No description available.

Pacific salmon migrate long distances to spawn as part of their life cycle. During this journey from sea to their natal stream, they undergo major endocrine, physiological and immune changes. Cortisol, the primary stress hormone, gradually increases during the journey. Persistent high cortisol levels have deleterious health effects, including suppression of the antibody response. However, pathogens encountered during their journey may stimulate antibody responses to overcome the infection. My main research question focuses on how salmonids balance the immunosuppressive effects of high cortisol levels with activation of the antibody response. A recent field study from our lab showed a transient increase in abundance of B cells during the spawning run which is suggestive of activation of the immune system during this journey. However, our field study had too many confounding variables. In this study, we investigated the activation of the antibody response under conditions of elevated levels of cortisol in rainbow trout under laboratory-controlled conditions. We looked at the effects of a) cortisol alone, b) fish pathogen Flavobacterium psycrophilum (Fp) alone and c) combined cortisol and Fp challenge on the gene expression of immunoglobulins IgM and IgT using qPCR. We have found that cortisol suppresses the IgM response in the spleens of Fp-susceptible line but not in Fp-resistant line of Rainbow trout. No significant effects on B cell development where observed in the anterior kidney. Taken together, our data suggest that the antibody response in Fp-resistant rainbow trout is less sensitive to increased cortisol levels compared to Fp-susceptible fish, confirming our hypothesis that Fp-resistant fish have in some way evolved to manage stress more successfully.

Human immunodeficiency virus (HIV) infects and destroys lymphocytes, leading to acquired immunodeficiency syndrome. Homosexual men were impacted disproportionately in the early years of the pandemic, while women are now the majority of those infected, making development of anti-HIV treatments and preventatives effective in the female reproductive tract (FRT) imperative. Here, we investigated HIV prevention in women through 1) discovery of antivirals effective in the FRT; and 2) determining a mechanism by which bacterial vaginosis (BV), a disorder of the FRT in which protective Lactobacillus are replaced by BV-associated bacteria (BVAB), increases HIV transmission. We identified a small molecule active against HIV from an extensive compound library and used its structure for an in silico screening, identifying a novel class of HIV inhibitors, Avirulins. Three were active in the low micromolar range and exhibited HIV-1 reverse transcriptase inhibition. Avirulins were not cytotoxic to FRT epithelial cells and maintained activity in human cervicovaginal fluid (CVF). With continued development, Avirulins could serve as additions to antiretroviral therapies or preventatives for the FRT. Next, we hypothesized that BV increases HIV transmission though disruption of the FRT epithelium at the endocervical monolayer, as most FRT lymphocytes reside below the epithelium. We determined that matrix metalloproteinases (MMPs) were secreted by endocervical epithelium in response to BVAB and could depolarize endocervical cell layers. When HIV infected lymphocytes were cocultured with endocervical cell layers, treatment with conditioned media from endocervical cells cocultured with BVAB increased HIV transmigration. Treatment with MMP inhibitors reduced this effect. We demonstrated that CVF from women with BV had greater MMP activity, and presence of certain MMP isotypes in CVF correlated with increased HIV transmigration through the endocervical epithelium. These results propose endocervical disruption by MMPs as a mechanism for BV-induced HIV transmission and suggest the potential of MMP inhibitors as HIV preventatives.

No description available.

Mycobacterium tuberculosis (M.tb) is the causative agent of pulmonary tuberculosis (TB). Over 25% of the world’s population is estimated to be infected with tuberculosis, yielding over 10 million new cases and over 1.5 million deaths in 2017 alone. This is all despite the near universal implementation of bacille Calmette-Guérin (BCG) vaccination across TB endemic areas. BCG is capable of preventing childhood disseminated forms of disease but fails to induce potent immunity within the lung. We expect this to in part play a role in the lack of protection against pulmonary TB. Our lab has developed a human adenovirus serotype 5 vaccine expressing the M.tb antigen Ag85A (AdHu5Ag85A). When delivered directly to the respiratory mucosa (RM), AdHu5Ag85A has proven to be safe and immunogenic, generating both CD4 and CD8 T cell responses within the lung. We have found that RM AdHu5Ag85A vaccination also generates a long-lasting population of memory macrophages in the airway and lung, that are primed to better control early M.tb infection. Importantly, this was a vaccination route-dependent mechanism. Memory macrophages express a trained innate immune phenotype as they express high levels of MHC II, are highly glycolytic, and produce more IL-12 upon re-stimulation. Importantly, utilization of an in vivo T cell depletion approach allowed us to show that these memory macrophages are capable of limiting early M.tb infection independent of T cells. Our findings indicate that RM vaccination may be advantageous to parenteral routes by not only drawing antigen specific T cells into immunologically restricted lung mucosa but also generating a memory macrophage population within the lung. Induction of memory macrophages within the airway helps overcome early innate immune suppression by M.tb. / Thesis / Master of Health Sciences (MSc) / Tuberculosis (TB) is a pulmonary disease responsible for 10 million new clinical cases and more than 1 million deaths annually. Over one quarter of the world is believed to be infected with TB. This is despite the near-universal administration of bacille Calmette-Guérin, a preventative TB vaccine, and an effective, but lengthy antibiotic treatment. With antibiotic resistance on the rise, developing a protective vaccine against TB is more important than ever. Tuberculosis has confounded researchers for decades and has thus escaped development of a more effective vaccine. One of the ways to improve a TB vaccine would be to inhale it, to have local effects at the main site of TB infection in the lung. We found that by aerosolizing our vaccine, we can impact local immunity within the lung in a way that has never before been described, opening new avenues for TB research.

One strain of Bacillus subtilis and three antibiotic-resistant strains of Staphylococcus aureus were employed to determine the biological effects of everninomicin B (EvB), a naturally occurring antibiotic produced by Micromonospora carbonacea (NRRL 2972) and M. carbonacea var. aurantiaca. Minimum inhibitory concentration (MIC) of EvB for B. subtilis was 1.2 x 10-3 µmole/ml of EvB in glucose minimal broth and 2.6 x 10-3 µmole/ml of EvB in nutrient broth. MIC values of EvB for S. aureus were 3.25 x 10-4 µmole/ml of EvB for strains resistant to penicillin or tetracycline and 6.5 x 10-4 µmole/ml of EvB for the aminoglycoside-resistant strain. The inhibitory effect of EvB was found to be reversible for all concentrations of EvB and all bacterial strains tested. The inhibitory effect of EvB for B. subtilis was not dependent upon the initial concentration of cells nor the stage in the growth cycle at the time the compound was introduced. The inhibitory effect was dependent upon the initial concentration of cells for S. aureus. Electron microscopy studies showed distinct morphological changes in treated cells of S. aureus. Cellular lysis in these cells was also detected.

The drug of choice for the treatment of Stieptococcal pharyngitis is penicillin G. However, a common home remedy prescribes the use of salt-water solutions for gargling. Members of Beta -hemolytic streptococcal groups A, B, and C were isolated from the upper -respiratory tracts of patients diagnosed as having streptococcal pharyngitis. These cultures we:e obtained from HCA Greenview Hospital (Bowling Green, Kentucky) and used to study the effects of sodium chloride on the isolates. The minimum inhibitory concentration of sodium chloride was determined for each of eight hospital isolates. Croup A streptococci were inhibited at a concentration of 7.2% sodium chloride while Group C streptococci were inhibited at a 7.0% concentration. Group P streptococci were more resistant, and inhibition of growth occurred at 12.0% sodium chloride concentrations. Scanning electron microscopic studies showed no significant differences in the external structure of cells treated with sodium chloride when compared to non-treated cells. Despite the lack of changes in the external structure of treated cells, fine structural alterations were observed with transmission electron microscopic studies. Treatment of the cells with sodium chloride resulted in a condensation of nucleoid deoxyribonucleic acid (DNA) and some loss of ribosomes. These changes were followed by a dissolution of the cytoplasmic cell contents resulting in an intact cell wall with capsule. Other parameters such as the rate of growth, minimum bactericidal concentrations, DNA content and protein content of cells treated with sodium chloride were examined and compared to control cells.

No description available.

The study of antibody responses in prominent aquaculture species such as the rainbow trout, Oncorhynchus mykiss, can facilitate vaccine development and contribute to producing useful paradigms of adaptive immunity in lower vertebrates. Thus, it is essential to identify genes responsible for antibody responses. In the mouse model, hybridoma technology allows for the association of monoclonal antibodies possessing various affinities for antigen with specific VH sequences, gene family utilization, and other molecular events (i.e. somatic hypermutation) that occur during the specific immune response. The absence of a comparable hybridoma technology in piscine systems has limited similar studies of fish immunogenetics to date. Molecular and serological experiments were performed in an attempt to obtain information regarding somatic mutation and VH gene utilization for trout antibodies without reliance on hybridoma technology. PCR primers recognizing consensus sequences of FR1 and FR3 were used to amplify antibody VH sequences from panned, antigen-specific B cells. to follow the development of the expressed VH repertoire, lymphocytes were obtained at weeks 0, 5, 10, and 20 post primary immunization with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) or infectious hematopoietic necrosis virus (IHNV). Lymphocytes were also collected 10 weeks post secondary immunization (week 35). These studies were conducted in parallel with serological analyses of plasma antibodies obtained from the same sample in order to correlate molecular data with serological data from individual trout. Antigen-specific lymphocytes were processed to isolate RNA templates to produce cDNA which was cloned and sequenced. This sequence analysis allowed us to report, for the first time, the temporal accumulation of potential somatic variants that correlate to the development of new, high affinity antibody subpopulations during the immune response, some with the emergence of new antibody heavy chain isoelectropherotypes as identified by 2D-IEF/SDS-PAGE. Southern analysis and gene titration using various antigen-specific cDNA probes allowed us to correlate trout antibodies possessing various affinities for antigen with specific VH sequence and gene family utilization. Thus, trout Ig VH gene family utilization appears to follow the mouse model of differential use for specific immune response. These results reveal a capability for fine-tuning the piscine immune response previously not recognized.

Leishmania infantum chagasi (Lic) is a pathogenic protozoan parasite and one of the etiological agents of human visceral leishmaniasis (VL). VL is a potentially deadly disease characterized by variable fevers, cachexia, hepatosplenomegaly, and global immune suppression. Many questions regarding the pathogenesis of VL and the mechanisms of host defense during Lic infection remain to be elucidated. The primary focus of this thesis is the relationship between Lic and the mammalian immune system. We studied parasite-host interactions during Lic infection at the molecular, cellular, and organismal level. We generated transgenic parasites that expressed firefly luciferase and/or fluorescent proteins to expand our capacity to detect, observe, and quantify the parasites in a variety of experimental settings with modern analytical methodologies. Using luciferase-expressing Leishmania, we developed an experimental infection model in which parasites were detected and the relative parasite burden in specific anatomical locations could be quantified in a live animal host using bioluminescence imaging. This method allowed the parasite burden to be assessed in the same host throughout the course of infection. Utilizing this model we have made some intriguing observations relating to the kinetics and distribution of the parasite burden over time. The parasite burden was observed primarily in the liver and bone marrow over the first few weeks and then shifts to the spleen and bone marrow. To gain a better understanding of the initial parasite-host immune interactions in vivo, we studied the early inflammatory response after intradermal (i.d.) inoculation. We observed a rapid and abundant influx of neutrophils into the inoculated ears. The neutrophil influx was transient, dose dependent and specific for the local inoculation site. While there was not a significant neutrophil influx into the draining lymph nodes (dLN), there was an increase in the total cellularity and a striking increase in the relative proportion of B cells to T cells over the first week after intradermal parasite challenge. By inoculating transgenic mCherry-Lic we found that neutrophils were the primary parasite-laden host cell in the dermal tissue during the first day, but macrophages harbored most of the parasites by 2 days. Neutrophil depletion using low-dose antibody treatment resulted in a reduced rate of parasite uptake initially at the site of inoculation, but no significant change in the dLN dynamics. We further examined the parasite-host relationship by studying molecular signaling and cellular interactions between Leishmania and human neutrophils. We investigated the nature of the chemotactic activity of Leishmania-conditioned growth medium for human neutrophils by testing physical properties of the activity and ruled out some of the major Leishmania surface molecules as potential candidates. We aim to identify the agent(s) responsible for the activity in on-going studies. To this end, we are collaborating with a group at the NIH and testing biochemical purification/separation samples. We conclude that intradermal Lic challenge induces a rapid innate immune response at the local site of infection, that neutrophils sense Leishmania-derived factors leading to directed migration, and that neutrophils function as a primary site for Leishmania entry into the mammalian host.