Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, has become a well recognized diarrheal disease of immunodeficient humans and other mammals throughout the world. Specific therapy and immunoprophylaxis are currently unavailable, but passive immunization with C. parvum-specific monoclonal antibodies (mAbs) has demonstrated efficacy in immunocompromised hosts. In particular, mAbs eliciting the circumsporozoite precipitate (CSP)-like reaction protected against C. parvum infection. The circumsporozoite-like antigen (CSL), an ∼1,300 kDa apical glycoprotein of sporozoites and merozoites, is the molecular species mechanistically bound by mAbs having the ability to elicit the CSP-like reaction. These findings indicated that CSL has a functional role in sporozoite infectivity. In the present study, a quantitative in vitro sporozoite infectivity assay was developed to evaluate neutralizing activity of mAbs. 3E2, a mAb which elicited the CSP-like reaction, demonstrated the greatest level of neutralizing activity against C. parvum infections. Here, I report that CSL has properties consistent with being a sporozoite ligand for epithelial cells. For these studies, CSL was isolated from sporozoites by isoelectric focusing (IEF). The 1,200-1,400 kDa Mᵣ region containing CSL in SDS-PAGE of sporozoites was comprised of 31 species when analyzed by two-dimensional electrophoresis. Eight species were present in IEF-isolated CSL. CSL specifically bound to Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner with high affinity. CSL bound to Caco-2 cells inhibited the attachment and invasion of sporozoites in a dose-dependent manner. Characterization of the epitope recognized by 3E2 in a dot immunoblot indicated a β-glucose component. Sporozoites having undergone the CSP-like reaction after incubation with CSL-reactive 3E2, did not attach to or invade Caco-2 cells. These findings indicated that at least 1 of 8 CSL species isolated by IEF was a sporozoite ligand. The Caco-2 cell receptor for CSL was comprised of 16, 51, and 85 kDa molecules. Further, sporozoites incubated with isolated Caco-2 receptors failed to attach to and invade Caco-2 cells. Finally, CSL bound distinctly to receptors present on calf ileum. Based on these findings, I concluded that CSL ligand function is amenable to targeted disruption by CSL-reactive mAbs and that it is a rational target for immunization against cryptosporidiosis.

No description available.

<p> <i>PTEN</i> and <i>TP53</i> loss are common molecular alterations in aggressive prostate cancer that progresses to castrate resistant prostate cancer (CRPC). <i>PTEN/TP53</i> loss contributes to regulation of self-renewal and differentiation in prostate progenitor cells, the presumptive tumor and metastasis initiating cells for prostate cancer. <i>TP53</i> plays an important role in regulating normal cellular metabolism, and loss of function is responsible for metabolic alterations in tumor cells, including increased aerobic glycolysis. We use a novel model of <i>Pten/Tp53</i> deleted prostate cancer to investigate properties of tumor and metastasis initiating cells, and metabolic alterations that contribute to the evolution of CRPC. </p><p> We employed a genetically engineered mouse model of <i>Pten^-/-Tp53^-/-</i> prostate cancer to develop an orthotopic model derived from a clonal cell line from the parental heterogeneous prostate carcinoma. We used histopathology and immunohistochemistry to characterize the orthotopic primary tumors and metastases. We performed metabolomic screening followed by focused analysis of HK II enzyme levels, activity, and cellular distribution in androgen replete and androgen deprived tumors. We also compared HK II levels in primary and metastatic human prostate cancer. </p><p> Tumor heterogeneity was due to transformation of tumor and metastasis initiating cells with biphenotypic potential capable of basal and luminal differentiation. There was epithelial-to-mesenchymal transition (EMT) in cells of the luminal lineage. The model was capable of androgen independent growth, which influenced the differentiation of metastasis initiating cells. CRPC had increased reliance on glycolysis with increased cytoplasmic and mitochondrial-associated HK II. These metabolic adaptations afforded CRPC increased ability to withstand metabolic stress. HK II levels in human metastases were markedly increased compared to primary tumors. </p><p> <i>Pten/Tp53</i> loss in prostate cancer contributes to lineage plasticity in both tumor and metastasis initiating cells, contributing to heterogeneity observed in primary tumors and metastases. Increased glycolysis due to increased total and mitochondrial HK II is a metabolic adaptation that contributes to the evolution of aggressive disease, with progression to androgen independence, providing increased energy and carbon precursors for anabolic processes. Mitochondrial bound HK II blocks apoptosis and contributes to survival in the androgen deprived environment. Targeting this metabolic adaptation may provide improved treatment for this deadly disease.</p>

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2838. Adviser: Peter D. Constable. Includes supplementary digital materials. Includes bibliographical references (leaves 215-240) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-07, Section: B, page: 4318. Adviser: Indrani C. Bagchi. Includes bibliographical references (leaves 86-93) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2839. Adviser: Ronald M. Weigel. Includes bibliographical references (leaves 120-128) Available on microfilm from Pro Quest Information and Learning.

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No description available.

Master of Science / Diagnostic Medicine and Pathobiology / Carol R. Wyatt / Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant disease affecting the swine industry. PRRSV is known for its restricted cell tropism, primarily infecting porcine alveolar macrophages (PAM) via receptor-mediated endocytosis. PRRSV infects only a portion of the PAM population both in vivo and in vitro, which suggests that not every macrophage is PRRSV-permissive. Three surface proteins that can act as receptors for PRRSV have been identified on PAM, however, little else is known about the regulation of macrophage tropism. Factors determining cellular permissibility or resistance to PRRSV infection remain largely uncharacterized, although a recent study from our laboratory demonstrated that 1) permissiveness to PRRSV infection increased with time in culture, 2) macrophages from infected pigs could be superinfected, and 3) addition of actinomycin D, which inhibits mRNA synthesis, blocked infection. These data suggest that a PRRSV-permissive subpopulation of cells derives from a non-permissive precursor population and depends on new mRNA synthesis. The current studies were designed to examine the effects of PRRSV on both infected and uninfected PAM cells in vitro, specifically focusing on the expression of MHC I, MHC II, CD14, CD163 and CD172a surface proteins. The results show upregulation of MHC II, CD14, CD163 and CD172a expression in PRRSV-infected cells and a downregulation on the uninfected cells within the PRRSV-inoculated cultures. The role of apoptosis in the PRRSV-inoculated cultures was investigated, with results showing similar, low levels of apoptosis in control and infected PAM. PAM cytokine responses to PRRSV and LPS were also examined and, although they were uniquely different relative to control PAM, no trends were detected in the responses of PAM infected with PRRSV compared to uninfected and classically stimulated PAM. These data confirm that there are at least two subsets of macrophages within the alveolar population and suggests that the subsets are differentially affected by PRRS virus. We also demonstrated that MHC I becomes undetectable on PAM as a result of the freezing process, and that PRRSV-permissiveness is greater in the cell population after freezing.

Corynebacterium pseudotuberculosis causes caseous lymphadenitis of sheep and goats and produces a phospholipase D (PLD) exotoxin which is putatively important in pathogenesis. Viability and function of ovine polymorphonuclear leukocytes (PMN) treated with crude and purified forms of PLD were determined by various assays. PMN viability by dye exclusion showed the PLD-treatment had a significant effect only after 24 hour incubation. Scanning electron microscopy of PLD-treated ovine erythrocytes revealed membrane alterations, but no such alterations were seen in PLD-treated PMN. Transmission electron microscopy revealed significantly fewer granules in PMN treated with PLD, although other facets of phagocytic function appeared to be normal, including phagosome-lysosome fusion. PLD-treated PMN were significantly reduced in their ability to internalize Corynebacterium pseudotuberculosis and to attach to or phagocytose Staphylococcus epidermidis. Purified PLD activated normal sheep serum, producing chemotactic factors. PLD treatment of PMN significantly reduced the ability of these cells to migrate toward activated sheep serum.