Johne�s disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne�s disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne�s disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne�s vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne�s disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.

Indole-3-carbinol (I3C) is a naturally occurring plant alkaloid, found in significant concentrations in cruciferous vegetables such as broccoli and Brussels sprouts. I3C is an unstable compound that undergoes rapid oligomerization in an acidic environment to form higher order condensation products (I3C-ACPs), such as 3-3'-diindolylmethane (DIM). Both I3C and DIM are marketed as dietary supplements and are under investigation as potential chemopreventive agents, despite limited data on the effects of chronic exposure. Previous studies have demonstrated that the chemopreventive potential of I3C and DIM in animal studies is dependent on species, strain, tissue and timing of treatment relative to carcinogen exposure, and long-term post-initiation exposure can even promote tumors. The majority of biological effects from I3C are the result of the abilities DIM and other I3C-ACPs to bind to the aryl hydrocarbon receptor and the subsequent induction of phase I and phase II enzymes. Phase I and phase II enzyme induction in many cases leads to protection from carcinogens by increasing the rate of metabolism and excretion but in some cases enhances carcinogenicity by increasing the rate of bioactivation. It has been demonstrated that modulation of enzyme levels can also result in altered metabolism of compounds that could affect efficacy and toxicity of pharmaceuticals and xenobiotics. The current work utilizes chronic dietary I3C and DIM exposures in rodent models to further elucidate the effect these compounds might have on health, drug metabolism and carcinogenesis. The reduced weight of Fischer 344 rats treated with 2500 ppm I3C for 1 year may be indicative of adverse effects but toxicity was not confirmed by blood chemistry or histopathological examination. Furthermore, no toxicity was observed after a comparable treatment of Sprague-Dawley rats. As observed after acute and sub-chronic exposures to I3C and DIM, we documented significant induction of cytochrome P450 enzymes and a related modification to drug metabolism in liver slice incubations. Evidence is also provided that may suggest that tumor modulation in mice may occur through an estrogenic mechanism. Further studies should be completed to determine the potential for similar responses in humans. / Graduation date: 2004

Proper immune function is indispensable, as failure to mount an immune response against a pathogen can lead to serious complications or even death. T cells act by enhancing the activation of phagocytic cells as well as the activation of B cells. Their widespread influence on an immune response makes optimal T cell activation vital. Maximal T cell proliferation and survival is accomplished by stimulation with antigen, a costimulatory signal, and an adjuvant. However, excessive T cell activation can lead to chronic B cell activation and the production of autoantibodies, a hallmark of autoimmune disease. In this thesis, optimal T cell stimulation was studied using an in vivo adoptive transfer model. Results showed that antigen stimulation of T cells along with ligation of the costimulatory molecule OX40 led to an accumulation of antigen-specific cells. OX40 ligation allowed the antigen specific cells to proceed through more cell cycles than cells stimulated with antigen alone. The addition of the adjuvant lipopolysaceharide (LPS) to this system allowed for increased cell survival. However, the continual presence of an adjuvant may also have injurious effects. This was highlighted with the appearance of "Toxic Oil Syndrome" (TOS) in which an adulterated rapeseed oil, an oil with known adjuvant activity, was sold for human consumption. People developed an autoimmune condition characterized by polyclonal B cell activation and autoantibody production. A genetic predisposition was implicated with TOS and was further investigated in this thesis. Although the A. SW mouse has the genetically susceptible genotype, these mice did not develop TOS following exposure to "toxic oil" indicating that other factors may be important in TOS susceptibility. Extending the techniques used in these studies and applying them to the canine immune system was the final topic investigated in this series of studies. Understanding immune pathways of the mammalian immune system is particularly important for comparative studies when dogs are used as models to investigate human immune system disorders. These studies combined will allow for a better understanding of the balance between an optimal immune response and an imbalance leading to hypersensitivity or immunosuppression, as well as interspecies relationships. / Graduation date: 2002

Ball State University LibrariesLibrary services and resources for knowledge buildingMasters ThesesThere is no abstract available for this thesis.

C57B1/6 mice bearing Lewis lung carcinoma CLLC) are suppressed in their ability to generate cytotoxic T lymphocytes against the LLC tumor associated antigens. Since LLC have previously been shown to secrete the immunosuppressive factor PGE2, indomethacin, a prostaglandine synthetase inhibitor, was administered to LLC-bearing mice in their drinking water to prevent the immunosuppression typical of tumor bearers. The indomethacin treated tumor-bearers displayed an increase in their cytotoxic responses to the LLC tumor associated antigens. Thus the immunosuppression found in mice bearing LLC could be due to prostaglandin synthesis. Production of PGE2 by tumor cells may be a mechanism by which the tumors escape immune mediated destruction.Ball State UniversityMuncie, IN 47306

Thesis (Ph. D.)--Mississippi State University. Department of Biochemistry and Molecular Biology. / Title from title screen. Includes bibliographical references.

Severe lymphopenia is a clinical feature of Severe Acute Respiratory Syndrome (SARS) patients. However, lymphocytes do not express receptor for SARS-CoV, neither the widely accepted viral receptor angiotensin converting enzyme 2 (ACE2) nor the putative receptors Dendritic Cell- and Liver/lymph-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN and L-SIGN). Our group previously showed in vitro that, SARS-CoV Spike pseudotyped particles (SARSCoVpp) could infect human B cells only when inoculated in presence of anti-SARSCoV Spike immune serum. Such observations raised concerns about the possible occurrence of antibody-dependent enhancement (ADE) of infection, a phenomenon during which a virus bounded by antibodies could gain entry into cells through mechanisms involving complement receptors or Fc receptors. Recently, we have demonstrated the participation of the human Fc gamma receptor II (hFcγRII) molecules in granting SARS-CoV an opportunity to infect human immune cells. The aim of this study was to decipher the molecular mechanism leading to antibodymediated, FcγRII-dependent infection of immune cells by SARS-CoV. By using transduction experiment, I highlighted that different members of the hFcγRII family (namely hFcγRIIA, hFcγRIIB1 and hFcγRIIB2) could confer susceptibility to ADE of SARS-CoVpp infection. I further demonstrated that purified anti-viral immunoglobulin G, but not other soluble factor(s) from heat-inactivated immune serum, was the determinant for occurrence of ADE infection. Additionally, with the development of a cell-cell fusion assay, I illustrated that in contrast to the ACE2- dependent pathway, ADE infection did not occur at the plasma membrane, but rather require internalization of virus/antibodies immune complexes by the target cells. In line with this hypothesis, my results using a panel of FcγRII-expressing mutants demonstrated that binding of immune complexes to cell surface FcγRII was a prerequisite but was not sufficient to trigger ADE infection. In these experiments, only FcγRII signaling-competent constructions conferred susceptibility to ADE of SARS-CoVpp infection. Altogether my results point toward a role of the anti-SARS-CoV Spike IgG in vitro in granting SARS-CoV an opportunity to infect cells bearing signaling-competent FcγRII receptors. If further confirmed, such observations could have implications for understanding SARS-CoV tropism and SARS pathogenesis, as well as warrant for careful design of SARS vaccines and immunotherapy based on anti-viral antibodies. / published_or_final_version / Microbiology / Master / Master of Philosophy

Chronic rejection is a major obstacle to long-term survival of organ transplants. PPAR-γ agonist rosiglitazone has been shown to reduce graft rejection but the underlying mechanisms remain unclear. Combined treatment of rosiglitazone and anti-IL-5 antibody prevented MHC class II histoincompatiblecardiac graft rejection with a reduction of cellular infiltration, vasculopathy and interstitial fibrosis in a heterotopic heart transplantation model. In particularly, rosiglitazone decreased CD8 T cells infiltration and luminal occlusion, while anti-IL-5 antibody reduced eosinophil infiltration and collagen deposition. Adiponectin gene (APN) is a PPAR-γ target gene, and the expression of APN receptor AdipoRII in grafts, dendritic cells (DCs) and T cells are increased by rosiglitazone. These findings prompted me to further examine the immunomodulatory role of APN in graft rejection. APN is an anti-inflammatory adipocytokine, and has been shown to inhibitimmunostimulatory function of monocytes and macrophages. Rosiglitazone suppresses DCs maturation, activation and proliferation;hence, it is possible that APN could protect graft rejection through immunoregulation of DCs. Here, using in vitro culture systems, I found that APN has only moderate effect on the differentiation of bone marrow derived DCs but itcould alter DC phenotypes. APN-treated DCs showed an increased expression of PD-L1, which is consistent with the increased PD-L1 expression in rosiglitazone treated cardiac allografts. APN-treated DCs led to a decreased proliferation and reduction of IL-2production of T cell. Moreover, APN-treated DCs increased the expansion of Tregs (regulatory T cells) which could be inhibited by the blockage of PD-1/PD-L1 pathway, suggesting that PD-1/PD-L1 pathway and expansion of Tregs played important roles in APN-treated DCs mediated immunomodulation. Further, I employed APN-/-mice for functional and mechanistic studies, and found that cardiac allografts were not rejected by APN-/-recipient mice even after 120 days post-transplantation. Histological analyses revealed very little eosinophils, CD4 and CD8 T cells infiltration; no collagen deposit and no vessel occlusion in the cardiac allografts. Furthermore, Th2 cytokines such as IL-4 and IL-5 were lower in cardiac allografts and in the serum of APN-/-recipient. Inhibition of AMPK signaling, a major APN mediated pathway, reduced the eosinophils infiltration in wild type recipient. In contrast, AMPK activation increased eosinophils infiltration in APN-null recipient. APN enhanced T cell proliferation. AMPK and P38MAPK inhibitors as well as anti-IL-4 antibody inhibited APN-induced T cell proliferation. P38 MAPK inhibitors reduced IL-4 production in mature DCs but enhanced IL-4 expression in immature DCs. In EL-4 T cells, APN increased nuclear expressions of GATA-3 and p-STAT6 and augmented IL-4 expression, and the phenomenon was suppressed by target specific knockdown of AdipoR I and II. In summary, current study provides new mechanistic insights of PPAR-γ activation and APN signaling in the modulation of adaptive and transplantation immunity, establishing a link between metabolism and immune regulation. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Dendritic Cells (DCs) are professional antigen presenting cells that play a crucial role in the induction of immune tolerance. Although DCs have been a potential target for immunotherapy, the amount of DCs in blood source is limited and ex vivo expansion has been inefficient. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a great source in cell-based therapy because of their self-renewal ability and pluripotency. My project focuses on generating tolerogenic DCs (tDCs) from human pluripotent stem cells (i.e. hESCs and iPSCs) and their characterization. Specifically, hESCs and hiPSCs were first differentiated to hematopoietic progenitor cells (HPCs) using three different methods (i.e. bone-marrow stromal cell co-culture and two previously reported defined medium methods). The hESC/iPSC-differentiated hematopoietic progenitor cells (HPCs) were characterized by their surface phenotype using flow cytometry. Then the hESC/iPSC-differentiated immature DCs were further expanded and differentiated from the hESC/iPSCdifferentiated CD34+ HPCs with the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4). Tolerogenic properties were introduced by treating hESC-differentiated DCs with rapamycin. The treated DCs were characterized for their tolerogenicity by examining their expression of PDL1, PDL2, ICOS and CD40 etc., and their ability to promote regulatory T cells (Treg) differentiation. All these were compared with monocyte-derived tDCs. In summary, this study has examined the potential of using pluripotent stem cells-derived DCs as a cell source for immune tolerance induction therapy. / published_or_final_version / Anatomy / Master / Master of Philosophy