-6517 PA0324 VIRAL CHARACTERIZATION AND COMPARISON OF WILDTYPE HIV-1 WITH RT INHIBITOR-RESISTANT MUTANTS. Katsushi Ilichi', Masatoshi Fujiwara*, Kenji Konno*, Shiro Shigeta**, Tomoyuki Yokota* and Masanori Baba**, Rational Drug Design Laboratories', Department of Bacteriology, Fukushima Medical College", Fukushima, Japan. Several nucleoside (e.g., AZT, ddl and ddC etc.) and nonnucleosid RT inhibitors (e.g., TIBO, nevirapine and HEPT etc.) markedly suppress replication of HIV-1. However, the drug sensitivity of HIV-1 changes during a long-term treatment with these compounds. We characterized drug-resistant mutants and wild-type virus and compared them. Mutant viruses from HIV/IIIB strain were obtained by treatment with AZT or HEPT derivative E-EBU. Activity of reverse transcriptase (RT) extracted from AZT-resistant HIV/IIIB showed slightly higher than that of wild-type RT. While the RT activity of E-EBUresistant HIV/IIIB was approximately half that of wild-type virus. The intensity of cytopathogenicity among these viruses in MT-4 cells was as follows: AZT-resistant mutants > wildtype > E-EBU-resistant mutants. These results suggest that the property of drug-resistant virus is different from that of wild-type. IJICHI, Katsushi, Rational Drug Design Laboratories, Fukushima, Japan Telephone (81)-245-67-3593; Telefax (81)-245-67-5554 PA0325 N VITRO ISOLATION AND CHARACTERIZATION OF HIV-1 MUTANTS RESISTANT TO BOTH AZT AND MKC-442. Yuasa. Satoshi*; Sadakata, Y.*; Seki, M.*; Hashimoto, K.**; Baba, M.**, *Mitsubishi Kasei Corporation, Yokohama, Japan; **Fukushima Medical College, Fukushima, Japan. Objective: MKC-442 (6-benzyl-1-ethoxymethyl-5-isopropyluracil) is a highly potent and specific inhibitor of HIV-1 reverse transcriptase (RT). To explore the possibility of combination chemotherapy with AZT and MKC-442, we isolated HIV-1 mutants that were co-resistant to these compounds. Methods: The mutants were isolated and cloned from A018C (a clinically isolated AZT-resistant strain of HIV-1) following serial passages of the virus in cell culture in the presence of increasing concentrations of MKC-442. Drug-susceptibility of viruses to various RT inhibitors was determined by the inhibition of viral antigen expression in MT-4 cells. Nucleotide sequence analysis of their RT genes was also carried out. Results: The mutants were about 200 fold less susceptible to MKC-442. Although they still retained resistance to AZT, their susceptibilities to AZT were several fold higher than that of A018C. The mutants and A018C were equally susceptible to DDI. Mutations at codons 74 (Leu to Ile), 103 (Lys to Glu), and 108 (Val to Ile) were identified in the RT of the mutants when compared to the A018C RT. Discussion and Conclusions: HIV-1 mutants co-resistant to AZT and MKC-442 could be established from an AZT-resistant strain. However, it seems to be more difficult to isolate such mutants from AZT-susceptible strains by simultaneous treatment with AZT and MKC-442. YUASA, Satoshi, Mitsubishi Kasei Corp., Yokohama, Japan. Telephone (81)-45-963-3397; Telefax (81)-45-963-3890 PA0326 A SIMPLE EIA FOR ASSAYING HIV PROTEASE ACTIVITY Syu. Wan-Jr Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, R.O.C. Objective: An economic and convenient method for measuring viral enzyme activity should accelerate the discovery of novel anti-HIV reagents. We aimed at developing an enzyme immunoassay for simultaneous monitoring of many HIV protease reactions. For simplicity and cost-effective reasons, uses of crude preparations of enzyme, substrate, and antibodies were the prior consideration. Methods: we took advantages of that the protease can be actively produced in E. coli and that the protease substrate, gag precursors, can be similarly generated. The substrate was single-biotin labeled so that it can be directly absorbed onto avidin-coated plates. The protease activity was then reflected by the decrease of anti-p24 MAb retained on plates. Results and Conclusion: The protease activity measured was specific and inhibited by a known inhibitor. Since the interactions between avidin and biotin, HIV protease and substrate, and MAb and antigen all have unique specificity, crude preparations of recombinant proteins and ascites fluid can be directly used without a single purification step. Adaptation of our method for monitoring a large number of HIV protease reactions in the presence of different compounds should be feasible. SYU, Wan-Jr, Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, R.O.C. Telephone (886)-2-8267112; Telefax (886)-2-8212880 -o O ) 0 WA 0)

Page  112 PA0327 BIOLOGICAL PROFILE OF ANALOGS OF THE HIV-1 PROTEASE INHIBITOR CGP 53 437 Schneider Peter, Alteri E., Bold G., Capraro H.-G., Faessler A., Lang M., Mestan J. and Roesel J., Research Dpt., Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland Objective: Screening of renin inhibitors led to the identification of CGP 53 437 as a potent inhibitor of HIV-1 protease. Our objective was to investigate structure-activity relationships in a series of close analogs of the leading compound in order to improve potency and specificity of inhibition. Methods: The novel compounds were prepared through the synthetic scheme which was presented at the IXth International Conference on AIDS in Berlin. The IC50 against HIV-l proteinase was determined at pH 6.0 in a peptide based cleavage assay as described by A. Faessler et al. [Bioorg. Med. Chem. Letters 1993, 3(12), 2837-42]. The cleavage was monitored by HPLC. The ED9o was determined in acutely HIV-1IAv infected MT-2 cells as described by E. Alteri et al. [Antimicrob. Agents Chemother. 1993, 37(10), 2087-921. The IC50's against renin and cathepsin D were determined as described by J. Wood et al. [Hypertension 1985, 7(5), 797-803 and J. Cardivasc. Pharmacol. 1989, 14,221-6]. Results: Various structural modifications in either the dipeptide isostere moiety or the C-terminal dipeptide sidechain afforded very potent derivatives. From this series, CGP 57 244, the bulky P'l benzyloxy substituted analog of CGP 53 437, emerged as the most potent (IC50: 3.4 nM; ED90: 100 nM) and specific inhibitor of HIV-1 proteinase. Discussion and Conclusions: Extension of the lipophilic substituent in P'1 towards P'3 offers the possibility to improve in vitro potehcy and selectivity. SCHNEIDER, Peter, Research Dpt., Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland Telephone (41)-61-6963298; Telefax (41)-61-6967826 PA0328 Objective: INHIBITORY EFFECTS OF TROPICAL PLANTS ON HIV-PROTEASE Miyashiro, H., Lim, J. A., Nakabayashi, T., Miyaji, M., Hattori, M., and Shimotohno, K.* Toyama Med. Pharm. Univ. and Nat. Cancer Cent. Res. Inst.* JAPAN For the purpose of developing new anti-HIV agents from natural sources, various tropical plant extracts were screened for inhibitory effects on HIV-1 protease. Methods: Various plants and plant materials collected in Sri Lanka and Panama were extracted with water and MeOH to give the respective extracts. Recombinant HIV-l protease activity was measured by cleavage of a synthetic substrate. Results and Discussion: The extracts of Areca catechu (seeds), Eugenia jambolana (bark), Saraca indica (bark), Terminalia arjuna (stem bark), Waltheria indica (branch), Erythroxylum citrifolium (trunk), Aegiphila anomala (leaves), Cordia spinescens (leave), Hyptis lantanifolia (aerial part) etc. showed an appreciable inhibition against HIV-1 protease. From the A. catechu extract, some procyanidins were isolated and arecatannin B I showed a significant HIV-PR inhibitory effect. Since some of these extracts also showed anti-HIV-1 activity, searching for HIV-protease inhibitors in tropical plants is worthy to be considered where effective anti-HIV drugs are really demanded. MIYASHIRO, Hirotsugu, Res. Inst. for Wakan-Yaku, Toyama Med. Pharm. Univ., 2630 Sugitani, Toyama, Japan Telephone (0764)-34-2281; Telefax (0764)-34-4656 0 CA) 0 CA) CA) 0 A0329 INHIBITION OF HIV-1 BY ANTISENSE DNA OLIGONUCLEOTIDE- M.S.Bochkova**, A.O.Zhigulin*, M.S.Petrova*, L.B.Kalnina*, V.V.Pokrovsky** D.N.Nossik*. *D.I.Ivanovsky Institute of Virology,Moscow;**Institute of Epidemiology, Moscow. The idea of selective inhibition of express of particular HIV genes is one of the most potential approach in antiviral therapy. We used antisense DNA complementary to the initial region of tat HIV-1 gene with lengh 30 nucleotides. For higly efficient transfection into mammalian cells grown in suspension antisense DNA oligonucleotide was entrapped in liposomes prepared by phosphotatidyl serine calcium-induced fusion method. The model of CEl cells infected b syncitium-inducing russian isolates HIV-1 IV3 arid HIV-1 IV17 were used. The replication of HIV was monitored by assaying the tissue-culture supernatant for HIV antigen. The results showed theinhibition of HIV reproduction in the first 20 h after the introduction of ant isense DNA oligonucleotide. There was no substanstial difference in level of inhibition between 2 tested isolates. The effect on panel of wild HIV-1 isolates with different biological characteristics is under the investigation. Marina Bochkova. 105275, Moscow, 8-Sokolinaya St., 15-2, Russia. TEL: 365-30-09. FAX: (095) 365-48-80 PA0330 MECHAN ISMS OF MODIFIED ANTISENSE OLIGONUCLEOTIDES, Hatta,T., Takaki,K., Nakashima,N., Yamamoto,N.', and Takaku H., Chiba Institute of Technology and 'Tokyo Medical and Dental University of School of Medicine Objective: Antisense oligomers have been targeted to RNAs but up to now biological studies have been mainly performed with oligomers complementary to either pre-mRNA or mRNA. In most of cases the DNA/mRNA hybrid prevented translation of the massage,. The inhibition of reverse transcriptase (RT) by antisense DNA can also be envisaged. This can be achieved in 3 different ways: interaction with enzyme, competition with primer, or arrested with elongation of the cDNA chain. We investigated the latter possibility. Methods: For studies performed with the rabbit It-globin mRNA, the oligomer complementary to region 113-129 of the message was used as the primer Modified olgomers, targeted to nucleotides were used as stopper. For primer extension reactions, mRNA, primer and stopper were incubated with RT and the reaction mixture was loaded on a 10% PAGE. Results: Reverse transcription of mRNA by AMV-RT or HIV-RT was primed with unmodified and modified DNA, giving rise to expected cDNA fragment. However, modified DNA (phosphorothioate) inhibited polymerization by binding to the AMV-RT rather than to the template RNA, whereas there are no competitive binding of the modified DNA on the HIV-RT during reverse transcription. Discussion and conclusions: The antisense phosphorothioate DNA participates through a mechanisms of interaction different from those of RT enzymes, in adherence and structural differences. TAKAKU,Hiroshi, Chiba Inatitute of Technology, Chiba, Japan Telephone (81)-474-78-0407; Telefax (81) -474-71-8764

Page  113 PA0331 ADDITIONAL ANTI-HIV ACTIVITY OF ANTISENSE WITH INTERFERONS, Matsukura Makoto*, Zon G **, *Kumamoto Univ. Med. School,**Lynx Therapeutics Objectives: Antisense phosphorothioate oligodeoxynucleotide against rev (S-anti-rev) has been shown to inhibit HIV expression. Interferons (INFs) (a,3,y) are also known to regulate HIV expression through many mecha-nisms including interaction with RRE(re v responsive element). Methods: Chronically HIV-infected T-cell line, H9 cells (H9/HIV), and promonocyte cell line, U1 cells, were cultured for 5 days in 96 -well culture plate with or without the compounds. Culture supernatants were collected and tested for p24 gag by ELISA. Results: In H9/HIV, S-anti-rev and INFs exhibited significant antiHIV activity and the combination of S-anti-rev and INFs resulted in additional anti-HIV activity without addition of their toxicity. In U1 cells, interferon y augumented the viral production, which could be inhibited by S-anti-rev. Discussion and Conclusion: The combination of S-anti-rev and INFs could be a potent remedy for AIDS and related disorders. MATSUKURA, Makoto, Kumamoto Univ. Med. School, Kumamoto, Japan. Telephone (81)-96-344 -2111; Telefax (81)-96-363-5125 PA0333 I'EPIDE T ANTAGONISES TIlE POTENTIATION OF HIV-1 REPLICATION BY TNFa Esther Race, Stein, C.A., Chargelegue, D., Oxford. J.S. and *Aston, R. Retroscreen Ltd., Lond Hosp Med Coll, London, UK. *Peptech UK. Objective: To investigate the mode of action of peptide T. Methods: TNF-a was obtained from Peptech (UK) as a 500 tg/ml solution with a specific activity of 3.2 x 107 units/mg. The laboratory strain HIV-lRF was quantified on C8166 cells, cultivated in RPMI 1640 with sodium bicarbonate plus 1 mM L-Glutamine, 50 u/ml Penicillin, 50 tg/ l Streptomycin, 25 mM Hepes buffer and 10% FCS. Peripheral blood mononuclear cells (PBMC) were isolated from blood packs by density gradient centrifuigation (Ficoll Paque, Pharmacia). PHA stimulated cells were maintained 10 u/ml of 112. Results and conclusions: The virus assay system was optimised for using varying multiplicities of infection to allow a sensitive detection of a potentiation of HIV-1 replication by TNF alpha. In the absence of Peptide T, TNF-a, at concentrations of 10 IU/ml and above stimulated the production of HIV-1 in culture after 6 days. The addition of Peptide T to the assay systems significantly reduced this effect even at low concentrations (0.01 mM). At concentrations of I mM and above Peptide T completely inhibited the enhancement of viral p24 levels by the TNF-a. Peptide T also showed some inhibitory activity against HIV-1 in the absence of TNF-ax with p24 levels in the culture containing 0.01 mM and above of Peptide T, being less than half of p24 in the control. Esther Race, Retroscreen Ltd, 64 Tumrner St, London El1 2AD, UK. Tel ++44-71-375 03 45, Fax ++44-71-375 25 97. PA0332 Multi-branched peptlde constructs (MBPC) of the V3 loop of gp120 Inhibit human Immunodeflciency virus Infection. Abdelaziz Benjouad', E. Fenouillet1, F.Chapuis1, J.C. Gluckman1, J.M.Sabatier2. (1) CNRS URA 1463, H6pital Piti6-Salp6tribre, Paris; (2) CNRS URA 1455, Facult6 de M6decine Nord, Marseille; France. Objective: The V3 loop of HIV-1 Env is assumed to be involved in HIV-1 mediated membrane fusion. V3 -derived monomeric peptides may either enhance (DeRossi et al. Virology 1991,184:187) or impair (Nehete et al. J. Virol. 1993,67:6841) HIV-1 infection. Based on these observations, MBPCs were designed and tested for their ability to interfere with HIV infection. Methods: MBPCs were derived from the V3 North American/European consensus sequence: MBPC-I comprises 8 GPGRAF branched copies, and MBPC-II 4 RKSIHIGPGRAFYT copies. The MBPCs were tested for their ability to alter HIV-1- or HIV-2-induced syncytia and to inhibit virus infection. Results: In contrast with their monomeric counterparts and with MBPCs based on HTLV-II Gag and calf thymus histone, MBPC-I and MBPC-II inhibited both HIV-1- and HIV-2-mediated syncytia at concentrations 510-5 M, which did not alter cell viability nor T lymphocyte allogeneic, antigen- or mitogeninduced reactivities. These MBPCs also impaired HIV-1 infection. Finally, V3 MBPCs bound to CD4+ cells, and the binding was specifically inhibited by sCD4 or by a mAb recognizing theCDR3 region of CD4 D! domain. Conclusion: MBPCs, which bind to CDR3 of CD4, may then be used to further delineate the role of V3 in HIV entry mechanisms into cells. A. Benjouad: CNRS URA 1463, 83Bd de I'H6pital, H6pital Piti6-Salpetribre, 75651 Paris cedex 13; France.Tel: 33 1 4217 74 50; Fax: 33 1 42 17 74 41 PA0334 NEW HIV-1 RT INHIBITORS BELONGING TO THE CLASS OF TIBO/NEVIRAPINE COMPOUNDS. D.Bellarosa'*,G.Antonelli**,F.Bambacioni',O.Turriziani',E.Riva',D.Giannotti*,A.Giachetti*,F.Dianzani'.'Institute of Virology, University La Sapienza,Rome. *Menarini Ricerche Sud, Department of Pharmacology,Pomezia.**Department of Biomedicine, University of Pisa, Pisa. Here we describe a new group of TIBO/Nevirapine-like compounds, the most active of which are the 6,11 -diidro-dipirido-benzotiodiazepin-5,5-dioxide (MEN 10690) and the 6,7-diidro-dipirido-furo-diazepin-6-one and thione derivative, respectively called MEN 10880 and MEN 10979. In C8166 cells infected with HTLV-IIIB they show a 50% inhibitory concentration (IC50) of 50 nM for MEN 10690, [ with a selectivity index (SI) of 3300], of 6 nM for MEN 10880 (SI>10000), of 2,5 nM for MEN 10979 (SI=15000). Interestingly, as reported for TIBO derivatives, they are inactive against HIV-2 (ROD strain) or SIV (MAC strain). An antiviral activity has been documented also in PBL infected with HTLV-IIIB, MEN 10690 showing an IC50 of 30 nM, (SI of 300), while MEN 10880 and MEN 10979 have an IC50 of 40 nM (SI>1500) and 10 nM (SI>6000), respectively.In order to to identify the drug sensitive phase in HIV-1 replicative cycle, experiments were performed comparing MEN products to other RT inhibitors in terms of antiviral activity at different times of drug addition. The results showed that MEN 10690, MEN 10880, and MEN 10979 lost their activity when added 6-10 hours post infection. The same results were obtained using TIBO R82913, while AZT and ddl lost their activity in a time frame of 3-5 hours. Furthermore these molecules inhibit recombinant HIV-1 RT in a way that is template-primer dependent; as in the case of TIBO derivatives, the inhibition is more effective (5-7 -fold higher) with Poly(rC)-oligo(dG) than with Poly(rA)-oligo(dT) template/primer. Preliminary data also indicate that these MEN compounds have a favorable activity against HIV-1 strains resistant to AZT and ddl. In conclusion, our data indicate that the newly sinthetized MEN 10690, MEN 10880 and MEN 10979 are new members of the TIBO/Nevirapine class of non-nucleoside inhibitors and probably they interact with HIV-1 RT at the same binding site of TIBO compounds. BELLAROSA DANIELA,Institute of Virology, V. Porta Tiburtina 28, 00185 Rome Italy. Tel.:39-6-4452846;Fax:39-6-4469024. "O 0 CA) -h) 0 wA wA

Page  114 PA0335 NEW ANTI-HIV DRUG WHICH TARGETS THE OLIGOSACCHARIDE CHAINS OF HIV ENVELOPE GLYCOPROTEIN Mizuochi Tsuguo*, Nakata, M.*, Oki, T.**, Murakami, T.***, Nakashima, H.***, Yamamoto, N.*** *Tokai University, Hiratsuka, Kanagawa, **Tamagawa Universit, Tokyo, ***Tokyo Medical and Dental University, Tokyo, Japan Objective: An extensive part of virion surface of HIV-1 is predicted to be covered with oligosaccharide chains of gpl20, especially high mannose type oligosaccharides. The aim of this study was to investigate whether or not some antifungal agents inhibited HIV infection since the surface of fungi is also covered with a lot of mannose residues. Methods: Antifungal agent pradimicin A and its derivatives were tested for ability to block HIV infection in the presence and absence of glycoconjugates which contain the oligosaccharide moiety of gpl20 and for direct binding to oligosaccharide chains of gpl20. Results: Pradimicin A and the derivative BMY-28864 blocked syncytium formation and the activity was inhibited by high mannose type oligosaccharides and mannose-rich glycoconjugates. BMY-28864 directly bound to gpl20, mannose-BSA, and neoglycolipids containing high mannose type oligosaccharides of gpl20, but not to natural mammalian glycoproteins. The binding was Ca2+-dependent and inhibited by mannose. Conclusion: BMY-28864 is a unique carbohydrate-binding antibiotic which has never been reported. It is possible to block HIV-1 infection by targetting oligosaccharide chains of the envelope glycoprotein. MIZUOCHI, Tsuguo, School of Engineering, Tokai University, Hiratsuka, Kanagawa, Japan Telephone: 0463-58-1211 Ext.4193; Telefax: 0463-58-1812 PA0336 ANTI-HIV EFFECTS OF REDOX REAGENTS ON HIV-1 INFECTED CELL LINES SHOJI Shozo, Furuishi K., Misumi S., Miyazaki T., Kino M., Yamataka K., Matsuoka H., and Tachibana K., Dept. of Biochem., Fac. of Pharm. Sci., Kumamoto Univ., Kumamoto, Japan Objective: We previously reported that the allyl disulfide compounds have selective cytotoxicity against the HIV1 infected laboratory cell lines. The purpose of the present study is to explore a potential anti-HIV reagent from redox compounds and redox-coupled medicines. Methods: HIV-1 producing cell lines (CEM/LAV-1, U937/IIIB, U937/RF, H9IMN, Molt-4/IIIB) and human T cell lines were maintained at 37 ~C in RPMI-1640 medium (10 % FCS) in 5 % C02. The efficacy of the compounds was primarily determined by anti-Tat assay with the reporter plasmid (pBC12/HIV/SeAP) and the activator plasmid (pBCl2/HIV/TAT), and the cytotoxicity was subsequently examined by tryphan blue dye exclusion. Finally, the viral infectivity (TCID50/ml) was estimated, and furthermore, the quantity of HIV-p24 was mesured by western immunoblotting analyses. Results and Discussion: Of the various mercapto- and disulfide compounds tested, N-acetylcysteine, a-lipoic acid, and thiamine disulfide significantly depressed both HIV-1 Tat activity and HIV-l infectivity. These compounds are active in acute and chronic infection. On the other band, the diallyl disulfide resulted in remarkable promotion of the cytopathic effect induced by HIV-1 virus, but it did not inhibit HIV-Tat activity. The mechanism of anti-HIV activity with the redox-reagent has been unknown, it may be responsible for HIV-1 replication in both cellular and viral redox regulation. SHOJI, Shozo, Dept. of Biochem., Fac. of Pharm., Kumamoto Univ., Kumamoto, Japan Telephone 096-344-2111; Telefax 096-362-7800 0 W CA) 0 PA0337 EFFECTS OF POLYCATIONS ON INFECTION WITH HUMAN RETROVIRUSES. Yuji Haraguchi, Dawei Yang, Nobuaki Shimizu, Yasuhiro Takeuchi and Hiroo Hoshino. Department of Hygiene and Virology, Gunma University School of Medicine, Showa-machi, Maebashi, Gunma 371, Japan. QOblectives: Polycations (DEAE-dexlran and polybrene) have been reported to enhance the plating efficiencies of many retroviruses, while the plaling of avian sarcoma virus subgroup A is not enhanced by polycations and infection of CD4-positive HeLa cells with HIV is enhanced or inhibited by them. We examined effects of polycations on infection with human retroviruses in vitro. Materials and methods: Effects of polycations on infection of HTLV-I and HTLV-II were examined by syncytium formation assay and the plaque assay using vesicular stomatitis virus pseudotypes. As for HTLV-I, infection was also assayed using PCR. Effects on infection with HIV-I was examined by indirect immunofluorescence assay. Amphotropic murine leukemia virus (MLV-A) and feline endogenious RD114 virus were used as the control. The human glioma cell line U-251 MG was used as the indicator cells for infection with HTLV-I, HTLV-II, MLV-A and RD114 virus. The human T cell line MT-4 and CD4-positive, brain-derived fibrobrast-like BT-3 cells and CD4-positive, human glioma cell line U87/CD4 were used as the indicator cells for HIVI infection. Results: Syncytium formation Induced by HTLV-producing cells was weakly enhanced by polycations. However, polycations inhibited the plating of VSV(HTLV-l) pseudotype and cellfree HTLV-I infection. On the contrary, polycations weakly enhanced the plating of VSV(HTLVII) pseudotype. Infection of MT-4 cells and the brain cells with HIV-I was slightly inhibited by polycations. Conclusions: Although polycations have been used to potentiate retrovirus infection, our results indicated that polycations had no marked effects on infection with human retroviruses and that infection with HTLV-I and HIV-1 was rather inhibited by polycations. Hiroo Hoshino; Department of Hygiene and Virology, Gunma University School of Medicine, Showa-machi, Maebashi, Gunma 371, Japan. Tel: (81)-272-31-7221 Extension 2590, Fax: (81)-272-32-8312 PA0338 THERAPEUTIC POTENTIAL OF FK 506 IN PATIENTS WITH HIV DISEASE Karpas Abraham, University of Cambridge, UK. Objective: To determine whether FK 506 is likely to benefit patients with HIV disease. Methods: We have compared the effects of FK 506 with its structural analogue, Rapamycin, on the replication of HIV-1 infected and non-infected T-cells. Results: FK 506 was found to selectively inhibit the growth of HIV infected cells at concentrations which did not inhibit the growth of uninfected cells. In contrast, Rapamycin inhibited the growth of HIV infected and uninfected T-cells to the same degree. FK 506 was also found to reduce cell-to-cell transmission of HIV and reduce viral production by infected cells. Discussion and Conclusion: Unlike Rapamycin, FK 506 was shown to be a selective inhibitor of HIV infected T-cells as well as reduce viral production and cell to cell transmission. It is, therefore, likely to suppress HIV induce immune activation in viva FK 506 also blocks TNF production (Goldfeld et al, PNAS 89: 12198, 1992) which is highly elevated in patients with HIV disease. Since TNF amplifies the viral cytopathic effect and induces cachexia which leads to emaciation, FK 506 is likely to delay the progression to AIDS through these two independent pathways. Since FK 506, but not Rapamycin, modulates Calcineurin activity and prevents formation of NF-AT (nuclear factor of activated T-cells), which are cellular rather than viral targets, HIV is less likely to develop resistant mutants. KARPAS, Abraham, University of Cambridge Dept Haematology, MRC Centre, CAMBRIDGE, UK. Telephone: 44) 223 336823; Telefax (44) 223 214788

Page  115 PA0339 HIV-1 RESISTANT TO PROTEASE INHIBITORS IN VITRO DUE TO ONE POINT MUTATION IN POL GENE. EL-FARRASH Mohamed A. Kuroda, M.J., Harada, S., Kumamoto University School of Medicine Kumamoto Japan Objective: To study the possibility of emergence of HIV-I strains resistant to protease inhibitors and explore the mechanism of resistance. Methods: A. An HIV-1 clone was maintained in gradually increasing doses of a protease inhibitor in vitro. The resulting resistant mutant was characterized in terms of: 1) sensitivity to the agent; 2) infectivity and cytopathogenicity; 3) genetic changes in gag and pol genes. B. An HIV-1 infectious DNA clone with the detected point mutation in pol gene was constructed and also characterized. Results: 1) The resistant mutant had one point mutation in pol gene resulting in one amino acid change in the protease enzyme. 2) The DNA infectious clone having this point mutation was also resistant to the protease inhibitor. 3) This point mutation resulted in a significant loss of the virus infectivity and cytopathogenicity. Conclusion: 1) HIV-l will be able to escape the effect of protease inhibitors by just one point mutation, so combined therapy must be used in vivo. 2) There is a relation between HIV-1 protease structure and its acute infectivity. EL-FARRASH, Mohamed A., Kumamoto University School of Medicine, Kumamoto, Japan Telephone (81)-96-344-2111 Telfax (81)-96-363-9686 PA0340 TODOXIN- HIV INHIBITORY AND IMMUNOSTIMULATING EFFICACY l*oyanol c-Todor~, JondaI-M", Cragg-G"* (*) Todoxin Study Group, Austria (*) Dept. of Immunology, Karolinska Institute, Sweden ("') National Institute of Health, NCI, Maryland, USA Objective: Todoxin Isa novel antiviral and knmunostimulating preparation based upon an ion carrier molecule with specific binding affinities and a carrier specific substrate formulation. Todoxin ' s formulation is unique in three aspects: 1. It comprises a very broad spectrum of physiologicaly active, cs-oriented and water soluable carbohydrates, carboxylic acids, vtamn fat soludable vitamrrins, coenzymes and enzymes such as Amylases, Phosphatases and Catalases 2. It contains both agonic and antagonic agents such as Ca and Fe in physiologicaly relevant concentrations. 3. It contains a specntrum of minerals in permanent solution. Induction of desired cell physiologic effects in vitro such as increase of celular exchange dffusion, uptake of substrates, metaboltes, oxydative phosphorylation, mitogen response of RET cels, have previously been described by author. We report here (a)Todoxh'seflrcacy to hmaseNaal Kier( NQcellachyInviro (b) Todon'sefficacytoitlt Hlbkatrectinhin viro. Methods: (a) Peripheral blood lymphocytes were treated with Todoxin for 17 h and tested for king of B cel lymphoma cell line DAUDI in a 3 h Cr51-release assay. Treatments were done in medium or L-2 (10 U/ml). The lghest Todoxin concentration was a final lution of 1:10 with 4 consecutive dilution steps 1:10. (b) HIV infected CEM-IW eels were incubated 7 days with fraction of Todoxin. Concurrent studies were performed on diffenrent sample concentrations. The Iwest sample concentration was 6.0 x 10-1 ug/ml with increasing steps of Log1a Results: (a) Todoxin increases NK kiling to the same levels as IL-2 does. (b) Todoxin prevents HIV infection in vitro 100% as per uninfected untreated control culture. s'o Results (a) ro 0 -,) Condusion. The desrted rests may hn Too 2 spat e lie pontUhrp fecsOfTos75 H donseenhvial deeasehckudslgsow s WOF F - Todoxin 25 infeciornreeMus and vieassociatedcard25H1 IognrI]s UI.1 o 1 2 3 4 (see also Track B29/B31 and 017/D18) 1 2 3 4 5 Cat 1 2 3 4 5 Codt LogsoofTodoxinConcentration(ug/m( JOVANOVIC MD PhD, Todoxin Study Group, Coordinating Office, 8463 Eichberg 15 Austria TEL 0043 3454 226-0 FAX 0043 3454 6314 PAO341 ESNARINONEA QUINOLINONE DERIVATIVE, INHIBITS THE CYTOKINE SECRETIONS BY HUMAN PBMCs. Koutoku Aihara*, H.Momota',', H.Okada', J.Thkamatsu, S.Akamatsu', S.Sogo',Y.Shindoh', Y.Ohmoto', T.Nishida', S.Nakai*, I.Kitajima5, I.Maruyamas, M.Adachi* ('Otsuka Pharm.Co.,Ltd.,Tbkushima,Japan, "Nagoya City Univ.,Nagoya, Japan.,'Nagoya Univ., Nagoya, Japan, $Kagoshima Univ.,Kagoshima, Japan) Objectivei: Vesnarinone, a synthetic oral positive inotropic agent that has been used for treatment of patients with congestive heart failure, was found to inhibit replication of HIV-1 in vitro. To investigate the mechanism of action of vesnarinone, we studied the effects of vernarinone on cytokine production by stimulated peripheral blood mononucleated cells (PBMCs) from patiean's infected with HIV-I and healthy volunteers. Methods: Blood was obtained from healthy volunteers and a total 21 HIV-1 sero-positive patients. PBMCs were separated and stimulated with mitogens or anti-CD3 antibody (UCH-TI). Cell culture supernatants were assayed for the amount of cytokines by using an ELISA kit. Results: Vesnarinone inhibited the production of TNF- a and IL6 by stimulated PBMCs from healthy volunteers in a dose-dependent manner Transcriptional factor NF- B is known to enhance HIV gene expression. Vesnarinone treatment of LPS-stimulated PBMCs from healthy individual reduced NF- K B binding activity of nuclear extracts by an electrophoretic mobility shift assay. So, we investigated the effect of vesnarinone on cytokine secretion of PBMCs from patients infected with HIV-1. As a result, vesnarinone inhibited TNF- a production by stimulated PBMCs from 8 of 9 patients infected with HIV1. Although spontaneous secretion of TNF- a from PBMCs was observed in 16 patients, vesnarinone reduced the production of TNF- a in 14 patients. These inhibitory effects were obtained at clinically achieved concentrations of vesnarinone. Discussions: These results suggest that vesnarinone inhibits the replication of HIV-1 in PBMCs enhanced by cytokines, such as TNF- a and IL-6. AIHARA, Koutoku, Cellular Tbchnology Institute, Otsuka Pharm. Co.,Ltd., lbkushima, Japan TeIblephone (0886)-65-2126; TIblefax (0886)-65-5662 PAO342 Inhibition of HIV infection by a novel soluble factor produced by nonlymphoid cell line. Keisuke Teshigawara', T. Hattori2 Y. Okubo, L. Sun, H. Fukata1 M. Honda3 A. Uchlda '.Kyoto Univ. Radiation Biology Ctr' Virus Inst2. NIH in Japan3. Objection: Soluble factors produced by hamster cell lines has been shown to prevent the infection of the amphotropic retrovirus into hamster cells itself. The present study was designed to investigate whether HIV infection into human T cell lines could be prevented by a soluble factor produced by non-lymphoid cell lines. Methods: The conditioned medium(CM) was prepared from various human non-lymphoid cell lines and concentrated by 5-6 folds. HIV-sensitive human T cell lines were cultured alone or with these CM for 24 hr. After culture, the cells were infected with HIV by the use of the supernatant of H9/IIIB. They were then tested for production of p24 and synthitium formation. Results: The supernatant produced by the EC-1 cell line inhibited the production of p24 in both supernatant and cells. The synthitium formation was also suppressed by the soluble factors. The fraction under 10,000 had no inhibiting activity. No known cytokines were detected in the CM except small amounts of IL-1 oa when tested by using enzyme-linked immunosorbent assays. Discussion and Conclusions: The EC-1 non-lymphoid cell line is found to produce a novel factor(s) which may prevent CD4+ T cells from the infection of HIV. The cloning of cDNA encoding the factor and the analysis of the mechanism responsible for its inhibitory effect are in progress. Since the factor did not affect the viability of normal cells, its administration might produce clinical benefit to patients with AIDS. Keisuke Teshigawara, Radiation Biology Center. Yshida-Konoe, Sa kyo, kyoto 606, Japan Telephone:+81-75-753-7558, Fax:+81-75-753-7564 0 CA) CA) 0 CA) N

Page  116 PA0343 HIV protease Inhibitor RO 31-8959: absence of Immunoregulatory effects. A. DULIOUST, C. GOUJARD, M. CROUVOISIER, C. WALLON, and J.F. DELFRAISSY PA0344 PROPOSAL FOR EXPERIMENTAL STUDIES TO EVALUATE SODIUM HYPOCHLORITE DIALYSATE IN RETROVIRAL TREATMENT. Avlicino, Aldo, A., Newton, C.L. The purpose of the study was to investigate possible immuno-modulatory effects of a HIV protease Inhibitor Ro 31-8959. We studied PHA- and anti-CD3- (lOT3) induced T cells proliferative response, antip-, IL2-, IL4-, and SAC- induced B cells activation and monocytes cytokines production stimulated by LPS, in the absence or presence of various concentrations of protease inhibitor (6.25, 25 et 200 nM). PBL were obtained from healthy volonteers after Ficoll-Hypaque sedimentation. In addition, we studied T cells apoptosis after 24 hours culture by propidium iodide incorporation and FACS analysis. This was performed in vitro in presence or absence of protease inhibitor on PBL obtained from healthy donors and untreated HIV-infected patients (CDC stage II and IV). Results: B and T cell activation induced by mitogens or cytokines were not influenced by the addition of RO 31-8959. In addition RO 31-8959 has no effect on B and T cell response when added alone in culture. TNF and IL6 production by LPS-stimulated monocytes were not modified by RO 31-8959. TH1 (IL2, IFNy) and TH2 (IL4, IL10) like cytokines are under investigation. Futhermore, apoptosis was neither inhibited nor stimulated by this protease inhibitor in PBL provided by HIV and non HIV infected subjects. In conclusion, these data Indicate that this protease Inhibitor used at concentrations reached in the blood of treated patients has no in vitro immunoregulatory effects. In vivo effects of RO 31-8959 have to be analyzed. These results suggest that the antiretroviral efficacy of RO 31 -8959 is due to direct anti-HIV activity. C. GOUJARD" INSERM U131 et Service de M6decine InterneHtpital Antoine Beclre - 92141 CLAMART - FRANCE - Objective: This proposal establishes the thesis that sodium hypochlorite and its related oxygen free radicals can be administered in minute quantities in vivo to achieve a reduction in retroviral titer within HIV seropositives. Methods: Infusion can include the ex vivo retroviral inactivation of plasma utilizing extracorporeal circulation through a continuous centrifugal plasma separator, and alternative modes for lymphatic infusion. Results: Sodium hypochlorite's protein depletion and oxidation of sulfhydrl compounds is reversible and possibly preventable by administration of disulfide reducing agents. The expected range of HIV particle reduction in human blood is from 2 to 6 log10, at infusion of 7 mEq/kg body weight of 10 ppm sodium hypochlorite in physiologic saline adjusted to pH 7.4 succeeded by dithiothreitbl @ a concentration of 2.5 mM. Discussion and conclusion: This application also correlates to HIV's effect on the Na+/K+ATPase and Na+/K+/2C1- cotransport system and the resultant increase in intracellular Na+ levels, postulating a connection to long term ingestion of chlorinated tap-water, high consumption levels of table salt, and exposure to other sodium compounds with low HIV incidence. Avlicino, Aldo, A., 4213 Mariner Bl. #202, Spring Hill, FL 34609 USA. (904)688-9669. 0 CA) 44) -o 0 CA) 0) PA0345 GENETIC IMMUNIZATION OF HUMANS FOR AOIDS IMMUNOTHERAPY USING DIRECT RETROVIRAL VECTOR ADMINISTRATION Wamner. John F., Irwin, M., Laube, L., Lee, V., Anderson, C-G., Chada, S., Peters, G., Ziegner, U., Merchant, B., Klump, W., Sajjadi, N., Jolly, D., Mento, S., and Galpin, J.*, Viagene, Inc., San Diego, California; *Shared Medical Research Foundation, Tarzana, California. The cytotoxic T lymphocyte (CTL) response plays an important role in controlling the severity and duration of viral infections. Gene-based vaccines, using retroviral vectors, represent an efficient means of introducing and expressing genes in mammalian cells and, hence, can be employed to provide foreign proteins to the appropriate antigen processing and presentation pathways for CTL activation. We have developed retroviral vectors encoding HIV-1 proteins and have examined these vectors for their ability to stimulate immune responses, particularly CTL activity, in several animal models including mice, Rhesus monkeys, and baboons as well as in HIV-infected humans. The direct administration of the retroviral vector consistently activates CD8+, Class I MHC-restricted anti-HIV1 CTL responses in these models. Moreover, the activated CTL show crossreactivity against target cells infected with many laboratory and clinical HIV-1 isolates. These studies demonstrated the ability of retroviral vectors encoding HIV-1 proteins to consistently stimulate the cellular immune response and suggest that retroviral vectors may provide an effective means of inducing or augmenting CTL responses in HIV-infected individuals. Based on these studies, HIV-infected patients have been injected with a retroviral vector encoding the HIV- I IIIB envelope/REV proteins. The results of the animal studies and interim results of human trials will be presented. John F. Warner, Ph.D., Viagene, Inc.. 11075 Roselle Street, San Diego, CA 92121. Ph: (619) 452-1288; Fax: (619) 625-7271. PA0346 INTERFERONI TRANSFECTED HUMAN CELLS ARE PROTECTED FROM HIV INFECTION IN VITRO AND IN VIVO FOLLOWING TRANSPLANTATION INTO SCID MICE. Sanhadji Kamel*, Firouzi R*, Leissner P", Laplace S*, Touraine J-L* & Mehtall M"**, *Facult4 A. Carrel, INSERM U80, Lyon, " Transgene, Strasbourg, France. Objective: To render human cells resistant to HIV1 infection, in vitro and in vivo, by transfection with interferon O (INFI) gene associated with LTR gene. Methods: Cells from human monocytoid line U937 were transfected with INFI gene associated with an HIV1 LTR sequence (LTR-HIVI-INF construction). They were subjected to HIVI infection in vitro or in vivo after being transplanted to severe combined immunodeficiency (SCID) mice. Results: Whereas U937 untransfected cells were readily infected by HIV1 (with cytopathogenic effect and increased reverse transcriptase activity in culture supernatants), the LTR-HIV1-INF constructed U937 cells were resistant to it. When U937 cells were injected into SCID mice, these human cells rapidly developed in blood, spleen, peritoneal cavity and liver, leading to abdominal tumor development. Inoculation of 1000 TCID50 of LAVl-Bru to these mice resulted in significant HIV infection. When LTR-INF constructed U937 cells were administered instead of naive U937 cells, no infection was detected in any mouse by cythopathogenic effect and reverse transcriptase criteria. Discussion and conclusions: Transfection of the INFO gene together with LTR protected human cells from infectivity with HIV1, both in vitro and in vivo. The use of protective genes against HIV for ex vivo cell alteration may lead to gene therapy of AIDS. SANHADJI Kamel, Transplantation & Clinical Immunology Unit, INSERM U80, Pay. P, Hop. E. Herriot, 69437 Lyon cedex 03, France Telephone: (00.33).; Telefax: (00.33).

Page  117 PA0347 GENE THERAPEUTIC APPROACHES TO AIDS SYMONDS Geoff, Sun L-Q, Pyati J, Smythe J, Gerlach W The RW Johnson Pharmaceutical Research Institute objective: Ribozyme, antisense and RNA decoy constructs are being employed to inhibit HIV replication. Methods: For the ribozyme constructs, accessible GUC or CUC sites were chosen and oligodeoxynucleotides coding for ribozymes with 12 mer complementary arms synthesised and cloned into expression vectors. For antisense constructs, different regions of the HIV proviral sequence were cloned into expression constructs in an antisense orientation. Results: Data has been obtained in a Moloney murine leukemia virus retroviral model system to show the efficacy of ribozymes in inhibiting viral replication. In this system ribozymes significantly decreased Virus production in chronically infected NIH3T3 cells and the level of viral suppression correlated with the efficacy of in vitro ribozyme cleavage. For HIV, certain of the ribozyme, antisense and decoy constructs targeted to different regions of the HIV genome inhibited HIV-1 replication in T lymphocytes after introduction by transfection and/or by retroviral transduction. This has been shown in clonal and pooled T lymphocyte cell populations. Discussion and Conclusions: Our results show that ribozymes, antisense and decoy constructs represent means by which to decrease virus production invivo. SYMONDS, Geoff, The RW Johnson Pharmaceutical Research Institute, GPO Box 3331, Sydney NSW 2001, Australia Telephone (61)-2-360 9377; Fax (61)-2-360 9813 PA0348 A REV MUTANT EXPRESSED IN CYTOPLASM RESCUED CELLS FROM HIV-1 INDUCED CELL DEATH. Furuta. Rika. A.. Kubota, S., Hattori, T., and Hatanaka, M. Institute for Virus Research, Kyoto University Objective: To apply intracellular immunization to AIDS therapy, we introduced a gene encoding Rev mutant, dRev, into CD4 positive cells and examined inhibitory effects of dRev against HIV-1 infection. Methods: dRev is a mutant Rev with 7 amino acid deletion in its nucleolar localization signal and strongly inhibits Rev function. The CD4 positive cells introduced with rvy gene were subjected to HIV-1 infection in vitro, then virus production, syncytia formation and cell viability were examined. To estimate cytotoxicity of dRev, we studied distribution of rRNAs and nucleolar deformity of the dRev expressing cells. Results: In dRev introduced cells, virus production and syncytium formation were suppressed compared to control cells. Many dRev cells could escape from cell death caused by HIV-1 infection. dRev did not cause nucleolar deformity, because it could not migrate into cell nucleus/nucleolus unlike wild type and another mutant Rev. In dRev cells, distribution of rRNA was the same as normal cells. Aberrant accumulation of rRNA in deformed nucleoli was, however, observed in cells expressing wild type or another trans-dominant Rev. Discussion and Conclusions: The cells introduced with drey gene show a phenotype resistant to HIV-I infection without any direct effects on nuclear events, because dRev worked in cytoplasm as an anti-HIV molecule. In consideration for safety of gene therapy, dRev appears to be a more prospective candidate than other trans-dominant mutants working in cell nuclei. Furuta, A. Rika, Institute for Virus Research, Kyoto University, Japan Telephone (81)-75-751-4034; Telefax (81)-75-751-3998 PA0O349 INTRACELLULAR ANTIBODIES "INTRABODIES" FOR AIDS GENE THERAPY Marasco, Wayne A., Chen, S-Y., Bagley, J. Mhashilkar, A.. Khouri, Y'., Jones, S.', and Ramstedt, U.' Division o Human Retrovirology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA and Virus Research Institute, Cambridge, MA.'. Objective: Effective gene therapy of AIDS may require a multi-targeted approach to attack the virus at different critical steps in the virus life cycle. Recent advances in antibody engineering have allowed antibody genes to be manipulated and antibody molecules to be reshaped. These advances have raised the possibility that antibodies directed against HIV-1 can be made to function intracellularly, to bind and inactivate HIV-1 proteins within cells. Methods: We have designed intracellular antibodies termed "intrabodies" against several HIV-1 structural and regulatory proteins and have used different intracellular trafficking signals to direct the "intrabodies" to different subcellular locations. Stable eukaryotic cell lines have been established and have been challenged with HIV-1. Results: Stable intracellular expression of anti-HIV intrabodies has been achieved. Intracellular trafficking signals can direct the intrabodies to different sub-cellular locations. Upon challenging these stable cell lines with HIV-1, marked inhibition of cytopathic effects and of HIV-1 replication and infectivity have been achieved. Discussion and Conclusions: Intracellular antibodies provide a powerful new tool that may be useful for the gene therapy of AIDS. Wayne A. Marasco, M.D. Ph.D. Dana-Farber Cancer inst., 44 Binney St. Boston, MA., USA-02115 #617-632-2153 Fax# 617-632-3889. PA0350 MONOCLONAL HUMAN Fab AGAINST HIV-1 rev and tat PROTEINS Pilkington, Glenn R., Duan, L*, Keil, W. and Pomerantz, R.*, Intracel Corp., Cambridge, Ma & *Thomas Jefferson University, Philadelphia. Objective: Combinatorial human Fab to HIV-1 rev and tat proteins have been cloned for passive and active immunotherapy (gene therapy) in AIDS. Methods: PCR amplification of the Fd IgG1 heavy chain fragments and light chains was performed on cDNA from peripheral blood lymphocytes from a donor infected with HIV who is asymptomatic after 10 years of infection. Primers contained restriction enzyme sites to allow the sequential cloning of Fd fragments and light chains into a phage display vector containing the cap protein of the M13 filamentous phage as a fusion protein to the Fd fragment. From a phagemid library of 107 clones, Fab to HIV-1 rev and tat proteins were enriched by panning 4 times against rev or tat proteins bound to plastic surfaces and amplifying the eluted phagemids. Cap protein DNA was excised to allow production of soluble Fab and positive clones identified by ELISA. Results: Three Fab clones have been isolated against HIV-1 rev and 4 isolated against HIV-1 tat. Fab have been purified, binding constants determined, DNA sequenced to allow cloning into vectors for antibody gene therapy and the binding sites epitope mapped. The anti-tat Fab bind to the cysteine rich tat functional domain and the anti-rev Fab bind, one to the sequence immediately adjacent to basic nucleolar localization domain and, two to the region adjacent to the activation domain. Conclusion: Work is continuing on the intracellular expression of these human Fab for gene therapy. PILKINGTON, Glenn, Intracel Corporation, Cambridge, Ma 02139, USA, Telephone (617) 547-5535; Telefax (617) 491-9015 0 CA) 4h -o 0 CA) 0

Page  118 PAO351 DEVELOPMENT OF A SHOTGUN-TYPE ANTI-HIV-I MULTIPE TARGEFED RIBOZYME EXPRESSION SYSTEM Jun Ohkawat.2, Noriko Yuyamal, Matthias Homann3, Georg Sczakiel3, Yutaka Takebe2 and Kazunari Tairal 1National Institute of Bioscience and Human Technlmology, Agency of Industrial Science & Technology, MITI, Tsukuba, 305, Japan, 2AIDS Research Center, National Institute of Health, Tokyo, Japan, 3Angewandte Tumnorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Objective: To protect the IlIlV-1I infection effectively by a ribozyme strategy, we developed a new type ribozymec expression system designated "shotgun-type ribozyme expression system". This system makes it possible to express multiple ribozymes without reducing of the activities of each ribozymes. In the case of a single ribozyme system, "escape phenomenon" due to a sequence mutation of the target site was a serious problem. Our system can supplement this weak point of a single ribozyme construct. Method: This system is basically composed of trans-acting ribozyme (offensive ribozyme) placed between two cis-acting ribozymes (trimmning ribozymes). At first, several kinds of mono unit, each possessing different target site against HIV-1, were tested by transient assay system in cultured cells to test whether this system was active enough against HIV-1. Then, those units were tandemly connected and tested by in vitro cleavage assay system. Result: All mono units showed suppressive effect. Especially one of them targeting at tat position showed strong suppressivity (up to 95%). In vitro cleavage assay indicated that tandemly connected system expressed multiple offensive ribozymes and precisely cleaved HIV-1 RNA at predetenrmined sites. Discussion and Conclusion: By using this system, it is expected that HIV-1 RNA would be cleaved at multiple position in vivo. Thus, (i) the means that shotgun-type system is more effective than a single ribozyme system and (ii) it may overcome the escape phenomenon. OHIKAWA, Jun, National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technlmology, MITI, Tsukuba, 305, Japan Telephone (81)-298-54-6086; Telefax (81)-298-54-6095 PAO352 MULTI-FUNCTIONAL ANTI-HIV-I RIBOZYME EXPRESSION VECTOR Noriko Yuyama,Jun Ohkawa and Kazunari Taira National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technology, MNITI, Tsukuba, 305, Japan. Objective: We have previously constructed a unique ribozyme expression system. This system is basically composed of trans-acting ribozyme (offensive ribozyme) placed between two cis-acting ribozymes (trimming ribozymes). Upon transcription, two cis-acting ribozymes liberate trans-acting ribozyme. However after having performnned their duties, both cis-acting ribozymes were to await for degradation by RNase. Inorder not to waste these fragments, we equipped cis-acting ribozymes with decoy activity against Tat and Rev. By this modification the ribozyme expression system has gained two functions and, therefore, an improved inhibitory effect against HIV would be expected. Method: TAR sequence was introduced into the stemll region of 5'-cis-acting ribozymes and RRE sequence was introduced into the corresponding 3'-. Inorder to check whether this modification might influence the ribozyme activity of cis-acting ribozymes, in vitro transcriptional cleavage assay was performed. Moreover, to examine the protein bainding activity of those decoy ribozymes, gel shift assay of 5'- TARcis-acting ribozymes to Tat and 3'-RRE-cis-acting ribozymes to Rev was perfonned. Result: Introduction of TAR and RRE sequences into cis-acting ribozymes did not significantly influence the ribozyme activity. Both kinds of cis-acting ribozymes could liberate trans-acting ribozyme at the same rate as the original one. On the other hand, gel shift assay showed that (i) TAR-cis-acting ribozymes could bind Tat and (ii) RRE-cis-acting ribozyme could bind Rev. Discussion and Conclusion: By using this system, HIV- I RNA would be cleaved by trans-acting ribozyme and Tat and Rev necessary for viral replication would be trapped by TAR- and RRE-decoy ribozymes. An improved inhibitory effect against HIV comparing to ribozyme alone system might be expected. TAIRA, Kazunari, National Institute of Bioscience and human Technology, Agency of Industrial Science & Technology, IlTI, Tsukuba, 305, Japan Telephone (81)-298-54-6086; Telefax (81)-298-54-6095 -o 0 I 0 CA) (in PA0353 DOMINANT NEGATIVE EN/V MUTANT OF FRIEND MURINE LEUKEMIA VIRUS MATANO, Tetsuro, Odawara,T., Ohshima,M., Iwamoto,A., Yoshikura,H. Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Objective: The purpose of this study is to inhibit replication of retrovirus including HIV by a dominant negative mutant. A model system was established by using Friend murine leukemia virus (FMLV). Methods: A mutant FMLV env gene was cloned from an immunoselected Friend erythroleukemia cell and was expressed in NIH3T3 cells. The processing of the mutant env gene product and the effect of its expression on the wild-type ecotropic Moloney MLV (MoMLV) infection was examined. Results: The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env formed abnormal multimers, was retained in the endoplasmic reticulum (ER), and accumulated because of its slow degradation. The NIH3T3 cells expressing the mutant env were resistant to MoMLV penetration. When the mutant env gene was expressed in the cells persistently infected with MoMLV, the wild-type Env was bound to the mutant Env and trapped in the ER, and the MoMLV production was suppressed. The inhibition of virus infection by the mutant Env was specific to the ecotropic MLV. Discussion and Conclusions: The mutant Env accumulating in the ER transdominantly and efficiently inhibited the ecotropic MLV infection at both the early and the late stages. These downregulations by protein-protein interactions may lead to the development of an antiviral strategy based on intracellular immunization and an animal model for gene therapy. MATANO, Tet suro, Ba:teriology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Telephone (81 )-3-3812-2111 (ext.3409); Telefax (81 )-3-5684-9374 PAO354 MOLECULAR THERAPEUTIC APPROACHES TO HIV-1 INFECTION AND AIDS LiSziewicz, Jul ianna, Sup D, Lori F, Peng B, Ensoli B, Agrawal S, Gallo RC LTCB, NCI, NIH, Bethesda, MD; Hybridon, Inc., Worcester, MA USA We have developed an autoregulated, dual function inhibitory gene, anti-Tat, which is expressed only in the presence of HIV-1 Tat. Anti-Tat blocks the function of intra- and extracellular Tat through expression of polymeric-TAR and antisense-Tat RNA. The anti-tat gene, transferred by retrovirus vectors, blocked the spread of HIV-1 in the T-lymphocytic cell line, and the extent and duration were dependent upon viral infectious dose. Virus replication was inhibited up to 7 months in two different cell lines after high multiplicity of infection, suggesting that the anti-tat gene is an effective long-term suppressor. In primary lymphocytes anti-tat gene transfer resulted in up to 4000-fold inhibition of a primary HIV-1 isolate and inhibition of the cytopathic effect of the virus, suggesting a potential use for anti-tat gene therapy to treat HIV-1 infection. Our other approach is the development of GEM91, a 25 nucleotides long antisense oligonucleotide phosphorothioate complementary to Gag mRNA. This compound inhibited HIV-1 replication in a dose-dependent and sequence specific manner in the T-lymphocytic cell line. GEM91 also completely blocked the growth of four different HIV-1 isolates in primary lymphocytes/macrophages. A clinical trial was initiated for the treatment of AIDS based on the antiviral activity, safety and pharmacokinetic profile of GEM91. LISZIEWICZ, Julianna, Laboratory of Tumor Cell Biology, NCI, NIH, Bethesda, Maryland USA Telephone (301) 496-1234; Telefax (301) 496-8394

Page  119 PA0355 Specific ablation of ATL cells using HIV vector carrying thymidine kinase gene. Obaru Kenshi, Matsushita S, Fujii S, Shimada T, Takatsuki K. The IeconIDepartment of Internal Medicine, Kumamoto University Medical School Objective: Adult T-cell Leukemia(ATL) is a leukemia or lymphoma of CD4 positive T-cells and has poor prognosis because of resistency to chemotherapy. To evaluate effectiveness of gene therapy for ATL, HIV vector that has specific infectivity to CD4 positive cells, carrying thymidine kinase gene(TK) was transduced to ATL cell lines. Specific ablation of transduced ATL cells by gancyclovir was evaluated. Methods: TK gene of HSV-I was inserted between LTRs of HIV-l and drived by the SL3 promoter(named HXBSL3TK). HXBSL3TK was cotransfected with HXBCAT into MT2 or HUT1O2 cells and incubated with gancyclovir. Chloramphenicol acetyltransferase activity (CAT) was analysed. HXBSL3TK was cotransfected with packaging signal defective HIV-1 proviral DNA into Cos cells. The supernatent was transfered to MT2 cell and incubated with gancyclovir and rate of cell death was measured. Results: CAT activity of MT2 cells and HUTIO2 cells, trasfected with HXBSL3TK was decreased dose-dependently with gancyclovir,comparing with non-transduced cells. Increased rate of dead cell of MT2 cell infected by HXBSL3TK, incubated with gancyclovir, was observed. Conclusion: Thus HIV vector carrying TK gene could ablate ATL cell lines effectively by gancyclovir. OBARU, Kenshi, The Second Department of Internal Medicine Kumamoto University Medical School, Honjo-l-1-1, Kumamoto, 860 Japan. Tel 096-344-2111, Fax 096-363-5263 PA0356 ENHANCING IMMUNOGENICITY OF HIV-1 PND BY USING FOREIGN T-h EPITOPE. A. KUMAR, GENETIC UNIT, AIIMS, NEW DELHI-29, INDIA. Synthetic peptides provide a neat and efficient alternative to the conventional vaccines, however, their potential has been under-utilized by the frequent nonresponsiveness in out-bred population with heterogenous background. In the present study, principal neutralization domain (PND) of IIIB and RF isolates of HIV-1 were found to elicit poor to moderate immune response in selective strains of mice. The universal T-helper epitopes tt830-844 and tt947-967 from tetanus toxin which had been earlier found to be recognized in association with different HLA-DR alleles were then used to construct chimeric peptides. These chimeric peptides evoked strong immune response directed against PND and moreover, the response was generalized. In carrier primed animals, use of only T helper determinant to provide carrier help was also possible. Substitution of tetanus toxin with small T-helper epitope, derived from it, was also able to overcome carrier-induced epitope specific suppression of immune response. These results suggest that a heterovalent molecule that contains an universal T-epitope and PND of HIV-1 may provide a viable means to be explored as therapeutic vaccine in individuals infected with HIV-l. A. Kumar, Genetic Unti, AIIMS, New Delhi-29, INDIA Tel: (91-11-6854434) Fax (91-11-6862663) PA0357 IMPROVED CONSTRUCTION OF SYNTHETIC HIV VACCINE CANDIDATES. Charles Sla, P. Chong, 'T. Matthews, D. Bolognesi and M. Klein. Connaught Labs. Ltd., 1755 Steeles Av. West, Ont., Canada M2R3T4.'Dept.ofSurgery, Duke University, NC27710, USA. PA0358 CONTROLLED RELEASE SUBUNIT VACCINE FOR HIV-I leffrey L Cleland, Michael F. Powell, Amy Lim, Lorena Barr6n, Phillip W. Berman, Jack H. Nunberg, Krishna K. Murthy, Genentech, Inc., SFBR OBJECTIVE: To optimize the construction of immunogenic synthetic HIV vaccine candidates. METHODS: Two synthetic constructs, p24E-PND-K and p24E-K-PND, containing the principle neutralizing determinant (PND) of the V3(MN) loop and the neutralization epitope (K) encompassing the amino acid residues 663-668 of gp41 described by Katinger linked in tandem to the C- terminus of a p24 T-helper epitope, p24E (residues 291-305), were synthesized. Immunogenicity studies were carried out in mice and guinea pigs. Humoral responses elicited by the constructs were measured by peptide-specific EIA, and the functional activity of the antibodies assessed by an in vitro HIV neutralization assay. RESULTS: The human neutralizing monoclonal antibody, 2F5, directed against K recognized peptide p24E-PND-K better than p24E-K-PND in EIA.. Both constructs were highly Immunogenic when administered in alum. Antibody responses elicited were directed against both the PND and a gp4l peptide containing K. Antisera raised against both constructs were able to strongly neutralize the MN virus. However, anti-K and neutralizing antibody titres were significantly lower in sera of animals immunized with the p24E-K-PND peptide, whereas anti-PND titres were comparable. DISCUSSION AND CONCLUSIONS: The relative orientation of tandem PND and K epitope in a synthetic construct significantly influenced its repective immunogenicity. Dr Charles Sia, Connaught Laboratories Limited, 1755 Steeles Avenue West, Willowdale, Ontario, Canada M2R 3T4. Telephone: (416) 667 2895 Fax: (416) 661 7960 Objective An efficient AIDS vaccine formulation should invoke a high and sustained immunological response with as few injections as possible. A vaccine formulation comprised of MN rgpl20 encapsulated in PLGA microspheres was developed to provide an in vivo autoboost of antigen. The protein was released in its intact native state. These formulations were designed to yield an in vivo autoboost at 1, 2, 3 or 4-6 months. PLGA microspheres containing the adjuvant QS21, were also prepared to provide an in vivo autoboost concomitant with antigen. These formulations yielded higher antibody and virus neutralization titers in guinea pigs than alum formulations. Different doses of encapsulated MN rgpl20 provided a well-defined dose response curve in antibody titers. The addition of soluble or encapsulated QS21 to MN rgpl20/PLGA greatly enhanced the immune reponse in both guinea pigs and baboons. These results also indicated the adjuvant properties of QS21 can be increased by microencapsulation in PLGA. Furthermore, the virus neutralization titers induced after one immunization with the encapsulated MN rgpl20/QS21 formulation were equivalent to those obtained with three immunizations of the soluble MN rgpa20/QS21 formulation having significantly higher antigen and QS21 doses. Baboon data also indicated that the PLGA formulations provided a longer lasting immune response and higher antibody and virus neutralization titers than soluble QS-21 and MN rgpl20 formulations. These studies validate the in vivo autoboost concept, reveal a method for improving the adjuvant properties of QS21, demonstrate that a controlled release formulation yields a sustained and higher immune response, and present the potential of future single shot vaccine formulations. leffrey L. Cleland. Genentech, Inc. 460 Pt. San Bruno Blvd. S. San Francisco, CA 94080. Telephone (415)225-3921; FAX (415) 225-2866 "U 0 CA) (J1 0 U'

Page  120 r$,|,N 0 PAH359 1E N PROLIFERATIVEMBINANAND GCYTOTOXIC ACTIVITY INDUCED IN ANIMALS BY GHEORGHIU Marina, GICQUEL Brigitte, LAGRANDERIE Micheline Recently it has been shown the Bacille Calmette-Guerin (BCG) can be used as live recombinant vector for several HIV type 1 virus proteins such as Gag, Env or Nef. The cytotoxic immune response against the Nef protein seems very important in a vaccinal strategy as it may kill infected cells before the release of the new virions. To check if a HIV 1 - Nef recombinant BCG induces CTL, mice and guineapigs were parenterally vaccinated with this BCG recombinant. The draining lymphnode T-cells from vaccinated guinea-pigs, in vitro stimulated with Nef protein, produced 70 % lysis of autologous alveolar macrophages. Similar cytotoxic activity was also induced in mice. T-cell proliferation and INF-gamma production specific of Nef protein were also found. These findings encourage research on BCG as a live vector in view of a candidate vaccine against AIDS. GHEORGHIU Marina, INSTITUT PASTEUR, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Tel:33-1-45 68 82 35 Fax:33-1-45 68 83 51 PA0361 PRIMING OF CD8+ CTL BY IMMUNIZATION WITH SYNGENEIC IRRADIATED HIV-1 DERIVED PEPTIDE-PULSED DENDRITIC CELLS. Takahashi. Hidemi. Nakagawa, Y., Yokomuro, K., Berzofsky, J.*; Nippon Medical School, Tokyo 113, Japan; *NCI, NIH, Bethesda, MD 20892, USA. Objective & Methods: Because HIV can be transmitted via infected cells and spread by passage between fused cells without a requirement for free virus that would be accessible to antibody, virus-specific class I-MHC molecule-restricted CD8+ CTL may contribute to recovery from and protection against such viral infection. To obtain such CTL responses usually requires in vivo priming with potentially dangerous replicating infectious agents. However, to prime such CD8+ CTL with a non-living antigen, especially with synthetic peptides, has been difficult to accomplish in comparison with induction of neutralizing antibodies or class II-MHC molecule restricted CD4+ helper T cells. To overcome this problem, we have been focusing on the fact that CD8+ CTL do not seem to distinguish between virusinfected cells and viral peptide-pulsed syngeneic cells. Without using any harmful adjuvant, we have developed a method for priming CD8+ CTL specific for the immunodominant determinant p18 (RIQRGPGRAFVTIGK) that we have identified in the HIV-1 IIIB gpl60 envelope glycoprotein as an epitope for not only CD8+ CTL but also CD4+ helper T cells in both mice and humans, that is also recognized by neutralizing antibodies. Results: We could prime in vivo such CTL with syngeneic 2200-3300 rad irradiated peptide-pulsed spleen cells, and much more efficiently with a very small number of splenic dendritic cells, by a single low-dose i.v. inoculation. And we could not prime such CTL if we depleted the dendritic cells before immunization. Interestingly, we could not generate CTL with unirradiated or low dose (less than 2000 rad) irradiated spleen cells. CTL generated by this method could kill both p18-pulsed syngeneic targets and targets infected with recombinant vaccinia virus expressing the whole gpl60 gene. Such priming lasted at least for 6 months. Conclusions: We conclude that dendritic cells bearing peptide may prime CTL in vivo, and these results may offer useful information for synthetic peptide vaccine development for preventing both HIV infection and disease progression. TAKAHASHI, Hidemi, Nippon Medical School, Tokyo, JAPAN; Phone: (81)-3-3822-2131 ext. 339; FAX: (81)-3-3316-1904 PA0360 INDUCTION OF CROSS-NEUTRALISING ANTIBODIES AND CTL BY A CHEMICALLY INACTIVATED WHOLE HIV VACCINE Race. E.. Addawe, M.D., Frezza, P*. and John S. Oxford Londlbsp Med Coll London UK and * ISI, Naples, Italy. Objectives: To investigate the immune response to an experimental whole virus chemically inactivated HIV-1 vaccine in small animals. Methods / Results: Groups of guinea pigs and mice (balb/c) were immunised with various doses (100pg to 0.1 g) of a whole HIV-I IIIB vaccine produced using a multiple step inactivation procedure comprising p-propiolactone, sodium cholate, ethylenimmine, and formaldehyde. Animals were boosted at monthly intervals. After three boosts sera was assayed for binding to whole virus and to gpl20 by ELISA and for virus neutralisation by syncytium reduction in C8166 cells. Tlymphocyte proliferation was determined by 3H-thymidine uptake and cytotoxic T lymphocytes were detected using a non-radioactive lactate dehydrogenase release assay (Promega CytoTox 96) with the mouse cell line p815 serving as a target. High antibody titres to whole virus and to recombinant gpl20 were detected in the sera of animals immunised with as little as 1 pg of vaccine. These antibodies also neutralised IIIB,RF and MN viruses at titres of between 20 and 240. Sera did not bind to antigens derived from uninfected C8166 cells. Splenocytes taken from mice immunised with 10pg of vaccine demonstrated gp120 specific cytotoxic activity. Conclusions: The experimental chemically inactivated HIV vaccine is highly immunogenic, inducing both Neutralising antibody and virus specific cytotoxic T-lymphocytes. The vaccine does not induce antibodies to antigens derived from the cell line in which the vaccine virus was produced. The vaccine is designed to contain a mixture of four virus strains to induce a broadly reactive immune response. John S Oxford, Department of Academic Virology, Lon H-Iosp Med Coil, Turner Street, London El1 2AD. Tel ++44 71 375 0345 Fax ++44 71 375 2597 PA0362 EXPRESSION OF HIV-1 CORE/ENVELOPE CHIMERIC VIRUS-LIKE PART CLES IN BACULOVIRUS-INFECTED CELLS TRUONG Catherine, BRAND D, ROINGEARD P, MALLET F, GOUDEAU A, BARIN F., CHRU Brelonneau, Tours, and ENS, Lyon, France. Objective: To obtain recombinant HIV1 gag polyproteins expressing neutralizing epitopes of HIV1 gpSU. Methods: Several chimeric genes have been constructed by an original procedure using the polymerase chain reaction procedure, and then cloned in a baculovirus transfer vector. The V3 consensus sequence (subtype B) or the conserved C4 region of the CD4 binding site (CD4bd) have been inserted in place of two exposed epilopes ol the p24 region (aa 196-226 and 303-317). An unmodified gag gene (wild type) was also cloned following the same procedure. After cotransfection of the recombinant vectors with wild type baculoviral DNA, recombinant viruses have been selected and purified. Protein expression has been analyzed by western blot using several monoclonal antibodies directed to V3, p17 and various epitopes of p24 (including regions of insertion). Auto-assembling into virus-like particles has been studied by electron microscopy examination. A simple procedure for purification has been developped and immunogenicity of the chimeric proteins is being evaluated in mice. Results and discussion: Wild-type p55 and chimeric core/envelope proteins (p55-V3, p55-CD4bd) derived from insertion at positions 196-226 were capable of auto-assembling. Chimeric proteins derived from insertions at positions 303-317 did not assemble into immature particles. However they were embedded into spherical structures that derived probably from the plasma membrane of infected insect cells. Results on immunogenicity of the various types of particles should be available. TRUONG Catherine, Laboratoire de Virologie, CHRU Bretonneau, 37044 TOURS Cedex, France. tel. (33) 47 47 47 47 ext. 3046; fax. (33) 47 47 36 10. 0 3 co 'o 0 CA 0

Page  121 PA0363 EX VIVO NEUTRALIZATION OF HIV QUASISPESIES BY A BROADLY REACTIVE HUMANIZED MONOCLONAL ANTIBODY MATSUSHITA Shuzg, EDA Y', YOSHIMURA K, MURAKAMI T',SHIOZAKI K*, TOKIYOSHI S*,TAKATSUKI K, Kumamoto Univ. and *CSTRI, Kumamoto Japan Objective: Because HIV-1 exsisted in each patient as a population of diverse yet related viruses (quasispecies) we attempted to determine the reactivity and the neutralizing activity of a broadly reactive monoclonal antibody (mAb) against the HIV-quasispecies in patients' peripheral blood mononuclear cells (PBM). Methods: By immunizing mice with several different V3-loop peptides we obtained a murine monoclonal antibody and its humanized counterpart (designated as C25 and RC25, respectively) which had a broad reactivity and a neutralizing activity against the variety of HIV strains. The epitope recognized by the mAb was mapped to an amino acid sequence IGPGRA by pepscan analysis. Neutralization of HIV-quasispesies by the mAb was determined by short-term ex-vivo culture of the CD8-depleted PBM(s) from seropositive patients in the presence or absence of the mAb. Inhibition of the HIV replication was monitored by detecting HIV-p24 antigen in the supernatant. Results: PCR-cloned DNA fragment that encoded the V3-loop was obtained from the patient's PBM and was expressed as the recombinant fusion peptides in E-coli. In 10 out of 15 patients' samples the mAb bound more than 92% of the V3-peptides. Inhibition of viral replication by RC25 was observed for 9 out of 13 patients' PBM. At a lower concentration of the mAb emergence of the escape mutants was observed. However, in combination with 4 tiM of 2'-3'-dideoxyinosine (ddl), complete inhibition of HIV replication was observed even at the lower concentration of the mAb. Discussion and Conclusions: These results may facilitate the clinical application of the "combination chemo-immunotherapy" using RC25 mAb and ddl together for the treatment of HIV infected individuals. MATSUSHITA Shuzo, The Transfusion Service Department, Kumamoto University School of Medicine, 1-1-1 Honjo Kumamoto 860, Japan. Tel. (81) 096-344-2111: Fax (81) 096-363-5265 PA0364 A NOVEL MULTICOMPONENT VACCINE (VC1): Hiroki Bukawa, Kenji Hamajima, Kenji Okuda: Department of Bacteriology, Yokohama City University Objective.: We have exploited the vaccine against HIV. Methods: Several V3 regions (consensus common amino acids sequenses, IIIB, Thai A, Thai B and Japan type) and one gag region (HGP-30) was synthesized using model 430A peptide synthesizer. Japan type and Thai B peptide were synthesized by MAP method. After synthesis, consensus common peptide was modified for constructing conformational type. These peptides were conjugated using glutaraldehyde method, were constructed as the macromolecular substance. The novel multicomponent vaccine was named VC 1. Seven rabbits were immunized by subcutaneus injection of 200.tg/kg of VC1 with or without adjuvant. Several mice were immunized orally with VCI plus cholera toxin. Results: VCI indicated strong immunogenecity, by ELISA enough antibody titers were obtained without adjuvant. Antisera immunized by VC1 inhibited cell to cell fusion approximately 5080%. Not only serum IgG but mucosal IgA were obtained by oral immunization. The results suggested the possibility of oral vaccination. Discussion/Conclusions: The synthetic peptide vaccine is advantageous for taking measures against HIV cell tropism. VCI which contains the conformational parts is the largest multicomponent peptide vaccine we have developed. The use of VC1 with DNA vaccine would be one of the strongest strategy against the HIV prevalence. Hiroki Bukawa, Department of Bacteriology, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan telephone 81-45-787-2602, telefax 81-45-787-2509 PA0365 ADJUVAvT SIRA'IEGY FOR TI-t1 IYPE IML"rNIIY M. Sugimoto, K. Ohishi, M. Fukasawa, K. Shilkata, H. Kawai, H. Itakura, R. Sakaklbara, M. Ishlguro, M. Nakada, T. Mlzuochi, and M. Hatanaka. Inst. of Trop. Med. & Dept. of Pharn. et., Nagasaki Unlv., and School of Engineering, T'okal Univ. Objective: Development of safe Ilposome aUdjuvant for the induction of TIl-1 type cell-mediated Immunity (CMI) applicable to AIDS vaccine. Methods: Construction of liposomnes coated with various ollgo- and polymannose containing ovalbumln and retrovlrus peptldes. Imnmtuiogenclty was assessed by a delayed-type hyperseinsltlvlty (DTH) response and In vitro lyrnphokine production. Results: The Inoculation of these liposomes in mice induced a U11 response, and the production of IFN- r, but not of IL- 4. It was shown that the coating of lilposomes with mannose-contalning sugar was essential for the activity. Discussion and Conclusions: Since liposomes made of physiologically ubiquitous components were effective, the present system Is considered to provide the basis for the development of a safe adjuvant for AIDS. SUGIIvKITo, Masanobu, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto-machl, Nagasaki, 852, Japan Telephone 0958-47-2111(ext. 3753); Fax 0958-47-6607 PAl366 lly Isolates repositories in the developing world: Establishment and I'reliminary Characterization _B igido Luis Fetmando de Macedo*;Beltbro P.C.B.*.Rossini,M.A.A.*;Oliveira,M.I.*:Santos,V.A. Wakin,V.L-I;Duarte, A.J.S.4 *Retrovinis Laboratory. Virolo Service, Instituto Adolfo Lutz &4mmunologv & Allergy Service, Hospital da ('linicas, Faculty of Medicine USP, Sao Paulo, Brasil Objective: The development and preliminary characterization of a repository of biological material and isolates fioun HIV positive individuals prospectively followed at the great Sao Paulo area. Patients and Methods: 176 Plasma(ACD) and 81 PBMCs(heparin). were obtained simultaneously with (-'4 counting by IF -'ells (FCS/DMSO and PCR solution) and plasma were stored 78 primary plasma or micro co-cultures were set (ACTG protocol adapted). Positivity was assessed in 16 of those by the detection of vital antigens in serial supernatant. PBMCs florn blood bank bags vohune inadequate fo transfusion were also used. Expansions were obtained according to the WHO protocol. Results: Plasma cultures from ansymptomatic. ASX tmean CD4 = 463) and from ARC/AIDS patients (mean ('D4 =412) showed mostly a first day positivity at the 2nd week of culture The latter had a higher positivity (78%,7/9), as compared to the ASX group (25%, 5/20). Positivity of female (3I 1) and male (2/9) was simnilar. Micro co-cultues from ASX showed a positivity of only 62%(16/26), but at least 1/3 of those were already positive on the first week of culture. 11 expansions have been obtained so far. Conclusions: We started a repository of plasma. cells, primary isolates and expansions from So Paulo individuals, mostly with sexual acquired HIV. Extended characterization, neutralization and DHMNIA are currently being evaluated. The inability to provide ideal culture conditions tluroughout the study curtail analysis, but both plasma and micro co-culture growth kinetics are relatively fast,even in asymptomatic patienls.The use of cells frliom discharged blood bank bags, retised due to their under optimal volume. allow the supply ofa cnici:al element for HIV cultures with the sparing of needed blood bank supply The infrastructure for this work is still insufficient and therefore consistent alternative international supporting mechanisms should be generated. luis Femrnando de Macedo Brigido Retrovirus laboratory, Servigo de Virologia - Instihtuto Adolfo lutz Ave Dr. Arnaldo 355, Sbo Paulo SP Brasil 01246 phone 55 11 8510111 ext. 170; Fax 55 11 853 35 05 0 A) CA) 0 CA) a)

Page  122 K3 AO36~I7 Buy-i V LtOOPS!QUENC FRO GUUDISTRCT (FANDA,. UM Girado3Gaetano, Buonaguro F.M., Greco4D., BethGirado E., Luwiya M., Sempala S.D K.. I.N.T., Naples, Italy4 I.S.S., Rome, Italy, Lacor Hospital, Gulu,Uganda; UVRI, Entebbe, Uganda. Objective: To determine the HIV-1 genetic variatility, necessary for development of anti-HIV-1 preventive vaccines and/or immunotherapies, we are sequencing the third hypervariable region (V3) of the HIV-1 env gene from an Ugandan rural area in the Gulu District. The nucleotide sequence is compared with the major US/European HIV-1 strain (HIV-1MN), which is also the most represented HIV-1 variant in Italy (L.Buonaguro et al., AIDS, in press). Methods: Peripheral blood leukocytes (PBLs) have been isolated from fresh blood samples by Fycoll-Hypaque centrifugation and lysed at 56~C in the presence of Proteinase K. The 7009-7331 region of the env gene is amplified by Polymerase Chain Reaction (PCR) and the products are subjected to direct sequencing analysis after a modified precipitation step in PEG (M.L. Tornesello et al., Biotechniuue, submitted). Results: HIV sequence analysis is performed on sibjects screened by serological (IFA, ELISA and Western Blot) and molecular techniques (PCR for the most conserved HIV-1 gjg gene sequences). The HIV-1 strains, characterized by direct sequencing, can be assigned prevalently to B (MN-like) and D subtypes; in progress is the evaluation, by sequencing subcloned fragments, of intra-atient variation frequency. Discussion and Conclusion: The data obtained should allow the identification of conserved peptides able to induce a broad immunologic anti-viral response. These studies could have a relevant impact on the establishment of efficacy trials for anti-HIV-1 preventive vaccines, reccomended by WHO in Uganda, one of the 4 world countries with the highest incidence rate of HIV-1 infection. GIRALDO, Gaetano, Div. of Viral Oncology Ist. Naz. Tumori "Fond. Pascale", cappella Cangiani 80131 Naples Italy Tel./Fax 39.81.5451276 PA0368 v3 EPITOPE-GRAFTED IMMUNOGLOBULIN AS A NEW AIDS VACCINE CANDIDATE, Yoshihiro KUMAGAI, Biomaterial Research Institute 244, Japan. Objectives: The synthetic peptides corresponding to the principally neutralizing determinant (PND) in the V3 loop domain of HIV-gpl20 are known to elicit the potential protective immune response but their immunogenicity is poor and their halflives in the body are generally short. The specific aim of this study is to induce the strong immune response against PND by the immunization of V3 epitope grafted immunoglobulins. Methods: The antigen-binding or complementarity-determining regions (CDR) of immunoglobulin (Ig) molecules represent an array of peptides which are as diverse as B and/or T cell epitopes and are also comparable in size to express epitopes. In order to present the V3 epitopes efficiently to the immune system, murine artificial self Ig, whose CDR loops of the hypervariable regions of heavy chains (CDR2 and CDR3 of VH) and those of light chains (CDR1 and CDR3 of VL) were replaced by part of the V3 loop region of HIV MN homology group by molecular grafting, were constructed. Results: The epitope-grafted immunoglobulins were found to carry V3-epitopes, and could not only induce anti-V3 antibody response, but also prime HIV specific T cells in vivo.. In addition, the antigen presentation efficiency was 500 to 1000 times more than free synthetic peptides by internalization with Fc receptors for Ig on antigen presenting cells (APC). Discussion and Conclusion: The immunogenic properties of V3 epitope-grafted immunoglobulin suggest that this version of immunogen can be an improved and safer vaccine for the induction of HIV-specific protective immune response. Yoshihiro Kumagai, Biomaterial Research Inst., 1 Taya-cho, Sakaeku, Yokohama 244, Japan. Tel: 045-851-9271, FAX; 045-853-3528 -o 0 0) mcJ -o ~0 0 PA0369 SYNTHETIC GP20-DERIVED PEPTIDE IMMUNOGENS INDUCE IL-4 AND IL-6 SECRETION IN VIVO Klinman Dennis Haynes, BF+, AND Conover, J. * CBER/FDA or + Duke Univ Med Cntr, USA OBJECTIVE: To investigate the capacity of synthetic gpl20 -derived peptides to induce murine spleen cells to release IL-4 and/or IL-6 under physiologic conditions in vivo. METHODS: Peptides were synthesized expressing the typespecific determinant of the V3 loop region of gpl20 (SP10) C-terminal to a conserved T helper epitope (Tl) on the same molecule. Cytokine-specific ELIspot assays were used to examine the number, kinetics and fine specificity of cells activated to secrete IL-4 and IL-6 in vivo in mice immunized with these Tl-SPlO peptides. RESULTS: i) IL-4 and IL-6 producing cells were activated by T1-SPO0 peptides in a dose-dependent manner; ii) boosting increased the number of lymphokine producing cells; iii) both the V3 loop region and the T1 epitope induced lymphokine secretion and iv) the order in which mice were immunized with T1-SP10 peptides influenced the magnitude and cross-reactivity of the ensuing antibody response. DISCUSSION AND CONCLUSIONS: Gpl20 subunit immunogens stimulate both lymphokine and Ab secreting cells. Early intervention with such an agent might induce an anti-HIV response optimized for cross-reactivity/protection. KLINMAN, Dennis M, Virology, Bldg 29A Rm 3 D 10, CBER/FDA, Bethesda, MD. USA 20892 Telephone (301) 496-8492 FAX (301) 496-1810 PAO370 IMMUNOGENECITY OF MACROMOLECULE SYNTHETIC PEPTIDE (AG3) CORRESPONDING TO THE EPITOPE OF V3 REGION IN HIV-I ENVELOPE gpl20: K. Hamajima! ), H. BukawaI ), A. Yamamoto2), K. Okuda": 1)Department of Bacteriology, Yokohama City University and 2)Terumo Corporation R and D Center. 1500 Inokuchi, Ashigarakami-gun, Kanagawa Objictive: The immunogenicity of synthetic peptide with the V3 in the envelope gpl20 of HIV-1 was examined. We developed 3V3 synthetic peptides of 21-24 resudies corresponding to PNDs of 3HIV subtype, i, e., IIIB, Thai A and Thai B and coupled these peptides with glutaraldehyde (AG3). Results: The injection of this macuromolecule (AG3) with FIA induced cytolytic activity to syngenic target cells pulsed with V3 peptides and rapid apperance of anti ELISA titer to each peptide and also rgpl120. Furthermore these sera showed the neutralizing and fusion blocking activity.Our macromolecular peptide was safity and useful immunogen when injected with Freund adjurant. Discussions/Conclusions: The macromolecular synthetic peptides corresponding to PNDs with or if possible, without adjuvant is to be as value for HIV peptide vaccine. Kenji Hamajima, Department of Bacteriology, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan Telephone 81-45-787-2602, Telefax 81-45-787-2509

Page  123 PAO371 IMMUNOGENICITY OF AN HIV-1-MULTIEPITOPE PEPTI DE(MEP).Montero,M.;Duarte,C.;Menendez Alfredo Navea,L*. et.al. CIGB,*AIDS Ref.Labs,Havana. Objective:To determine whether a protein comprising epitopes from V3T region of env from several HIV-1 isolates is able to elicit an immune response against all these isolates. Methods: A gene was synthesized which includes 15 aa from env V3 loop of six isolates (MN, BRVA, JY1,RF, IIIB,LR150) joined by an spacer sequence. The gene was expressed in E. coli and the protein TAB-4 purified and inoculated subcutaneously to rabbits. Antibody titers and HIV(MN) neutralization capacity of the sera were evaluated. Results: All rabbits seroconverted after the second dose. After four inoculations the animals receiving 200ug/dose developed antibody titers from 1:6000 to 1:1000000 against all six peptides and neutralized the MN isolate (1:80,1:320). ELISA and neutralization titers of the animals immunized with 50 ug/dose were lower (<1:100 to 1:100000 for antibodies and 1:40 for neutralization). Conclusions: It was demonstrated that our MEP elicit antibodies against all the isolates included in the protein when inoculated to rabbitts and that the sera from these animals are able to neutralize at least the MN strain "in vitro". PA0372 DNA VACCINE INDUCES STRONG CTLS AGAINST IIIV-I TARGET CELLS. Okuda Kenii', Bukawa, I.*, Ilamajima, K.', Fukushima, J.', Yamamoto, A." Kawamoto, S.'*Dept. Of Bacteriol. Yokohama City Univ. Sch. or Med., Yokohama, Japan, "*Terumo Corporation R and D Center, Kanagawa, Japan. Objective: DNA vaccines for IIIV-I infection was studied by using env and rev genes. Method: Mice and guinea pigs were intramuscularly injected at one time in. The construct was the mixture of Cytomegalovirus promotor-region DNA, which was donated from Dr. B. Cullen, Duke Univ., N.C., and gpl60 DNA. In addition, we added Rev trans-activation gene for env. gene expression. Results: After 8 days, we could observe anti-IIIV antibodies, including antigpl20 and anti-V3 loop antibodies, by ELISA. After 5 weeks the antibody titer was 51200x and the titer is still increasing. We could also observe the strong both anti-fusion and neutralizing activities using guinea pig sera. The delayed-type hypersensitivity using foot-pad swelling response was also positive, when we injected gpl20 or V3 peptide antigens. In addition, a strong level of cytotoxic T lymphocytes (CTLs) against HIV-1 infected target cells was also observed. DiscussionlConclusions: DNA inoculation mimics the aspects of attenuated vaccine. Because it has been reported that in vivo synthesis of specific proteins and binding the antigenic peptides with class I molecules in the cell are essential for inducing CTLs. Our present results are suggesting that these DNA vaccine could induce both humoral and cellular immunity. In addition, studies of the combined procedure with muliticomponent peptide and DNA vaccines are also now progress. OKUDA, Kenji, Dept. of Bacteriol., Yokohama City Univ., Sch. of Med., 3-9 Fukuura, Kanazawa-ku, Yokohama 236. Tel. (81)-45-787-2600; Telfax (81)-45-787-2509 PA0373 COMBINED MODALITY IMMUNIZATION ELICITS SIVSPECIFIC CTL. Yasutomi. Yasuhiro', Alving, C.R.2, Wassef, N.2, Conard, P.', Conley, A.J.', Emini, E.A.', Madsen, J.', Woods, R.', Koenig, S.', Letvin, N.L.' 'Harvard Medical School, New England Regional Primate Research Center, Southborough, MA, 2Walter Reed Army Institute of Research, Washington, D.C., 'Merck Research Laboratories, West Point, PA, 'Medlmmune, Inc., Gaithersburg, MD. Objective: We have assessed the capacity of combined modality approach using two types of novel vaccines, recombinant BCG-SIVmac gag priming and peptide/liposome boosting, to elicit SIVmac gag-specific CTL in rhesus monkeys. Methods: We have previously shown that SIVmac-infected rhesus monkeys possessing the MHC class I allele Mamu-A*01 develop a gag-specific CTL response limited to a single 9 amino acid epitope (11p IC). Two Mamu-A*01+ rhesus monkeys were immunized with BCGSIVmac gag and 13 months later were boosted with pllC formulated in a lipid A-containing liposome. Control animals included 2 Mamu-A*01O- monkeys similarly immunized, and pairs of naive Mamu-A*01+ and Mamu-A*01- monkeys immunized with p1 IC/liposome alone. Results: The SIVmac gag-specific effector cell response in Mamnu-A*01 + monkeys elicited by combined modality immunization was CD8+ and Mamu-A*01-restricted. The combined modality immunization approach elicited a higher frequency of SIVmac gag p1l IC-specific effector cells than was elicited by single modality immunization. In fact, the CTL frequency elicited by this approach was similar to that of SIVmac-infected animals. Discussion and Conclusions: The capacity of such CTL to contribute toward protective immunity against an AIDS virus infection is presently being evaluated in this model. Yasuhiro Yasutomi, NERPRC, One Pine Hill Drive Southborough, MA 01772. 508-624-8057 FAX:508-624-8161 PA0375 PHASE I STUDY OF HIV-1 MN V3 OCTAMERIC PEPTIDE VACCINE Qgr. Geoffrey J', Keefer M, Weinhold K, Matthews T, Stablein D, Potts B*, Forrest B+, and the NIAID AIDS Vaccine Clinical Trials Network (AVEG) "Saint Louis University and VAMC, St. Louis, MO, USA; *United Biomedicals, Inc., Hauppauge, NY, USA Objectives: To evaluate the safety and immunogenicity of an HIV-1 MN V3 octameric peptide vaccine (United Biomedicals, Inc., UBI) in low-risk, uninfected volunteers. Methds: In a double-blind, randomized clinical trial, 36 volunteers were enrolled and completed follow-up. Volunteers received either 20g, 100 g, or SOug per dose of vaccine (10 volunteers per group) or alum adjuvant (6 volunteers) at months 0, 1 and 6. Measured were clinical and laboratory parameters of safety; and immunologic tests including binding and neutralizing antibodies, lymphocyte proliferation, and anti-HIV-1 cytotoxic T-cell activity (CTL). Results: The vaccine was safe. Antibody responses post third vaccination by AVEG labs were as follows: Number Positive/Number Tested (Mean Value of Positives) Vaccine Monomeric V3 Peptide Octameric V3 Peptide HIV-I MN Dose (ue) (O.D.-Cut-off) (O.D./Cut-off) Neutralization (GMT) 20 3/10 (0.37) 4/10 (6.35) 3/9 (30) 100 1/8' (0.50) 318' (2.95) 1/7'(27) 500 2/10 (0.42) 7/7' (4.39) 1/3'(87) "Additional results pending. Preliminary results at UBI suggest larger numbers of higher vaccine dose recipients developed binding and neutralizing antibodies afterthe third immunization. Lymphocyte proliferation was detected to V3 octameric peptide in 14/18 and to rgpl60 MN in 6/18 volunteers tested. CTL activity was present transiently in 2 volunteers. Discussion and Conclusions: The vaccine was safe, and induced T-cell memory and binding antibody for vaccine antigen, and neutralizing antibody to HIV-1 MN virus in some vaccinees. Geoffrey J. Gorse, M.D., Div.of Infectious Diseases Saint Louis University Health Sciences Center 3635 Vista Avenue, FDT-8N, St. Louis, MO 63110 USA telephone 314-577-8648; fax 314-771-3816 -o 0 CA) 0 CA) *4

Page  124 PA0376 PPD-V3 LOOP PEPTIDE CONJUGATE VACCINE. Rubinstein Arye, Goldstein H, Cryz S*. Albert Einstein College of Medicine, Bronx, ew York; Swiss Serum and Vaccine Institute, Berne, Switzerland* Objective: To further assess the toxicity and immunogenicity of the PPD-V3 loop peptide conjugate vaccine in human volunteers. Methods: A 13 aminoacid MN V3 loop peptide was coupled to PPD by glutaraldehyde. Submicrogram doses were administered intradermally to HIV negative volunteers on days 0, 14, 28, 82 and 383. Results: No systemic adverse reactions were noted. Local skin reactions were mild and transient. Immune responses were restricted to PPD skin test positive volunteers and last for over 1 year. High titer and high affinity serum IgG (mainly lgG3) and IgA antibodies were detected. Specific IgA antibodies were present in the saliva. Antibodies neutralized the HIVI MN prototype. HLA-B7 restricted primary cytotoxic T cells (CTL) were also elicited. Discussion and Conclusions: The MN-V3 loop-PPD conjugate vaccine is well tolerated and induces at submicrogram doses immune responses in HIV-I seronegative PPD skin test positive volunteers. Long lasting humoral (lgG and IgA) and mucosal (IgA) immune responses were associated with HLA-B7 restricted primary CTL. Dr. Arye Rubinstein, AECOM, F401, 1300 Morris Park Ave, Bronx, NY 10461; Phone: 718-430-4227; Fax: 718-931-9213 PA0377 RAPID DETECTION OF HIV-1 DNA BY FLUORESCENCE IN A SEALED CONTAINMENT CASSETTE HIELDS, Robert Objective: To develop a sensitive, high-speed PCR technology for quantitation of plasma HIV, the number of infected cells, and the level of HIV replication, using a PCR technique that eliminates the risk of "carryover" DNA" contamination. Methods: Amplifications were carried out using a high speed air thermal controller, 5'Iluorescently labeled primers, with hybridization probes covalently attached to the inner surface of sample tubes. Tubes were placed in a containment cassette having compartments capable of forming connections with the sample tubes. After thermal cycling, fluorescently labelled target amplified DNA products, captured by the hybridization probes, were detected by fluorescence. Results: Samples containing HIV-1 DNA were amplified by the PCR (30 cycles) in under 10 minutes and amplification products were detected in the sample tube using fluorescence, without opening the sample tube or exposing the environment to the DNA amplified products. Discussion and Conclusions: The use of a DNA containment cassette with fluorometric detection of products provides a format for the development of automated clinical PCR to quantitate the number of infected cells or the level of viral replication in a clinical sample in under 20 minutes. Elimination of "carryover DNA" allows the PCR instrument to be used in any location with no requirement for separate sample-preparation facilities or for special ventilation. FIELDS, Robert, Institute for Therapeutic Immunology, 11 Waverly Place, New York, NY 10003, USA Telephone: (212) 673-3405; Fax: (212) 673-3969 "o 0 01 0 CA) 00 PA0378 THE EFFECTS OF CY OKINrES ON ANTIBODY-DEPENDENT ENHANCEMENT OF HIV-1 INFECTION. A.Takeda, U.Kontny*, 1.Kurane*, J.E.Monroe**,and F.A.Ennis*. Div. of Clinical Immunol., Jichi Med. School, *Div. of Infectious Dis. and Immunol., and **Dept. of Pharmacol., University of Massachusetts Med. School. Objective: We have reported serial studies on antibody-dependent enhancement of HIV-I infection and have elucidated some of the mechanisms. This study was conducted to analyze the effects of cytokines on the enhancement of HIV-1 infection mediated by FcR. Methods and Results: U937 cells, a human monocytic cell line, and human monocytes, both of which possess FcRI and FcRll, were infected with lIV-I in the presence of IgG from an HIV-1 antibody positive serum, and the virus replication was monitored by the assay for p24 antigen, the indirect immunofluorescence assay, and the detection of viral RNA using cytoplasmic dot-blot hybridization. The enhancement of infection was found at a 106 or 106 serum dilution equivalent of IgG. Pretreatment of U937 cells with IFN-gamma suppressed infection by the virus alone, whereas this pretreatment augmented infection by the virus complexed with antibodies (the enhancement indices increased from 2 to 6). The augmentation appears to be due to the increase in the number of FcRs after IFN-gamma treatment. Next, we examined the effects of IFN-gamma, GM-CSF and MCSF, on the infection of peripheral blood mononuclear cells in the presence of antibodies. Pretreatment of PBMC with IFN-gammma or M-CSF increased the antibody-mediated infection, but GM-CSF treatment decreased it. Conclusions: Cytokines known as the activator of macrophages modulate the antibody-mediated infection enhancement, which may be important to understanding the in vivo role of enhancing antibodies and require careful consideration concerning the administration of cytokines to HIV-1 infected individuals. TAKEDA, akira, Division of Clinical Immunology, Jichi Medical School, Minami-kawachi-machi, Tochigi, 329-04 Japan Phone: 0285-44-2111 Ext. 3463; FAX: 0285-44-2779

Page  125 PBO512 USE OF INTRAOCULAR GANCICLOVIR IN EYES WITH CMV RETINITIS PREVIOUSLY H AVING UNDERGONE SILICONEOILTAMPONADEOF A RETINAL DETACHMENT Ussey.Fred M. I. Park Plaza Hospital, Special Diseases Unit, Houston, Texas. USA QObjective: To determine the safety of using intraocular ganciclovir in eyes having undergone prior vitrectomy with silicone oil tamponade for repair of cytomegalovirus-induced retinal detachment. Method: An eye with sight-threatening CMV retinitis and optic neuritis that had been unresponsive to maximum-dose intravenous foscarnet therapy for three weeks was treated with serial injections of 200 micrograms of ganciclovir into the silicone oil-filled vitreous space every 72 hours for 4 weeks. Visual actuity and retinal appearance were monitored, and retinal status was documented by fundus photography. Results: Gradual suppression of the retinitis was achieved. Visual acuity remained "Hand Motion" throughout the course of therapy. Conclusions: The use of an aqueous solution of ganciclovir in a silicone oil-filled eye has been the subject of widespread debate. This case demonstrated that emulsification of the ganciclovir solution in the silicone oil can be avoided, allowing the "bubble" of ganciclovir to settle to the silicone oil-retinal interface and then disperse uniformly over the surface of the infected retina. This knowledge makes it feasible to use intra-ocular ganciclovir in eyes with sight-threatening CMV retinitis that were previously thought to be ineligible for such therapy. Fred M. Ussery, III, M.D. 1213 Hermann Drive # 380 Houston, Texas, USA 77004 713/790-1954 713/523-0423 PB0514 FOSCARNET AND GANCYCLO' R ASSOCIATED THERAPHY FOR CYTOMEGALOVIRUS RETINITIS IN AIDS PATIENTS Ursitti M.A. Zotti C.A'. Gaf S. Giudici M.G. Testa L. Corradinil R. Chiaravalloti F'. Bonazzi L. Div. Malattle Infettive. Div Oculistica'. USL 9. Reggio Emilia. Italy Introduction: Cytomegalovirus Retinitis (CR) develops in a high percentage of patients with advanced AIDS, although it may be frequently the first opportunistic infection. The median survival lime after the first diagnosis of Cytomegalovirus (CMV) infection is 10 months. Gancyclovir and Foscamet demonstrated high efficacy on CR with a response rate of more than 80%. Howewer relapses are commonly seen, with both the therapeutic regimens, 1-5 months after the induction treatment, inspite of the maintenance therapy, and, as a consequence, the surviving patients esperience a number of relapses that may determine a total visual loss. Gancyclovir and Foscamet are commonly employed as monotherapy even if a synergistic activity have been shown in vitro and their combined use has been episodically described in vivo. Objective: to evaluate the tolerance of the associated therapy in patients that experienced more than one relapse with both the drugs on maintenance monotherapy, or exhibited no response of CR on induction therapy although the drug was changed after the demonstration of no efficacy. Methods: The patients with advanced AIDS and CR, after a second relapse on maintenance therapy following a reinduction with the same drug, or when a poor response was evident on Induction therapy with one of the two drugs, were treated with the other one for reinduction and maintenance. The employed doses were Gancyclovyr 5 mg/Kg/bid. and Foscamet 90 mg/Kg/bid. for induction or reinductions after relapses, Gancyclovyr 5 mglKg/d. or Foscamet 90 mglKg/d. as maintenance. When both the single drugs had been used with evidence of no response to the induction treatment or repeated relapses on maintenance, the patients were considered elegible to the associated therapy. Unresponsive were defined the patients who had an active lesions persisting on the posterior pole and/or ncreasing in size after 21 days of induction treatment and/or a development of blateral CR after a prior monocular involvement. AN the patients had weekly clinical and biochemical controls and retinal photographs during the folow up. The ophthalmological examination was performed every week by the same ophthalmologist. The treatment schedule was Gancycdovyr 5 mg/Kg/bid. + Foscamet 90 mg/Kg/d. As maintenance Gancydovyr was reduced to 5 mg/Kg/d. associated with unchanged doses of Foscamet. Results: 7 patients were considered elegible for the associated treatment. The mean follow up was 98.7 days. In 7/7 patients the progression of CR was stopped; in 4/7 a complete response was evident. In 217 a retinal detachment was observed, in one of them during the Induction treatment, in the second after a complete regression of lesions. In 217 patients the associated treatment was interrupted for a severe transient decrease of creatinine clearance after 21 and 60 days respectively. 4 patients showed severe neutropenia: Fllgrastim was episodicaly administered. Relapses were seen in 3 patients, while were treated with associated maintenance therapy, after 92, 23, 42 days respectively from the end of the induction treatment. PB0513 A LONGITUDINAL COMPARISON OF RETINAL FUNCTION OF INTRAVENOUS GANCICLOVIR VS. THE CHIRON INTRAOCULAR IMPLANT IN AIDS PATIENTS WITH CMV RETINITIS (P. Latkany', K. Frost', W. Seiple',K. Holopigian') New York University Medical Center, NY, NY',Treatment Action Group/NY2. Purpose: Clinical, electrophysiological and psychophysical measures were used to compare retinal function in AIDS patients with CMV retinitis receiving either intravenous ganciclovir or the ganciclovir intraocular implant. Methods: Complete clinical histories, routine laboratory tests, and dilated fundoscopic exams were obtained. In addition, rod-mediated psychophysical thresholds were measured as a function of retinal eccentricity. Rod-mediated full-field flash electroretinogram (ERG) responses were then obtained as a function of flash intensity (Viogl). Following ten minutes of light adaptation, photopic ERGs were obtained. Retinal function was measured pre-operatively, and then two weeks, three months and six months post-operatively. Results: At baseline, in all patients with CMV retinitis, the ERG b-wave amplitudes were reduced, b-wave implicit times were delayed and psychophysical thresholds for detection of light were elevated at all retinal locations, indicating widespread retinal dysfunction. Preliminary results of an ongoing study suggest psychophysical and electrophysiological parameters do not indicate any difference in retinal function after insertion of the intraocular implant when compared to intravenous ganciclovir. Conclusion: Patients with AIDS and CMV retinitis have globally decreased retinal function. This preliminary data suggests no additional detriment in retinal function attributable to either intravenous ganciclovir or the ganciclovir intraocular implant. Kevin Robert Frost One Bank Street, #4L New York, NY 10014 Tel & Fax (212) 675-6459 L'SER TREATMENT ON RET rOCALLEtESIS AS PROPtLAXIS OF RETW4AL DETACHMENT CAUSED P B0515 sY CYTOMEGALOVIRUS AND HERPES SNP.EX VIRUS RETNIS 5N PATIENTS WITH AIDS. ZotDCA'" -M S.ki5 MAT eslL BancE.usd M.O. Coadn R. BonA L FL CrawO F.' t, Ocoltsia,'. DDv. Mae, a Ik f tUSL 9 R.a aRs0m, Rely Introduction: Cytomegalovirus (CMV) and Herpes simplex (HSV) retinitis frequently develope in patients with advanced AIDS and in more than 29% of them may lead to retinal detachment (RD) and consequently to a total visual loss. A retinal detachment surgery is under dicusslon because of the poor life expectancy of such patients, the observed postoperative optic-nerve atrophy and the severe retinal damage associated to the infection. RD is determined by the virus induced necrotic lesions. In most Istances early focal lesions predictive of RD may be discovered by frequent ophthalmologic controls. Objective: to evaluate the tolerance of laser treatment on retinal focal lesions as a prophllaxis of complete RD. Methods: 6 patients affected by AIDS, 5 with CMV retInitis, 1 with HSV retinitis, that completely responded to the nduction therapy (3 had been treated with Gancyclovir 5 mgJkgJbid., 2 with Foscamet 90 mgJkgbid. 1 with Acyclovir 15 mg./kg/tid l.v.), had a laser treatment because initial RD was observed during the manteinance therapy (Gancyclovir 5 mg./kg.Id., Foscamet 90 mgJkg/d., Acyclovir 1600 mg/d./p.o.). The patients have had an ophthalmological examination every week from the onset of the retinitis and the weekly examination was pursuived by the same ophthalmologist for a mean follow up period of three months after the laser treatment. Results: no intraoperative or postoperative complications were encountered; a regular scar formation was observed on laser spots. None of our patients demostrated recurrence of retinitis in correspondence of laser scars. No RD was observed over a three months period of folow up. Conclusion: laser treatment on focal RD seems well tolerated in patients with HSV or CMV retinitis. More studies are needed to assess the long term efficacy of laser therapy on preventing complete RD and on prolonging vision in such patients; moreover we believe that a further studly to evaluate the usefulness of laser treatment on larger lesions or focal RD located on the posterior pole is warranted. -I 0 "' MO 0 MA Cor Dr. Sergio Gafa' Divisone Malattie Infettive - Arcispedale Santa Maria Nuova - VIale Risorgimento, 80 - 42100 REGGIO EMILIA (Italy) Tel: 0522/296407 Fax: 0522/296266 F; U'

Page  126 SPB0516 CMV ANTIGENEMIA IN AIDS PATIENTS RECEIVING GANCICLOVIR FOR CMV RETINITIS. Magno M, Sebastiani G, Folgori F, Mastroianni CM Vull Vincenzo, *Volpi A. Infect. Dis. La Sapienza University, and *Tor Vergata University, Rome, Italy Objective: To evaluate CMV antigenemia in AIDS patients with CMV retinitis treated with ganciclovir. Methods: Fourteen consecutive patients with a first episode of retinitis (9 males and 5 females, median age at diagnosis=31; median CD4+ cell count at diagnosis=21/mm3) were followed up for CMV antigenemia for 3-16 months. Peripheral WBC samples were tested for pp65 antigen of CMV by IIF using a specific monoclonal antibody. Eight patients have been evaluated from the time of diagnosis, the remaining were already on therapy when the monitoring began. Results: CMV pp65 antigen was detected in a high number of WBC from all patients (8/8) examined before starting ganciclovir, but from only one of the patients on treatment. After the beginning of therapy, all the patients improved and became antigenemia-negative. After 3-5 months of therapy three patients presented with clinical reactivation of retinitis and responded to increased dosage of ganciclovir. In one of them CMV antigenemia was again detected 2 weeks before clinical evidence of progression. Conclusions: CMV antigenemia seems to be a useful marker for monitoring ganciclovir therapy in patients with CMV retinitis. Its role in predicting disease reactivations remains to be established. Vincenzo Vullo, Dept. Infectious Diseases, La Sapienza University, Policlinico Umberto I, 00161 Rome, Italy. Tel. +39 6 491749; Fax: +39 6 4453760 PB0518 EFFECTS OF SERUM STARVATION ON CYTOMEGALOVIRUS INFECTION OF CULTURED HUMAN RETINAL PIGMENT EPITHELIAL CELLS ANDO Yasutakat,2, SOUSHI S.,.3, ARAO Y.1, SATA T.1, and KURATA T.1 I Dept. of Pathol., National Institute of Health, 2 Dept. of Ophthalmol., Keio Univ. School of Med., 3 Dept. of Ophthalmol., Tokai Univ. School of Med., Tokyo, Japan Objective: Cytomegalovirus (CMV) retinitis is the most frequent and significant ocular opportunistic infection in AIDS. CMV retinitis is preceded by the appearance of cottonwool spots in AIDS patients. The disturbance of retinal circulation may influence some serum factors and relate frequent occurrences of CMV retinitis in AIDS. We studied the effects of serum starvation on CMV infection of retinal pigment epithelial (RPE) cells, which are suspected to play an important role in CMV retinitis. Methods: CMV(AD169)-infected RPE cells were cultured in serum-free medium or medium supplemented with 10% fetal bovine serum. Cytopathic effect (CPE) characteristic for CMV was observed. Viral antigens were detected by immunofluorescence and Westernblotting method. Sensitivities of RPE cells to CMV were determined by TCID50 method. Viral replication was assayed by a plaque forming method. Results: The condition of serum starvation prominently enhanced the appearance of CPE and expression of viral proteins in the RPE cells. The sensitivity of RPE cells to CMV was enhanced 13 times by serum starvation. Infectious titers were similar in both conditions. Discussion and Conclusions: The disturbance of circulation may decrease some serum factors affecting the RPE cells. The result in this study indicates that the decrease changes the RPE cells more permissive for an initiation of lytic infection of CMV. ANDO, Yasutaka, Department of Pathology, National Institute of Health, Tokyo, Japan Telephone (8l)-3-5285-1111, Ext.2626; Telefax (81)-3-5285-1150 PB0517 LONG-TERM VISUAL OUTCOME IN CMV RETINITIS. Olmari, Maya, Gabriel V, Sansonetti A, Hirschel B. Infectious Dis. and Ophthal., HCUG, Geneva, Switzerland Objective: To follow the visual acuity of patients with CMV retinitis until loss of follow-up or death. Methods: Review of medical records of all 48 patients with a diagnosis of CMV retinitis at Geneva University Hospital diagnosed since 1983. Survival analysis (Cox-Mantel). Follow-up was complete until death or =7/31/93 for 44/48 = 92%. Results: The Table presents results for 48 affected patients (best eye considered), and for 69 affected eyes, showing the probability of a given outcome in percent at various times after the diagnosis (dx) of CMV retinitis. Patients Eyes Months after dx of CMV Months after dx of CMV 0 1 3 6 12 0 1 3 6 12 Blind or light only 0 2 2 2 7 0 4 12 14 36 Less than 6/10 vision 2 11 21 34 39 0 25 38 50 68 Dead 0 11 39 50 77 At the last examination before death (median time before death: 21 days), 4/42 = 8% of patients were blind. Conclusions: Median survival after CMV retinitis was 6 months. Surviving patients have more than 90% probability of keeping ambulatory vision. Dr M. Olmari, Infectious Diseases Unit, HCUG, CH-1211 Geneva 14. Phone 41 22 372 98 12, Fax 41 22 372 98 20. PB0519 Foscarnet 5 vs. 7 days a week in gastrointestinal CMV-disease Salzberaer Bernd,Stoehr A,Heise W,Jablonowski H,Ewald U,Peters K,Schrappe M UK KSIn, AK St.Georg Hamburg, AVK Berlin, UK Diusseldorf, Astra Chemicals FRG Objectives:To determine the efficacy and safety of foscarnet therapy 5 days weekly vs. 7 days weekly. Patients and Methods: 36 patients were included in a randomized prospective study in four centers, all HIV-infection CDC C3, 36m, 24-49 years, CD4 0-400, median 40/mcl. Patients were randomized to 2 x9Omg/kg Foscarnet i.v. for 5 days a week vs. 7 days a week for 3 weeks, stratified according to upper/lower gastrointestinal tract involvement. At the present time 33 patients are evaluable, 19 with esophagitis, 14 with colitis. A score was used consisting of symptoms, endoscopic evaluation for mucosal ulcers and histology to determine treatment efficacy. Results:9 of 15 (60%) patients treated in the 5 day treatment group had complete or partial remission of symptoms and mucosal inflammation vs. 14 of 18 (78%) in the 7 days group. This difference was not statistically significant. Relevant side effects were reversible nephrotoxicity in 5, penile ulcers in 7 patients, nausea and headache in 7 patients and minor laboratory abnormalities in 8 patients not differing between both groups. Conclusions: 5 days a week treatment with foscarnet 90mg/kg bid i.v. is a safe and efficient treatment for gastrointestinal cytomegalovirus disease. In this study its efficacy was slightly lower than treatment for 7 days a week, though not statistically significant. Outpatient treatment with foscarnet 5 days a week so is facilitated. An update of the data will be presented. Dr. Bernd Salzberger, Med.Klinik I, Univ.-klinik Koln, JosefStelzmann-Str. 9, 50924 KOIn. Tel. 00492214781, Fax 00492215956 00 0 0 J 0 U' -hl...

Page  127 PB0520 COMBINED THERAPY WITH GANCICLOVIR AND FOSCARNET FOR PCMV POLYRADICULOMYELITIS. Karmochkine M*, Lesprit P**, Coutellier A*, Scieux C***, Welker Y**, Ch&rin P*, Modai J**, Herson S*.*Internal Medicine, Salptritre; **Infectious Disease, ***Microbiology, St-Louis, Paris, France. Objective: To evaluate the efficacy of a dual anti-cytomegalovirus (CMV) therapy in 3 cases of CMV polyradiculomyelitis. Patients and Methods: Three men with AIDS, presented with progressive paraparesis in their lower limbs, together with a cauda equine syndrome. Two were previously treated for CMV retinitis: Pt 2 has been treated with Foscarnet for one year, and Pt 3 with Ganciclovir for 6 months. Cerebrospinal fluid (CSF) contained polymorphonuclear leukocytes, hyperproteinorachia and normal or low glycorrhachia. CMV was detected in blood and CSF using culture and PCR, confirming the diagnosis. The 3 patients were treated with combined therapy with full dose Foscarnet and Ganciclovir, instituted 2 weeks after the initial symptoms. Results: 1/Neurological status dramatically improved (i.e. able to walk) after 3 weeks of treatment. 2/ CSF was cleared in blood and CSF in all cases after 3 weeks, as shown in the Table:. Patient 1 Patient 2 Patient 3 CMV viraemia CSF CSF viraemia CSF CSF viraemia CSF CSF culture PCR culture PCR culture PCR JO + + + + - + + + + J21......... 3/ Renal insufficiency was observed in one patient during the fourth week, leading to reduce the dose of Foscarnet; neutropenia in another, leading to institute granulocyte colony stimulating factor. Discussion and Conclusions: After early recognition of CMV polyradiculomyelitis, a combination of antiviral regimen appears as an attractive therapy. It is not ascertained that improvement is due to the early institution of the treatment and/or to the dual anti-CMV regimen. KARMOCHKINE Marina, Department of Internal Medicine, Piti6-Salp triere, Paris, France. Tel: 33 1 42 16 10 62; Fax: 33 1 42 16 10 58 PB0521 CYTOMECALOVIRUS OLYRADICULOPATHY (CMV-PR) IN AIDS: = 5IMPROVEMENT VITH THERAPY. Espinoza LA, Jayaweera DT*, Maiewski S, Fischl MA. University of Miami, Miami FL, U.S.A. Objectives: Therapy for CMV-PR in AIDS patients is ill defined. We present a series of four cases and their responses to therapy. Methods: Admissions to our AIDS unit between 1/92 and 2/94 were reviewed. Criteria for CMV-PR included clinical evidence of polyradiculopathy, characteristic MRI findings, and exclusion of other infections or malignancies (CSF and autopsy). Outcome measures were improvement of motor activity using the Medical Research Council Grading (MRG-CD) and sphincter control. Results: Four patients with AIDS and CMV-PR were identified. All patients were treated with gancyclovir (GCV) or foscavir (FCV). All had severe leg weakness and conus medullaris syndrome at presentation. Patient HRC-GD (time) Treatment MRC-GD (time) Sphincter control 1 1 (0 wks) CCV 4 (12 wks) normal 2 0 (0 wks) GCV 2 (12 wks) absent 3 0 (0 wks) GCV/FCV@ 5 ( 8 wks) normal 4 0 (0 wks) FCV 4 (12 wks) absent @Induction with GCV/FCV for 4 wks then GCV alone. Two patients have died (#1 and 2) at 32 and 16 wks, respectively. Autopsy findings included CMV of the spinal cord and CNS lymphoma (#2). Discussion: This series suggest prolonged therapy with GCV or FCV may result in measurable clinical improvement in AIDS patients with CMV-PR. University of Miami School of Medicine, Miami, FL Telephone (305) 548-4598; Telefax (305) 547-4037 PB0522 GANCICLOVIR THERAPY IN EXTRAOCULAR CMV INFECTION Manata MJ, Forte M, Quaresma MJ,Infectious Diseases Dep. Hospital de Santa Maria, Lisboa Portual INTRODUCTION:CMV infection is almost universal among HIV infected patients, although only a few of them develop specific organ damage requiring treatment. Retinitis is the most commonly recognized disorder caused by CMV in AIDS, with a response to therapy with ganciclovir. Cases of extraocular CMV infection have also been documented to respond to ganciclovir therapy. We present two clinical cases of hepatic CMV infection documented by liver biopsy, in AIDS patients, with a response to ganciclovir confirmed by histology. CLINICAL CASES:The patients were 25 and 50 years-old, male, homosexuals, admited to the hospital with a 3 and 2-week history of fever and generalized symptoms, both of them with a CD4+ lymphocyte count of 10 cells/mm3 and CMV serology positive for IgG and negative for IgM.The diagnostic of CMV infection was done by liver biopsy and they were started on ganciclovir (250 mg 12/12 h for 3 weeks, followed by 250 mg/d, 3 times a week). The 1st patient improved clinicaly with apyrexia and nearly normal laboratorial tests. The 2nd patient remained febrile and with abnormal liver function tests. A liver biopsy, done on the 5th week of ganciclovir, showed regression of hepatic lesions in both patients (complete regression in one patient and parcial in the other). CONCLUSIONS:Systemic therapy is clearly beneficial in CMV retinitis. These two cases of clinical and histologically documented improvement of CMV hepatitis with systemic ganciclovir therapy suports that, in face of a diagnostic of extraocular CMV infection, therapy should be started. Miguel Forte,Servigo de Doenras Infecciosas,Hospital de Santa Maria, Av. Prof.Egas Moniz, 1600 Lisboa,Portugal PBO523 ABSOLUTE BIOAVAILABLITY OFACYCLOVIR IS SUBSTANTIALLY INCREASED FOLLOWING ORAL VALACICLOVIR (VALTREXTM) Bentley Stephent, Soul-Lawton J2, Rolan P2. 'Nippon Wellcome KK, KitaKu, Osaka, Japan, 2Wellcome Research Laboratories, Beckenham, Kent, UK. Oral administration of valaciclovir (VACV), the L-valyl ester of acyclovir (ACV), to healthy volunteers results in plasma levels of ACV that are estimated to be 3-5 times higher than those following 800 mg oral ACV. Formal measurements of bioavailability had not previously been carried out, however, and so the aim of this study was to determine the absolute bioavailability of ACV following oral administration of 1000 mg of VACV. In an open, randomized, balanced two period crossover study 12 fasted healthy volunteers (aged 23-50 years) received 2 x 500 mg VACV tablets or 350 mg ACV as a 70 ml intravenous Infusion over 1 hour. Plasma samples were taken at intervals up to 24 hours following each dose and all urine was collected during this period. Both plasma and urine ACV levels were assayed by RIA. Following oral VACV the mean (tSD) C=, T and AUC of ACV were 29.5 ~ 12.5 M, 1.7 ~ 0.7 h and 89.4 t 19.4 MM.h, respectively. Following intravenous ACV the mean (~SD) Cm,Tmax and AUC were 41.0 ~ 8.4 M, 1.0 ~ 0.1 h and 84.0 ~ 14.2#M.h, respectively. Mean (95% CI) absolute bioavailability of ACV from oral VACV, calculated from the ratio of AUCs after adjusting for molar dose, was 54.2% (49.4-59.5%). The estimated bioavailability of ACV from the ratio of urinary recovery was 52.8%. The mean (~SD) t,2 of ACV following VACV was 2.6 ~ 0.4 h and following ACV was 2.4 + 0.3 h. The 95% Cl of the difference in tilr for VACV and ACV was 0.03 h to 0.45 h. This slight increase in ACV t1/2 following VACV is not clinically significant and is probably due to a small component of continuing absorption of VACV. It is expected that the significantly higher levels of ACV in the plasma that are achieved following oral VACV will provide improved clinical benefit to the patient over and above that seen with oral acyclovir and may extend the range of herpesvirus infections amenable to oral therapy. STEPHEN BENTLEY, C/O JENNY GRICE, PPS EUROPE LTD, WICKER HOUSE, HIGH STREET, WORTHING, WEST SUSSEX, BN 11 1DJ. UK. Tel +44 903 205884 Fax +44 903 234862 0 O W -o 0 UW oA

Page  128 PB0524 Incidence of CMV Infection and Disease in AIDS patients. Claramonte X, Gatell Jose M, Moreno A, Zamora L, Vidal J, Mallolas J and Soriano E. Hospital Clinic. Barcelona. Spain. Objectives: To asses the incidence of Cytomegalovirus (CMV) infection and disease in AIDS patients (Pts) with a CD4+ cell count <200/mm3 and a positive serology (IgG) for CMV. Methods: Blood and urine cultures for CMV (Shell-vial technique) were performed at baseline and every 2 months. Fundoscopic examination, liver function tests, serum amylase and lipase were done at least every 2 months. Results: A cohort of 21 consecutive Pts was recruited. The baseline mean CD4 count was 125t64/mm3, 13 (62%) were drug addicts and 14 (67%) were males. One or more episodes of CMV viremia was detected in 13 (62%) and CMV was isolated at least once in blood or urine in 18 (86%). Two Pts (9.5%; 10 episodes per 100 Pt-years), developed a CMV disease (one episode of hepatitis and one of retinitis). Both Pts had a CD4 count < 100/mm3. CMV viremia was detected only in one of these 2 Pts. Two Pts died during the follow-up. Conclusions: The incidence of CMV infection in AIDS Pts with <200 CD4ns and a positive baseline serology (IgG) for CMV was very high but was not a good predictor for CMV disease. Jos4 M. Gatell. Infectious Disease Unit. Hospital Clinic. Villarroel 170. 08036 Barcelona. Spain. Telf. Int+34+3+4534261. Fax. Int+34+3+4514438. PB0525 (:Y!OMEGALOVIRIIS INFECTION IN PAT11-NTS WIlH AI). 10 RPIAZIL: SIIDY OF 119 CASE=S RY AuTOPSY FXAMINAIION Maria T Edelweiss,A Sfair, LtGolbspatn,M.ronfeld, F Sprint. Hlospital dPe Clinicas de Porto Alegre (HCPA), Brasil1 Objective: Deteremiep the incidnnce and most affected;ite!; by cytromegalovirus (CMV) infection in patients with AlI)S. Patients_& Methods It has been studied 300 consecutive cases of patientwith AIDS who were rlhrnrit.ted to autopsy examinal ion at tC1'A, from Argqust 1989) to May 1993. or.t ions wer sta inl wit, th heeatoxy li in-Ros i, per tiod ic acid Schiff, G rocott, and 7iell-Neolsen. Diagnosis of (:MV was established from direct hi ito lnlical confitmation or)f cvtor rnmaa1ic inc I usion on cytoplasm and nucl eus. CMV encephalitis (dlue to logistic problemsI and retinitis (due to law difficulties) are not included in the samnple. Results:CMV was diagnosed in 119(40%) cases. In 16 cases it was diagnosed in more than one organ. the most frequent involved siteRs were: adrenal, 85 cases; lung, 65; bowels, 41; esoplhagus, 27; liver, 10; pancreas, 7; and stomach and spleen with 5 each. Conclusions:Even without immune-his tochemical techniques or considerirq r et initis and encephalitis C(MV is an important and common opportunistic disease in Brazil. Any physician must considered this pathology whet dealing with patient.s with AIDS. We strengthen the importance of havin accessible drug against CMV to the people with AIDS living in Rrazi 1 who suffers from CMV infect ion. partitlly supported by internutinnal AlI training grant Ifl3-i)ffl0-03, 'Frgartv irtrrpnti.nal Cnter,Nl Sde lweiss, Maria 1. Hnpit.alI do Cl inicas de Porto Alegre, RS, Br.zil. Ruta Fernandes Vieira 2:19/701. lelephone 55-51-2243362 Telefax 55-51-3328324 -Q w O 0 N r40 W 0 Nl PBO526 pMV INFECTION ON AIDS PATIENTS.Potsch DV, Perez MA, Peixoto CAM, Martins MMC, Fonseca IVT. HUCFF/NESC/DI/UFRJ,Rio de Janeiro Brazil. INTRODUCTION: Study clinical and therapeutical spectrum of the Cytomegalovirus (CMV) on AIDS patients attended at the AIDS PROGRAMME. METHODS: A retrospective study in 96 patients with CMV and AIDS was carried out between 1991 and 1993. RESULTS: Mean age of 36.8 (SD=9.16);78 (81.2%) were men and 18 (18.8%) were women. Concerning clinical presentation we have found 60 cases with retinitis (62,5%), 16 with esophagitis (16.7%), 4 with colitis (4.2%), 2 with pneumonitie (2.1 %) and 14 had CMV disease in more than one site. Serological test for CMV antibodies were positive in 50 (64.9% patients and all were already group IV (CDC-1986) when a CMV diagnose was performed (mean of 271.9 days, SD=242.4). 74.5% patients had clinical improvement (in wich 67.2% were diagnosed as retinitis cases).Granulocytopenia bellow 1000 cells occured in 31 (56.4%) of all patients during GANCICLOVIR therapy. Survival after CMV was diagnosed had a mean of 140.3 days(SD=134.1). When data ceased to be gathered for this study, 87 (91.6%) patients were dead or had abandoned follow up. CONCLUSION: In third world countries, there are relatively few studies focusing CMV diasease/AIDS. In our study, we have found the same clinical and therapaeutic behavior described in several studies in developed countries i.e., it points out a terminal condition for AIDS patients with limited chances on its therapy. Peixoto CAM. Fagundes Varela 515/602, Icarai Niteroi CEP: 24210-520; FAX: (021)270-2193. PB0527 CLINICAL IMPLICATIONS OF CYTOMEGALOVIRAL VASCULITIS IN AIDS PATIENTS WITH COLITIS McNamara. Patrick. Garland, J., Gathe. Jr., J., Bernal, A., Clemmons, J., Bockmon, K., Piot, D. Park Plaza Hospital. Special Diseases Unit, Houston, Texas, USA Objective: To describe the finding of cytomegalovirus (CMV) vasculitis in colon and small bowel biopsies of AIDS patients with colitis, and the possible advantage of adding high dose steroid therapy in this patient group. Methods: Retrospective chart review of four patients with colitis and endoscopic biopsy proven CMV vasculitis. Results Four cases of colitis with CMV vasculitis were demonstrated in gastrointestinal biopsies of patients with full blown AIDS between October 1991 and November 1992. The clinical presentation was not distinctive from that of patients without demonstrable CMV vasculopathy, ranging from abdominal pain to chronic diarrhea and gastrointestinal bleeding of variable severity. The age range was 25 to 38 years, and there were no statistically significant differences in clinical laboratory parameters between patients. While the endoscopic appearance in some cases was suggestive of CMV, the histologic correlation was not uniform. Three of these four patients succumbed to progressive disease. In one patient, high dose steroids were added at the onset of conventional antiviral therapy. Significant, sustained clinical improvement was apparent after this addition. Conclusions: Addition of high dose steroid therapy may be of clinical benefit in AIDS patients with colitis having demonstrable CMV vasculitis. Further prospective studies seem warranted based on these findings. Patrick McNamara, M.D. 4101 Greenbriar Drive #200 Houston, Texas 77004 USA 713/520-5537 713/520-1672

Page  129 PB0528 CORRELATION BETWEEN DETECTION OF CMV DNA IN 5 SERUM BY PCR AND ONSET OF CLINICAL SYMPTOMS: Hansen,KarenKaae*,Richsten,A# Hofmann,B et al *Dept of infectious diseases,Hvidovre University Hospital, Copenhagen, Denmark. Objective: To investigate the correlation between a presumed clinical diagnosis of cytomegalovirus chorioretinitis and the detection of CMV DNA in serum samples from individuals with AIDS. Methods: In this study the PCR technique was used to detect CMV DNA in serum from patients with AIDS. Five consecutive serum samples from a total of 52 HIV seropositive male subjects attending an AIDS clinic from january 1991 to december 1992 were investigated. Nine-teen of these individuals had a clinical diagnosis of CMV chorioretinitis (obtained by ophthalmoscopy). Sixteen HIV negative blood donors were also examined. Results: Eleven of the patients with a clinical diagnosis of chorioretinitis were positive for CMV DNA in serum samples taken 3 to 6 months before the clinical diagnosis. Three retinitis patients who initially were negative for CMV DNA became positive after the onset of clinical symptoms. Detection of CMV DNA in serum was, therefore, found to precede clinical symptoms. In contrast 29 of 33 HIV seropositive patients without clinical CMV retinitis were negative for CMV DNA in all five serum samples. Discussion and conclusions: This study shows that the presence of CMV DNA in serum is a useful and sensitive predictive marker of CMV retinitis. Therefore, failure to detect CMV DNA in five consecutive serum samples from five patients with a clinical presumed diagnosis of CMV chorioretinitis should raise questions about the correctness of adiagnosis of CMV chorioretinitis based only on ophthalmoscopy. Karen Kaae Hansen, department of Infectious diseases, Hvidovre University Hospital, Copenhagen, Kettegaard Alle 30, 2650 Hvidovre Denmark. Fax 45 31474979, Telephone 4536322142 PB0529 CYTOMEGALOVIRUS BLOODANTIGENTEST (CMV-AG TEST) AS A MARKER OF FAILING TREATMENT OF CMV DISEASE AND OF CMV RESISTANCE IN AIDS PATIENTS HG Sprenger, J Schirm*, M vd Giessen. TH The, J Welts. University Hospital, Public Health Lab.*,Groningen, The Netherlands Objective: to determine the value of monitoring the CMV-Ag test during antiviral therapy for CMV disease. Methods: CMV disease was treated in 19 patients. Nine patients had retinitis, 2 gastritis, 1 encephalitis and 7 patients systemic CMV disease (wasting with fever). Ganciclovir and/or foscamet was given. Prospectively the CMV-Ag test was performed on blood leucocytes with monoclonal antibodies (against the 65 kD CMV lower matrix protein) and immunoperoxidase staining. The number of CMV Ag positive cells was expressed as negative, low (<15 per 50.000 polymorph nuclear leucocytes), intermediate (<70) or high (>70). Simultaneously CMV was cultured from blood, urine and saliva. The sensitivity of CMV for gancicovir and foscarnet was tested using a shell vial CMV culture system by counting the reduction of the number of CMV antigen positive cells at different concentrations of the drugs. IC50s were determined for both drugs. Results: The 12 patients without systemic CMV disease had always a negative or low CMV-Ag test at the start of therapy. All 7 patients with systemic CMV disease had a high CMV-Ag test. After the first induction therapy the CMV-Ag test became negative in all patients. Maintenance therapy was given to 13 patients: 4 with systemic disease. In 3 out of these 4 patients the result of the CMV-Ag test increased to a high level during maintenance therapy. The rise of the CMV-Ag test preceded clinical deterioration. If a new induction therapy did not decrease the CMV-Ag test treatment with the other drug was started. The course of the CMV-Ag test correlated also well with the detection of resistant virus, In 1 patient to ganciclovir and in 2 both to gancicovir and foscamet. Conclusions: In AIDS patients the CMV-Ag test seems to be an important tool for the early identification of failing CMV therapy and of emerging ganciclovir- and foscamet-resistant virus. H.G. Sprenger, Department of Internal Medicine, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen. Tel.:050-619111. Fax.:050-613312 PB0530 EBVREPLICATIVE CYCLE IN ORAL HAIRY LEUKOPLAKIA MC Meyohas,D Rea,O Ovaguimian,V Marechal,A sergeant,JC Nicolas Service de Maladies Infectieuses,H6pital St Antoine,Paris,France Laboratoire de Microbiologie,H6pital Rothschild,Paris,France Objective: OHL is an EBV productive infection-related epithelial lesion occuring in human Immunodeficiency virus infected patients. Previous studies showed that EBV lytic gene expression was linked with epithelial differentiation. Methods: To characterize the different steps of EBV lytic cycle we analyzed 5 biopsies of OHL with immunofluorescent techniques on frozen sections with the following monoclonal antibodies: AZ125 (Z DNA binding domain), AZ130 (Z nuclear transport domain), 5A9D6 (immediate early transactivator R), H140 (MA) and F3.23 (VCA). Results: All biopsies showed nuclear fluorescence for Z from the stratum spinosum (SP) to the superficial parakeratotic layers. A cytoplasmic signal located in the SP was seen with AZ125. A nuclear signal for R was detected from the upper part of SP to parakeratotic layers. VCA and MA were present in vacuolated spinous cells and in parakeratotic layers. Discussion and Conclusions: We show that the initiation of EBV lytic cycle starls in the lower layers of SP and may require Z transport from the cytoplasm to the nucleus. Virions production takes place in the upper part of differentiated layers. We suggest that Z translocation from the cytoplasm to the nucleus may be required for EBV lytic cycle activation and may involve cellular factors. We confirm that EBV lytic gene expression is influenced by the state of keratinocyte differentiation Meyohas Marie Caroline,Service de Maladies infectieuses,H6pital St Antoine,Paris,France Phone: 49 28 24 38; Fax: 40 19 33 35 PB0531 HERPES SIMPLEX VIRUS INFECTION IN 2 POPULATION GROUPS WITH HIV DISEASE. DEL AMOJ, H BENZ-TIN, HILLMAN J, FORSTER GE. AMBROSE KING CENTRE, ROYAL LONDON HOSPITAL, LONDON El 1BB, UK Objective: To study the clinical presentation of HSV Infections in Black African heterosexual men (BAHM) and White Caucasian homosexual men (WCHM) with HIV disease. Methods: Retrospective study' of 26 consecutive BAHM and a control group of 150 WCHM seen between 1986- 1993. HSV infection was diagnosed only if the culture was positive Results Eight (31%) of 26 BAHr- and 30 (20%) of 150 WCHM had HSV related disease. All BAHM had penile lesions compared to WCHM-1 where 24 had perianal lesions of whom 2 also had penile lesions, 3 had penile lesions alone and 3 had lesions elsewhere Including 2 oral lesions. 4 (50%) BAHM compared to 3 (10%) WCHM had chronic mucocutaneous HSV infection. 6 (75%) of the BAHM compared to 17 (57%) WCHM had a past history of HSV infection. HSV infection led to HIV diagnosis In 4 (50%) of 8 BAHM, in 3 (38%) of whom it was their first AIDS defining illness, whilst in 1, HIV testing was offered in view of frequent recurrences. In WCHM, HSV infection led to HIV diagnosis in 5 (17%), 3 of whom it was their first AIDS defining illness and in 2 cases, increased frequency led to HIV testing. The epidemiological data and relationship to CD4 count will be discussed. Conclusions: There were differences in the clinical manifestations of HSV in BAHM and WCHM: HSV infection in BAHM were all penile and more likely to lead to initial HIV or AIDS diagnosis. 0 H, Ben-Tin, Ambrose King Centre, Royal London Hospital, LondonEl 1BB,UK. Telephone: 071-377 7310 Fax: 071-377 7648 "0 0 0 N -MA r

Page  130 PBO532 HHV-6 AMONG HIV INFECTED INDIVIDUALS AND BLOOD DONORS Zapiola Ines, Savy V, Cando 0, Bouzas MB, Cahn P, Muchinik G, Wainstein C. Hosp. de Ninos R. Gutierrez; IDEFI; Hosp. Fernandez; Secretaria de Salud. Objective: To determine the incidence of HHV-6 and to investigate any association between seropositivity to HHV-6 and HIV. Methods: In 1993, 116 serum samples from 59 blood donors, 29 HIV positive individuals and 28 HIV negative individuals at risk for HIV infection, were analyzed for the presence of HHV-6 antibodies. AntiHHV-6 were studied by IFA using HSB-2 cell line infected with HHV-6o. (NIH AIDS RRRC 8350) at a screening dilution 1:20. Results: 51.7% of the HIV + individuals, 57. of the HIV - individuals at risk for HIV infection and 57.6% of the blood donors were anti-HHV-6 positive at 1:20 serum dilution. Titers from 24 HHV-6 positive blood donors ranged between 1:20 and 1:160, 50% of them with a titer of 1:80. A titer 1> 1:320 was obtained in only one sample. Conclusions: There was no association between HHV-6 and HIV. Individuals infected with HIV or at risk of infection showed seroreactivity to HHV-6 comparable to blood donors. The incidence of HHV-6 infection and antibody titers observed among blood donors is similar to the reported by other groups. Zapiola Ines. IDEFI. Aguero 2117. 1425 Buenos Aires, Argentina. Telephone (54-1) 805-8145; Telefax: (54-1) 806-2078. PB0533 HUMAN PAPILLOMAVIRUS TYPE 64 INDUCES A CHARACTERISTIC DYSPIALSTIC LESION IN VAGINAL MUCOSA IWASAKI. T.1, SATA, T.1,2, KURATA, T.', MATSUKURA, T.3 and SUGASE, M.4 I Dept. Pathol. 2AIDS Research Center and 3Dept. Virol., N.I.II., Tokyo, and 4Dept. Obstet. Gynecol., Nagano Red Cross Hospital, Nagano, JAPAN Objective: Genital infections of human papillomavirus (IIPV) are known to increase in seropositives for HIV. More than 27 types of HPV have been identified in the genital tract of general population. Some lesions such as condyloma acuminatum (HPV-6 and -11) and bowenold papulosis (IIPV-16), are known to be induced by specific types of IIPV. We cloned a genome of HPV-64 from a vaginal intraepithellal neoplasla and studied the pathology of HPV-64 infection. Methods: The biopsy specimens obtained from the genital lesions were fixed in 10% buffered formalin for histological examinations. The localization of the capsid antigen (GSA) and viral nucleic acid was examined by immunohistochemistry and by in situ hybridization, respectively. Extracted DNA was used for typing of HPV-DNA by the Southern blot hybridization after digestion with appropriate restriction enzymes. Results and conclusion: We identified the IPV-64 DNA in 4 cases of vaginal intraepithelial neoplasias. Macroscopically, these were flat aceto-white lesions. Histologically, the lesions positive for GSA showed a rather specific dysplastic changes characterized by presence of multinucleated giant keratinocytes and atypical mitosis. IliPV-64 infections are seemed to induce a pathognomonic histological change and this must be confirmed by further studies. IWASAKI Takuya, Dept. of Pathol, National Institute of Health, Tokyo, JAPAN Telephone (81)-3-5285-1111 ext. 2626; Telefax (81)-3-5285-1150 0 U' W N -o 0 0 U' C0) U' PB0534 HPY INFECTION AlONG HIV INFECTED WOMEN.CLINICAL EVALUATION, CORRELATED WITH HIV(-) WOMlEN Authors: Rodriguez Rios,E.; Macias,J.R.; Masini,R.; Franchi, MfjLloveras, S. and Navarro L. OBJECTIVE: To investigate the prevalence of HPV genital infection in females HIV+/AIDS and to pinpoint the difference in prevalence in HIV negative patients. METHOD: Between 11/92 and 12/93, 160 HIV+ females were studied, and compared to 160 HIVi-) women in a concurrent study. RESULTS: 1) AGE HIV+pts HIV(-)pts 16-25 years 71 (44.4X) 74 (46.31) 26-35 years 73 (45.21) 67 (43.11) +36 years 16 (10) i17 (10.6X) 2) LESIONS HIV+pts HIV(-)pts Vulva 37 (24.4X) 4 (2.51) Vagina 23 (14.4X) 0 Cervix 64 (401) 2 (1.2X) No apparent HPV inf 34 (201) 154 (96.21) Total 160 160 3) INHUNE COiMPROMISE in HIV+ females with HPV genital infection (126 pts) CD,/mm -200 201-497 9+500 48 (381) 431 (34.11) 35 (27.7X11) CONCLUSION: Our data confirm the high incidence of HPV genital infection in females with AIDS with no significative relationship to the inmune compromise. Do not type in this box Macias Jost Rafael. Tel. 571-3641 Jost Cubas 3194. (1419) Buenos Aires. Argentina PB0535 B12 PARVOVIRUS INFECTION IN AIDS Ramratnam, Bharat*, Gollerkeri A, Flanigan T, Schiffman F, Ueda P, Gottlieb F, Deutsch J *Brown U. USA Objective: To characterize the clinical course of B19 parvovirus infection in AIDS. Methods: Retrospective chart review at multiple sites to identify HIV+ individuals with B19 parvovirus infection. Presentation, treatment, and clinical course were reviewed. Results: Four individuals were identified. All presented with profound anemia (Hct < 23.0) and reticulocytopenia. All had CD4 < 60. B19 parvovirus infection was diagnosed by bone marrow biopsy and aspiration and/or by isolation of viral DNA in serum or in bone marrow aspirate. All responded to IVIG (Intravenous immunoglobulin:.1-.4g/kg/day X 2-10 days) with reticulocytosis and resolving anemia. Two required no additional IVIG and had no recurrence of anemia with reticulocytopenia. Two individuals have required bimonthly IVIG over 3 years. In one of these patients anemia and reticulocytopenia requiring transfusions have recurred when IVIG has been interrupted. Follow up ranged from 9 to 51 months. Conclusion: B19 parvovirus infection in AIDS can be successfully treated with IVIG. Persistent infection may require prolonged IVIG therapy. 401-331-8501 fax Bharat Ramratnam, M.D. 401-331-8500 phone The Miriam Hospital 164 Summit Avenue Providence, RI 02906

Page  131 PB0536 DIAGNOSIS, DETECTION AND GENOTYPING OF HEPATITIS C VIRUS IN HIV INFECTED PATIENTS IN A NORTH-WEST CITY IN FRANCE. Clotteau Laurent 1, Mendel I.1, Pfaff F.1, Prevost M.1. Buffet-Janvresse C.1,. Caron F.2, Humbert G2.1.Virology, 2.CISIH, Hosp.C.Nicolle,Rouen.France. Objective To determine HCV viremia (PCR and genotyping) among a random HIV- positive population and to correlate HCV infection with the serological profiles. Methods 161 HIV (+) patients (123 males, 41 females; mean age 36 ~ 10: CDC II (85) and IV(76); 58 D.A., 48 homosexuals, 55 others) were enrolled in the study. HCV markers were tested using 2nd generation Elisa tests (Ortho, Abbott) and RIBA-3 (Ortho). RT-PCR and nested-RT-PCR, using primers from the 5' -NTR of HCV, were performed on 100 samples (50 Elisa (+), 50 Elisa (-)). HCV genotyping and subtyping of the first round PCR positive was done using the HCV LiPA (Innogenetics). Results" 1. The HCV serological follow-up was independent of the HIV clinical stage. 2. ALT levels were normal (<45 IU/I) except in 15 cases PCR (+) Riba (+). 3. PCR results according to the HCV antibody profiles: patterns PCR (+) 22 5 11 15 11 PCR (-) 28 2 1 2 3 4. HCV Riba (+) /viremia (+) patients were more frequent in D.A. than in homosexuals (60% vs.19%). Concldusion These preliminary results suggest: 1. Serology and ALT are not reliable markers of HCV infection in HIV (+) patients. 2. The viremia can be positive independent of the HIV clinical status. - RIBA (+) sera are generally correlated with PCR (+) but RIBA(-) sera are not correlated with PCR (-). - HCV RNA positivity seems to be independent from the ALT increase. 3.The genotypes found in our group seem to be similar to those generally found in Normandy, France. Pr. C. Buffet-Janvresse, Virology. Faculte de Medecine, C.H.U. C.Nicolle 76000 Rouen France.Tel: PB0537 HEPATITIS C VIRUS GENOMIC SEQUENCES IN CEREBROSPINAL FLUID OF HIV-1 INFECTED PATIENTS Morsica Giulia. Gianotti N., Novati R., Bernardi M.T., Migone T.S., Accordini A., Castagna A., Lazzarin.A. Divisione di Malattie Infettive, IRCCS San Raffaele, Milan, Italy. Objective: hepatitis C virus (HCV), the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis, showing homology with the flaviviridae (bovine viral diarrhea, tick-borne encephalitis virus, yiellow fever virus). Recently it has been suggested that HCV may infect pheripheral blood mononuclear cells but there are no information about its tropism for other tissues. Aim of this study was to investigate the presence of HCV RNA in the cerebrospinal fluid (CSF) of anti-HIV-1 and anti-HCV positive patients (pts) with or without HCV viremia. Methods: twenty one anti-HIV-1 and anti-HCV positive pts. suspected to have opportunistic infection of central nervous system (CNS) were subjected to lumbar puncture. Serum samples and CSF were collected in the same day. In 5 pts 2 or 3 following CSF were analyzed. HCV RNA was extracted from 150 ul of serum and 150 ul of CSF by phenol-chloroform extraction, retrotranscribed into cDNA with specific primer and amplified by Nested PCR with primers spanning the highly conserved 5' untraslated region of HCV genome. Amplification products were revealed upon agarose gel electrophoresis by ethidium bromide staining followed by liquid hybridization using an internal 32 P labeled oligonucleotide probe. Results: HCV genomic sequences were detected in 14 out of 21 serum samples tested. HCV RNA was shown in CSF of 4 out of 21 pts studied; in these 4 pts HCV RNA was also present in the serum sample. In 5 pts for whom following CSF were analyzed, HCV RNA persisted always negative in 3 pts, while in the other 2 pts HCV RNA was detected in CSF in 2 subsequent samples. Discussion and conclusions: the presence of HCV genomic sequences in CSF of pts with HCV infection suggests that the CNS may be a very important reservoir for HCV. Further investigations will clarify the tropism of hepatitis C virus for neural tissue. 'oR C A Glltl tv ^, tt" 1 1tet-'A cc.SAA' RAF-A (L, ' c7, HIPFA,b',jNCOA/I eo. "0 2 L 114,.- 'r fe-. 3S-2. 26(,3 905 7'x 3 '.2-.~A, 3 Jo PB0538 COURSE OF HEPATITIS C IN HIV-COINFECTED HEMOPHILIACS Rockstroh Ju0rgen*, Spengler U*, Hammerstein U**, L0chters G**, Brackmann HH**, Oldenburg J**, Sauerbruch T' * Dept. of Medicine and * Institute for Experimental Hemotology, University of Bonn, Germany Objective: To determine the effects of different stages of HIV-infection on the course of HCV liver disease in HIV-coinfected hemophiliacs. Methods: We examined amlnotransferases (ALT, AST), cholestatic parameters (y-GT, AP) and cholinesterase (CHE) from 1990 to 1993 In four groups of patients: A: HIV- + HCV-seropositive hemophiliacs with progressive Immunodeflclency (defined as CD4-counts in 1993 < 150/pI or AIDS); B: HIV- + HCV-Infected hemophiliacs with stable Immune function; C: HCV-positive hemophiliacs without HIV-Infection; D: HIV-Infected homosexuals with progressive immunodeficiency without HCV-coinfection. Results: Pat. n A AST A ALT A y-GT A AP A CHE Group (U/I) (U/I) (U/I) (U/I) (U/I) A 32 13 18 42* 81* -313 B 67 -3 0 4 12 -46 C 71 -1 -3 1 6 -221 L D 24 4 4 7 3 -365 A: difference 1993 vs 1990; * p < 0.01 Conclusion: Deteriorating immune function in HIV- and hepatitis C coinfected hemophiliacs leads to a cholestatic course of liver disease. br *l:v... Ro c ks,ok r'. 4f Mc.).- IU v _r,CA'-; N,1rd Fr e-*R E S & r:V>S 3 eJvs13 -, Gv.,7 PB0539 ANTIBODY RESPONSE TO HEPATITIS B VACCINE IN A HIV CLINIC COHORT Broisman, Larry B., Hartford Hospital, Hartford, CT, USA Objective: A retrospective study to determine the efficacy of Hepatitis B vaccination in a HIV clinic population. Epidemiologic and lab parameters are examined as possible determinants of efficacy. Methods: All patients in the HIV Clinic are screened for Hepatitis B surface antigen and Hepatitis B surface antibody. Patients who test negative for both are offered HepatitisB vaccine. All patients receive recombinant Hepatitis B vaccine according to CDC recommendations as to dose, route and interval. Post-vaccination immunity testing is performed between 1 and 6 months after the third dose. Anergy screening, Hepatitis C antibody and CD4 counts are also determined. Results are analyzed using Chi-square testing. Results: Forty-one patients have received all 3 doses of vaccine with post-vaccination antibody titers available for 20 patients. Ten of these patients are Injectable Drug Users's (IDU's) and 10 infected sexually (6 by heterosexual contact and 4 by homosexual contact). Only 2 patients (10% overall response rate) have seroconverted: both homosexual (50% rate for homosexuals). Of note is a 0% response rate for IDU's despite a 55% response rate for DTH skin testing and 70% with CD4 counts > 200. No statistically significant predictors emerged. Discussion and Conclusions: Our results show much lower response rates for IDU's than previously reported, despite markers indicating relatively intact immune function. The cost-effectiveness of Hepatitis B vaccination in HIV-infected IDU's must be questioned. Modifications in the regime (boosters, intradermal injections) should be evaluated. Additional data will soon be available to add to the above results. Larry Broisman, Hartford Hospital, 80 Seymour St., PO B ox 5037, Hartford, CT, USA, 06102-5037 Telephone (203)545-4012; Telefax (203)545-3149 0 O 'O C.) 0 N c0

Page  132 PB0540 ANTIBODY RESPONSE TO HEPATITIS B VACCINATION OF HIV-INFECTED PATIENTS. Garcia S(I), del Romero J(1), Prez A(I), Rodriguez C(), Garcia A(I), Oil A(2). (I) Centro Sanitario Sandoval, CAM, Madrid. (2) Medicina Preventiva, UAM, Madrid OBJETIVE To evaluate the response of 1HIV infected patients to hepatitis B vaccine. POPULATION ANALYSED Between February/92 and January/94 a total of 70 HIV positive patients (ELISA and Wester-blot positive) negative for anti-HBe antibody, were vaccinted against HBV. There were 44 males and 26 females, with a mean age of 29.7+ 7.0 years. They were clasified as CDC group Al (41), A2 (16), A3 (1), BI (3), B132 (5), 133 (3) or C2 (1) METI IODS: A 40 meg dose of recombinant hepatitis B vaccine( Engerix B) was given intramuscularly in the deltoid regions at 0,1,2 and 6 months. Blood samples were collected between 30 and 60 days after the last dose for anti-HBs measurement (ELISA, IMx Abbott). Seroconversion was defined as anti-HBs titers > 10 IU/L. RESULTS: Seroconversion to anti-HBs occurred by the end of the immunization series (month 7) in 59/70 (84.2%).The Geometric mean titers on seroresponders was 184.5 UI/L. Antibody response was not significantly associated with age, sex, CDC group, and mode of transmission. The distribution of the antibody titers on the studied patients was as follow: No patients >10 U/L >100 U/L >1000 U/L (%) (%) (%) 70 84.2 61.4 15.7 (%) = Proportion of subjects with antibody titers DISCUSSION AND CONCLUSIONS: Hepatitis B vaccination is recommended in HIV infected patients. However the optimal regimen of vaccination has not been establised. A fourth dose regimen (40 meg per dose) seems to dramaticaly improve the response. The seroconversion rate obtained (84.2%) is higher than the reached with other doses and schedules evaluated in these patients. Soledad Garcia. Centro Sanitario Sandoval. C/ Sandoval 7. 28010-Madrid Spain Tf 34-1-4452328 Fax: 34-1-5931004 PB0541 HTLV PREVALENCE IN PRIJGtANTT WOMEN IN SOUTHERN FRANCE Catherine Vignoli, C. Tamalet, C. Zandotti, S. Rousseau, P. de Micco. H6pital de la Timone, Marseille, France. Objective: Determine HTLV-1/ll prevalence in a general population of pregnant women. Methods: Between April and November 1992, blood samples from pregnant women in two public maternity wards in Marseilles were collected. A short questionnaire focusing on possible risk-factors was established. Detection of HTLV-I/ll antibodies was performed by EIA (Organon) and confirmed by Western blot (DBL). Seropositive women were then assayed by DNA amplification (PCR) to discriminate type I from type II. Results: A total of 921 women has been tested (mean age: 26.8 years, HIV-1 positive: 1.3%). Positive results for HTLV antibodies with EIA test were 10 (1.1%). After WB, 3 (30%) were negative, 3 (30%) were confirmed positive by WB and PCR and 4 (40%) had an indeterminated WB and were PCR negative. The differentiation by PCR identified 2 HTLV-1 and 1 HTLV-ll. They all presented a risk factor: a region of origin endemic for HTLV. Discussion and Conclusions: This study presents a seroprevalence of 0.32% of HTLV In a general population of pregnant women. Although this prevalence rate is low, it's significantly higher (a hundred times more) than those observed in the blood donors population in Marseilles (0.37 per 10 000) or in France (0.46 per 10 000). It also demonstrates that the principal variable associated with HTLV seropositivity is region of origin. These data provide a powerful basis for devising an extension of the HTLV-1/I! screening to high-risk pregnant women. C. Vignoli, Laboratoire de Virologie. H6pital de la Timone, 13005 Marseille, France. Tel:, Fax: -0 0 0 0 N 44b W CMV IgG, SV IgG, HSV2 IgG and T.gondlT IgG PREVALENCE AMONG PRIMARY BLOOD P B0542 DONORS IN LOoI,,PREVALENCE COUNTRY. MilOnavdute Ruta, Grlkevlclus A., Valanclauskalte V., Capllnskas S. AIDS Centre of Lithuania, Vilnlus, Lithuania. Objective: To determine the prevalence of anti-CMV, HSV1/2, and T.gondii IgG among primary blood donors representing healthy population in Lithuania. Methods: We tested blood sera of 83 primary blood donors (giving blood for the first time) from two cities of Lithuania - 34 men and 49 women. The following test kits were used: CMV IgG IMx, Toxo IgG IMx ("Abbott"), Herpes simplex 1/2 Bioservis EIA. Results: CMV, HSV1, HSV2, T.gondii prevalence by age roups 20-29(n=18) 30-39(n=27) 40-49(n=29) 50-59(n=9) CMV IgG 17(94.4%) 26(93.3%) 24(82.8%) 8(88.9%) HSV1 IgG 18(100%) 27(96.3%) 29(100%) 7(77.8%) HSV2 IgG 10(55.6%) 20(74.1%) 19(65.5%) 5(55.6%) T.gondii IgG 7(38.9%) 10(37.1%) 12(41.4%) 2(22.2%) Conclusion: The prevalence of CMV, HSV1/2 is very high and it is similar to the prevalence of these infections in other countries with low socio-economic status. The prevalence of T.gondii is lower but in all cases it is a possibility for HIV infected patients to get ill with AIDS indicator diseases caused by CMV, HSV, T.gondii. MILINAVICIUTE, Ruta., Moletu plentas 40, Vilnius, 2021, Lithuania, tel.+3702 350465, fax.+370-2-350225 PB0543 PREVALENCE OF HBV, HCV AND HDV IN 500 HIV + PATIENTS. F. Denis', C.C. Adjide, S. Ranger-Rogez", J.P. Rogez**, P. Weinbreck". Departments of *Virology and " Infectious Diseases, Limoges, France. Objective: To determine if there was a difference in the prevalences of markers to hepatitis B, C and delta viruses (HBV, HCV and HDV) in HIV infected patients according to risk factors. Methods: HBV, HCV and HDV markers were investigated in a cohort of 500 French HIV infected patients and in 1000 controls. HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe, HBV DNA, anti-HCV, HDVAg and anti-HDV were all tested using Abbott tests. Anti-HCV was confirmed by RIBA II immunoassay. Results: Sera of 344 out of the 500 patients (68.8%) were positive for at least one HBV marker, 69 (13.8%) being HBsAg positive. Among the HBsAg carrier group, 48% were HBeAg positive (DNA+: 69%), 24% anti-HBe positive (DNA+: 35%) and 28% without e marker (DNA+: 16%). HBV markers were often associated with IVDU risk (HBsAg: 11.5%, at least one HBV marker: 89%), and with sexual risk, homosexual, bisexual or heterosexual (respectively HBsAg: 15%, 24%, 17%, at least one HBV marker: 63%, 80%, 45%). HDVAg and anti-HDV were investigated in HBsAg carriers and found respectively in 3% and 19% of these sera. The prevalence of anti-HCV was 31.1%: 82% of IVDUs, 46% of transfused patients and only 1.5% of the patients with sexual risk were seropositive for HCV. HBV markers were associated with HCV in 25% of HIV patients. ALT were 22x the normal level in the sera of 31% of HBsAg carriers, in 16% of anti-HCV positive patients and In 5% of the patients without B or C markers. No significant difference was observed in HBV, HDV or HCV prevalences according to clinical CDC classification states. Conclusion: IVDUs were the more exposed to infection by HBV, HDV and HCV. F. Denis, Department of Virology, CHU Dupuytren, Limoges, France. Phone N~: Fax N~:

Page  133 PB0544 SEPOPREVALENCE OF CHRONIC COINFECTION IN HIV(+) PATIENTS IN ARGENTINA. Bortolozzi Raul; LupoS.; TabordaM.; Fcrnandez,E.; Viglianco,R.;Fay,O.;First Clinical MedicneDepatrment,Faculty of Medicine. Center for tecluiology in Public Health, Nac. Univ. of Rosario Argentina Objectives: To determinate the seroprevalence of chronic infections no HIV, in HIV(+) patients and compare it with the general population. Methods: Between 1989 and 1994 we studied 231 IIIV(+) patients (182 (78%) male and 49 (21%) 0e1) age range between 15 and 55 ( mean: 27 years). all residents of the area of Rosario city in Argentina. The infections ways were IVDU 140 (60.60%), 47 (20.3%) heterosexual. 41(17.34%) homosexual, 3(1.29%) blood transfusion. We utilized the following laboratory tests: ELISA;RIBA; and LIA for HCV, ELISA for HBV, Toxoplasm g.,CMV, Chagas disease at HTLV I/ll, and serology tests for Brusellosis and syphilis. Control groups were voluntary HIV(-) blood donors. The stadistic methods applied was Chi-square test. Results: The table summarize the data Population HCV HBV TOXO/IgG CMV lgG Hudd. Chagas VDRL HTLV I/Ill HIV(+) STUD. N~/ 119/63 176/40 136/79 95/61 95/0 32/3 111/15 18/0 +N~(%) (52.94) (22.72) (58.51) (64.21) (9.37) (13.51) CONTROL 5000/ 5000/ 5000/10 500/0 STUD. N~/ 5000/50 5000/2.5 200/64 200/90 0.05 250 (0.002) +N~(%) (1) (0.05) (32) (45) (0.001 (5) Conclusions: 1) The seroprevalence for HCV. HBV, Toxoplasmosis, CMV, and syphilis was higher in V(+) population than in controls groups with statistical significance (p<0.05). 2) The Possibility of a reactivation and transmission of these infections jusitifies the serological in our HIV(+) population 3) The seroprevalence for Brusellosis was higher in the general population with statistical significance (p<0.05) The prevalence of rural population in these group should explained the result. 4) There were no statistical signicative differences for Chagas infection in the groups considered. RAUL BORTOLOZZI FIRST CLINICAL DEPT. RODRIGUEZ 1215 2000 ROSARIO ARGENTINA PB0545 Viral Markers in HIVI+ Children Sheltered in an Orphanage Sell Sfartz', Cristiana Dragomir', Florin Zugun-E.2, Monica Balteanu3 'Clinica Puericultura, 2Lab. imunol. Turnmor., UMF las, 'Inst Cantacuzino, Bucuresti, Rominla Clinical and immunological investigation of HIV1+ child (aged 1-5 years) sheltered in a closed community, orphanage type, allowed us to identify some particular pathological patterns, non described in the literature. The discovery of HIV I infection was done using the classical methodology. The investigation of opportunistic bacterial infection was done using a personal methodology. In those 27 HIVI+ children that represent the subject of this paper, we succeeded to detect 13 infected with Cytomegalovirus (Welcozyme detection kit for IgG antibodies anti CMV), 12 children with C hepatitis virus (Monolisa Diagnosis Pasteur for IgM antibodies anti HCV), and 6 infected with B hepatitis virus (using an Abbott ELISA II kit for HBsAg and HBeAg). 5 of those HIVI+ children cumulate CMV and HCV infection; 3 cumulate HCV and HBV infection, and 2 the CMV and HBV infection. The coexistence of HIVI with CMV, HCV and HBV produces the accumulation of symptomatology, resulting in difficulties in clinical classification and also in the interpretation of immunophenotyping values (CD4+ lymphocytes, CD4/CD8 ratio; CDI9+ cells) and serum markers investigation. The interference of other pathogenic entities, especially caused by opportunistic and pathological bacteria, sometimes by parasites and infrequently by protozoa, appeared in episodic waves in the community of children, results in supplementary variation in evolution patterns of the HIVI+ children that distinguish the HIV pathology described here from the pattern currently shown in literature. Dr. Sell Sfartz, B-dnl Copon nr. 26 A, Bloc BS, Ap. 30, Tel 098-140956, Fax 098-117845 6600 - IASI, ROMANIA PB0546 FINAL RESULT S OF A TWO YEAR, MULTIPLE OPPORTUNISTIC PATHOGEN PROPHYLAXIS STUDY (MOPPS) PERSONS WITH CD4<200/MM3. Sullivan hdig*, Henry K*, Weiser J*, Melroe H', 'St. Paul Ramsey Med. Ctr., MN, USA Objective: To gain experience in providing primary prophylaxis for multiple HIVrelated pathogens (fungal, herpes viruses, MAI, PCP) in persons CD4<200/mm3. Methods: Beginning in 8/91 and finishing in 1/94, 24 HIV infected persons with CD4<200/mm3 were enrolled in an open-label 24 month MOPP study and started on the following drugs: acyclovir (3200mg/D), fluconazole (100mg/D), ciprofloxacin (500mg/D), and TMP/SMX (1 DS/D). If intolerant, alternatives were provided. All were receiving mono or combo nucleosides. Patients were followed monthly for compliance, tolerance, HIV events, and lab evaluations. Results: Eleven patients discontinued study medications early (5 HIV progression, 3 non-compliance, 2 personal reasons and 1 adverse rxn). Nine patients experienced 15 adverse rxn (8T/S, 4 cipro, 2 rifabutin, 1 flu). Thirteen patients progressed with 8 patients developing 14 targeted Ols (CMV 7, Candida Esoph 3, MAC 2, PCP 2). The mean CD4 count was significantly higher for patients who did not progress both at study entry and at end of study (163 vs 65, p=.01, and 142 vs 20, p=.0001) when compared to patients that had AIDS defining progression. Conclusion: Results indicate that persons with CD4 counts<50 remain at high risk for targeted Ols and other disease progression/death despite our MOPP regimen and nucleoside therapy. Patient interest and acceptance was high. Further MOPP studies are needed to assess cost/benefit of various MOPP regimens and optimal time to initiate. Christopher Sullivan, St. Paul Ramsey Medical Center 640 Jackson Street, Suite 125 HIV Programs St. Paul, MN 55101 612 221-1280 Fax 612 221-8616 PB0547 DREASED E GAMMA GLOBULIN CONCENTRATION AND DECREASED CD4+ COUNT ARE PROGNOSTIC FOR THE OCCURRENCE OF CMV DISEASE: A LONGITUDINAL STUDY St.A. Geier, I. Sadri, J.R. Bogner, V. Klauss, F.-D. Goebel; Medizinische Poliklinik und Augenlinik, 80336 Munich, Germany; Objective: To validate the prognostic power of gamma globulin concentration and CD4 + count for the occurrence of CMV disease because both variables were identified as prognostic factors by means of Cox regression analysis*. Methods: Gamma globulin concentration and CD4+ count were measured at the time of diagnosis of AIDS and at the time of diagnosis of CMV disease in 27 patients. 25 patients were homosexual men. Median time between diagnosis of AIDS and CMV disease was 14.3 months. Results: Mean gamma globulin concentration was 1.74 g/dl (SEM: ~0.11), and mean CD4+ count was 84.11 cells/pl (~13.58) at the time of diagnosis of AIDS. Gamma globulin concentration significantly decreased to 1.36 g/dl (~0.12; p<0.001), and CD4+ count significantly decreased to 28.19 cells/ l (~8.42; p<0.001) at the time of diagnosis CMV disease. Conclusion: Our results validate the finding that gamma globulin concentration, CD4 + count, and homosexual life-style are prognostic factors for the occurrence of CMV disease. The finding that gamma globulin concentration decreases in patients with CMV disease needs further consideration. Geier SA, Sadri I, Goebel FD et al.: Prognostic Factors for the development of CMV disease in patients with AIDS. VIIIth Int. Conf. on AIDS, Amsterdam. Abstractbook, Abstr. PoB 3177, pB116, 1992. This study was supported by Bundesministerium ftr Forschung und Technologie, and by Bundesministerium firt Gesundheit, Germany, grant FKZ BGA III-02-089/FVP. Stephan A. Geier, Medizinische Poliklinik and Augenklinik der LMU, 80336 Munich, Germany. Tel. 0049895160-3653, FAX -3659 0 -o 0 (in 44h V

Page  134 PB0548 FALSE POSITIVE IgM SEROLOGIES ASSOCIATED WITH RECENT HIV DIAGNOSIS. Rusnak, Janice M., Blatt S.P. Wilford Hall Medical Center, Lackland Air Force Base, TX, USA Objective: HIV seroconversion may be clinically indistinguishable from mononucleosis syndromes due to toxoplasma (toxo), EBV, and CMV. Following identification of 2 patients during the HIV seroconversion syndrome with a false positive toxo IgM, a false positive RPR (1 patient), and positive EBV and CMV IgM serologies, we reviewed IgM serological testing in the United States Air Force HIV Natural History study cohort. Methods: Toxo, EBV, CMV serologies, and immunoglobulin levels of 1352 HIV patients obtained yearly from January 1986-89, and 1992 were reviewed (toxo 1986-87, 92 only). Results: Toxoplasma EBV CMV # Patients (+) IgM serology (total) 22/420(5%) 31/1000(3%) 133/889(15%) # Patients (+) IgM at HIV diagnosis 15/140(11%) 22/804(3%) 90/749(12%) # Patients (+) IgM post HIV diagnosis 7/299(3%) 9/599(2%) 43/477(9%) 51-70% of IgM serologies were obtained post diagnosis of HIV. Seven patients with proven false positive toxo IgM's had higher total serum IgM levels than IgM negative patients (340 mg/dl ~ 44 vs 213 mg/dl + 16, P =.03) as did patients with positive EB VCA IgM's (203 mg/dl + 17 vs 160 mg/dl ~3, P =.01). The median HIV seroconversion time of 4 known seroconvertors with (+) toxo IgM's was lower than 121 1gM (-) patients (155 vs 990 days). Discussion and Conclusions: Given the prevalence of positive IgM serologies in early HIV infection (particularly near seroconversion) which may represent false positives, relapses, or initial infection, one must be cautious in diagnosing a mono-like illness if there are risk factors for HIV. Nonspecific elevation of total IgM levels may explain this predisposition for false positive IgM serologies. JANICE M. RUSNAK, M.D.,WHMC/PSMI-H, 2200 BERGQUIST DR STE 1, LACKLAND AFB TX 78236-5300, TEL: 210-670-7897 FAX: 210-675-0173 PB0550 ANALc0NDIOMI 3ISREOJRNCEINHIV (+) PATIETS.0 I BR,THREE MO ITIES OF T F,rEA R.ALFUNZO NH VAIB,B J lE2, A.HAVTISTA, M.(X*SN, J.FE24ANLEZ G.FIIAN(X. HOSPTIAL DE (LINICAS CARACAS. EE IO DE FEREL INFECCIOSAS. CARACAS - VE2BUELA OBJECTIVES We compared the outcomes of HIV(+) patients treated for Anal Condilomatosis Three diferent modalities of teatment were used A) Electrof ulgwnation alone. B) ElectrofRlpuration + Intralesional al&-2 Interfrron (I. al&-2I) and C) Blectrof lgration + Sub-cutaneous ald-2 Interferon (SC alf-21). The purpose of the study is to evaluate the rate of recurrences in the different groups. Recurrence of Anal Cendiloatosis is a ecuentproblem in IV (+)patients. MEgTHODS 4511 HIV (+) patients (diagnosed by Eliss and confimed by Western-Blott) ranging between 20 and 39 years were included The CD4 count varied from 200 to 50 cells x mm3.Three groups of 15 patients each, who were randomly asigned, underwent the three modalities of treatment In the group A Elecdrofulguration (E) alone was usedIn the group B we used B + IL ald-21 in a dose of 4.500.000 Uonce a week for 4 weeks. The group C received E+ SC alfa-21 in a dose of 4.500.000 U. three times a week for 4 weeks. The sine of the lesions were comparable for the three groups We used T Student to compare the results. The patients were controled weeldy for I month and then monthly to complete 4 months after the begining oftretsent RESULTS AND DISCUSSION In the group A recurence ocurred in 14 of 15 patients (93,3%). In group B reosrence ocurred in 13 of 15 patients (86.6%). and in group C recurence was seen in I patient of 15 (6.66%) The relation between recurrence and CD4 counts could not be established in this study (CD4 counts from 200 to 50 cell x mm3) for lack of statistically significant sample to reach a conclusion in this point There were no statistical difference between group A and B (P=0552), but statistacall dierence was signifieant for group C compared to A and B (P=0,000 and P=0,.000 respectively) CONCLUSION Ins o p erienc. e InHIV (+) patients with Anal Condilomatnis and CD4 cout <200., the lowest recurrence rate om ed using E+ SC alff-21) (three times a week for 4 weeks) 4 months after the begining of treatment More studies are needed to find out the minimal dose of SC al-21) required Futhermore a larger follow up is necessary. R. A5UN N. ltTPM L CE CLTIC S @R CAS. CfLUITCRID 1100 PISD 11 AVE. PANIKN (1N ALA SAN R\ER)W RAS - VEIPLELA 1011 PB0549 REPORTED CASES OF HIV I IN DIABETES MELLITUS PATIENTS ALONG WITH HUMAN HERPES VIRUS. By: DR. BAGALKOTE, S.G. and DR. J.P.S. MEHTA PHYSIOLOGY DEPARTMENT, KENYATTA UNIVERSITY BOX 43844, NAIROBI & MEHTA NURSING HOME, PANGANI, NAIROBI.. Objective: To determine potential confounding factors in the development of HIV I in a cohort of diabetis patients. Methods: Prospective cohort study of 15 Diabetes mellitus patients, later HIV I infected men and woman (3+1) enrolled in this study for a mean 24 months, obtaining physical exam. Glucose level, Cholesterol and triglycerides, HCT and detailed information on HIV I related symtoms and reproductive sexual behavior, using routine laboratory tests. Results: The implications of said findings will be presented and discussed with respect to the role of Herpes Zoaster and Diabetes mellitus. Conclusion: Important prefactors of HIV I and related implications in Diabetes mellitus patients along with Human Herpes Virus were older age, recent weight loss and low haematocrit and interestingly reversal phenomenon of HIV I infection. DR. BAGALKOTE, S.G. SENIOR LECTURER, PHYSIOLOGY DEPT., KENYATTA UNIVERSITY,P.O. BOX 43844, NAIROBI, KENYA. TEL. NO. 810901-19 EXT. 57305/57307. FAX 810759. PB0551 Asteroid nocardia infection in two HIV infected patients as the first episode of opportunistic infection. Daniele A., Masini R., Maclas J. Room h10. "F.J.Muiz" Hospital. Buenos Aires, Argentina. Purpose: Description of the clinical and therapeutical characteristics of two patients native from Argentina with asteroid nocardia infection and HIV infection. Patient 1: Age: 29. Sex: male. Cause of hospitalization: febrile syndrome, productive cough, dyspnea and loss of weight. Formerly adicted to intravenous and breathing in drugs. Heterosexual. First opportunistic infection suffered by the patient. The physical examination shows a patient who seems severely ill. Condensation syndrome in the right lung middle field and left middle and lower field. Tachypnea. Hepatomegaly. Mucopurulent expectoration, once hemoptoic. Skin and mucosa jaundice. Splenomegaly. Thorax X-rays with mixed interstice-alveolar-type infiltrate in the right middle lung field and left middle and lower fields. CD4 less than 50/mm3. Asteroid Nocardia sputum examination. Sulfadiazine and imipenem treatment. Patient 2: Age: 19. Sex: male. Cause of hospitalization: febrile syndrome, diarrhea and subcutaneous nodules on the left upper limb, head and left pectoral area. Formerly addicted to intravenous drugs. Heterosexual. First opportunistic infection. First physical examination shows a patient who seems severely ill. Nodules on the left upper limb left parietal area and left pectoral area of about 5 cm to 8 cm in diameter; painful, soft, seem slightly swelling. Drainage of pectoral tumor and upper limb with abundant purulent secretion. Thorax X-rays with extended bilateral micronodulillar infiltrate from vertex to base. CD4 less than 50/mm3. Patient develops a Lyell syndrome in his evolution. Asteroid nocardia isolated in the purulent material of the subcutaneous nodules. Treatment with sulfadizine and amikacine. Conclusions: Nocardiosis is not commonly found in HIV patients. It must be included in the differential diagnostic of HIV patients with fever mainly showing respiratory, neurologic and cutaneous evidences. It was present among our patients as the first episode of opportu nistic infection, although these were patients with advanced immunosuppression. We further evaluated the efficiency of the sulfadiazine association to imipenem and amikacine. Daniele A. Jose Indart 401. Lomas del Mirador (1752). Buenos Aires, Argentina. 441-3046. w 0 U' 0 U' U' (in

Page  135 PB0552 BACTERIAL PNEUMONIAS AS A MARKER OF DISEASE PROGRESSION R.Teira,M.Virosta,J.Mufioz,Z.Zubero,J.Baraia-Etxaburu, JUAN M. SANTAMARIA. Hospital de Basurto, Bilbao SPAIN Objective:Bacterial infections are a major cause of morbidity and mortality in patients infected by the HIV. We wanted to test wether if, as it's been suggested, they can be a marker for risk of disease progression. Methods:In 1989 we conducted a longitudinal descriptive study of bacterial pneumonias (BP) in HIV-infected patients. We present their follow-up data and compare them with those of a historical cohort of controls matched according to sex, CD4-cell count, previous HIV-related disease (asymptomatic,non-AIDS HIV-related,AIDS) and zidovudine intake (the latter,as possible).Two outcome events were defined to be analysed together: first AIDS diagnosis or death. Results:98 patients were available for analysis (49 in each group).Matching was successful with regard to CD4 count (means:398 and 378,p=O.81) and zidovudine treatment (percentages:51 and 61,p=O.41).Length of followup ranged from 0 to 61 months. 33 patients in the BP group reached an out come event (67%) as compared with 27 (55%) controls (Relative Risk: 1.22, 95% CI: 0.89-1.68, p=O.3). The log-rank test of the event-free survival yielded a non-significant value (p=0.96). It remained so in a post-hoc su bgroup analysis, except for patients with previous AIDS, among whom event -free survival was found to be greater for controls (p=O.008,log-rank). Conclusions:The hypotesis that BP are a marker of worse prognosis (as defined by the risk of AIDS or death) in HIV-infected patients could not be proved. Juan M. Santamaria.Seccion Enfermedades Infecciosas Hospital de Basurto.Av.Montevideo 18, 48013 Bilbao Spain. 4-4418800 fax:4425804 PB0553 RESPIRATORY INFECTION BY P. AERUGINOSA IN HIV PATIENTS. Arrizabalaoa Julio, Mentxaka B, Iribarren JA, Oyarbide J, Rodrfguez FJ, Garde C., Von Wichmann MA. Unidad de Enf. Infecciosas. H. Aranzazu. San Sebastian.Spain. OBJECTIVE: To settle the frecuency and recurrence of this infection, as cause of admittance to hospital in HIV patients, because of the increase of respiratory infection by Pseudomona aeruginosa that we have noticed, in order to evaluate the usefulness of making a secondary prophylaxis. METHODS: We have made a retrospective study of the number of admittances for this infection, in the period of time between january 1990 and december 1993. All of them were treated with to antibiotics sensitive to Pseudomona. RESULTS: There were 45 episodes in 20 patients, in this period. The recurrence was of 60%. There were 6 episodes in two patients, and both of them died because of this infection. CONCLUSION: There has been an increase of the respiratory infection by Pseudomona aeruginosa in HIV patients, who despite of receiving a correct treatment with two sensitive antibiotics, have made a great number of recurrences. This make us think that it would be opportune to carry out prophylaxis of this infection. The medicament to utilize needs further investigation, but in our experience, we have obtained very good results with tobramicin in aerosol. JULIO ARRIZABALAGA.Unidad de Enf.Infecciosas. Hospital Aranzazu.Paseo del DR Beguiristain.20080. San Sebastian.Spain.TF 453800 Fax 460758 PB0554 AZITHROMYCIN VERSUS AMOXICILLIN/CLAVULANATE IN COMMUNITY ACQUIRED PNEUMONIAS OF HIV-PATIENTS. Ciaffi L., Franzetti F.*, Gervasoni C.*, Romaniello A.*, Maillard M., Moro M., Ruggieri A, Cernuschi. M., Lazzarin A.. Infectious Diseases Department HSR and * Infectious Diseases Clinic H. Sacco, Milano, Italy. PB0555 PULMONARY INFECTION IN AIDS PATIENTS CAUSED BY THE "RHODOCOCCUS EQUI": A CASE DESCRIPTION AND A REVIEW OF THE LITERATURE. COMES, V.R.; POZZOBON, E.; LIMA, D.B.; PINHO, K.G. (ESTADUAL UNIVERSITY OF RIO DE JANEIRO - BRAZIL). STUDY DESIGN: open, randomized, comparative study of azithromycin versus amoxicillin/clavulanate (AC) in community-acquired pneumonias (CAP) in non hospitalized HIV-positive patients (pts). PATIENTS AND METHODS: we enrolled 60 HIV-positive pts (46 males and 14 females) with 62 evaluable episodes of CAP. Age range varied from 24 to 55, CD4 cells count from 1 to 585, WBC count from 1000 to 14000, PO2 from 40 to 117. 34 pts. without known intolerance and/or allergy to study drugs received azithromycin 500 mg/day for 6 days and 28 AC 1000 mg/tid. A microbiological cofirmation was obtained in 14 cases (8 S. pneumoniae. 2 H. influenzae, 1 H. parainfluenzae, 1 S. beta haemolvticus gr.B, I P. aeruginosa. 1 M. pneumoniae). RESULTS: we observed a good clinical response in 22/34 cases (64.7%) and a radiological response in 20/34 cases (58.8%) receiving azithromycin; in pts. receiving AC 19/28 cases (67.8%) showed a good clinical and radiological response. Side effects have been observed mainly in AC group: I diarrhea, 7 allergic reactions (fever and/or skin rash), while in azithromycin group we noted only one case of mild increase in liver function tests. CONCLUSIONS: In empirical treatment of CAP in non hospitalized HIV pts the two study drugs showed the same efficacy. Failure of therapy was mainly due to resistant bacteria and pulmonary opportunistic infections, diagnosed after the enrollement in the study. Azithromycin caused less side effects and allowed easier dosage schedule, owing in the future to a wider use in CAP. L ~1U f C IAFFI - I tec'iou,S bise.Se xbe. V-AWe.At R - \ - o2 Adc'Av c a 2o ot24 'MIL r o - I TLY INTRODUCTION AND OBJETIVE: The authors describe the case involving a patient suffering from a long-lasting pulmonary abscess caused by the "R. equi" and take the opoortunity to discuss how difficult in was to make the diagnosis and to carry out the treatment and to review the existing medical literature on the subject. CLINICAL CASE: The patiente was a white, homosexual man, 32 years old and with AIDS (group IVC3, CDC) who was admitted into our hospital in january, 1993. At the time, he complained he had been coughing, having fever, and losing weight for six mounths. The image of an X-ray of his chest slihowed something compatible with a pulmonary abscess in his left lung. An empirical treatment was iniciated using crystaline penicillin, oxacillin and garamicin which did not produce any positive result. Ile, then, was submitted to bronchoscopy and to a pulmonary biopsy by means of puncture. These procedures did not improve his situation, either. He underwent apical segmentoectomy whose material showed that there was an abundant growth of R. equi which was sensitive to penicillin and garamicin. In spite of the bacterial susceptibility, the patient ended up dying ina few days. DISCUSSION: There are 51 cases of pulmonary infection caused by the R. equi registered in the medical literature and the majority of them involved immunocompromised patients by several causes and 20 of them had AIDS. The author describe their experience with an AIDS patient at an advanced stage, who progressed into a chronic pulmonary stage wi ch was difficult to diaginose. A long treatment, combinicy clinical and surgical therapies, is necessary and, in most cases, the results are not satisfactory. In our case, the R. equi was sensitive to every antibiotic procedure wich the patient underwent. In our opinion, the lack of sucess in the treatment was due to the delay in the diagnosis and the advanced immunodeficiency stage at which the patient was. We call everyone's attention to this new and opportunistic infection that is on the rise in AIDS patients. GOMES, Valeria, Rua Lauro Muller 96 cob 7 Urca Rio de Janeiro Brazil CEP 22290-160 Telephone (021) 295-5852; FAX 284-5033 0 (31 (31 N 0 (31 (31

Page  136 PB0556 RHODOCOCU EOUIINFECTION IN AIDS PATIENTS: PROECTIVE ROLE OF HUMORAL IMMUNITY Mastroianni C.M., Vullo V., Delia Slvatore. Dept. of Infectious and Tropical Diseases, La Sapienza University, Rome, Italy Objective: In previous investigations, we demonstrated that clinical severity of R.eaui pneumonia in AIDS patients was correlated with lower specific antibody response.The aim of this study was to further evaluate the humoral immune response to R. eui infection in AIDS patients as well as in healthy subjects. Moreover, the immunoreactivity of human intravenous immunoglobulin (IVIG) to R. eaui antigens was also examined. Methods: Specific antibody response to whole cell antigens of Legui was analyzed by enhanced chemiluminescence immunoblotting in 22 serum samples from 5 AIDS patients with R. equi pneumonia, in 20 from AIDS patients without R.egui infection, in 30 from healthy controls and in a commercial lot of IVIG (Ig VENA, lot 192, Sclavo, Italy). Results: The sera from 3 AIDS patients, who fully recovered from R. eagui pneumonia, had a stronger antibody response toL R. eaui antigens than that found in AIDS patients without R. eui infection and healthy controls. Conversely, the sera from 2 patients with severe R. Qi infection showed little or no reaction with R. eui antigens. Finally, immunoblotting experiments showed that IVIG exhibited an intense reactivity with all antigenic components of R. eui. Discussion and Conclusions: Our findings suggest that: i) R. egaui antibodies naturally occur in humans; ii) humoral immunity may play an important role both in the protection against and recovery from RL.egui infection in AIDS patients. Further studies are in progress to investigate the value of IVIG or other immunotherapies in AIDS patients with R. eui.infection. Delia Salvatore, Infectious Diseases, La Sapienza University, Policlinico Umberto I, 00161 Rome, Italy. Tel. +39 6 490672; Fax: +39 6 4453760 PB0557 CLARITHROMYCIN 500mg BID AS PRIMARY PROPHYLAXIS IN PATIENTS WITH AIDS Grossman, Howard A. New York, New York USA Disseminated MAC (dMAC) Infections are a common complication in Patients With AIDS (PWA) occurring almost exclusively in patients with 100 lymphocytes/mm3. The diagnosis of dMAC among PWA's is now being made with increasing frequency. Disseminated MAC is a devastating and costly disease requiring multiple drug therapy, therefore there is great need for new effective regimens for prophylaxis. Clarithromycin (CLA) a semi-synthetic macrolide with documented in-vitro activity against MAI recently received FDA approval for the treatment of dMAC. A retrospective study of PWA's with significantly low CD4 counts who started Clarithromycin 500mg BID was conducted to evaluate CLA for primary prophylaxis. A total of 30 evaluable patients with a mean age of 40.1 (range 28 to 69) were enrolled. Mean CD4 count shortly before or during prophylaxis was 22.3 cells/mm3 (range 0-92). Twenty patients are known to remain dMAC free. Two patients were lost to follow-up after 9 and 14 months of prophylaxis. Eight patients died of AIDS related events. All were clinically free of dMAC disease at the time of death. The average MAC free interval was 10.1I months with an average of 10.8 months for those still alive. Clarithromycin 500 BID seems to be quite effective in preventing dMAC disease in severely immunocompromised patients. Howard A. Grossman, MD; 285 West 11th Street, Suite 1A New York, NY 10014 Tel: 212-929-2629;Fax:212-929-4971 -Q W 0 Un U' a) W 0 c0 PB0558 CLARITHROMYCIN 500mg BID FOR MAC PROPHYLAXIS IN PATIENTS WITH AIDS GOLDEN, Samuel W., Homestead, PA, USA PB0559 CLARITHROMYCIN 500 mg BID FOR dMAC PROPHYLAXIS Coulston, Daniel R., Wood, C.D., Spokane, WA, USA Clarithromycin's (CLA) recent approval for treatment of disseminated Mycobacterium avium complex (dMAC) resulting from its clinical success even as monotherapy and its excellent in-vitro microbiologic activity suggests that it may yield potential as a dMAC prophylaxis agent. A retrospective study of immunocompromised AIDS patients who started CLA 500mg BID was conducted to evaluate CLA for primary prophylaxis. A total of 18 evaluable patients with a mean age of 38.6 years (range 31 to 47) were enrolled. Mean CD4 count shortly before or during prophylaxis was 33.7 cells/mm3 (range 3-98). Nine patients continue to be successfully prophylaxed with CLA. One patient was discontinued to enter a rifabutin clinical trial after 13.2 months of successful prophylaxis with CLA. Eight patients died of AIDS related events without clinical evidence of dMAC at the time of death. Two of the eight were transferred to a rifabutin clinical trial prior to death. Mean MAC free duration (N=18) is 9.1 months. Clarithromycin 500mg BID seems to be well tolerated and successfully prevent onset of dMAC in immunocompromised AIDS patients. Further evaluation is encouraged. GOLDEN, Samuel W., 220 East 8th Avenue Homestead, PA 15120 USA Telephone 412-461-7220; Telefax 412-461-4626 Disseminated MAC (dMAC) Infections are associated with high mortality and morbidity in patients with AIDS (PWA). Consistent, effective agents for prophylaxis would be of great benefit as the incidence of diagnosing dMAC is ever increasing. The effectiveness of the recently FDA approved Clarithromycin (CIA) as treatment for dMAC prompts evaluation of this agent for prophylaxis. A retrospective study of PEWIA's from a community-based practice was conducted to evaluate Clarithromycin 500mg BID monotherapy for primary prophylaxis. Fifteen severely immunocompromised evaluable male patients (mean age 34.8) were enrolled. Mean CD4 count prior to initiation of CILA was 60.5 cells/nrn3. One patient died of a non-related AIDS event after 19 months of successful prophylaxis and was clinically free of dMAC at the time of death. Fourteen patients continue to receive CIA without untoward effects or evidence of dMAC. Current mean MAC free survival time is 17.7 months with a current mean CD4 count of 46.6 Cells/nrn3. Seven of the fourteen patients have a mean MAC free survival time exceeding two years. Clarithromycin is well tolerated and appears to be very effective in preventing dMAC disease in severely immnunocompromised patients. Coulston, Daniel R. 509 Deaconess Medical Building, Spokane, WA, USA Telephone 509-623-1456 Telefax 509-623-2939

Page  137 PB0560 MAC PROPHYLAXIS WITH CLARITHROMYCIN 500 MG BIG IN PATIENTS WITH AIDS Greiger-Zanlungo, Paula Alaie-Petrillo, A.,MtVernon NY USA The frequency of diagnosing Disseminated Mycobacterium avium-intracellulare (dMAC) is increasing and the effect of dMAC on mortality and morbidity has been documented. Therefore, regimens for consistent effective prophylaxis would be of great benefit. Clarithromycin has documented in-vitro activity against MAI and is currently approved as treatment for dMAC. Thirty severely immunocompromised AIDS patients (26 male, 4 female; mean age = 38.7 years) were studied in a retrospective fashion to evaluate clarithromycin as a potential agent to prevent or delay the occurrence of symptomatic dMAC disease. Clarithromycin was administered orally 500mg twice daily. The mean CD4 count at or prior to the initiation of prophylaxis was 39.8 /mm3 (range 02 to 112). Twentyseven patients continue to receive clarithromycin and remain free of dMAC disease with a current mean CD4 count of 48.6/mm3 and mean MAC free survival of 12.8 months. Two patients died of pneumonia and pulmonary KS respectively both of whom were clinically MAC free at the time of death. One patient was lost to follow-up after 9 months of successful prophylaxis. Clarithromycin 500mg BID is well-tolerated and appears to be significantly effective as primary prophylaxis for dMAC in severely immunocompromised AIDS patients. Paula Greiger-Zanlungo, MD; The Mount Vernon Hospital 12 N. Seventh Ave, Rm 501; Mt. Vernon, NY 10550 Tel: 914-664-8000 Ext. 3451; Fax: 914-664-0788 PB0561 COMMUNITY AND NOSOCOMIAL BACTEREMIA IN HIV-PATIENTS BernardEvelyne. Caries M., Ozouf N., De Salvador F., Pradier C., Dellamonica P. Infectious Diseases Department Nice, France. Objective:To determine characteristics of community and nosocomial acquired bacteremia in HIVpatients Patients and method: We included in a retrospective study all the HIV positive patients hospitalized at Nice Infectious Diseases Department between january 1992 and december 1993 in whom diagnosis of bacteremia was established. Chi2-sqare and t student tests were used for statistical analysis. Results: We identified 78 episodes of bacteremia in 63 patients (53 male, 10 female, mean age 34.3 years (range25-63)). HIV risk factors were IVDU (41), homosexuality (18), heterosexual contact (2), and unknown(2); CDC stages: A(1), B(18), C(49). Of the 78 episodes, 47 (60.3%) were community acquired (C), and 3 1( 39,7%) nosocomial (N). Characteristics of the C and N bacteremia are the followings: _C N C N CD4/mm3 48.9 45.4 NS S. arus 5 6 NS Pulmonary EP* 17 5.003 CNS**** 1 11 <.001 Gastrointestinal EP* 10 2 NS S.neumoniae 13 1.002 IV devices** EP* 3 16 <.0001 Salmonella s 12 0.001 septicshock 10 8 NS Klebsiela-Enteroacter 0 6.005 death*** 13 10 NS P.aeruinoa 4 4 NS * EP: entry point ** IV devices: central venous catheter (7), implantable venous access system(12) *** related to bacteremia**** CNS: Coagulase Negative Staphylococci. Conclusion:Bacteremia are increasing in HIV patients with advanced stage.N bacteremia are mainly due to CNS and associated with IVdevices;Salmolnelja and S.p numoniae are mainly isolated in C bacteremia. BERNARD Evelyne HOPITAL DE L'ARCHET BP 79 06202 NICE CEDEX 3 Tel 92 03 55 15 FAX 93 96 54 54 PB0562 Blood Cultures in AIDS patients in South of Brazil. Silbert,S; Sprinz,E; Mallmann,R; Rarcellos,S... Hospital de ClITnicas de Porto Alegre. OBJECTIVES:Determine the incidence of positive blood cultures and the most common pathogens in HIV inpatients. METHODS:From February 1993 to February 1994, 686 blood samples from 159 HIV inpatients were studied at Hospital de Clinicas de Porto Alegre - AIDS Reference Center. These samples were inoculated in blood culture medium, incubated at 379C during 7 days with every day examination. The positive samples were identified at "Vitek Systems". RESULTS:From the 159 patients, 123(77%) had all samples negative,28(18%) had in one hospitalization, at least bne positive sample and 8(5%) had in two or more hospitalization, at least one positive sample. In the 686 samples, 596(87%) were negative and 90(13%) were positive. The most ' common pathogens were: Staphylococcus aureus (29%), Staohylococcus epidermidis (23%) and Klebsiella pneumoniae (13%). CONCLUSION:lIt is possible to conclude that these patients commonly have blood stream bacterial infections and as in other conditions, Staphylococcus aureus is the most common pathogen. NAME: Suzane Silbert TELEFAX:(051) 224-0967. ADDRESS: Rua Joao Telles 440/301-Porto Alegre,Rrasil. PB0563 ANTIBIOTIC PREMEDICATION - PREVENTION OF SUBACUTE BACTERIAL ENDOCARDITIS IN METHADONE MAINTENANCE PATIENTS. Reich.Daniel, McEachrane D, Quart A. North Central Bronx & Montefiore Hospitals, Bronx, New York, USA Objectives: To assess prevalence or oral risk factors associated with subacute bacterial endocarditis in heterosexual HIV(+) and HIV(-) methadone patients. Methods: Two hundred and ninety seven patients at a predominantly minority indigent methadone maintenance treatment center were examined for oral, dental and periodontal disease. 118 were HIV(+), 179 HIV(-). Medical history was obtained from patient questionnaires, review of medical chart, and consultation with patients' medical providers. Patients identified as being at risk for subacute bacterial endocarditis: 1)past medical history of rheumatic fever 2)endocarditis or 3)valvular heart disease) were given antibiotic prophylaxis prior to, and post, invasive examination (e.g. periodontal) and procedure (e.g. injections, scaling and extractions). Results: Number of patients requiring antibiotic premedication SEX BLACK HISPANIC WHITE TOTAL % of TOTAL PATIENTS Male 7 23 2 32 18 Female 10 14 4 28 24 Total 17 37 6 60 20 Thirty-five patients had one risk factor, while 25 had multiple risk factors. 30 of these patients were HIV(+), 30 HIV(-). Conclusions: Prior to performing invasive exam/procedures on patients with a history of intravenous drug abuse, dentists must have a complete history and physical pertaining to risk factors for subacute bacterial endocarditis for each patient. The 20% of patients requiring antibiotic premedication was much greater than that found in the general population (4%). Of these patients, 25.4% were HIV(+), 16.8% HIV(-). Daniel Reich, North Central Bronx Hospital 3424 Kossuth Avenue, Bronx, New York 10467 USA 718-519-4997, 718-519-4902 0 (J1 0 0 N OU W 0 U' 0) CA)

Page  138 PB0564 INFECTIOUS MORBIDITY ASSOCIATED WITH LONG TERM VENOUS ACCES DEVICES IN AIDS. Capdevila JA, Barbers J, Garcia Quintana A, Gasser I, Ocaia I. Hospital General Vall d Hebr6n, Barcelona, SPAIN. Objective: To asses the morbidity due to infections of central venous catheters in patients with AIDS. Method: Prospective study of 46 tunneled central lines (Vygon'), that during a period of two years were inserted in 36 patients for seconday prophylaxis of CMV desease. Patients and catheters were monthly evaluated with clinical and microbiological assessments. Also, controls were done in each febrile episode whithout apparent etiology. Microbiological assessment consisted in quantitative blood cultures and cultures of skin swab and hub. When catheter related infection was documented, it was treated with catheter in place with endoluminal antibiotics. The catheter hub was removed when it was the source of infection. Results: The mean duration of patient catheterization was 160 days (14 -565), with a mean time of a catheter in place of 120 days. There were 15 episodes of catheter infection, 0.25 episodes for 100 days/patient. The germs isolated were S.epidermidis (7), S.hemolyticus (3), S.hominis (1), Corynebacterium sp. (2), S.aureus (1), Ps. plitida (1), Achromobacter (1). The hub was the source of infection in 13 catheters. All but three episodes were satisfactorily treated without catheter removal. Discussion and conclusions: Catheter infections in AIDS patients are similar to that occur in others diseases. These infections could be managed succesfully with catheter in place. Efforts should be made to avoid infections with a great care of hub manipulation. Long term catheterization is a safe and useful procedure in AIDS patients. 11,1. vat as. p. e by " g,.a DGICYl n'93-1210. Capdevila JA. Hospital General Vail d Hebron. Unitat de Malalties Infeccioses. 08035. Barcelona, Spain. Telefax: 34.3.4282637. PBO565 INFECTIVE ENVOCARVITIS IN INTRAVENOUS VRUG USERS ANV HIV POSITIVE.Setnano A,Man tin T, Mac.ado J.*P,MaLtez F, CoutZinho F, Mongado A, Proenca R.H.CNurty Cabral/SeL. 1/Li.sboa-Potugal The authors reviewed the cases o 20 patients (6 woman and 14 men) 4 atta venous drug uses HIVi seAopositive (Ebsa and W.8Btot) with in6ective endocarditids. The age o the pattient6 tanged 6tom 17 to 38 year. in all cases vegetation~ were demonstrated by echocadiLogaphy. The ticuspid valve was involved in 65% o6 cases (13 patients) and Sta phytocoecus auteus was the most frequent in6ecLive micootganism, occ i ring in 12 caes (60%). The n.ieat outeome was favotable in 18 patients and 2 patients died, both with aorticl. involvement and in one od them HIV in6ec tion was in the group CDC IV. In ths study, the indection HIV does not appear to change substancialy the clin.icat manifestations, the bacteiZology and the outcome o6 indec tive endocarditi in intavenou.ls drug ulses. even in HIV in6ected pat.i;t tb. Machado J, HospitaZ Cury Cabra/Servico 1 Te l:71933080 rua da BeniicenciLa 1000 Lisboa-Portugal Fax: 7969515 -o 0 0) 0 0) N, PB0566 MICROBIOLOGY OF SINUSITIS IN AIDS Goncalves Denise U., Greco, DB, Toledo Jr AC,Tupinambas U Immunodeficiency and ENT sectores, UFMG, BRASIL PBO567 PNEUMOCOCCAL ENDOPHTHALMITIS IN AN HIV + PATIENT Lionnet F*, Pulik Marc*, Genet P*, Petitdidier C.* Le Turdu F**. Departements of *Haematology and **Microbiology, Argenteuil, France. Objective: To investigate sinusitis in 22 patients with AIDS. Methods: Patients were seem from March to December of 1993 and had confirmed diagnosis of sinusitis. When treatment with standard antibiotic regimens was ineffective, maxillary antral puncture was performed and sinus contents were cultured for fungi, Mycobacterium tuberculosis, aerobic, anaerobic germs and other opportunistic agents. Results: The majority were male (19=86,4%), between 21 and 48 years. The most common symptoms were nasal discharge (90%), nasal congestion (77%), fever (50%), cough (45,5%), postnasal drainage (30%), headache (30%) and facial pain (10%). The antral wash-out was performed in 11 patients. The cultured germs were Staphylococcus aureus in 6 cases, Streptococcus pyogenes in 5, Haemophilus influenzae in 3, Pseudomonas aeruginosa in 2, Moraxella catarhalis in 1, Proteus mirabilis in 1 and Mycobacterium tuberculosis in 1 case. All culture showed more than one microorganism. Conclusion: Sinusitis in AIDS patients have distinct clinical and microbiologic when compared to HIV negative individuals. These findings must be considered when evaluating ENT manifestation in infected individuals. GON(CALVES, Denise U. Rua Cristina, 1120 ap. 201 Bairro Santo Antonio - Belo Horizonte/BRASIL CEP.30330-130 Phone. (55)31-342-3852 Fax: (55) 31-224.8801 Objective: Streptococcus pneumoniae (SP) is the most common respiratory pathogen with associated bacteremia in patients with HIV. Extrapulmonary manifestations are rare and endophthalmitis (E) has never been described. Case report: A 28-year-old HIV + man was admitted with fever, arthritis and ocular pain. He underwent a splenectomy 2 years earlier for HIV-related thrombocytopenia, preceded by the administration of 23-valent pneumococcal polysaccharide vaccine, and subsequent oral penicillin with bad observance. The CD4 cell count was 287/mm' Cultures of articular fluid and blood yielded SP (serotype 9). He was treated with intravenous penicillin. Within 24h., he had a marked decrease in vision, due to a bilateral E. High level resistance to penicillin was found, and penicillin was changed to imipenem and rifampicin and intravitreal vancomycin. Apyrexia occured, but complete blindness developped. Discussion: E is uncommon, and occurs most often after ocular surgery or penetrating trauma. In 2 recent series, SP was found to be the organism responsible in 8 % and 9,4% Our patient had an increased risk, because of HIV infection and splenectomy. The failure of the vaccine is not surprising, because patients with <500 CD4 cells/mm' are less likely to respond to pneumococcal antigens. Resistance to penicillin is increasing in France (12% in 1990), with high-level resistance (> 1mg/I) in 48%. We think that E should be added to the list of unusual manifestations of SP in HIV patients, and should be precocely recognized, in order to propose early specific management. M. PULIK, Departement of Haematology, Victor Dupouy Hospital, F 95107 Argenteuil, France. Tel:(1) 34 23 24 05 Fax:(1) 34 23 23 15

Page  139 PBANT568 IMICROBIAL SUSCEPTIBILITY OF STREPPOCOCCUS PNEUMHONIAE IN HIV PB0568 ATENTS. de la Rubia F. Clavo A, L6pez Prieto D, de Cueto M. Prez Ramos S,Shncbez Porto A, Lopez Sdnchez A. Group of Infectious Diseases, Adiz (GIDC), Spain. OBJECTIVE: This work was performed in order to define the antimicrobial susceptibility of Streptococcus pneumoniae in HIV+ patients and to analyze the possible differences with a control group HIV-. PATIENTS AND METHODS: From January 1993 to February 1994, 76 cases of pneumococcal infection (PI) were attended at the seven Hospitals included in GIDC, 17 of them affecting to HIV+ patients (CD4/mm3: 15.50 +/-7.51). Every one of HIV+ patients'was receiving antirretrovirals and cotrimoxazole prophylaxis. Antimicrobial susceptibility was performed by disc testing. CMI to penicillin (micrograms/ml) was performed by the E-test. RESULTS:There was no significative difference between HIV+ and lHIV- groups when we considered the percentage of patients with resistance to antimicrobials chloramphenicol, tetracyclin, vancomycin, erythromycin, cefalotine, cefotaxime, ceftriaxone or teicoplanin. However the percentage of HIV+ patients with resistance to cotrimoxazol (HIV+ 75%, lIIV- 61.5%, p < 0.04) or clyndamicin (HIV+ 57.1%, HIV- 13.1%, p < 0.002) was significantly increased when compared with HIV- patients. CMI to penicillin is shown in the table: CMITO HIV + (17) HIV - (59) PENICILLIN LESS THAN O.1 64.7% 44.1% p < 0.008 0.1 TO 1 29.5% 30.6% MORE THAN 1 11.8% 25.3% CONCLUSIONS: 1) The antimicrobials erythromycin and cephalosporins, habitually employed in the treatment of the PI, are eficacious in the treatment of this entity in HIV+ patients. The resistance to cotrimoxazol may be related to the continuous use of the drug in the prophylaxis of Pneumocystis pneumonia. 2) In our area, there is no increment in the resistence to penicillin in HIV+ patients. PB0569 CAMPYLOBACTER AND YERSINIA IN HIV+ PATIENTS. L. Franzin, A. Sinicco, F. Scramuzza, T. Zaccaria Inst. of Infect. Dis., Univ. of Turin, TURIN, Italy. In a study of enteritis in HIV+ patients (pts) Yersinia and Campylobacter were both isolated from feces of 3 pts with diarrhea, abdominal pain and fever. Campylobacter was isolated using selective media (Preston, CCDA, Goossens) and blood agar (membrane filtration technique) microaerophilically at 37~C. Yersinia isolation was done directly on HacConkey and CIN agar at 25~C and after cold enrichment. In 1 pt (59ys,Sbisex,CD4+ 12/Nl) antibiotics multiresistant strains of C.jejuni subsp. jejuni(Lior biotype II) were repeatedly isolated with Y.enterocolitica. In 1 pt (48ys,comosex CD4+ 108) C.lari and Y.frederiksenii were found, while in another (55ys, fIDU, CD4+ 30) atypical Campylobacter with Y.frederiksenii, Salmonella, Aeromonas. C.jejuni subsp. jejuni (biotype II and I, nalidixic acid resistant) was also isolated in 1 pt (36ys,fIDU,CD4+25), while in 2 Y.frederiksenii (associated in 1 [33ys,&omosex,CD4+33] with Salmonella and in 1 [48ys, omosex,CD4+35] with microaerophilic-like Treponema). Conclusions: The isolation rate of Yersinia and Campylobacter was higher than that of Salmonella. The association of Yersinia and Campylobacter was frequent. Unusual strains were isolated. Even if the etiopathogenic role of atypical strains need to be further studied, Yersinia and Campylobacter can be considered important cause of gastrointestinal infections in HIV+patients. L, FRANIN INsT. OF INgFEcriOUS tIEA.sGS, UN/. oF FURiIN, OSP AMEbEO bI SAVOIA,.so Siltap-RA -11- oAJg roRINO, IrA Ly.:33-4-/383859 PBO570 SURGICAL DISEASES IN THE AREA ANORECTUM IN AIDS PATIENTS AT THE UNIVERSITY HOSPITAL IN BUTARERWANDA. G. GATERA*, G. BUGINGO*, D. BIZIMUNGU*, A. RURANGIRWA* University Hospital in Butare, Rwanda. * Comittee IEC/AIDS Butare, Rwanda. OBJECTIVE: To show the frequency of surgical diseases in the area anorectum in AIDS patients examined and/or admitted to the surgical service. METHODS: From 1987 until 1992, we have observed that certain surgical diseases in the area anorectum were much more frequent in AIDS patients. Because of this, we began to register all cases examined. Since then, we have registered 320 cases. In addition, the bacteriological and histological examinations of the lesions observed also have been done. RESULTS: The following diseases have been registered: abcesses(52,5%), anal fistula(20%), hemorroids(11,3%), anal fissure(8,1%), anal condyloma (5,6%) and proctitis due to gonorrhoea. DISCUSSION AND CONCLUSION: Our results show that AIDS favours surgical diseases to prevent these surgical diseases we recommend strict physical hygiene. In addition sanitary education must be given in order to favour the change of behaviour in sexual practice. PBO571 DALACIN C IN THE TREATMENT OF INFECTIOUS DISEASES OF THE UROGENITAL TRACT.Zudin Boris, Borisenko K., Kisina V. "SANAM". Moscow.Russia In recent years, infectious diseases of urogenital tract have been found to be rather often of a polymicrobial nature. We have studied the efficacy of clyndamycin in the treatment of infectious diseases, mainly combined ones, of the urogenital tract in three dosage forms, namely:clindamycin hydrohloride for parenteral use, clindamycin hydrochloride per os, and clindamycin phosphate as a 2% vaginal cream of the "Upjohn Company". 62 patients (women -48, men -14) aged from 18 to 42 with the diagnosis of gonorrheal-chlamydeal, chlamydeal-gardnerella infections and BV have been subjected to clinical and laboratory examination and treatment. A repeated laboratory exam was carried out 7-10 days after the end of the treatment. The conducted studies have shown different dosage forms of clindamycin to be highly effective against associated diseases of the urogenital tract, as well as a possibility to apply it only locally in pregnant women. w O 0 a> 0w 0 "0 PROF. GEOFFROY GATERA, UNIVERSITY HOSPITAL OF BUTARE, RWANDA B.P. 30 BUTARE, RWANDA.-FAX(0O0250)30870

Page  140 -= PB0572 SPINAL EPIDURAL ABSCESS IN 8 PATIENTS INFECTED WITH HIV. ANDRADE, A AKDAR, N ROTHMAN,M RIGAMONTI,D WHEELER,D. UNIVERSITY OF MD. OBJECTIVE: To determine the clinical course, microbiology, management, and outcome of spinel epidurel abscess (SEA) in patients (pts) infected with the human immunodeficiency virus type 1 (HIV). METHODS: A retrospective chart review of all adults presenting with HIV and SEA, searching computerized discharge data and radiographic records at the University of Maryland Medical Systems over the last four years. RESULTS: Eight SEA were found among 2054 HIV admissions (.38%). Four females and 4 males were identified, all African Americans, mean age-42 range (r) = 35-51. All were injection drug users (IDU), 2 injected in the neck, 1 had a previously infected dialysis catheter, and 5 had skin ulcers. The mean CD4 count (5 pts) was 400 (r = 200-584). One had thrush and 1 prior PCP. On admission 4 patients had paralysis (Heusner IV. NEJM 23:845.1948), 3 had weakness (Heus. tIll and 1 had beck pain (Heus. I). SEA was diagnosed by magnetic resonance imaging (7) or computed tomography (1l, 3 were cervical, 2 thoracic. 2 both and 1 lumbar. Staphylococcus aureus was isolated from all patients: blood and abscess (4) blood only 13) and abscess drainage (1). Coagulese negative Staphylococcus was also isolated from 2 blood cultures. Mean white blood cell was 9,125/mm3 (range 1,700-14,000), mean erythrocyte sedimentation rate was 61mm/hr (6 pts). Four pts underwent surgical decompression. Post-operative IV antibiotic therapy averaged 11.5 weeks (r = 6 -12), 3 Nafcillin, 1 Vancomycin. Three pts survived, 1 improved and 2 were stable. One died after relapse of SEA for medical non-compliance. The mean length of stay (3 pts) was 37 days (range 7-42). Four patients received medical management. Mean IV therapy (3pts) was 9 weeks (r= 4-1 2), 3 Vancomycin 1 Nafcillin. Three patients improved, 1 was stable. DISCUSSION: The incidence of SEA in HIV is 5 times the rate of SEA in our hospital (.076%). Most patients did not have advanced HIV, all were IDU's, 50% were females, end 62.5% involved the cervical spine, probably associated with IDU in the neck. S. aureus was implicated in all cases. Pts presented with advanced neurologic findings, yet 50% received medical management. Prolonged antibiotic therapy was associated with poor medical compliance. ADRIANA SOUZA DE ARAUJO ANDRADE. MEDICAL BIOTECHNOLOGY CENTER. 618 WEST LOMBARD STREET 2 FLOOR. BALTIMORE,HD. PB0573 AUTOPSY FINDINGS IN A SERIE OF 56 AIDS PATIENTS IN BRAZII, Veloso, Valdilea; Grinszlejn,B; Wemrneck-Barroso, E; Freitas,W; Scrapiao, M.; Bottino,A; Chicarino, J. - Evandro Chagas Hospital/IOC/FIOCRUZ Objective: To assess the diagnosis provided by autopsy that were not clinically suspected in a serie of 56 AIDS patient and evaluate their clinical significance. Methods: The autopsy findings from 56 AIDS patientes (CDC-87) (49 male, 8 female, mean age 38 f SD 9.6, 29 hom/bisexual, 13 heterosexuals, 2 IVDU, I blood receptor and 11 with unindentified risk; mean survival was 12,5 months i SD 11) from 1989 through 1993 were evaluated in order to record Ihose that were not clinically suspected. Ultimate cause of death (CD) was also analysed. Results: Bacterial pneumonia was the most frequent autopsy finding 50% (28/56), CD in 9 cases and the main unrecognized infection (26/28) followed by CMV infection (22/26), toxoplasmosis (6/12), histoplasmosis (5/7), cryptosporidiosis (4/7), tuberculosis (3/9), criptococosis (3/12), P.carinii pneumonia (2/6), MAC (2/3), intestinal zygomicosis (1/1), herpetic esofagitis (111), bacillary angiomatosis (1/1). Neoplasic disease was not suspected in 2/7 cases (SK). The ultimate cause of death was not properly identified in 41.07% (23/56) patients. Comments: The high number of deaths determined by bacterial pneumonia clinically unsuspected indicate a necessity of a high index of suspection of this condition as it is potentially treatable. CMV remains the most frequent clinically undiagnosed opportunistic infection in AIDS patients. The low number of undiagnosed tuberculosis in our serie probably represents a high index of suspeclion of this infection in Brazil and its occurrence early in the progression of the immunodeficiency. Valdile a G. Veloso dos Santos - Rua Alzira Cortes 5/308 CEP 22260050 Tel/Fax 55212460493 Rio de Janeiro - Brazil 0 01,,4 w 0 (31 N) I 01 PB0574 Neutropenia is associated with Bacterial Infections Philip Keiser, Elizabeth Higgs, Dolores Peterson N Universi tv.o.foTexas. Southwestern nallas. Texas _ Neutropenarin -voinfection is causea oy orugs, invasive'bbone mat'row diseases, and HIV itself. Studies have not associated AIDS neutropenia (AN) with bacteremia but these have been confounded by high rates of bacteremia in patients with low CD4 counts. We performed a case controlled study of AN to determine whether it is an independent risk for bacteremia. 29 patients with AN were paired with a control and matched for age, sex, and CD4 count. Each group was evaluated for bacteremia, central venous catheters (CVC), invasive bone marrow disease (IBD), drugs causing AN, and deaths. There were 8 bacteremias in the cases and 1 in the controls (p=0.016). There were 7 CVC's in the cases and 6 in the controls (p= 0.5). IBD was similar in both groups (cases= 17, conts= 13, p=0.61) and included mycobacterium avium complex, cytomegalovirus infection, disseminated histoplasmosis and lymphoma. Antiretroviral use was similar in both groups (cases=22, conts= 30, p=0.13) but zidovudine was less frequently associated with neutropenia (cases = 10, controls=21, p =0.01). Trimethoprim / sulfamethoxazole prophylaxis was less frequently associated with neutropenia (cases= 10, conts=21, p=0.01). Dapsone, ganciclovir, pentamidine, and foscarnet usages were similar in each group. Total deaths were similar (cases= 21, conts = 16, p= 0.13). Bacteremia occurred more frequently in the cases suggesting that AN is an independent risk factor for bacteremia. There was a no association between drug use, IBD and AN suggesting that an unmeasured factor, such as HIV itself may the etiologic agent. PB0575 "Factor-R - A Peroral Polybacterlal Vaccine for Immunostimulation Against AIDS" by B.Petrounov, P.Nenkov, R.ShekerdJiski, J.Cvetanov LifeChoice International AD, 96a Rakovski St., Sofia 1000, Bulgaria OBJECTIVE: LifeChoice immunostimulant "Factor-R ", a polybacterial vaccine, designed to boost the human immune system, to control the onset of AIDS-related secondary Infections and to prevent and to reverse AIDS-related Immunological disorders. METHODS: Factor-R is a polybacterial vaccine composed of freeze-dried lysate and killed bacterial bodies of microbial species causing the occurrence of non-specific respiratory diseases. It has a stimulating effect on the cells of the immunocompetent system, evokes the production of corresponding antibodies and enhances the non-specific defence mechanisms of the body: phagocytosis, secretory IgA, complement, gamma-interferon, alveolar surfactant. RESULTS: Factor-R is largely used with great success in Bulgaria for peroral immunotherapy and immunoprophylaxis of non-specific and viral infections. Its efficiency has been confirmed by many experimental studies, 9 clinical trials with over 800 patients with different non-specific respiratory infections and over 7 years of use by hundreds of thousands of persons. It has an excellent tolerability and no side effects have been declared. DISCUSSIONS AND CONCLUSIONS: Factor-R Is proposed as a scientifically based immunostimulant for treatment and prophylaxis of AIDS-related secondary bacterial and viral infections.To prove this we started clinical studies with AIDS patients and HIV positive persons in order to give them better quality of life and a new chance. Our first results are promising. Prof.Bogdan Petrounov, MD, PhD, DSc 96a Rakovski St., Sofia 1000, Bulgaria Tel: *359-2-442875; Fax: *359-2-801236

Page  141 PB0576 NUCLEAR MEDICINE and AIDS 1994 a REVIEW SILVESTRI M.. TARCHI R., AMBU S., LEONCINI F., MENNUTI A. NUCLEAR MEDICINE and INFECTIOUS DISEASE DEPT. PRATO - ITALY The aim of this work is to stigmatize the actual role of Nuclear Medicine in the management of patients affected by HIV Infection. Nuclear Medicine offered its contribution In the clinical evaluation of Immunodeficlency patients since 1982. Sensibility and specificity of these examinations were and remain variable according to the clinical question; nevertheless we can confirm that they are now better than in the past The Improvement of the accuracy is due, for the same tracers, to the technological evolution of gamma-cameras and the better knowledge of the tracer's blodistribution. Very important results are due to the new radiopharmaceutlcals that are disposable for detection of various pathologies In HIV seroposltive patients. Remarkable results were obtained in the evaluation of the opportunistic infections in AIDS: the first experiences with Gallium were followed from an In vivo evaluation of Human Immunoglobulin -Tc marked that showed a good accuracy in the detection of pulmonary Infections and in their follow-up, giving Important Informatlons about differential diagnosis. Now are available a new radiopharmaceutlcal: monoclonal antibodies against white cells (granulocytes), that allows to mark the granulocytes In vivo, prevaenting from the manipulation of large quantity of Infected blood. We will be able, using this tracer, to give Informations weven on the abdominal Infections that were difficultly detectable with Nuclear Medicine techniques till now. The evaluation of infections is only one of the procedures available in these patients, in fact every organ may be detected for his function with Nuclear Medicine examinations and we remember that no preparation is requested, no adverse reactions are usually described and the scintigraphy may be performed in every patient's clinical condition. Dott. MASSIMO SILVESTRI, U.O. MEDICINA NUCLEARE, OSPEDALE DI PRATO - ITALIA PHONE 0574-494212; FAX 0574-494212 PB0577 BACILLARY ANGIOMATOSIS HOOI A.Y.S. LAWLOR F. MEADWAY J.V. WISDOM A.R. NEWHAM GENERAL HOSPITAL LONDON ENGLAND. Object Jvrte: To illustrate with serial photographs the diagnosis, treatment and clinical outcome of bacillary angiomatosis and concomitant peliosis hepatis in a male patient with acquired immunodeficiency syndrome (AIDS) and mycobacterium avium intracellulare infection (HAI). Methods: The patient developed widespread cutaneous nodules and papules in association with pyrexia, weight loss, severe anaemia and hepatomegaly. Skin biopsy showed histological features of bacillary angiomatosis. Ultrasound scanning revealed an enlarged liver and serial liver enzymes showed raised alkaline phosphatase, aspartate aminotransferase and bilirubin. Results: Treatment with erythromycin in a dose of 500 mg four times daily for four weeks resulted in resolution of skin lesions with a decreased in liver size and an improvement in liver enzymes. Discussion and Conclusion: Bacillary angiomatosis has emerged as a new infection in patients with AIDS. Constitutional symptoms due to disseminated infection are similar to late human immunodeficiency virus (HIV) disease. As bacillary angiomatosis is readily treatable, recognition of this infection hinges on clinical suspicion and histological studies. HOOI ALEX Y.S. NEWHAM GENERAL HOSPITAL LONDON