The contributions of peptide antigen affinity for TCR in driving T cell memory is unclear. Effector CD4 T cells must recognize cognate antigen again at an effector checkpoint, 5-8 days post-infection, to generate an optimal memory population. In this thesis, we examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We used an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP- peptide variants that bind FluNP TCR with a broad range of avidity. Varying peptide avidity in vivo at the effector checkpoint revealed that higher affinity interactions yielded greater numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. These memory cells demonstrated enhanced protection against lethal IAV infection with a robust early day 5 secondary effector response in the lung. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors while IL-2R expression was unaffected. However, IL-2Ra expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory and can instruct future vaccine design.

T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.

Rhodococcus equi (R. equi) is one of the most serious causes of pneumonia in young foals. The clinical disease is of great concern to breeding farms worldwide due to the impact of mortality on economic losses. While adult horses are resistant to R. equi, foals exhibit a distinct age-associated susceptibility. The mechanism underlying this susceptibility in foals is not well understood. Interferon-gamma (IFNg) plays an important role in the clearance of R. equi, but its expression is impaired in neonatal foals. Moreover, the regulation of this age-related IFNg expression in foals remains unknown. In humans, IFNg expression has been shown to be regulated by DNA methylation, lymphoproliferation, and influenced by environmental exposure. Therefore, we hypothesized that environmental exposure promotes IFNg expression through regulation of DNA methylation and lymphoproliferation. The objectives were: (1) to estimate the relevance of IFN-g production and R. equi infection in foals; (2) investigate the role of lymphoproliferation and DNA methylation in the regulation of IFN-g expression in foals; (3) to evaluate the effect of environmental exposure on IFN-g expression by housing foals in a barn environment verses pasture.; (4) to investigate the effect of environment exposure on antigen-presenting cells (APC), which sensor the environmental antigens and modulate IFN-g production by T cells. The results demonstrated that the IFN-g expression was inversely correlated with the age-related susceptibility to R. equi infection. lymphoproliferation promoted IFN-g expression in foals, whereas, DNA methylation repressed IFN-g expression. The IFN-g expression was augmented in foals exposed to the barn air which contained higher numbers of aerosol miroorganisms. DNA on the IFN-g promoter was demethylated and the lymphoproliferative activity was elevated in foals with barn-air exposure. The barn-air exposure also promoted the maturation and activation of APC to prime IFN-g expression by T cells in foals. Overall, this body of work demenstrated a relationship between IFN-g expression and R. equi infection, provided novel information on mechanisms that regulate IFN-g expression, and identified the effect of environment on mechanisms responsible for IFN-g expression.

The effect of ultraviolet (UV) radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV-irradiated mammalian virus was tests. An action spectrum for radiation enhanced reactivation (RER) Is presented. The shape of the action spectrum points to a combined nucleic acid protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in the RER does not allow the identification of the macromolecule which is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.

Human metapneumovirus (HMPV) is a respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped RNA virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. Despite its prevalence and importance to human health, no therapies are available against this pathogen. Entry of paramyxoviruses into host cells generally requires the coordinated activity of the attachment glycoprotein, G, which interacts with a cell receptor, and the fusion glycoprotein, F, which promotes subsequent fusion of viral and cellular membranes. However, HMPV F is the primary viral protein mediating both binding and fusion for HMPV. Previous work that showed HMPV F mediates attachment to heparan sulfate proteoglycans (HSPGs), and some HMPV F fusion activity can be promoted by acidic pH. The work presented here provides significant advances in our understanding of the fusion and binding events during HMPV infection. We demonstrated that low pH promotes fusion in HMPV F proteins from diverse clades, challenging previously reported requirements and identifying a critical residue that enhances low pH promoted fusion. These results support our hypothesis that electrostatic interactions play a key role in HMPV F triggering and further elucidate the complexity of viral fusion proteins. Additionally, we characterized the key features of the binding interaction between HMPV and HSPGs using heparan sulfate mimetics, identifying an important sulfate modification, and demonstrated that these interactions occur at the apical surface of polarized airways tissues. We identified differences in particle binding related to the presence or absence of the HMPV G and SH glycoproteins. Lastly, we characterized paramyxovirus infection in cystic fibrosis bronchial epithelial cells, identifying a potential specific susceptibility to HMPV infection in these individuals. The work presented here contributes to our understanding of HMPV infection, from mechanisms of early events of entry to clinical scenarios.

This study demonstrates original findings of statin effects on IL-33 stimulated mast cells. Statins are a class of drugs used to lower cholesterol production by targeting HMG CoA reductase. These commonly prescribed drugs have been shown to be immunomodulatory. In this study, we have found that pretreatment with statins has a variety of effects on IL-33 stimulated mast cells. Atorvastatin suppresses TNF and IL-6 production, while fluvastatin significantly enhances release of these proinflammatory cytokines in BMMCs. Although they have differing effects on cytokine production, both statins lowered ST2 expression on the cell surface, decreased cell viability, and enhanced expression of the transcription factor KLF2, a negative regulator of NFκB. Blocking isoprenylation by using geranylgeranyl transferase inhibitor, but not farnesyl transferase, mimicked the effects of atorvastatin, while neither mirrored the effect of fluvastatin. Furthermore, fluvastatin effects were not reversed by mevalonic acid, the product of HMG-CoA reductase. These data indicate that fluvastatin effects are distinct from its activities as an HMG CoA reductase inhibitor. Fluvastatin effects required the presence of stem cell factor (SCF), and were enhanced by increasing SCF concentrations. Finally, fluvastatin enhanced IL-33-induced cytokine production and neutrophil recruitment in vivo. Collectively, these data suggest that statins can alter the mast cell response, and that drug choice can have divergent effects on outcome.

Statin drugs are widely employed in the clinic to reduce serum low density lipoproteins (LDLs) in patients with hypocholesteremia. In addition to their cholesterol-lowering effects through HMG CoA reductase antagonism, isoprenyl lipids necessary for membrane anchorage and signaling of small G-proteins are abrogated. We previously found that statins suppress mast cell activation in murine and human cells, suggesting these drugs might be useful in treating allergic disease. While mast cell function is critical to allergic inflammation, mast cell hyperplasia and survival also impact these diseases, and were not studied in our previous work. In this study, we describe Fluvastatin-mediated apoptosis in both primary and transformed mast cells. An IC50 was achieved between 1-5μM in both systems, and apoptosis was preceded by mitochondrial dysfunction and caspase release. In addition to apoptosis, our work also uncovered evidence of autophagy, which can serve as a compensatory mechanism during apoptosis. Interestingly, autophagy appeared to be cyto-protective in the primary cells yet cytotoxic in transformed mast cells. These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases.

No description available.

No description available.

Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a substrate. Comparisons with other ACPs included the following items: biological activity, pI, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule. ACPs from E. coli and Euglena have been shown to interact with melittin, a cationic peptide from bee venom. E. coli ACP is a small (Mr, 8847), acidic, Ca$\sp{++}$-binding protein which possesses some characteristics resembling those of regulatory Ca$\sp{++}$-binding proteins including interaction with melittin. Melittin inhibited activity of the nonaggregated FAS from Euglena using either E. coli or Euglena ACP as a substrate. The peptide also inhibited activity of the aggregated FAS from Euglena. Antibodies against melittin were raised. Anti-melittin inhibited activity of both the nonaggregated and aggregated FAS enzyme systems from Euglena relative to nonimmune antibody. Investigation of inhibition of the nonaggregated FAS enzyme system demonstrated that acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and keto-acyl-ACP synthetase activities were inhibited to different degrees by anti-melittin antibodies, while keto-acyl-ACP reductase and enoyl-ACP reductase enzyme activities were not inhibited.