Background Up to 59% of tuberculosis (TB)/human immunodeficiency virus (HIV) co-treated patients develop paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) after addition of antiretroviral (ARV) therapy to anti-tuberculous therapy (ATT). The course can be prolonged and the average mortality rate is 2% (75% for TB-IRIS involving the central nervous system (CNS)). Immune elements – including neutrophils - involved in the anti-Mycobacterium tuberculosis (Mtb) response are implicated in pathogenesis, which remains incompletely understood. Diagnosis is one of exclusion, no reliable laboratory markers exist, corticosteroid-mediated prophylaxis and therapy are only partially effective, and no treatment targets tissue damage. Disentangling cause and effect in complex disorders such as TB-IRIS requires techniques capable of interrogating complex biological systems. Neutrophils are the major circulating leukocyte population, the earliest innate system responders, and exhibit various unusual immunometabolic functional specialisations. Proteins represent the most functionally-proximal and commonly pharmacologically-targeted cellular biomolecules. Label-free high-performance liquid chromatography-coupled tandem mass spectrometry (HPLCMS/MS) is well-suited to differentially profiling the ex vivo neutrophil proteome in an unbiased manner, in order to investigate TB-IRIS predisposition and pathogenesis. Methods Applying first principles to existing human literature, the most parsimonious holistic hypothetical model regarding paradoxical TB-IRIS predisposition and pathogenesis was inferred. A clinical cohort of control (CTRL) and matched TB-IRIS case (IRIS) study participants was assembled. Demographic, clinical, and biochemical characteristics were analysed for statistically-significant differences and to identify potential risk/protective factors (relative risk (RR) with 95% confidence interval (CI)). A small group (n = 9) of TB-HIV- healthy volunteers (HVs) was also assembled. Phlebotomy occurred at two timepoints: just prior to ARV initiation (week 0) and at the typical time of IRIS manifestation (week 2). Neutrophils were isolated and lysed, proteins underwent on-filter protein trypsinisation, peptide salts and detergents were removed, and neutrophil-optimised HPLC-MS/MS was conducted. Spectra were submitted to MaxQuant for parent protein identification and quantitation. Comparisons of (a) CTRL0 and IRIS0 to HV (and resultant differences) identified class-differential impacts of partial ATT-treated coinfection (IRIS predisposition) and of (b) CTRL2 to CTRL0 and IRIS2 to IRIS0 (and resultant differences) identified class-differential impacts of ARV therapy (IRIS pathogenesis). Class-discriminating proteomic differences were visualised using principal components analysis (PCA), protein differential expression analysis was performed (including for detectable/undetectable and significantly differentiallyexpressed (SDE) proteins), and results informed differential functional profiling via gene ontology overrepresentation analysis (GO-ORA) and pathway activation state prediction. To address shortcomings of current knowledgebases and automated tools, a novel deep manual analysis approach focused on key inference-friendly proteins, convergent findings, and neutrophil-specific functional modules. Integrated findings extended the literature-derived TB-IRIS model, generating testable novel hypotheses, one of which was partially validated using live-cell fluorescence microscopy. Proteomic data were additionally analysed to detect Mtb proteins, preliminarily analyse variable post-translational modifications (PTMs) of interest, and identify candidate prognostic and diagnostic biomarkers. Finally, mechanistic hypotheses facilitated identification of novel potential prophylactic and therapeutic targets. Results (1) Literature suggests advanced TB/HIV-coinfection (including a higher Mtb/antigen load) as the major TB-IRIS risk factor. Attendant significant immunometabolic state perturbations include myeloid overactivation, metabolic stress (possibly including adaptogen depletion), a lack of regulatory receptors, impaired pro-inflammatory signal transduction, and impaired antigen clearance. These likely predispose to lytic cell death - including release of host- and pathogen-derived inflammatory/cytotoxic molecules and proteolytic enzymes - and less restrained/more abnormal inflammation as well as tissue damage, on restoration of HIV-suppressed inflammatory signalling pathways by ARV therapy. (2) Regarding the clinical cohort, a sample size of > 42 participants per characteristic-matched comparison class provides > 95% power to detect a two-fold change with 99% confidence. Cases exhibited known TB-IRIS risk factors, and largely expected white cell count (WCC), body mass index (BMI), and C-reactive protein (CRP) level changes in response to ARV therapy (e.g. WCC increase and BMI decrease). None of the few participants on alternate (efavirenz (EFV)- or tenofovir (TDF)-lacking) regimens developed TB-IRIS. Pre-ARV prednisone (or incidental antihistamine/anti-fungal) use was associated with a non-significantly decreased TB-IRIS risk. Interestingly, smoking is associated with a significant decrease in TB-IRIS risk by 60%. (3) Regarding sample processing and analysis, the average sample collection to neutrophil isolation interval was within recommended limits. Isolation yield exceeded 15 x 106 and 25 x 106 per sample in the CTRL and IRIS groups, respectively. Isolated neutrophil purity exceeded 80% in both groups; the few low-purity samples were excluded from subsequent proteomic analysis. Lysates from 5 x 106 neutrophils routinely yielded over 100μg total protein, tryptic digestion was efficient (on average < 96% missed cleavages), and equivalent peptide injection volumes yielded comparable total ion chromatogram (TIC) profiles and intensities. An average 23% spectral identification rate resulted in a total of 2532 protein group identifications, the deepest neutrophil proteome coverage achieved to date without pre-fractionation, representing ~12% of human protein coding genes, and ~25% of the detectable human proteome. Samples were analysed in two (randomised) batches, producing independent datasets A (N = 37) and B (N =74). Withindataset technical replicates exhibit excellent agreement in protein identities and quantities; betweendataset protein identities and functional inferences also exhibit excellent agreement. We identify a number of proteins apparently not known to be expressed by human neutrophils, as well as one predicted human protein never before observed empirically. Overall, parent pathways of level-altered proteins suggest perturbation of nine major neutrophil function modules at both time-points: (a) signal transduction, (b) pattern recognition receptor (PRR) and cytokine signalling, (c) the eicosanoid cascade, (d) neutrophil antimicrobial functions, (e) carbon-energy metabolism, (f) protein homeostasis, (g) integrated nitrogen-sulfur-B-vitamin and redox/xenobiotic/glyoxal metabolism, (h) gene expression, and (i) cytoskeletal dynamics. (4) Regarding impacts of partially ATT-treated co-infection (week 0), neutrophil proteomic profiles successfully distinguish between HV and IRIS0 or CTRL0. Many differences from HV are shared between IRIS0 and CTRL0 (i.e. driven by partially ATT-treated co-infection), but some are class-unique (i.e. driven by factors predisposing to or protecting from TB-IRIS). Findings are supported by a head-to-head comparison of the CTRL0 and IRIS0 proteomes, including changes suggesting: more prevalent type I IFN, TGFβ, and Th2-type cytokine signalling; poorer capacity for restraint of alternate complement activation; mitochondrial and oxidative stress (including proneness to necrosis); impaired function (e.g. microbicidality, TLR/IL-1R-MyD88-NFκB signalling, and caspase 1- mediated IL-1β and IL-18 maturation) of activated neutrophils; and enhanced lipid and upstream (but inhibited downstream) isoprenoid synthesis (including decreased steroidogenesis). Candidate biomarkers distinguish CTRL- and IRIS-class partially ATT-treated neutrophils from HV neutrophils and from each other. (5) Regarding impacts of ARV therapy (week 2), both IRIS and CTRL neutrophil proteomes exhibit significant changes in response to ARV therapy. Many changes are shared between IRIS and CTRL (i.e. driven by ARV therapy and declining viral load (VL)), but some are class-unique (i.e. driven by factors preventing/contributing to TB-IRIS pathogenesis). Findings are supported by a head-to-head comparison of the CTRL2 and IRIS2 proteomes, including changes suggesting: a slower decline in type I IFN signalling; increased inflammatory cytokine (e.g. IL-6, TNFα, and IFNγ) signalling and protease (e.g. MMP-8) activity; decreased sensitivity to immunoregulatory glucocorticoids and vitamin A; and increased mitochondrial, endoplasmic reticulum (ER), and oxidative stress. Candidate biomarkers distinguish CTRL- and IRIS-group ARV-exposed neutrophils from baseline and from each other. (6) Livecell fluorescence microscopy of HV neutrophils suggests that in vivo-equivalent levels of EFV rapidly alter mitochondrial, lysosomal, and aggresomal architecture in a manner consistent with organelle and protein folding stress, and suggesting cell death commitment. (7) Integrated neutrophil immunometabolic changes suggested by proteomic findings support and extend the biologically compelling literature-derived model. Model highlights include more advanced baseline TB/HIV (including higher type I IFN, TGFβ, and possibly Th2-type cytokine levels), with consequent impaired myeloid-mediated Mtb antigen clearance and depletion of cellular adaptogens. The resultant abnormal immunometabolic state produces myeloid cells less able to counteract metabolic stress and primed for less-restrained inflammation. Introduction of mitotoxic ARV drugs and rapid lifting of HIVmediated immune embargoes escalates myeloid metabolic (including oxidative) stress and overactivation (including via NLRC4, CASP4/5, TLR/IL-1R-MyD88-NFκB, and MAPK-AP1 signalling), producing - instead of Mtb clearance - inflammatory cell death with release of immune-activating and tissue-damaging host- and Mtb-derived molecules. Reactivation of Mtb lymphocyte memory responses likely only produces clinically-apparent inflammation (TB-IRIS) when multiple simultaneous but incompatible immune programmes (e.g. overzealous myeloid activity, Th1, Th2, Th17, and Treg) coexist. Based on this model, existing compounds with the potential for rational, safe, effective TB-IRIS prophylaxis/therapy are identified (e.g. glutathione, vitamins B-complex and A/D/E, rapamycin, and metformin) which may assist in restoring system homeostasis.

<p>The progression of an inflammatory response is largely dictated by soluble<br />factors termed cytokines reknown for their redundant and pleiotropic nature in<br />modulation of both immune and stromal cells. Individual members of the<br />interleukin-6 (IL-6)-type cytokine family possess both unique and shared biological<br />activities. These cytokines may participate in tissue remodelling by promoting<br />reconstruction ofextracellular matrix (ECM) following nonspecific tissue damage by<br />inflammatory cells. Consistent with this view, these cytokines upregulate expression<br />of an ECM protease inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) and<br />thus may alter net enzymatic degradation of ECM. The overall goal of this thesis<br />is to examine mechanisms by which TIMP-1 is regulated by IL-6-type cytokines,<br />especially by the cytokine oncostatin M (OSM). The promoter of TIMP-1 has<br />therefore been studied in detail to address the mechanisms by which OSM (and IL-6<br />type cytokines) regulate the transcription of the TIMP-1 gene. The approaches<br />undertaken have included deletion analysis of the TIMP-1 gene nucleotide<br />sequences proximal to the start of transcription to define DNA sequences<br />necessary/sufficient for cytokine-induced TIMP-1 promoter activity. In addition, the<br />binding of nuclear factors to these DNA elements and cytokine-response elements, their expression and involvement in the regulation of TIMP-1 transcription have been explored.</p> <p>We have identified sequences proximal to the start of TIMP-1 transcription<br />that are necessary for maximal responsiveness to OSM and IL-6. Deletion analysis<br />of the proximal TIMP-1 promoter (-95 to +47 TIMP-1 sequences) has identified a<br />nucleotide sequence within -59 to -53 ofthe murine TIMP-1 promoter that harbours<br />an AP-1 consensus DNA binding element. This element is necessary for maximal<br />OSM or IL-6 induced promoter activity of TIMP1-CAT reporter gene constructs<br />transfected into human hepatoma HepG2 cells. OSM is the most potent stimulus<br />(approx. 11-fold for OSM, and 4-fold for IL-6) of this response and additional<br />sequences 3-prime to +1 ofthe TIMP-1 gene are also necessary for maximal OSM<br />responsiveness.</p> <p>Electrophoretic mobility shift assays demonstrated two gel-shifted complexes<br />which bind the TIMP-1 AP-1 site. An AP-1 gel-shifted complex is present in the<br />absence of cytokine stimulation ("complex 1"), while OSM and not other IL-6-type<br />cytokines, stimulated the formation of a second AP-1 gel shifted complex.<br />Nuclear factors binding to TIMP-1 AP-1 complex 1 include junB, junD and fosrelated<br />antigens. However, unlike complex1, c-fos is present and necessary for the<br />formation of the OSM-induced TIMP-1 AP-1 complex2. Consistent with this, OSM<br />is a potent inducerofc-fos protein expression among IL-6-type cytokines. Both the<br />formation of complex2 and c-fos expression require new protein synthesis. JunB<br />and junO are constitutively expressed, while the expression of fos-related antigens are induced in response to OSM. In addition, although PMA was also a potent<br />inducer of c-fos expression, induction of TIMP-1 promoter activity by the<br />combination of PMA and IL-6 was comparable to IL-6 alone and did not equal the<br />significantly higher induction by OSM. Within the same cells, OSM and IL-6 equally<br />induced STATDNA-binding activity. An Ets-consensus site (nucleotides -45 to -40)<br />flanking the 3-prime end of the AP-1 site is a weak binding site for Ets-related<br />nuclear factors, and an SP-1 site near +1 (-11 to -6) is a strong binding siteforSP1<br />nuclear factors and related SP-1 site binding proteins. No STAT nuclear factor<br />binding to the proximal TIMP-1 promoterwas detected. Taken together, the TIMP1<br />AP-1 site and c-fos represent a unique target of OSM signalling and activation of<br />AP-1 complexes (possibly containing c-fos) by OSM as well as sequences<br />downstream of TIMP-1 +1 contribute to maximal responsiveness of the promoter<br />to this cytokine among IL-6 family members.</p> <p>The contribution of c-fos to OSM-induced TIMP-1 expression was further<br />explored in murine cells. OSM stimulates the expression of c-fos and activates<br />STATs 1, 3 and 5 DNA-binding activity in murine fibroblasts. As observed in<br />human cells, OSM but not other IL-6-type cytokines upregulated c-fos expression<br />which participated in complexes binding the TIMP-1 AP-1 site. OSM was also<br />unique among IL-6 family members in activating STAT5 DNA-binding activity in<br />murine fibroblasts. However, in contrast to observations in human HepG2 cells,<br />deletion analysis of the TIMP-1 promoter showed that the AP-1 site (-59/-53) was<br />v not necessary for OSM-mediated upregulation of the TIMP-1 proximal promoter<br />activity over basal levels in murine NIH3T3 fibroblasts and co-transfection of a<br />dominant-negative of AP-1 had no effect. However, transfection of dominant-negative<br />STATs1, 3 or5 (especially STAT3) could diminish cytokine-induced TIMP1<br />promoter activity. In addition, c-Fos was dispensable for OSM-mediated<br />upregulation ofTIMP-1 mRNA levels as TIMP-1 expression was detected in wildtype<br />and c-fos knockout murine lung fibroblasts. Consistent with deletion analysis<br />ofthe TIMP-1 promoter in human cells, dominant-negative AP-1 expression vectors<br />abrogated OSM-mediated TIMP-1 promoter activity, while Stat-dominant negative<br />expression vectors did not. Taken together, the examination of murine and human<br />systems suggests that AP-1 and STAT nuclear factors can contribute to the<br />regulation of the TIMP-1 promoter.</p> / Doctor of Philosophy (PhD)

Dengue fever is a rapidly growing concern to human health and is currently the most prevalent mosquito-borne viral disease worldwide. Although there are several vaccine candidates being tested in clinical trials, there are no vaccines publicly available to prevent this disease. Plant-based vaccines are rapidly becoming viable alternatives to traditional animal-based vaccines because they are safe, easy to manufacture, and more cost-efficient. The purpose of this project is to develop a vaccine against the dengue virus by producing a recombinant DENV2 protein, engineered by Dr. David Lo and his lab at University of California Riverside, in Nicotiana benthamiana plants through Tobacco Mosaic Virus (TMV) infection. Initial attempts to ligate the complete DENV2 epitope, a combination of hybrid flagellin sequences and the envelope protein from dengue viral serotype 2, into the pJL TRBO vector were incompatible with established protocols. However, a proof of concept test that replaced the DENV2 envelope protein with Green Fluorescent Protein (GFP) successfully inserted the new sequence into pJL TRBO. In the future, the DENV2 envelope protein sequence will be re-inserted into the construct and updated protocols will be repeated for DENV2 protein expression. The recombinant DENV2 proteins will be extracted from the plants after signs of infection become apparent and tested for their ability to induce an immunogenic response that produces pathogen-specific antibodies.

No description available.

No description available.

Periodontitis is a biofilm-initiated inflammatory disease, resulting in soft tissue damage and alveolar bone loss. A nucleic acid sensor, toll-like receptor 9 (TLR9), has been recently implicated in periodontal inflammation. This study utilized an in vivo periodontitis model using TLR9 knockout (TLR9-/-) mice to assess the role of TLR9 in periodontitis. Significant bone loss was observed in wild type, but not in TLR9-/- mice. Further experiments using ex vivo assays revealed significantly higher IL-6 production in splenocytes of wild type mice compared to knockout cells in response to Porphyromonas gingivalis (a keystone pathogen for periodontitis) challenge. In conclusion, TLR9 contributes to periodontal inflammation through promoting a heightened inflammatory response. Therapeutics targeted to TLR9 may be beneficial to control periodontal disease.

Leishmaniasis refers to the group of diseases caused by pathogenic protozoan parasites of the genus Leishmania. Nearly all human Leishmania spp. infections are initiated in mammalian skin through the bite of the phlebotomine sand fly vector. However, clinical manifestations vary greatly with infecting species. Leishmania major establish infection locally within the skin and cause chronic ulcerating skin lesions at the local cutaneous site of inoculation, in a syndrome known as Cutaneous Leishmaniasis (CL) Leishmania infantum parasites metastasize from the site of skin infection via unknown mechanisms, and establish infection within visceral organs usually without inducing skin pathology, resulting in the potentially fatal disseminated disease, Visceral Leishmaniasis (VL). Mouse studies suggest early responses at the skin infection site are critical determinants of subsequent adaptive immune responses in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant immunoactive cell in the epidermis. We hypothesize that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with L. infantum or L. major elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of pro-inflammatory genes IL6, IL8, TNF, and IL1B by RT-qPCR; whereas keratinocytes exposed to L. major did not. Similar to live parasites, L. infantum-derived exosomes induced more IL8 mRNA compared to control or L. major-derived exosomes. Western blotting confirmed NFkBp65 phosphorylation in keratinocytes exposed to L. infantum but not L. major. However, no evidence of L. major inhibition of TNF-induced NFkBp65 phosphorylation was observed in simultaneously treated keratinocytes. To examine whether keratinocytes influence proximal host cells, L. infantum-infected human monocytes were co-cultured with keratinocytes across a transwell membrane. These studies suggested L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication. L. major-exposed keratinocytes had no comparable effect. These data suggest L. infantum and L. major differentially activate keratinocytes to release factors that limit infection in monocytes. Microarray analyses performed on human keratinocytes exposed to either L. infantum or L. major promastigotes identified a limited number of transcripts increased by parasite exposure. Consistent with RT-qPCR observations, several inflammatory cytokine and chemokine genes were more strongly induced in L. infantum-exposed keratinocytes compared to L. major-exposed keratinocytes. Pathway analyses of genes induced by L. infantum-treated keratinocytes suggested that this interaction may induce neutrophil recruitment. Notably, AP1 transcription factor subunit genes were significantly down regulated in L. major-treated compared with L. infantum-treated or control keratinocytes. This suggests L. major may actively inhibits this keratinocyte activation, which might affect its ability to establish infection within host skin. In addition, ex vivo intradermal infection of human skin explants was explored as a method to compare keratinocyte responses to L. infantum or L. major in the context of whole skin tissue and the effects of vector salivary gland components are considered. The response of keratinocytes found in these studies using L. infantum and L. major may give insight into the local host pathologic responses to different Leishmania species leading to visceralizing versus cutaneous manifestations to infection. We propose that Leishmania spp. elicit or evade a pro-inflammatory response by keratinocytes at the site of infection, generating a microenvironment uniquely tailored to each Leishmania species.

Tim-3 (for T cell immunoglobulin and mucin domain 3) is a surface molecule expressed throughout the immune system that appears to mediate both stimulatory and inhibitory effects. Tim-3 is expressed by activated CD4 and CD8 T cells, which suggests a direct role in the regulation of T cell responses. Consistent with this possibility, previous studies have shown that blockade of interactions between Tim-3 and ligands in the context of mouse models for autoimmunity and chronic infection can augment T cell responses, which indicates that Tim-3 functions as an inhibitory receptor for T cells. However, other studies have provided evidence that Tim-3 can function to promote T cell responses, which suggests that Tim-3 acts as a stimulatory receptor. In addition, biochemical and cell culture studies have shown that Tim-3 can induce both inhibitory and stimulatory signaling pathways. These conflicting findings highlight that the role of Tim-3 in regulating T cell responses remains unclear and warrants further investigation. My studies sought to determine the role of Tim-3 in regulating T cell responses to microbial infections in vivo and to acute stimulation in vitro. Using Tim-3 KO mice and Tim-3 KO cells, I demonstrate that Tim-3 can directly enhance CD8 T cell responses to Listeria monocytogenes (LM). I also provide evidence that Tim-3 indirectly regulates CD4 T cell responses to LM. Alternatively, I show that Tim-3 may inhibit CD4 and CD8 T cell responses to the chronic viral infection LCMV-Clone 13, and that Tim-3 may be dispensable for T cell responses to the acute viral infection LCMV-Armstrong. Additionally, I demonstrate that Tim-3 expression in response to acute stimulation in vitro or in vivo marks populations of CD4 Th1 cells that are enriched for cells with effector function as measured by cytokine production and markers for degranulation. Collectively, these data suggest that Tim-3 may differentially regulate T cell effector function in a manner that is dependent on infectious environment encountered.

The immunosuppressive effects of chronic alcohol abuse are profound, wide-ranging and readily apparent at the body's barriers. In the skin, alcoholism is associated with an increased incidence and severity of infection; yet the precise immunologic alterations responsible remain poorly understood. Cutaneous homeostasis and immunity are afforded via coordinated efforts of tissue-specific immune cell networks. Here, the Meadows-Cook murine model of alcoholism was used to investigate the impact of chronic ethanol (EtOH) exposure upon the following: 1) the composition and function of skin-resident dendritic cells (DCs) and T cells in preimmune mice 2) infection outcome and host defense following Staphylococcus aureus skin infection 3) the induction of cutaneous oxidative stress Chronic EtOH feeding caused a baseline reduction in skin DCs and T cells with the most pronounced effects occurring in self-renewing compartments (i.e, Langerhans cells and γδ T cells). In addition, we found that EtOH-induced immune cell subset loss was often associated with dysfunction of the remaining population. For DCs, EtOH-induced hyporesponsiveness was observed in both in vitro and in vivo migration assays. Defects in the former system could be corrected via TNFα restoration. EtOH-induced dysfunction in skin T cells was evident by the decreased upregulation of JAML and diminished production of IL-17 by epidermal and dermal γδ T cells respectively. In a murine model of EtOH withdrawal, some but not all of the EtOH-induced defects occurring in skin DCs and T cells recovered after cessation of EtOH exposure. Prior to this work, the impact of chronic EtOH exposure upon the cutaneous immune system had not been investigated in a murine model of infection. Using a novel method of cutaneous S. aureus challenge, evidence of exacerbated staphylococcal disease in EtOH-fed mice included skin lesions that were larger and contained more organisms, greater weight loss and increased bacterial dissemination. Infected EtOH-fed mice demonstrated poor maintenance and induction of PMN responses in the skin and draining LNs respectively. Additionally, altered PMN dynamics in the skin of these mice corresponded with reduced production of IL-23 and IL-1β by CD11b+ myeloid cells and IL-17 production by γδ T cells, with the latter defect occurring in the draining LNs as well. In addition, IL-17 restoration via intradermal injection improved bacterial clearance defects in EtOH-fed mice. Taken together, these findings show that the EtOH-induced increase in S. aureus-related injury/illness (i.e., weight loss, bacterial burden, lesion size) corresponds with defects in the IL-23/IL-17 inflammatory axis and poor PMN accumulation at the site of infection and draining LNs. Finally, two complementary tools (mice deficient in molecules that promote or inhibit reactive oxygen species induction) were used to investigate the role of oxidative stress as a driver of cutaneous immune dysfunction. In these studies intriguing evidence was obtained indicating that some but not all of the mechanisms by which oxidative stress contributes to cutaneous immune dysfunction are initiated through the Thurman cascade. In conclusion, this report offers new information about the impact of EtOH on cutaneous host defense pathways and provides potential mechanisms of explaining why alcoholics are predisposed to severe skin infections.

The immune system has evolved to fight off numerous pathogens. The first line of defense against these pathogens are innate immune cells. Innate immune cells ingest pathogens and a family of cytosolic proteins, NOD-like receptors (NLR) and AIM2-like receptors (ALRs), recognize conserved sequences on pathogens. Recognition of pathogens by NLRs and ALRs alerts the immune system to the presence of an invader and subsequent control of infection. NLRs and ALRs also recognize endogenous cell danger signals that are released during cell stress. Pathogens and tumor cells are capable of growing in a host with limited detection by the immune system. This evasion leads to a suboptimal immune response to both the bacteria and cancer cells resulting in enhanced infection and tumor growth. We investigated how the bacteria Francisella tularensis escapes recognition by the AIM2 receptor. We identified a novel gene important in bacterial folate metabolism that helps the bacteria escape immune recognition. The identification of this gene will help in the development of treatments for F. tularensis infection. In addition, we investigated the role of the NLRC4 receptor in a mouse model of melanoma. We found that mice lacking NLRC4 developed significantly larger tumors and had a diminished immune response compared to mice that expressed NLRC4. We also observed decreased expression of NLRC4 in metastatic melanoma tissue in humans. Thus demonstrating that NLRC4 is important for initiating an immune response to melanoma and down regulating expression of NLRC4 is a way for the tumor to evade the immune response.