Mast cells are tissue resident, innate immune cells that provide protection against parasitic and bacterial infections and venom poisoning. Mast cells also play a pathogenic role in atopy and allergic diseases. Atopy and allergic diseases are increasing in the developed world and are predicted to continue to increase at an alarming rate. Current treatment options include corticosteroids, anti-histamines, anti-IgE and avoidance of allergen. These interventions have limitations: some patients are steroid resistant; anti-histamines have low efficacy since they need to be administered early during allergen exposure; and anti-IgE is costly. Thus there is a clinical need for new treatment options. An efficient approach is to re-purpose FDA-approved drugs. Selective serotonin reuptake inhibitors (SSRIs) are a class of anti-depressants used to treat depression and other psychiatric disorders. SSRIs have been shown to possess anti-inflammatory properties, but the mechanism of action is unclear. The possibility to treat allergic diseases with SSRIs has not been studied. Using primary mouse bone marrow derived, ex vivo cultured mouse peritoneal, and primary skin derived human mast cells, we show that the SSRI fluoxetine suppresses IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion. Several other SSRIs showed similar effects on mouse mast cells. Cytokine suppression occurs at a transcriptional level, as evidenced by decreased signaling downstream of the IgE receptor and reduced cytokine mRNA induction. We found that fluoxetine-mediated suppression requires the purinergic receptor, P2X3. Furthermore, we show that IgE stimulation elicits rapid ATP release from mast cells, and that ATP and purinergic signaling is a positive feedback regulator of mast cell activation. Fluoxetine can also suppress ATP-mediated cytokine production, degranulation, and lipid production most likely via NFkb suppression and diminished purinergic receptor expression. Importantly, fluoxetine effects are consistent in an in vivo passive systemic anaphylaxis (PSA) model and in a house dust mite (HDM) airway hyperresponsiveness and lung inflammation model of asthma. In the PSA model, fluoxetine reduced hypothermia and cytokine production. In the asthma model, the drug suppressed bronchoresponsiveness as well as pulmonary mast cell hyperplasia and eosinophilia, and the recruitment of Th2 cells, neutrophils, eosinophils, and lymphocytes to the bronchoalveolar space, as well as cytokine levels in the bronchoalveolar fluid in sensitized mice. Overall, we show that fluoxetine broadly suppresses mast cell activation in vitro and in vivo, most likely by impeding an ATP-P2X3 positive feedback loop.

CD4 and CD8 T cells are constantly exposed to inflammatory signals that influence diverse functional outcomes during infections and certain autoimmune disorders. One of the signals controlling CD4 and CD8 T cell functions is the inflammatory cytokine IL-12. Previous studies have focused on how IL-12 regulates CD4 and CD8 T cell functions when present during or after the activation of the T cell receptor (TCR). However, based on murine studies, we have only recently begun to appreciate that exposure to inflammatory signals, driven in part by IL-12, could alter how CD4 and CD8 T cells respond to TCR stimulation. Although intriguing, these studies have left several questions unanswered. Does IL-12 similarly regulate the function of human T cells? If so, what is the exact molecular mechanism by which IL-12 mediates these effects? To address these critical questions, we examined how IL-12 pretreatment altered human CD4 and CD8 T cell responses to subsequent TCR stimulation. In CHAPTER III, we examined how prior exposure to IL-12 alters the responses of human CD4 T cells to subsequent TCR stimulation. Some of our key findings were that IL-12 pretreatment increased the production of IFN-γ, TNF-α, IL-13, IL-4 and IL-10 after TCR stimulation, suggesting that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. Based on the intracellular staining and mRNA expression data, we concluded that the IL-12-mediated increased production of a range of cytokines was a consequence of at least two separate mechanisms, increased mRNA expression for IFN-γ and increased release of TNF-α, IL-13, IL-4 and IL-10. In CHAPTER IV, we explored the mechanisms by which IL-12 pretreatment altered human CD4 T cell responses to TCR stimulation. We observed that IL-12 pretreatment increased the phosphorylation of AKT, P38 and LCK following TCR stimulation without altering other TCR signaling molecules, suggesting that this potentially mediates the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that were not transcriptionally regulated was partially driven by increased oxidative metabolism. Collectively our results uncovered a novel function of IL-12 in regulating human CD4 T cell responses; specifically, it enhanced the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism. Then, in CHAPTER V, we examined the effects of IL-12 pretreatment in altering the responses of human CD8 T cells to subsequent TCR stimulation. Our key finding was that pretreatment of human CD8 T cells with IL-12 resulted in increased IFN-γ and TNF-α cytokine mRNA and protein production following subsequent TCR challenge. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules. Together our results suggest that prior exposure to IL-12 potentiates human CD8 T cell responses to TCR stimulation possibly by altering the activation of TCR signaling pathways. In the end, our results increase our understanding of the physiologic properties of human CD4 and CD8 T cell and provide mechanistic insight into novel functions for IL-12. Our results also provide insights into potential avenues to improve the current uses of IL-12 in therapeutics.

Plasma cell neoplasms (PCN), including multiple myeloma, are tumors of terminally differentiated B cells. Despite a significant research effort, and numerous advances in therapy, most tumors of this B cell lineage remain incurable. To this end, understanding factors which are critical for the development of PCN may lead to new avenues for therapy. Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine which supports the growth, proliferation, and survival of myeloma cells. We found that inflammation, and in particular, IL-6 is critical for the development of PCN. In order to determine if tumor microenvironment (TME) or B cell-derived IL-6 was more important in PCN development, we utilized an adoptive transfer system of tumor formation. By adoptively transferring premalignant B cells into recipients, and then providing the B cells with an inflammatory microenvironment through the use of pristane, we were able to generate donor tumors in recipient mice. Utilizing this method, a series of adoptive transfers were performed to determine the primary source of IL-6 in murine PCN development. We discovered that TME-derived IL-6, and not B cell-derived IL-6, is most critical for PCN development. Furthermore, in studying the lesions in B cell development which lead to tumor formation, we discovered that IL-6 collaborates with the proto-oncogene c-Myc in spontaneous germinal center (GC) formation. The spontaneous GCs were accompanied by a robust follicular T helper cell response. In characterizing the genetic lesions which lead to the GC formation, we discovered that Myc-transgenic mice develop a significant increase in the population of B1a B cells. Furthermore, these B1a B cells infiltrate the spontaneous GCs of double transgenic Myc/IL-6 mice. Lastly, utilizing our adoptive transfer method, we determined that the germinal center response is necessary for the development of PCN in mice. Lastly, we focused our efforts on another oncogene which collaborates with IL-6, BCL-2. Double transgenic BCL-2/IL-6 mice develop PCN and spontaneous GCs. Of interest however, the adoptive transfer of BCL-2/IL-6 B cells results in tumor formation without the use of pristane. Furthermore, the adoptive transfer recipients develop bone lesions, hind limb paralysis, and a monoclonal gammopathy. This model closely recapitulates many of the pathophysiological features seen in human PCN. This new model promises to be important for future studies into PCN development and treatment.

No description available.

Three potential driver genes were identified by use of retroviral mutagenesis in the newly developed iMyc model of B cell malignancy. To do so, iMyc mice, which bear an inserted copy of c-Myc in the IgH locus, were treated with MOL4070LTR to favor the development of B cell malignancies. After tumor development, B cell origin tumors were identified by use of immunohistochemistry and selected for downstream analysis. Three genes were chosen as potential driver genes and validated in mouse or human disease as involved in disease or directly in malignancy. These genes are CD82, IRAK2 and DNMT3a.

Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.

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.

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.