No description available.

No description available.

No description available.

No description available.

No description available.

<p>Inflammatory bowel disease (IBD) is chronic, relapsing, intestinal inflammation characterized by periods of remission and exacerbation. Pathogenesis of IBD is a complex process and the exact aetiology is unclear yet. Studies have provided evidences that IBD is a result of a genetic predisposition that leads to a mucosal immune regulatory cell defect, barrier defects, and susceptibility to environmental triggers like luminal bacteria and specific antigens. Innate and adaptive immune responses to commensal bacteria are balanced by the multiple regulatory cells like T regs and B regs. Monoclonal-antibody-based therapies have been identified for the treatment of IBD but, they have side effects and the efficacy is not stable. Hence, great expectations lie towards finding a successful cure for IBD.</p> <p>Recent studies have proved the involvement of several different genes in the pathogenesis of IBD and there expressions were highly reduced in IBD. Homeodomain only protein- Hopx is a nuclear protein required to modulate heart growth and function. Expression of Hopx has been reported in various types of normal tissues but not in malignant tissues. Recent study on Hopx revealed that Hopx is needed for the function of regulatory T cells induced by DCs. However the correlation between Hopx gene and IBD remains unanswered. In this study, we examined the role of Hopx expressing regulatory B cell in IBD.</p> <p>The aim of this study is to investigate whether the expression of Hopx in regulatory B cell play a key role in suppressing colitis in murine model.</p> <p>The expression of Hopx was studied using cellular and molecular techniques including flowcytometry, immunohistochemistry, western blotting and real time RT-PCR.</p> <p>As a first step, we investigated the Hopx expression in colitis and control murine model. After we found the possible involvement of Hopx gene in regulating intestinal inflammation, we further our study to investigate whether Hopx is expressed by regulatory B cell and function together to inhibit intestinal inflammation. After establishing Hopx and B regs association, we used probiotics to modulate Hopx expression in regulatory B cells in order to prevent/reduce intestinal inflammation. Finally we elucidate the importance of Hopx expression by injecting neutralizing anti-Hopx antibody to block Hopx.</p> <p>Together, studies on human intestinal tissue and murine model revealed that Hopx expression is suppressed during inflammation condition. Probiotic administration helps to increase Hopx expressing Breg cell thereby, prevent IBD. Hopx deficient group expressed high frequency of proinflammatory cytokines and reduced immune regulatory cells. This particular study proposes that the down regulation of Hopx contributes to the development of intestinal inflammation.</p> / Master of Health Sciences (MSc)

Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by a lowered platelet count (50% from baseline) 4-10 days after heparin exposure. Autoantibodies specific for platelet factor 4 (PF4) bind PF4 and heparin complexes (PF4/H) and activate platelets through the FcgammaRIIA receptor. Severe cases of HIT can result in thrombotic complications including deep vein thrombosis, pulmonary embolism, and death. Pathogenic class-switched antibodies against PF4/H (IgG) are detectable in circulation as early as five days post-heparin exposure and peak at 14 days. The timeline and class of antibody found in HIT patients suggest that there must be pre-existing immunity against PF4/H. Thus, B cells producing anti-PF4/H antibodies must exist prior to heparin exposure. Cardiac surgery patients are disproportionately prone to anti-PF4/H seroconversion (up to 70%) and thus are utilized in this study as a model patient group. Research objective: The objective of this study is to determine whether the preoperative immune profile is associated with postoperative anti-PF4/H antibody production in a cohort of patients undergoing cardiac pulmonary bypass (CPB) surgery. Materials and methods: To characterize the preoperative immune profile, we used 1) a peripheral blood mononuclear cell (PBMC) enzyme linked immunospot (ELISPOT) assay to measure the prevalence of preoperative anti-PF4/H specific antibody secreting cells (ASC) and 2) a PF4/H-dependant enzyme immunoassay (EIA) to measure the anti-PF4/H antibodies produced by PBMCs in vitro. To characterize postoperative anti-PF4/H seroconversion in CPB patients, we used a PF4/H dependent EIA to measure in vivo levels of anti-PF4/H antibodies produced postoperatively. We also utilize a functional assay, 14C-serotonin release assay (SRA) to determine if seroconverting patients produced platelet activating antibody. Results: All patients were able to produce anti-PF4/H spots in the ELISPOT; however, this did not correlate with the titer of antibody production in vitro nor did it correlate with antibody production in the postoperative period. Instead, we found that pre-operative in vitro anti-PF4/H IgM production was associated with post-operative IgG anti-PF4/H seroconversion (Spearman’s r=0.39, P=0.018). We observed that 92.1% of CPB patients produced PF4/H antibody at postoperative week 3 with some combination of IgA, IgG, and IgM. Of the anti-PF4/H seropositive patients, 26% developed platelet activating antibody and were found seropositive when the SRA was supplemented with PF4 instead of heparin, while 15.7% were seropositive in the original SRA. It was noted that 4 of 10 patients that caused the most robust platelet activation were also seropositive for anti-PF4/H IgA antibody. Lastly, throughout this serosurveillance study, several patients that demonstrated unique immunological features are presented in this study as case studies. Specifically, we report the preoperative, surgical, clinical and postoperative characteristics for 3 patients of interest: 1) in a preoperative setting, a CPB patient’s PBMC were able to be activated and produce anti-PF4/H IgG antibody in vitro, 2) the second patient had platelet-activating antibodies in circulation prior to intraoperative heparin challenge and early post-surgery 3) the third patient who developed probable HIT. Conclusions: Based on our findings, we conclude that preoperative PF4/H ELISPOTs were unable to predict post-operative production of anti PF4/H antibodies. However, preoperative in vitro production of anti-PF4/H IgM may be associated with postoperative production of anti-PF4/IgG antibody and should be investigated further as this may help to elucidate the mechanisms for anti-PF4/H production related to HIT. / Thesis / Master of Science (MSc)

Systemic lupus erythematosus (SLE) is a chronic multifactorial inflammatory autoimmune disease with heterogeneous clinical manifestations. Among different manifestations, lupus nephritis (LN) remains a major cause of morbidity and mortality. There are few FDA approved treatments for LN. In general, they are non-selective and lead to global immunosuppression with significant side effects including an increased risk of infection. In the past 60 years, only one new drug, belimumab was approved for lupus disease with modest efficacy in clinic and not approved for patients suffering for nephritis. Therefore, it is urgent to develop new treatments to replace or reduce the use of current ones. Histone deacetylase 6 (HDAC6) plays a variety of biologic functions in a number of important molecular pathways in diverse immune cells. Both innate and adaptive immune cells contribute to pathogenesis of lupus. Among those cells, B cells play a central role in pathogenesis of lupus nephritis in an anti-body dependent manner through differentiation into plasma cells (PCs). As a result, HDAC6 inhibitors represent an entirely new class of agents that could have potent effects in SLE. Importantly, the available toxicity profile suggests that HDAC6 inhibitors could be advanced into SLE safely. We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. ACY-738 is a hydroxamic acid HDAC6 inhibitor that is highly selective for HDAC6. In our current studies, we tested if an orally selective HDAC6 inhibitor, ACY-738, would decrease disease pathogenesis in a lupus mouse model with established early disease. Moreover, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. In order to define the mechanism by which HDAC6 inhibition decreases disease pathogenesis in NZB/W mice by using RNAseq to evaluate the transcriptomic signatures of splenocytes from treated and untreated mice coupled with applied computational cellular and pathway analysis. In addition, we sought to bridge between the transcriptomic data obtained from the HDAC6 treated mice and human gene expression information to determine the relevance to this target in possibly controlling human lupus. We treated 20-week-old (early-disease) NZB/W F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 4~5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by ELISA, and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells, and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. Our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. RNA sequence data showed that PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was in part due to inhibition of B cell development and response. RNA sequence data analysis show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggests that a critical event might be modulation of cellular metabolism. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease by which immune cells mistakenly attacks healthy self-cells in different organs. Kidney inflammation occurs in nearly 50% of patients with lupus resulting in kidney damage leading to end stage renal disease. Lupus nephritis (LN) is major cause of morbidity and mortality associated with SLE. Current treatments for LN consist primarily of immunosuppressants that block the immune response and leave the patients with unwanted side effects including an increased risk of infection. To circumvent the unwanted side effects, we explored a novel mechanism to target the immune response. My project was to determine whether histone deacetylase 6 (HDAC6) inhibition would suppress the autoimmune inflammatory response in lupus. We found that inhibition of HDAC6 was effective at attenuating early LN, probably by down-regulating innate immune response, which suppressed subsequent adaptive immune responses downstream. HDAC6 inhibition affected the innate immune response by inhibiting type I interferon production by plasmacytoid dendritic cells. HDAC6 inhibition affected the cell mediated immune response by decreasing T helper cell and B cell activation. To determine the mechanism by which HDAC6 inhibits immune cells activation, we used RNAseq to reveal HDAC6 inhibition on multiple signaling events associated with the induction of lupus disease. These results suggest that HDAC6 could be a potential therapeutic target in the early stage of LN.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13609号 / 論医博第2319号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 永井 純正 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.