Immune responses to Epstein-Barr Virus (EBV) encoded Latent Membrane Protein 1 (LMP1) peptides in seropositive donors are dominated by the induction of IL-10 secretion. These IL-10 responses, characteristic of T regulatory 1 (Tr1) cells, were able to inhibit T-cell proliferation and interferon-γ (IFN-γ) section induced by other antigens. Furthermore, this inhibition was specific to the co-presented antigen and persisted after LMP1 peptide had been removed. Thus, it may be possible to exploit such specific induction therapeutically to inhibit pathogenic responses in immune-mediated diseases. The current study was initiated to confirm and extend these findings and then to investigate the ability of LMP1 peptides to inhibit pathogenic responses to Rh autoantigen in AIHA patients <i>in vitro</i>. Inhibitory properties of LMP1 peptides were confirmed, although most such inhibition appeared non-specific and was not associated with IL-10. Inhibition appeared to involve an effect on antigen presenting cells. When autologous red cells or RhD peptides were used as stimulating antigens in patients with AIHA, IL-17 responses were more frequent than IFN-γ secretion. Furthermore, disease activity correlated better with IL-17 responses than TH1 responses. These data therefore suggest that the pathogenesis of AIHA has a substantial Th17 component. Finally, these autoreactive responses could be inhibited by LMP1 peptides.

Introdução: O aumento no consumo mundial de imunoglobulina humana tem desafiado os sistemas de saúde no estabelecimento de padrões de utilização adequados a esta terapia. O conhecimento das políticas públicas pelos profissionais de saúde, aderidos a uma conscientização ao uso racional e estudos baseados em evidências científicas é fundamental para assegurar o acesso adequado e uma maior segurança e efetividade de tratamento. Objetivo: Avaliar a utilização de imunoglobulina humana em hospital universitário de alta complexidade do sul do país e suas indicações relacionando as mesmas aos protocolos clínicos estabelecidos. Métodos: Trata-se de um estudo transversal, retrospectivo, baseado na busca de informações através do prontuário eletrônico dos pacientes do Hospital de Clínicas de Porto Alegre no período de janeiro à dezembro de 2015 Resultados: Foram identificadas 191 prescrições de imunoglobulina humana endovenosa, totalizando 116 pacientes. Desses pacientes, 23% apresentaram síndrome de Guillain Barré, púrpura trombocitopênica idiopática, miastenia gravis, transplante renal, imunodeficiência com aumento de IgM e outras anemias hemolíticas autoimunes. Todas essas situações clínicas tem indicação de uso de acordo com os protocolos estabelecidos pelo Ministério da Saúde. Os demais casos identificados (77%) não constam nas indicações previstas nos protocolos do Ministério da Saúde. Conclusão: Foi possível identificar a utilização de imunoglobulina humana endovenosa em hospital de alta complexidade e quantificar os casos clínicos que fazem uso desse medicamento e que apresentam protocolos nacionais orientando os profissionais de saúde quanto a correta administração desse medicamento. Observou-se que a maioria dos casos identificados no estudo não apresentam regulamentos oficiais que autorizem a sua administração. / Introduction: The increase in the world consumption of human immunoglobulin has challenged the health systems in establishing appropriate standards of use for this therapy. Knowledge of public policies by health professionals adhering to rational use awareness and evidence-based studies is critical to ensure adequate access and greater safety and effectiveness of treatment. Objective: To evaluate the use of human immunoglobulin in a university hospital of high complexity in the South of the country and its indications relating them to the established clinical protocols. Methods: This is a cross-sectional, retrospective study based on the search of information through the electronic medical records of patients from the Hospital de Clínicas of Porto Alegre from January to December 2015 Results: 191 prescriptions of intravenous human immunoglobulin were identified, totaling 116 patients. Of these patients, 23% had Guillain Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, renal transplantation, immunodeficiency with increased IgM and other autoimmune hemolytic anemias. All of these clinical situations are indicated for use according to protocols established by the Ministry of Health. The other cases identified (77%) are not included in the indications provided for in the protocols of the Ministry of Health. Conclusion: It was possible to identify the use of intravenous human immunoglobulin in hospital of high complexity and to quantify the clinical cases that use this medicine and that present national protocols guiding healthcare professionals about the correct administration of this medicine. It was observed that the majority of the cases identified in the study do not present official regulations that authorize its administration.

Doctor of Philosophy / Profound perturbation of epithelial structure is a characteristic feature of the immunopatholoical response to bacterial antigens considered to be central in the pathogenesis of the destructive lesion of periodontitis. The pathological basis for the disturbance of epithelial structure is not understood. It was demonstrated that the structural integrity and functional differentiation of the lining epithelium is compromised in relation to inflammatory changes associated with destructive periodontitis. In the pathological lining epithelium of the periodontal pocket there was a marked reduction of epithelial cadherin important in intercellular adhesion, of involucrin, a marker of terminal differentiation, and of the gap junction connexions that form intercellular communication channels. These changes were associated with alterations of filamentous actin expression, collectively indicating profound perturbation of epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised (Ye et al., 2000). In addition, a recent study (Ye et al., 2003) by Western analysis of serum IgG from all 22 patients with chronic periodontitis tested indicated recognition of multiple epithelial components in individual patterns. In contrast, subjects with a healthy periodontium displayed only trace recognition of epithelial antigens. Levels of epithelial-reactive antibodies were significantly correlated with attachment loss as an indication of disease activity. To investigate a possible relationship between the bacterial flora adjacent to the diseased sites and the presence of epithelial-reactive antibodies, subgingival plague samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens potentially cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony lifts with affinity-isolated (epitheial-specific) antibodies. The bacteria were identified as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone) by 16S rDNA sequence homology. Recognition by affinity-isolated antibodies of antigens from the captured organisms was confirmed by Western analysis. Conversely, absorption of affinity-isolated antibodies with bacterial species specifically reduced subsequent recognition of epithelial antigens. To identify the auto-antigens, a human keratinocyte cDNA expression library in Lambda phage was probed using a pooled sera. Groups of responders were detected for CD24 (a recently described adhesion molecule also known as P-selectin ligand), antioxidant protein 2 (a newly recognised member of the thiol-dependment anti-oxidant proteins), lavtate dehydrogenase A, the transcription factor NFAT5, and for three genes encoding novel proteins. Six identified bacteria, especially S intermedius were demonstrated to absorb antibodies reaching with identified auto-antigens in patterns varying between individuals. This evidence indicated that during the course of periodontits, subjects develop increased levels of antibodies to common oral bacteria amongst which are included tissue cross-reactive antigens. Periodontitis could therefore present a risk for the subsequent initiation or exacerbation of a broad spectrum of disease processes including autoimmune, inflammatory, proliferative and degenerative disorders.

Thesis (M.S.)--University of Toledo, 2009. / Typescript. "Submitted as partial fulfillment of the requirements for The Master of Science in Biology." "A thesis entitled"--at head of title. Bibliography: leaves 88-97.

Although B cells are well-known for their functions in antibody production and antigen presentation, certain B cell subsets have been recently identified as regulatory B cells to modulate immune responses through cytokine production. However, the microenvironmental factors involved in the induction of regulatory B cells remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines produced by myeloid cells, is a key regulator for B cell maturation and function. However, it remains unknown whether BAFF plays a role in modulating the generation of regulatory B cells and how regulatory B cells suppress autoimmune pathogenesis. In this study, treatment with BAFF significantly increased IL-10-producing B cells in culture of mouse splenic B cells, an effect specifically abrogated by neutralization with TACI-Fc. BAFF-induced IL-10-producing B cells showed a distinct CD1dhiCD5+(B10) phenotype. Phenotypic analysis further indicated that these BAFF-induced B10 cells were marginal-zone (MZ)-like B cells. Interestingly, BAFF treatment in vivo also increased the number of IL-10-producingB cells in splenic MZ regions. Moreover, chromatin immunoprecipitation analysis revealed that BAFF activated the transcription factor AP-1 for binding to IL-10 promoter, demonstrating a novel function for BAFF in inducing IL-10 production. Furthermore, those BAFF-induced B10 cells exhibited significant suppressive effects on CD4+T cell proliferation and Th1 cytokine production in culture. To explore whether these BAFF-induced B10 cells possess a regulatory function in suppressing autoimmune progression in vivo, collagen-induced arthritis (CIA) mouse model was employed. In vitro-expanded B10 cells and other control B cells were intravenously transferred into DBA/1J mice on the day of 2ndcollagen II (CII)-immunization. After adoptive transfer of BAFF-induced B10 cells, CII-immunized mice exhibited a delayed onset of arthritis and substantially reduced severity of clinical symptoms. The pathogenesis of IL-17-producing CD4+T cells (Th17) in the development of arthritis has been well-recognized, which has led me to test the hypothesis whether B10 cells ameliorate the development of arthritis via modulating Th17 cells. During the progression of CIA, IL-10-producing B cells were decreasedwhereasTh17 cells were significantly increased at the acute phase of CIA. Upon transfer of BAFF-induced B10 cells, a substantially reduction ofTh17 cells in both lymphoid organs and inflamed joints were detected. To verify whether B10 cells inhibit Th17 cell generation in culture, CFSE-labeled na?ve CD4+T cells were cocultured with B10 cells in Th17 cell polarization medium. It was found that B10 cells suppressed Th17 cell differentiation via reducing STAT3 phosphorylation and RORt expression. Although adoptive transfer of Th17 cells triggered the development of CIA in IL-17-/-DBA mice, cotransfer of B10 cells with Th17 cells profoundly delayed the onset of delayed the onset of arthritisand remarkably reduced the infiltration of Th17 cells in synovial fluid. Taken together, I have identified a novel function of BAFF in the induction of IL-10-producing regulatory B cells. My findings that adoptive transfer of BAFF-expanded B10 cells can effectively suppress the development of experimental arthritisin mice via the inhibition of Th17 cell generation may contribute to the development of new therapeutic strategies in treating human rheumatoid arthritis. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Leptin, a protein hormone encoded by obese (ob) gene, is mainly produced by adipocytes. Leptin plays an important role in regulating neuroendocrine function and energy metabolism. As a cytokine, leptin is involved in modulating the hematopoiesis and lymphopoiesis. Although leptin has been found to promote T cells activation, it is largely unclear whether and how leptin regulates T cell differentiation and function. Leptin has been associated with disease severity in rheumatoid arthritis (RA). Elevated leptin levels have been detected in the sera and synovial fluid of active RA patients. Th17 cells play key roles in synovitis and joint damage during arthritis development. However, the role of leptin on Th17 cells has not been investigated so far. In culture, leptin promoted Th17 cells differentiationfrom naïve 〖CD4〗^+ T cells via upregulating ROR-γt expression. Moreover, Th17 cells and IL-17 levels were significantly increased in leptin-treated naïve CD4+ T cells. Moreover, this study found that synoviocytes and chondrocytes produced large amounts of leptin especially during acute and chronic stages of mice with collagen-induced arthritis (CIA). Furthermore, leptin levels, Th17 cells in the joint and IL-17 levels in the synovial fluid were closely related to disease activity. Leptin intraarticular injection led to earlier onset of disease, higher clinical score, and more severe joint destruction compared with PBS-treated control mice. Importantly, leptin-injected mice had higher percentages of Th17 cells and cell numbers, elevated IL-17 levels in the synovial fluid, and increased infiltration of Th17 cells in the inflamed joint tissues compared with PBS-treated mice. T follicular helper (Tfh) cells are indispensible for pathogenic autoantibodies production. However, whether leptin receptor (ObR) signaling has a role on Tfh cells and its implication in CIA remain elusive. Upon a T cell-dependent antigen TNP-KLH immunization, germinal center (GC) response, plasma cells (PCs) and memory B cells formation were impaired in db/db mice compared with wild-type (WT) controls. In coculture of Tfh cells from db/db and WT mice with WT GC B cells, anti-TNP IgGs titers in supernatants of db/db Tfh cells were significantly reduced. Intravenously transfer of naïve CD4+ T cells from db/db and WT mice to BoyJ recipient mice, donor CD4+ T cells from db/db mice showed impaired Tfh cells generation in spleens of BoyJ recipient mice compared with mice received with WT CD4+ T cells. These data indicated that ObR-mediated signaling intrinsically modulate Tfh cells generation. In culture, leptin promoted Tfh cells differentiation via inducing Bcl6 expression, and increased IL-21 production in Tfh cells in a dose-dependent manner. Leptin significantly enhanced the phosphorylation of STAT3, upregulated Bcl6 expression, and increased p-STAT3 binding to the Il21 promoter in CD4+ T cells with leptin receptor b (Ob-Rb) overexpression. Upon CIA induction, db/db mice exhibited ameliorated disease severity with impaired Tfh cells response. However, WT Tfh cells transfer to db/db mice restored GC responses, PCs formation, antibody production, and exacerbated synovium inflammation and joint damage in db/db recipient mice. Together, these findings demonstrate that leptin modulates arthritis development via enhancement of Th17 and Tfh cells responses. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

No description available.

No description available.

Research Doctorate - Doctor of Philosophy (PhD) / Background: Autoimmune diseases arise from the breakdown of central tolerance resulting in the escape of self reactive T-lymphocytes from the thymus to the periphery. As a group of conditions, autoimmune diseases occur in approximately 5% of the general population and represent the third most common cause of morbidity, placing considerable expenses on the health care system and society. Understanding the underlying pathogenesis and pathophysiology of these diseases is therefore important for the correct diagnosis and treatment of these patients. While some autoimmune diseases have been paid particular attention, little is known about the pathogenesis of the pituitary autoantibodies. Aims: To identify target autoantigens in the pituitary autoimmune disease lymphocytic hypophysitis and autoantigen(s) relating to pituitary manifestations in APS1 patients. Methods: A pituitary cDNA expression library was immunocreened with lymphocytic hypophysitis and APS1 patient sera to identify target autoantigens. These were then tested in an ITT assay for autoantigen specificity to relating to the disorders. Immunofluorescence of pituitary tissue was performed to determine the cell types targeted in the disorders. Results: Two APS1 autoantigens were identified, a major autoantigen ECE-2 and a minor autoantigen TSGA10, although neither apparently correlated to pituitary manifestations in APS1. T-box 19 was also identified as a significant minor autoantigen in 10.5% of lymphocytic hypophysitis patients. Immunoreactivity in a single lymphocytic hypophysitis patient against cells of the intermediate lobe of the guinea pig pituitary is also reported. Discussion: Immunoscreening a target organ cDNA expression library is a valuable method for identifying novel autoantigens, with immunopreciptation assay a quick and reliable method for analysing a large cohort of patients for autoantibodies. We have identified another two APS1 autoantigens and the first significant autoantigen in lymphocytic hypophysitis. While further characterisation of these autoantigens are required, these novel findings broaden our current understanding of pituitary autoimmunity.

Doctor of Philosophy / Profound perturbation of epithelial structure is a characteristic feature of the immunopatholoical response to bacterial antigens considered to be central in the pathogenesis of the destructive lesion of periodontitis. The pathological basis for the disturbance of epithelial structure is not understood. It was demonstrated that the structural integrity and functional differentiation of the lining epithelium is compromised in relation to inflammatory changes associated with destructive periodontitis. In the pathological lining epithelium of the periodontal pocket there was a marked reduction of epithelial cadherin important in intercellular adhesion, of involucrin, a marker of terminal differentiation, and of the gap junction connexions that form intercellular communication channels. These changes were associated with alterations of filamentous actin expression, collectively indicating profound perturbation of epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised (Ye et al., 2000). In addition, a recent study (Ye et al., 2003) by Western analysis of serum IgG from all 22 patients with chronic periodontitis tested indicated recognition of multiple epithelial components in individual patterns. In contrast, subjects with a healthy periodontium displayed only trace recognition of epithelial antigens. Levels of epithelial-reactive antibodies were significantly correlated with attachment loss as an indication of disease activity. To investigate a possible relationship between the bacterial flora adjacent to the diseased sites and the presence of epithelial-reactive antibodies, subgingival plague samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens potentially cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony lifts with affinity-isolated (epitheial-specific) antibodies. The bacteria were identified as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone) by 16S rDNA sequence homology. Recognition by affinity-isolated antibodies of antigens from the captured organisms was confirmed by Western analysis. Conversely, absorption of affinity-isolated antibodies with bacterial species specifically reduced subsequent recognition of epithelial antigens. To identify the auto-antigens, a human keratinocyte cDNA expression library in Lambda phage was probed using a pooled sera. Groups of responders were detected for CD24 (a recently described adhesion molecule also known as P-selectin ligand), antioxidant protein 2 (a newly recognised member of the thiol-dependment anti-oxidant proteins), lavtate dehydrogenase A, the transcription factor NFAT5, and for three genes encoding novel proteins. Six identified bacteria, especially S intermedius were demonstrated to absorb antibodies reaching with identified auto-antigens in patterns varying between individuals. This evidence indicated that during the course of periodontits, subjects develop increased levels of antibodies to common oral bacteria amongst which are included tissue cross-reactive antigens. Periodontitis could therefore present a risk for the subsequent initiation or exacerbation of a broad spectrum of disease processes including autoimmune, inflammatory, proliferative and degenerative disorders.