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Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3296. Advisers: Clifford A. Lowell; Eric J. Brown.

Cryptosporidium parvum is a ubiquitous parasite affecting a wide range of mammals. In immune competent hosts it causes profuse self-resolving diarrhea. In those with impaired immunity, diarrhea persists while the deficiency prevails because there are no effective therapies available. Previous studies reported the benefits of polyclonal antibodies for treating cryptosporidiosis. Data from preliminary studies supported the anti-parasitic efficacy of bovine and hen egg yolk polyclonal antibody preparations. Preliminary in vivo testing showed significant reductions in the parasite loads (p < 0.05) of animals treated with yolk antibodies when compared to controls. Preparations of anti-C. parvum polyclonal antibodies from human breast milk, bovine colostrum and chicken egg yolks were obtained and tested in vitro for activity and potency and subsequently tested for efficacy in comparative in vivo studies. Hen egg yolk preparations were significantly more efficacious (p < 0.01) and subsequently evaluated in human clinical trials. Analysis of data from the trial showed beneficial therapeutic value and also the need for improvements in the egg yolk formulations. Further work directed towards antibody concentration and lipid reductions were performed. The dilution of egg yolks with water precipitated the lipids and the lipid-reduced supernatant fluids were lyophilized resulting in an enhanced formulation with increased anti-cryptosporidial potency and about 90% reduction in the lipid contents. The efficacy of the enhanced preparations was also tested in vivo with parasite reductions in the order of 90%. Hen egg yolk antibodies could be a valuable component for treating or controlling enteric cryptosporidiosis.

The virulence of Mycobacterium tuberculosis (Mtb) depends on its ability to multiply and survive within host macrophages. In screening for Mtb genes that play a role in the intracellular survival, a Mtb gene ( eis) was identified that enhanced survival of Mycobacterium smegmatis in both human monocytes and in the human macrophage-like cell line U-937 when introduced on the multi-copy plasmid pOLYG. When a single chromosomal copy of eis was introduced into M. smegmatis, using an integrative vector, the construct still exhibited increased intracellular survival in U-937 cells. The eis gene was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other mycobacterial species. Western blot analysis showed that the eis gene could produce a 42-kD protein product in both M. smegmatis and M. tuberculosis. The role of the eis gene in M. tuberculosis intracellular survival and multiplication was investigated by inactivation of the eis gene via allelic exchange. A mutated eis allele ( eis::hyg) was delivered at the eis locus, using the suicide vector pMJ10, in both Mtb strains H37Rv and H37Ra. Complemented mutants were also constructed by reintroducing a wild-type eis into the chromosome attB site. Southern and Western blot analysis demonstrated that the eis gene was disrupted and no Eis protein was produced, and that complemented strains regained the ability to produce Eis. Wild-type M. tuberculosis, eis knockout mutant and complemented strain were then evaluated for their capacity to survive and multiply within U-937 cells. The eis mutant survived and multiplied, as its parental strain, in U-937 cells over a 7-day period. These findings suggest that eis may not be required for the short-term multiplication of M. tuberculosis in U-937 cells. Further in vivo study needs to be done to clarify the role of eis in the pathogenesis of tuberculosis.

In the United States greater than 6 million dry tons of biosolids are produced nationwide, with greater than 60% being land applied. Although most counties utilizing land application are practicing this beyond nearby homes, the increase in population has begun to blur the line between rural and urban communities. This study was conducted to investigate the occurrence of biological aerosols (bioaerosols) containing microorganisms and endotoxins, and assess the human health risk involved in these practices. Aerosol samples were collected for 2 years from land application sites located at various locations throughout the U.S.A., which represented different climatic conditions and different application practices. Land application practices involved the use of liquid biosolids spray and "cake" biosolids applicators depending on location. Bioaerosols were collected via the use of six SKC Biosamplers, impinging air at a rate of 12.5 L/min for a total of 20 minutes. Samples were collected from both downwind of land application and background sites from distances ranging between 2 m and 70 m downwind. Microbial concentrations were measured within these aerosols, measurements included: heterotrophic plate count bacteria (HPC), coliphage, Clostridium perfringens, total coliforms, Escherichia coli, endotoxin (lipopolysaccharide), enteroviruses, norovirus, and Hepatitis A virus (HAV). In addition a model was developed to predict viral transport. Overall the levels of aerosolized indicator bacteria and phage were at or below detection limits. Three samples were positive for the presence of norovirus viral RNA via reverse transcriptase polymerase chain reaction, although their viability was unable to be determined based on current available techniques. Calculated microbial risks of infection were determined to be at or below the acceptable risk of annual infection from drinking water proposed by the Environmental Protection Agency, 1:10,000. Biosolids loading scenarios presented the greatest risk of infection, partly due to the point source of exposure. All other portions of biosolids land application operations yielded risks of infection well below the annual 1:10,000 risk of infection. Overall the microbial aerosol exposures brought about by land applied biosolids are minimal and hence minimal overall risks of infection.

This dissertation contains a mathematical study of the growth and division of the bacterium Bacillus subtilis. We have developed methods which allow us to study individual based models by finding a relationship between the growth and division rates and the steady state length distribution of the bacterial population. This has allowed us to draw conclusions about the life cycle of B. subtilis such as the fact that a minimum age, as well as a minimum size is required of cells before division can commence. We have shown that successive divisions of of the minicell producing mutant divIV-B1 are not independent, in the sense that whether a minicell was produced as a result of a division is not independent of minicells appearing in later divisions. Finally, we have shown that individual based models are a much more powerful tool for studying bacterial kinetics than traditional population based models.

Acquired immune deficiency syndrome (AIDS) and aging are associated with significant immune dysfunction and increased oxidative stress, resulting in increased susceptibility to opportunistic infections, cancers, autoimmune diseases, and death. Human immunodeficiency virus (HIV) infection and aging lower host defenses by modulating cytokine production to alter T and B cell functions. The overall objective of this study is to determine effect of Murine AIDS (MAIDS), its cofactors and aging on opportunistic infection, and the potential of immunomodulation roles of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) on ameliorating the immunological abnormality caused by retrovirus infection and aging. DHEA(S) was previously shown to have immune enhancing properties during (MAIDS) and the aging. DHEA(S) significantly increased T-cell proliferation, restored secretion of Th1 cytokines production, and normalized secretion of Th2 cytokine. Survival was significantly increased in the 29-month-old mice treated by DHEA. DHEAS plus antioxidants significantly normalized immune function, as well as maintained hepatic vitamin E levels nearer control. In addition, DHEAS supplementation increased resistance to cryptosporidiosis in aged mice. Our study suggests that DHEA(S) alone and especially DHEAS plus antioxidant nutrients can prevent immune dysfunction in aging as well as aging, retrovirus infected mice. Chronic ethanol (EtOH) consumption causes immune dysfunction and further exacerbates immune dysfunction and cytokine imbalance during MAIDS. DHEAS supplementation during MAIDS with EtOH consumption partially restored immune function, prevented the further dysregulation of cytokines and hepatic lipid peroxidation, and maintained hepatic vitamin E levels to near normal levels. Coxsackievirus B3 (CVB3) initiates myocarditis especially in the immunologically deficient. EtOH or cocaine as a cofactor may exacerbates CVB3 cardiomyopathy in AIDS patients. In our study, the resistant mouse strain to CVB3 become susceptible due to the immune dysfunction in MAIDS. EtOH consumption or cocaine injection during MAIDS greatly exacerbated the pathogenesis of CVB3 infection and showed significant heart lesions. Our data suggest that EtOH consumption or cocaine injection shifted the cytokine balance in favor of a Th2 response, by enhancing Th2 cytokine and/or by suppressing Th1 cytokine function. MAIDS facilitated severe cardiotoxicity during CVB3 infection, MAIDS with EtOH consumption or cocaine injection was more susceptible to cardiotoxicity of CVB3 than the retrovirus infection alone.