Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 139-154).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

No description available.

Les fièvres hémorragiques à virus Lassa (LASV) et Ebola (EBOV) représentent un important problème de santé publique en Afrique. Les réponses immunes et la pathogenèse associées à ces maladies sont peu connues. Les cellules NK ont un rôle important dans la réponse immune innée par leurs propriétés cytotoxiques, mais également dans l'induction des réponses adaptatives par leur production de cytokines et leurs interactions avec les cellules dendritiques (DC) et les macrophages. Ce projet s'attache à comprendre le rôle des cellules NK dans le contrôle de la réplication virale et dans l'induction des réponses immunitaires au cours de ces infections. Un modèle in vitro de coculture de cellules NK humaines avec des DC et macrophages autologues a été développé. L'activation des cellules NK et leurs fonctions ont été analysées après l'infection par LASV et EBOV. Par ailleurs, les réponses des cellules NK en réponse à LASV ont été comparées avec celles induites lors de l'infection par le virus Mopeia (MOPV), très proche de LASV mais non pathogène pour l'homme. Les macrophages, mais pas les DC, infectés par LASV ou MOPV induisent l'activation et l'augmentation des capacités cytotoxiques des cellules NK. Toutefois, les cellules NK ne sont pas capables de lyser les cellules infectées et ne produisent pas d'IFN-γ. Les cellules NK s'activent et sont capables de lyser les cellules infectées en présence de macrophages mais également de DC infectés par des LASV mutants. Cependant, les IFN de type I sécrétés en grande quantité en réponse à ces virus ne sont pas impliqués dans l'activation des cellules NK. L'infection par EBOV n'induit qu'une très faible activation des cellules NK en présence de DC ou macrophages et ne conduit pas à la sécrétion de cytokines, ni à la modification du potentiel cytotoxique.Ces résultats permettent d'améliorer la compréhension des réponses immunes et des mécanismes de pathogenèse mis en place lors des fièvres hémorragiques Lassa et Ebola.

La candidose oro-pharyngée (COP) est l’infection opportuniste la plus répandue chez les patients infectés au VIH-1. Un modèle de COP chez la souris transgénique (Tg) exprimant une partie du génome du VIH-1 (CD4C/HIVMutA) est maintenant disponible. Grâce à ce modèle, il est possible d’étudier les perturbations quantitatives et fonctionnelles des macrophages exprimant les gènes nef, rev et env du VIH-1 dans le contexte d’une COP. Cette étude démontre que la présence du transgène n’influence pas le pourcentage des macrophages dans la muqueuse buccale et le petit intestin, malgré le fait que la charge buccale de C. albicans soit significativement plus élevée chez les souris Tg. Cependant, l’expression du transgène cause une diminution de la production de H2O2 par les macrophages, ainsi que l’augmentation de la production de la cytokine proinflammatoire IL-6 et de la chimiokine MCP-1. / Oro-pharyngeal candidiasis (OPC) is the most common opportunistic infection in HIV-1 infected patients. An OPC model using transgenic mice (CD4C/HIVMutA) expressing selected genes of the HIV-1 genome is now available. Using this model, it is now possible to study potential quantitative and functional disturbances in macrophages expressing the nef, rev and env genes of HIV-1 in the context of OPC. This study shows that transgene expression does not affect quantitative percentage values of macrophages in the oral mucosa and the small intestine, although burdens of C. albicans loads are increased in Tg mice. Transgene expression does induce diminished H2O2 production in macrophages, while increasing production of the proinflammatory cytokine IL-6 and the chemokine MCP-1.

Influenza virus is one of the major respiratory pathogens of humans as well as animals, including equines. There is an increasing evidence that bacterial infections are the most common cause of the death during influenza. In horses also, secondary bacterial pneumonia can lead to death, and surviving horses may take up to six months for the complete recovery resulting in heavy economic loss to the equine industry. Interleukin (IL)-23 mediated innate immune response has been shown to protect the host from various respiratory bacterial infections. However, studies to investigate the role of host and viral factors in the regulation of IL-23 are limited. Endoplasmic reticulum (ER) stress-induced transcription factor CHOP-10 and IFN-β has been shown to participate in the regulation of IL-23. Primary hypothesis for the current study was that influenza A virus (IAV) NS1 protein downregulates the IL-23 expression via inhibition of CHOP-10. In order to test our hypothesis, we infected the RAW264.7 cells - a murine macrophage cell line, and primary murine alveolar macrophage cells either with the wild type Influenza A virus (PR/8/34, PR8) or isogenic mutant virus lacking NS1 (delNS1). Quantitative analysis of mRNA expression revealed a significantly higher mRNA expression of IL23p19, IFN-β and CHOP-10 in delNS1 virus infected cells as compared the PR8 virus infected cells. Additionally, overexpression of CHOP-10 partially restored the expression of IL-23p19 in PR8 virus infected cells and knockdown of CHOP-10 resulted in downregulated expression of IL-23p19 in delNS1 infected cells. Taken together, these results suggest that IAV NS1 protein mediated inhibition of CHOP-10 expression leads to downregulation of IL-23 expression in macrophage cells in-vitro. Similar results were also observed in-vivo using IAV and Streptococcus zoooepidemicus (S. ze) co-infection model. In a co-infection mouse model delNS1 virus co-infection resulted in significantly higher expression of the IL-23 and IL-17. Considering the role of IL-23 in protection against respiratory bacterial pathogens, effect of exogenous supplementation of IL-23 was also investigated in the influenza and S. ze co-infection mouse model. We found that a single intranasal dose of recombinant murine IL-23 significantly improved the survival of mice co-infected with PR8 and S .ze. Overall, our study suggests that IAV infection subverts the IL-23 mediated respiratory innate immune response and restoration of IL-23 could protect from influenza-associated respiratory bacterial infections.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

Aphanomyces invadans is the pathogen that causes epizootic ulcerative syndrome (EUS), an economically devastating fish disease in southern Asia. The present thesis considered possible improvements to current methods of monitoring EUS, and examined the mechanisms of the host immune response to A. invadans in order to establish whether they could be enhanced to reduce the impact of EUS on aquaculture. Monoclonal antibody (MAb) technology was considered as a possible improvement to the histopathological methods currently used in diagnosis of EUS. Five MAbs were raised to day-old A. invadans germlings. Four gave weak reactions to A. invadans and cross-reacted with other Aphanomyces spp, though they may be useful for future studies on A. invadans. The other, designated MAb 3gJC9, only cross-reacted with the crayfish plague pathogen, A. astaci, and was used for the development of an immunohistochemistry protocol that may be of use in diagnosis. Immunohistochemistry with MAb 3gJC9, which recognised an extracellular product (ECP) of A. invadans, was specific to A. invadans in fish tissue, although it also recognised A. astaci in plague-infected crayfish. It also recognised the mycelium in fish infected with ulcerative mycosis, indicating that ulcerative mycosis is synonymous with EUS. Preliminary observations indicated that both ECPs and what appeared to be a hitherto unreported early stage of the mycelium are important in the pathology of EUS. Studies in vitro on the macrophages of EUS-susceptible giant gourami Osphronemus gouramy and silver barb Barbodes gonionotus, and EUS-resistant Nile tilapia Oreochromis niloticus, found that their macrophages were able to inhibit the growth of A. invadans. The macrophages of striped snakehead Channa striata did not inhibit A. invadans, which may account for their high EUS-susceptibility, especially as A. invadans strongly inhibited the respiratory burst of snakehead macrophages. Studies on humoral immune responses revealed that complement inhibited A. invadans in the case of snakeheads, gourami and barbs but not tilapia or swamp eels Monopterus albus. The humoral responses of the latter were very different to the four other species, and not elucidated. Low levels of anti A. invadans antibodies were found in tilapia and gourami from an EUS-endemic region, and high levels in snakehead. Snakehead antibodies appeared to be able to inhibit A. invadans even when complement was removed, but lower levels were produced at the low temperatures typically associated with EUS. A range of potential immunostimulants were screened for the ability to enhance resistance to EUS. The two successful products were administered as feed supplements to snakeheads and barbs that were subsequently injected intramuscularly with A. invadans. One, the algal extract Ergosan, showed some beneficial effects on snakeheads although the challenge was inconclusive. The other, the vitamin supplement Salar-bec, accelerated the cellular immune response and reduced mortality in snakeheads and barbs, and enhanced antibody production in snakeheads. The antibody response of snakeheads was further studied by comparing the anti- A. invadans antibody level, inhibitory activity of sera in vitro and protective capacity of sera from EUS-naïve snakeheads to that of snakeheads recently exposed to EUS and those subject to long term EUS-exposure. Sera of populations recently exposed to EUS showed an increased level of antibodies, but little improvement in inhibitory or protective activity. Sera from snakeheads that had endured long term exposure showed a wide range of antibody levels, but marked increases in inhibitory and protective activity. Antibodies cross-reacted with non-pathogenic Aphanomyces spp. in all cases.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.