Browsing by Subject "cytotoxicity"
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Item A Study of the Process and Causes of Abeta(25-35) Amyloid Formation(2011-02-22) Ridinger, Katherine V.Amyloid fibrils results from a type of ordered polypeptide aggregation that is associated with ailments such as Alzheimer's disease (AD). Annually, millions of people in the United States alone develop and die from AD. Therefore, it is necessary to understand not only the process of amyloid formation, but also the causes of this specific type of aggregation. This study used ABeta(25-35) since it is a fragment of the Alzheimer?s peptide that behaves like the full length peptide found in patients with AD. To study the process of amyloid formation, several methods were used so that a more complete picture of the stepped aggregation process could be realized. Several oligomeric species were detected and described many of which could not have been observed without using the complete battery of methods utilized here. The oligomeric species detected included a novel 'rolled sheet' that appeared to be the immediate precursor of amyloid fibrils, and two supermolecular species that appear after amyloid fibrils were formed. In determining the causes of amyloid formation, two significant discoveries were made. First, by partial sequence randomization, truncation, and Ala scanning mutagenesis, the critical amyloidogenic region of ABeta(25-35) was found to be residues 30-35. This critical core region is important because it is thought to be the region that initiates amyloid formation, therefore knowing the residues involved in the region is a useful tool for developing methods of fibril formation prevention. Second, by inserting all naturally occurring amino acids into position 34 of ABeta(25-35), three distinct classes of variants were observed and the effect of several physiochemical properties on amyloidosis were examined. Hydrophobicity, solubility, and ?-strand propensity were found to affect aggregation to the greatest extent. Also within these two studies, our results suggest that early oligomers are the cytotoxic species as opposed to amyloid fibrils or other larger macromolecular assemblies.Item Effector CD4+ T lymphocyte resolution of acute HSV infection at genital and neuronal sites, and the manipulation of CD4+ T cell responses via TLR ligand-induced proinflammatory cytokine milieus(2010-05-17) Alison Joy Johnson; Gregg N. Milligan; Rolf Konig; Nigel Bourne; Lawrence Stanberry; Janice EndsleyIn primary infection, CD8+ T cells are important for clearance of infectious HSV from sensory ganglia. We present evidence of CD4+ T-cell-mediated clearance of infectious HSV-1 from neural tissues. In immunocompetent mice, HSV-specific CD4+ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance and remained detectable at least 8 months post-infection. Neural CD4+ T cells isolated at the peak of neural infection secreted IFN-ã, TNF-á, IL-2, or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8+ T-cell-deficient and CD8+ T-cell-depleted mice, suggesting CD4+ T cells could mediate clearance from neural tissue. Clearance of infectious virus from neural tissues was not significantly different in CD8+ T-cell-depleted, perforin-deficient or FasL-defective mice compared to wild-type mice. Virus titers in neural tissues of chimeric mice expressing both perforin and Fas or neither perforin nor Fas were significantly lower than in controls. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results further define the HSV-specific CD4+ T cell response. To determine the influence of differential TLR activation of DCs in development of appropriate CD4+ T cell phenotype, magnitude, and memory, we established bone marrow-derived DCs that were 92.6% CD11c+CD11b+, and 94.0% CD11c+B220-Ly-6c- in vitro. Ligands for TLR3, -4, or -9 were applied to DCs, and cytokine and chemokine secretion was examined. Particular interest was paid to IL-12 and IFN-g (important for TH1 differentiation thought critical against HSV), and antiviral type I interferons. Proliferation and activation of the CD4+ T cells co-cultured with TLR-ligand-stimulated DCs were assessed. CD4+ T cell magnitude, effector function, and establishment of memory generated upon injection of TLR-ligand-stimulated peptide-pulsed DCs were examined. Stimulation of DCs through TLR3 enhanced CD4+ T cell production of large amounts of TH1-type cytokines and cytolytic molecules. Stimulation of DCs through TLR4 did drive this phenotype, and also enhanced memory CD4+ T cell population formation within the genital tract. A vaccine able to elicit a vigorous, long-lasting CD4+ T cell response may prove important in limiting disease and transmission of virus.Item Polyphenol-induced Anti-inflammatory and Cytotoxic Activities in Breast and Colon Cancer: Potential Role of miRNA's in Cell Survival and Inflammation(2013-12-11) Banerjee, NiveditaCancer is a major cause of death worldwide. Hence, there is a great need to develop novel therapeutic agents for the prevention and treatment of cancer in addition to conventional therapies. Dietary polyphenols are known to be effective in the prevention and treatment of several chronic diseases such as cancer, cardiovascular diseases, and diabetes. Particularly in carcinogenesis, polyphenols are known to suppress cancer growth, angiogenesis, and metastasis. Several studies have demonstrated that polyphenolics, ellagitannins, gallotannins, and chlorogenic acid from pomegranate, mango, and plum juice, respectively, are potent inhibitors of cancer cell proliferation and induce apoptosis and cell cycle arrest as well as decrease inflammation in vitro and vivo. The therapeutically relevant compounds in pomegranate are pelagic acid, ellagitannins, flavonoids, and 3-glucosides/3,5-diglucosides of the anthocyanins delphinidin, cyanidin, and pelargonidin that exerted antioxidant, anti-inflammatory, and anticarcinogenic activities in vitro and vivo. Mango pulp extract contains gallotannins, gallic acid, galloyl glycosides, and flavonoids such as quercetin and kaempferol glycosides, which showed antioxidant, anti-inflammatory, and anticarcinogenic activities in vitro and in vivo. Chlorogenic acid and neo-chlorogenic acid are contained in plum juice and are also known to function as chemoprevention and chemotherapeutic agents. The overall objective of this work was to investigate the underlying anti- inflammatory and cytotoxic mechanisms involving miR-27a-ZBTB10-Sp and miR-155- SHIP-1-PI3K axes, miR126-VCAM-1, miR126-PI3K/AKT-mTOR and miR143/PI3K/AKT/mTOR axes in polyphenol-mediated anti-inflammatory and anticarcinogenic activities in vitro and vivo. Pomegranate and Mango polyphenols exhibited antioxidant, anti-inflammatory, anticarcinogenic, and antiproliferative activities in vitro and in vivo. Polyphenols inhibited cell proliferation of breast cancer cell line BT474 and suppressed tumor growth in athymic BALB/c nude mice with BT474 xenografts. Interactions of Pg with miR-27a- ZBTB10-Sp and miR-155-SHIP-1-PI3K axes and mango miR126/PI3K/AKT axis were identified. In addition, pomegranate and plum polyphenols exerted cytotoxic and anti- inflammatory effects in azoxymethane AOM-treated rats and colon cancer cells. Interactions of Pg with miR126/VCAM-1 and miR126/PI3K/AKT/mTOR axes and plum with miR143/PI3K/AKT/mTOR were identified as mechanisms that at least in part appear to be involved in the anti-inflammatory and antiproliferative activities of pomegranate and plum polyphenolics. The presented research was conducted in order to understand the efficacy of polyphenols present in pomegranate, mango and plum and their underlying molecular mechanisms in different cancer models.Item Role of new virulence mechanisms/factors (type 3 secretion system and TOX-R regulated lipoprotein [TAGA]) in the pathogenesis of the emerging human pathogen Aeromonas hydrophila(2006-08-10) Lakshmi Pillai; Dr. Ashok K. Chopra, Ph.D. C.Sc.; Dr. Vladimir Motin, Ph.D.; Dr. Golda A.K. Leonard, Ph.D.; Dr. Eric M. Smith, Ph.D.; Dr. Chandrasekha Yallampalli, Ph.D.; Dr. Amy Horneman, Ph.D., SM(ASCP)Aeromonas hydrophila, a gram-negative bacterium that causes gastroenteritis, wound infections, septicemia, and other diseases in humans, produces many different virulence factors. A clinical isolate SSU of A. hydrophila possesses a cytotoxic enterotoxin Act, a potent virulence factor that is secreted into the environment through the bacterium’s type 2 secretion system (T2SS) and possesses several biological activities, including cytotoxicity, enterotoxicity, and lethality in a mouse model. The purpose of this study was to identify new virulence factors that contribute to the pathogenesis of this bacterium. We identified and characterized a type 3 secretion system (T3SS) in A. hydrophila SSU. By marker-exchange mutagenesis of the aopB gene, a crucial gene involved in the formation of the translocon apparatus, the functionality of the T3SS was elucidated, both in in vitro and in vivo models. Further, the characterization of the regulatory gene DNA adenine methyltransferase (Dam) from SSU and its role in modulating the function of both the T3SS and Act was investigated. The role of the T3SS in influencing the phenomenon of quorum sensing (QS) in A. hydrophila SSU was also conducted. This study highlights a unique link between the T3SS and Act of A. hydrophila and the production of QS molecules or lactones. While searching for potential effector proteins secreted through the T3SS of A. hydrophila SSU, the identification of a new virulence factor, ToxR regulated lipoprotein (TagA), was revealed. TagA is a zinc metalloprotease which has only been identified in the gram-negative pathogens, Escherichia coli O157:H7 and Vibrio cholerae. In A. hydrophila, TagA has been shown to play a role in the inhibition of complement by binding to and cleaving the serpin C1-INH. By recruiting C1-INH to the surface of the bacteria and cleaving it, TagA is able to significantly prevent the activation of complement at the cell surface, ultimately increasing the serum resistance of the pathogen. TagA can also target C1-INH to erythrocyte surfaces and decrease the lysis that occurs in the presence of serum. Confocal fluorescence microscopy revealed that the serpin C1-INH binds to TagA on the surface of the bacteria.