Asthma is a multi-faceted chronic inflammatory disease accompanied by loss of airway epithelial integrity leading to remodeling of the airways. Perturbations to the lung redox environment, including alterations in glutathione (GSH) content, have been reported in asthma. GSH can be conjugated to protein cysteines, controlling protein function in an oxidant-dependent process known as protein S-glutathionylation (PSSG). The thioltransferase, glutaredoxin-1 (Glrx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups of target proteins. Glrx1 is emerging as a critical player in settings of allergic airway disease, but its function in regulating epithelial cell responses to asthma-relevant cytokines has not been examined. Furthermore, the role of Glrx1 in controlling the extent of airway remodeling in response to house dust mite (HDM) in vivo is still not well understood. Interleukin-17A (IL-17A) is a potent cytokine that stimulates epithelial cells to produce pro-inflammatory mediators, in part by activating the nuclear factor kappaB (NF-κB) pathway, a key regulator of inflammation. We demonstrate that interleukin-17A (IL-17A) induces rapid activation of both classical and alternative NF-κB, while simultaneously resulting in protein oxidation and PSSG. In particular, we show IL 17A induces S-glutathionylation of RelA (RelA-SSG) and IKKα (IKKα-SSG), which is enhanced following siRNA-mediated knockdown of Glrx1. We also demonstrate that absence of Glrx1 leads to increased nuclear content of RelA and RelB and enhanced production of NF-κB-driven pro-inflammatory genes, KC and CCL20 while decreasing IL-6 expression. Finally, we show that siRNA-mediated knockdown of IKKα attenuates nuclear RelA and RelB and dampens pro-inflammatory gene production. Together, these data indicate a crucial role for the Glrx1/PSSG axis in controlling RelA-SSG, IKKα-SSG and epithelial cell responsiveness to IL-17A. Mice lacking Glrx1 were previously shown to display enhanced resolution of allergic airway disease induced by ovalbumin (Ova) challenge. In this study, we determined the role of Glrx1 in a HDM model of allergic airway disease. Wild type (WT) mice and Glrx1 deficient (Glrx1-/-) mice demonstrated similar total lung cell counts, but Glrx1-/- mice displayed fewer neutrophils than WT mice. Conversely, mice overexpressing Glrx1 specifically in CCSP positive cells in the lung (Epi-Glrx1) showed attenuated total lung cell counts and lung eosinophils compared to control mice. Immunohistological analysis of remodeling markers revealed that Glrx1-/- mice displayed increased HDM-induced mucus metaplasia, α smooth muscle actin (αSMA) positivity and collagen staining compared to WT mice. Evaluation of total lung collagen showed that Glrx1-/- mice had significantly higher collagen content compared to WT mice. In Epi-Glrx1 mice, attenuation of mucus metaplasia, αSMA content and collagen staining was observed compared to control mice. Furthermore, Epi-Glrx1 mice also demonstrated significantly impaired collagen production compared to control mice. We also demonstrate that Glrx1 absence results in decreased expression of the epithelial cell marker, E-cadherin, and increased expression of αSMA, a mesenchymal marker. Together, these studies demonstrate a critical role for Glrx1 in controlling epithelial cell responses to IL-17A and in mediating in vivo collagen production in response to chronic allergen exposure.

Doctor of Philosophy / Department of Industrial & Manufacturing Systems Engineering / Chih-Hang Wu / The topic of infectious disease epidemics has recently attracted substantial attentions in research communities and it has been shown that the changes of human behaviors have significant impacts on the dynamics of disease transmission. However, the study and understanding of human reactions into spread of infectious disease are still in the very beginning phase and how human behaviors change during the spread of infectious disease has not been systematically investigated. Moreover, the study of human behaviors includes not only various enforced measures by public authorities such as school closure, quarantine, vaccination, etc, but also the spontaneous self-protective actions which are triggered by risk perception and fear of diseases. Hence, the goal of this research is to study the impacts of human behaviors to the epidemic from these two perspectives: spontaneous behavioral changes and public intervention strategies. For the sake of studying spontaneous changes of human behaviors, this research first time applied evolutionary spatial game into the study of human reactions to the spread of infectious disease. This method integrated contact structures and epidemics information into the individuals’ decision processes, by adding two different types of information into the payoff functions: the local information and global information. The new method would not only advance the field of game theory, but also the field of epidemiology. In addition, this method was also applied to a classic compartmental dynamic system which is a widely used model for studying the disease transmission. With extensive numerical studies, the results first proved the consistency of two models for the sake of validating the effectiveness of the spatial evolutionary game. Then the impacts of changes of human behaviors to the dynamics of disease transmission and how information impacts human behaviors were discussed temporally and spatially. In addition to the spontaneous behavioral changes, the corresponding intervention strategies by policy-makers played the key role in process of mitigating the spread of infectious disease. For the purpose of minimizing the total lost, including the social costs and number of infected individuals, the intervention strategies should be optimized. Sensitivity analysis, stability analysis, bifurcation analysis, and optimal control methods are possible tools to understand the effects of different combination of intervention strategies or even find an appropriate policy to mitigate the disease transmission. One zoonotic disease, named Zoonotic Visceral Leishmaniasis (ZVL), was studied by adopting different methods and assumptions. Particularly, a special case, backward bifurcation, was discussed for the transmission of ZVL. Last but not least, the methodology and modeling framework used in this dissertation can be expanded to other disease situations and intervention applications, and have a broad impact to the research area related to mathematical modeling, epidemiology, decision-making processes, and industrial engineering. The further studies can combine the changes of human behaviors and intervention strategies by policy-makers so as to seek an optimal information dissemination to minimize the social costs and the number of infected individuals. If successful, this research should aid policy-makers by improving communication between them and the public, by directing educational efforts, and by predicting public response to infectious diseases and new risk management strategies (regulations, vaccination, quarantine, etc.).

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.

Dexamethasone has been shown to inhibit IgE-mediated mast cell activation, and the present research investigated its role in suppressing IL-33-mediated mast cell activation. We have found that micromolar concentrations of Dexamethasone are capable of suppressing IL-33-mediated mast cell cytokine production, on several genetic backgrounds, and in not only bone marrow derived mast cells, but also peritoneal mast cells. Intracellular staining demonstrated that Dexamethasone significantly reduces expression of the IL-33 receptor, T1/ST2, in mast cells; however, the cytokine suppression is independent of T1/ST2 downregulation. At the same time, Dexamethasone pretreatment significantly reduced ERK phosphorylation, but our data suggests that inhibition occurs even prior to ERK blockade. Finally, Dexamethasone treatment in vivo reduced IL-33-mediated cytokine production and neutrophil infiltration in the murine peritoneum. Thus, Dexamethasone, a well-established therapy for inflammatory disease, can suppress IL-33-mediated mast cell activation, and may therefore be effective for treating diseases now being attributed to IL-33 effects.

Kainate receptor knockout mice have unique differences within their immune system. They exhibit an attenuated TH2 branch, while maintaining a robust TH1 response. Specifically, blocking the formation of functional kainate receptors affects mast cells and their related pathologies. While they seem to develop and activate normally in vivo and in vitro, KAR KO mast cells release more inflammatory mediators upon degranulation. These mice experience severe anaphylactic shock due to two compounding abnormalities. First, KAR KO mast cells release significantly more histamine in vivo upon IgE-mediated activation. Second, the animals over-respond to exogenous histamine with drastic temperature drops compared to WT. This report shows that the kainate receptor plays an important role in mast cell-mediated immune responses.

Recent events have thrown the spotlight on infectious disease outbreak response. We developed a data-driven method, EpiGro, which can be applied to cumulative case reports to estimate the order of magnitude of the duration, peak and ultimate size of an ongoing outbreak. It is based on a surprisingly simple mathematical property of many epidemiological data sets, does not require knowledge or estimation of disease transmission parameters, is robust to noise and to small data sets, and runs quickly due to its mathematical simplicity. Using data from historic and ongoing epidemics, we present the model. We also provide modeling considerations that justify this approach and discuss its limitations. In the absence of other information or in conjunction with other models, EpiGro may be useful to public health responders. (C) 2016 The Authors. Published by Elsevier B.V.

Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood stage malaria and several clinical trials using mostly protein-in-adjuvant vaccines have shown limited success. This thesis describes the development of recombinant adenoviral (AdHu5) and poxviral (MVA) vectors encoding AMA1 from Plasmodium chabaudi murine parasites. In this murine malaria model, AdHu5 and MVA encoding AMA1 when used in a heterologous prime boost regime showed excellent immunogenicity, both humoral and cellular. The vaccination regime was protective against blood stage challenge and both antibodies and CD4+ T cells found to be important for vaccine induced blood stage protection. In parallel to this novel P. falciparum vaccines encoding AMA1 were also developed and administered in a similar prime boost regime to mice and rabbits. The vaccination regime induced cellular immune response and high titre antibodies against AMA1 and these antibodies showed growth inhibitory activity against the homologous parasite strain. In an effort to overcome the issue of antigenic polymorphism and to circumvent pre-existing immunity to human adenovirus, biallelic simian and human adenoviral vectors and MVA encoding AMA1 vaccines were also developed and administered to mice and macaques. These vectors also induced high titre antibodies and the serum from macaques was found to have growth inhibitory activity. These vaccine candidates are now being taken forward to Phase I/II clinical trials in Oxford. This work also described the attempt to improve MVA as a antibody inducing vector to allow better antibody mediated immunity to blood stage malaria.

Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of lives in Africa and could become a global health threat. Because of the danger of infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress due to both the cost and expertise required to operate these laboratories. The development of a safe surrogate would speed research and reduce risk to researchers. Two highly conserved Ebola gene segments—from the glycoprotein and nucleoprotein genes—were designed with modifications preventing expression while maintaining sequence integrity, spliced into high copy number plasmids, cloned into E.coli, and tested for stability, safety, and potential research applications. The surrogates were stable over 2-3 months, had a negligible mutation rate (<0.165% over the experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL. These protocols could be used to safely simulate other pathogens and promote infectious disease treatment and detection research. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Orientador: Luís Antonio Mathias / Banca: Samir Issa Samara / Banca: Edvirges Maristela Pituco / Resumo: O presente estudo teve como objetivo avaliar a influência da brucelose, leptospirose, rinotraqueíte infecciosa bovina (IBR), diarreia viral bovina (BVD) e neosporose no indicador de reprodução mais comumente utilizado por pecuaristas brasileiros, a taxa de concepção (ou taxa de prenhez) após a estação de monta. Dois estudos epidemiológicos analíticos foram feitos: um estudo caso-controle, utilizando uma amostra das matrizes do rebanho que foram submetidas à estação de monta durante o período chuvoso, e um estudo de coortes retrospectivo, utilizando todas as novilhas submetidas à estação de monta durante a estação de "inverno". A determinação do estado gestacional foi realizada por palpação retal entre 60 e 90 dias após o término da estação de monta. A pesquisa de anticorpos foi realizada pelos seguintes métodos: antígeno acidificado tamponado (AAT) e reação de fixação de complemento (RFC) para brucelose; soroaglutinação microscópica (SAM) para leptospirose; virusneutralização (VN) para IBR e BVD; e reação de imunofluorescência indireta (RIFI) para neosporose. A significância estatística da associação entre a doença e o fracasso na concepção foi avaliada pelos métodos de qui-quadrado ou teste exato de Fisher (p < 0,05) e pelo intervalo de confiança a 95% para o risco relativo (RR) ou a odds ratio (OR). Associações com relação causal possível foram encontradas entre falha na concepção e: infecção considerando qualquer sorovariedade de Leptospira spp. (Título ≥ 400) em primíparas (OR = 7,3636, IC 95%: 1,3373-40,5479; significativo), todas as quais foram infecções causadas por L. Wollfi; e infecção por L. Autmumnalis (título de ≥ 200), considerando todos os animais da estação de monta de "verão" (OR = 8,4058; IC95%: 1,0377 - 68,0882; significativo). Brucelose, neosporose IBR, BVD... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study aims to evaluate the influence of brucellosis, leptospirosis, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD) and neosporosis in the reproductive indicator most commonly used among Brazilian ranchers, the conception rate (or pregnancy rate) after breeding season. Two analytical epidemiological studies were made: a case control study using a sample of matrices of the herd that were submitted to the breeding season during the rainy season, and a retrospective cohort study using all heifers submitted to the breeding season during the dry season. The determination of pregnancy status was performed by rectal palpation between 60 and 90 days after the end of the breeding season. The antibody search was performed by the following methods: rose bengal test and complement fixation test for brucellosis; microscopic agglutination test for leptospirosis, virus neutralization for IBR and BVD, and indirect immunofluorescence assay for neosporosis. The statistical significance of association between the disease and failure in the conception was held by the methods of chi-square or Fisher's exact test (p < 0,05) and by the 95% confidence interval for the relative risk (RR) or odds ratio (OR). Associations with possible causal relationship were found between conception failure and: Infection considering any serovar of Leptospira spp. (titer ≥ 400) in primiparous (OR = 7.3636, 95% CI: 1.3373 to 40.5479; significant), all of which were infections caused by L. Wollfi; and infection by L. Autmumnalis (titer of ≥ 200) considering all the animals of the rainy breeding season (OR = 8.4058; IC95%: 1.0377 - 68.0882; significative). Brucellosis, IBR, BVD, neosporosis and leptospirosis caused by other serovars did not influence the rate of conception in the breeding season... (Complete abstract click electronic access below) / Mestre

Most studies of the induction of cytokine synthesis in monocytes have used an exogenous triggering agent such as Lipolpoysaccharide (LPS). However, during nonseptic chronic inflammatory responses (e.g., rheumatoid arthritis) monocyte activation occurs as a result of T cell generated signals. This report demonstrated that plasma membranes from anti-CD3 activated peripheral CD4$\sp{+}$ T cells (Tm$\sp{\rm A}$) but not from resting CD4$\sp{+}$ cells (Tm$\sp{\rm R}$) induced monocytes to synthesize IL-1 in the absence of costimulatory cytokines. The expression kinetics of the molecule(s) unique to activated T cells which interact with monocyte receptors to induce IL-1 demonstrated that optimal expression occurred at 6h post activation. This matched Lederman's, et al., (1992) previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of IL-1 induction in monocytes. In this work, it was demonstrated that the signal could be reduced up to 85% by addition of 5c8, a monoclonal anti-CD40L antibody. In addition, a monoclonal anti-CD40 IgM (BL-C4) induced resting monocytes to synthesize IL-1. Experiments demonstrated that crosslinking the CD40 molecules on monocytes was critical for IL-1 induction. Tm$\sp{\rm A}$ but not Tm$\sp{\rm R}$ also up-regulated cell surface expression of adhesion/costimulatory molecules on monocytes including CD40, ICAM-1, and LFA-3. Anti-CD40 signaling up-regulated expression of ICAM-1 and LFA-3. Experiments suggested that signaling through CD40 may utilize a protein tyrosine kinase (PTK) mediated pathway but not a protein kinase C mediated pathway and studies using THP-1, a premonocytic cell line, indicated that the transcription factor, NF-$\kappa$B, was activated through anti-CD40 signaling. Since CD40 ligand-transfected cells alone did not induce IL-1 but Tm$\sp{\rm A}$ did, it was considered that an additional costimulatory cell surface molecule was required. Preliminary experiments suggested that CD69 may be required. In summary, these results indicate that contact-dependent T cell-monocyte interactions, alone, can activate inflammatory cytokine production by resting monocytes and that a critical component of this interaction is the CD40-CD40L signaling event.