Department of Health and Human Services Public Health Service, Grant Application

Principal Investigator/Program DirectorLast, first, middle):

Duesber, Peter H.

described by Al-Bayati, Raabe and Teague (Al-Bayati et al., 1992).
The animals will be observed daily and clinical signs will be recorded and evaluated. Animals
will be weighed twice a month. 5 to 10 exposed, uninfected and exposed, infected mice and 5 to 10
uninfected and infected controls will be sacrificed monthly. Thymus, lymph nodes, spleen, liver,
kidney, lung, and brain will be taken for gross and microscopic examination as described by Al-Bayati
et al. (Al-Bayati et al., 1989). Blood and serum samples will be taken from each animal for standard
hematology and serum chemistry analysis as described by Al-Bayati et al. (Al-Bayati et al., 1990).
After six month of inhalation, 5 to 10 exposed, infected and uninfected mice and 5 to 10 infected and
uninfected control mice will be challenged with Listeria monocytogenes and will be sacrificed after 1 to
2 weeks.
Depending on the outcome of this experiment, in the second year similar groupings of mice
will be exposed at lower or higher nitrite concentration. In one of these groups the highest tolerable
dose will be used to determine the shortest exposure time to achieve disease. Another group will be
subjected to lower nitrite doses than are necessary to achieve pathogenicity within 6 months. These
mice will be exposed to nitrite chronically for two years to examine nitrite pathogenicity at low doses
over long periods of time. Longer exposure times may be particularly necessary to observe
carcinogenic potential of nitrites.
The envisioned endpoint of the study would be pneumonia, lowered T-cell counts, increased
susceptibility to infection, and perhaps cancers analogous to AIDS-Kaposi's sarcoma and AIDSlymphoma.
Potential problems with the experimental plan. We hope to achieve clinical nitrite toxicity in
mice within 6 months by exceeding the daily duration and dosage of nitrite exposure used by others,
and by approximating "recreational" use by humans. However, it is possible that the duration of the
experiment may have to be extended from 6 months to 9 or 12 months, based on estimates for the
average 10-year "latent periods" from the onset of "risk behavior" to AIDS in humans. According to
our hypothesis, this "latent period" is the cumulative time of drug consumption that leads to AIDS.
The onset of disease in mice could be further accelerated by subjecting the mice to malnutrition.
Malnutrition is a typical consequence of drug consumption in humans, and in rodents can accelerate
immunotoxic effects. Thus malnutrition would be a further adjustment of our experiment to the human
model. Indeed, psychoactive drugs are often used as "diet pills."
It is possible that the AIDS indicator diseases resulting from nitrite-induced immunodeficiency
in mice are different from those of human models, e.g. bacterial pneumonia instead of Pneumocystis (a
fungal) pneumonia. This could be controlled experimentally by further adjusting our experimental
system to the human model by treating mice subjected to immunosuppressive nitrites with antibiotics.
Many persons at risk for AIDS regularly use antibiotics, such as Bactrim etc., as a prophylaxis against
AIDS (Callen, 1990). This treatment would shift the balance of expected opportunistic infections from
bacterial to fungal and viral pneumonias-exactly as observed in AIDS patients. Indeed, young rats
treated for several weeks simultaneously with antibiotics and immunosuppressive cortisone all
developed Pneumocystis pneumonia spontaneously (Weller, 1955).
In vitro studies. a) In addition to the in vivo studies we propose to test the effects of nitrites
on the growth of human T-cell lines in vitro. For this purpose micromolar doses of amyl nitrites, that
have been observed in human recreational users (Osterloh and Olson, 1986), will be included into the.growth media of two human T-cell lines, RH9 and CEM (Rasheed et al., 1986). Our studies will
extend previous analyses that measured the effects of nitrites on cells after short-term exposures
(Hersh et al., 1983; Jacobs et al., 1983). The human T-cell lines are currently growing in our
laboratory. The system would provide a direct measure of the immunotoxic potential of nitrites on
human T-cells in vivo.

2) In a second line of experimentation we will test the Kaposi sarcomagenic potential of amyl
PHS 398 (Rev.9/91) Page 29
Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.