Introduction: The two subtypes of Antineutrophil Cytoplasmic autoantibody associated systemic vasculitis (AASV) cANCA and pANCA associated vasculitis are the commonest causes of rapidly progressive glomerulonephritis. In spite of recent advances in the pathogenesis and development of new therapeutic agents, long term outcomes are still poor with five year mortality of 25%. There are epidemiological, histological, clinical and outcome related differences between these two conditions. This strongly suggests that there must be differences between genetic factors and pathogenesis of these two conditions. There was also a perception amongst the clinicians that AASV is more common in the Greater Manchester area. Hence in this study I calculated the incidence of pauciimmune glomerulonephritis in Greater Manchester and analysed the genetic differences between cANCA and pANCA associated vasculitis. Methods: Five year incidence of pauciimmune glomerulonephritis was calculated in Greater Manchester between 1/1/1999 to 31/12/2003. I recruited 147 patients with ANCA associated vasculitis. Clinical data was collected. I studied single nucleotide polymorphisms (SNPs) of tumour necrosis factor alpha(TNFα), interleukin 8 (IL-8), transforming growth factor beta (TGFβ), platelet endothelial cell adhesion molecule 1 (PECAM-1), Chemokine (CC motif) ligand-5 C chemokine (CCL-5), interleukin 10 (IL-10) and interleukin 18 (IL-18) genes and compared the frequencies of genotypes and alleles in patients with cANCA and pANCA associated small vessel vasculitis and healthy volunteers. I also studied circulating cytokine profiles of IL-10 and IL-18. Results of IL-18 SNPs were validated in AASV cohort from South-East USA. Further I studied the gene expression patterns of active and remission state of AASV and metabolomics profile of cANCA and pANCA positive patients during active and remission state of vasculitis. Clinical outcomes (relapses and renal survival) were correlated with the genotypes. Results: I found a significantly higher incidence (9.8/million population) of pauciimmune glomerulonephritis in Greater Manchester compared to the previously published data from UK and USA (2.73 to 4.6/million). Renal function at the time of diagnosis predicted the long term renal survival. I also found a novel genetic association of increased frequency of high producer IL-18 SNPs 113T, 127C and 137G in pANCA positive patients compared to normal volunteers (p=0.04) and cANCA positive patients (trend- p=0.08). This was associated with increased levels of circulating IL-18 levels in these patients. This association was further confirmed in an independent cohort of AASV from USA. I also found a lower frequency of low producer GG genotype of IL-10 -1082 SNP (p=0.05) and this was associated with lower levels of circulating IL-10 in these patients compared to pANCA positive patients. I found significant difference in the metabolomics profiles of cANCA andpANCA positive patients. In paired plasma samples, levels of some metabolites were high during remission state compared to active vasculitis. Conclusions: These findings strongly support the hypothesis that there is an increased incidence of pauciimmune glomerulonephritis in Greater Manchester. There are genetic differences in cANCA and pANCA positive patients which may explain the different observed outcomes. Genome wide association study would strengthen these findings and should guide the vasculitis community to reclassify, assess and perhaps treat these two conditions separately.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13266号 / 論医博第2180号 / 新制||医||1038(附属図書館) / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1) resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune response to Salmonella infection, thereby bringing about varied outcomes in the infected host.

Signal propagation across the membrane by cytokine receptor signalling involves a complex interplay of receptor-ligand interactions, allostery and conformational changes. Type I interferons (IFNs) exert their biological activities through binding to a shared receptor consisting of the type II cytokine receptor subunits IFNAR1 and IFNAR2. The aim of this thesis was to establish biochemical and biophysical approaches for exploring in vitro interactions and conformational changes accompanying the formation of type I IFN receptor complex. For these purposes, in this work a versatile combination of covalent vs. non-covalent reversible site-specific protein modification chemistries was exploited for their surface immobilization, incorporation of fluorescence probes or crosslinkers. The generic bioorthogonal strategy of PPTase-catalysed covalent modification of ybbR short peptide tag fused to a protein of interest enabled highly efficient site-directed fluorescence labelling of wild type IFNα2 and mutants, IFNAR1 and IFNAR2 receptors as well as their functional immobilization onto surfaces. These modified proteins were confirmed to be active by studying their interactions in ligand-receptor surface binding assays in real time by total internal reflection fluorescence spectroscopy and reflectance interference. A rapid quantitative surface assay for probing binding kinetics of proteins captured directly via His-tags from cell supernatants was established and employed for screening of IFNAR1 mutants in order to identify the residues responsible for differential recognition of various IFN subtypes. Thus the fine-tuned IFN binding chemistries through few ligand-specific interaction points as the basis for receptor plasticity was identified. Site-specific covalent immobilization allowed exploring cooperativity in ligand recognition by the receptor subunits. The observed small allosteric effect is apparently not related to the potential contact of membrane-proximal receptor domains but probably mediated through conformational cross-communication of binding sites on the ligand. Substantial conformational changes of IFNAR1 upon ligand binding were exploited as fluorescence readout to monitor the assembly of ternary complexes on artificial membranes. This enabled exploring the life times of ternary complexes with IFNα2 combined mutants targeting binding to IFNAR1 and IFNAR2 and corroborated to the suggestion that the differential physiological activity of IFN subtypes is related to the total ternary complex affinity and not to ligand affinity towards individual receptor subunits. Finally, in vitro stabilization of dual-colour labelled weakly interacting IFNα2/IFNAR1/IFNAR2 complex by means of an entropic clamp was implemented, enabling to analyze ternary complexes by fluorescence cross-correlation spectroscopy Förster resonance energy transfer on the single molecule level. These novel tools will prove valuable for unravelling the subtle interplay of interactions and conformational changes in cytokine receptor complexes.

The pathophysiological mechanisms of pain in small fiber neuropathy (SFN) are unclear. Based on experimental and clinical studies, sensitized nociceptors in the skin are reported to be involved in pain development. These nociceptors may be sensitized by cutaneous and systemic pain mediators e.g. pro- and anti-inflammatory cytokines. The aim of our study was, to measure the systemic and local gene expression of pro- and anti-inflammatory cytokines in white blood cells (WBC) as well as in primary fibroblasts and keratinocytes obtained from human skin of patients with SFN. Furthermore, gene expression levels of axon guidance molecules and their receptors, as potential regulators of the intraepidermal nerve fiber density (IENFD), were investigated. 55 patients and 31 healthy controls were prospectively recruited. Participants underwent extensive clinical phenotyping and blood sampling, 6-mm skin punch biopsies were taken from the right lateral calf and the upper thigh. Systemic relative gene expression levels (ΔG) of the interleukin (IL)-1β, IL-2, IL-6, IL-8, and tumor necrosis factor (TNF) was measured in WBC. Skin punch biopsies were taken to determine the IENFD and to obtain primary fibroblast and keratinocyte cell cultures. Skin cells were then used for investigation of ΔG in axon guidance molecules netrin 1 (NTN1) and ephrin A4 (EPHA4) as well as their receptors Unc5b receptor, and ephrin A4 (EFNA4) as well as cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, TNF, and transforming growth factor (TGF). Systemically, gene expression of IL-2, IL-8, and TNF was higher in SFN patients compared to healthy controls. In keratinocytes, higher expression levels of NTN1 and TGF were found when comparing the SFN patients to the controls. In fibroblasts higher gene expression was shown in NTN1, Unc5b, IL-6, and IL-8 when comparing patients to healthy controls. The systemically and local elevated levels of pro-inflammatory, algesic cytokines in SFN patients compared to healthy controls, confirms a potential pathophysiological role in the development of neuropathic pain. Data also indicate fibroblasts and keratinocytes to influence subepidermal and intraepidermal nerve fiber growth through the expression of NTN1 and Unc5b. Thus, skin cells may contribute to the development of neuropathic pain through local denervation. / Der Pathomechanismus von Schmerz bei Small fiber Neuropathie (SFN) ist unklar. Auf Grundlage tierexperimenteller und klinischer Studien wird die Einwirkung kutaner und systemischer Schmerzmediatoren auf sensibilisierte Nozizeptoren in der Haut als mögliche Ursache diskutiert. In diesem Zusammenhang gab es Hinweise auf die Bedeutung von pro- und anti-inflammatorischen Zytokinen in der Pathophysiologie neuropathischer Schmerzen. Ziel der Studie war es, die systemische und lokale Genexpression pro- und anti-inflammatorischer Zytokine in Leukozyten sowie kutanen Fibroblasten und Keratinozyten von Patienten mit SFN zu messen. Ferner wurde untersucht, inwieweit die Expression repellierender Axon Guidance Moleküle und ihrer Rezeptoren in Hautzellen die intraepidermale Nervenfaserdichte (IENFD) regulieren könnte. Insgesamt konnten 55 SFN PatientInnen und 31 gesunde KontrollprobandInnen prospektiv rekrutiert werden. Nach ausführlicher klinischer Phänotypisierung und Blutentnahme wurden bei allen StudienteilnehmerInnen 6-mm Hautstanzbiopsien am lateralen Unter- und Oberschenkel entnommen. Die Messung der systemisch relativen Genexpression (ΔG) der Zytokine Interleukin (IL)-1β, IL-2, IL-6, IL-8 und des tumor necrose factors (TNF) erfolgte aus Leukozyten. Aus den Hautstanzbiopsien, die u.a. zur Bestimmung der IENFD verwendet wurden, wurden außerdem Primärzellkulturen von Keratinozyten und Fibroblasten angelegt, aus denen die lokale ΔG von Axon Guidance Molekülen Netrin 1 (NTN1) und Ephrin A4 (EPHA4), deren Rezeptoren Unc5b, und Ephrin A4 receptor (EFNA4) sowie der Zytokine IL-1β, IL-4, IL-6, IL-8, IL-10, TNF und des transforming growth factors (TGF) erfolgte. Systemisch zeigte sich eine höhere Genexpression für IL-2, IL-8 und TNF bei SFN Patienten im Vergleich zu gesunden Kontrollen. In Keratinozyten konnten höhere Expressionen von NTN1 und TGF-β1 bei Vergleich der Patientengruppe mit der Kontrollgruppe nachgewiesen werden. In Fibroblasten zeigte sich im Gruppenvergleich eine höhere Genexpression für NTN1, Unc5b sowie für IL-6 und IL-8. Die systemisch und lokal bei SFN Patienten nachgewiesene höhere Expression algetischer, pro-inflammatorischer Zytokine verglichen mit Kontrollen unterstützt eine mögliche pathophysiologische Rolle bei der Entstehung von neuropathischen Schmerzen. Ferner weisen die Daten darauf hin, dass Fibroblasten und Keratinozyten durch die Expression von NTN1 und Unc5b Einfluss auf das subepidermale und intraepidermale Nervenfaserwachstum nehmen und durch lokale Denervierung bei der Entstehung neuropathischer Schmerzen mitwirken könnten.

Bluetongue virus (BTV) is a pathogenic member of the Reoviridae family. BTV does not cause disease in humans, but is capable of selectively infecting and killing certain transformed human cell lines. Understanding BTV's oncotrophism may lead to new therapeutics for treating cancer. This study focused on the underlying mechanisms of BTV-induced cell death in carcinoma cell lines. It was our hypothesis that BTV infects human carcinoma transformed cells, produces mRNA and protein, induces a strong inflammatory response, induces mitogen activated protein kinase (MAPK)-based pro-apoptotic signaling, inhibits PKB-based signaling, and eventually kills the cell by inducing apoptosis. Three carcinoma cell lines (A498, HEP-G2, and A549) were independently infected with BTV. In each cell line we determined: (1) cell viability over the course of infection; (2) BTV induced cytokine expression profile and magnitude of expression; (3) BTV viral RNA expression profile and magnitude of expression; (4) BTV viral protein expression profile and magnitude of expression; (5) changes in BTV induced cell death and cytokine expression in cells with protein kinase B (PKB), p38-MAPK, extracellular receptor kinase (ERK-1/2), stress-activated protein kinase (SAPK-JNK), Src kinase, platelet-derived growth factor receptor (PDGFR) kinase, epidermal growth factor receptor (EDGFR) kinase, or Janus kinase (JAK) activity inhibited; (6) intracellular changes in PKB, p38-MAPK, ERK-1/2, and SAPK-JNK phosphorylation as a result of BTV infection; and (7) BTV-induced changes in tyrosine phosphorylation. We determined that BTV infects and kills all three cell lines in a cell line dependent manner. Relative cell death between cell lines was proportional to cytokine expression, but inversely proportional to viral protein expression. Only tyrosine kinase inhibitors influenced BTV-induced cell death and cytokine expression. Both A498 and A549 cells constitutively expressed phosphorylated PKB and p38 MAPK, of which both were de-phosphorylated during BTV infection. Tyrosine phosphorylation remained active, with elevated tyrosine phosphorylation exclusively in infected cells. We conclude that BTV-induced cell death and cytokine expression are a function of the cell's response to infection and are directly related through intracellular signaling. These pathways are only partially poly I:C inducible, but include PKB and tyrosine kinase signaling.

Indiana University-Purdue University Indianapolis (IUPUI) / Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or tolllike receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18901号 / 医科博第57号 / 新制||医科||4(附属図書館) / 31852 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長澤 丘司, 教授 長田 重一, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bovine embryos produced in vitro consistently display decreased quality in terms of their potential to reach the blastocyst stage as well as post-transfer survival. Media formulations, oocyte quality, and inferior expression of needed transcripts are all causes of this reduced developmental potential commonly present in in vitro-produced (IVP) bovine embryos. Recently our lab has confirmed interleukin-6 as an embryokine whose capabilities include increasing inner cell mass (ICM) numbers and promoting bovine blastocyst development. LIF is another family member of the IL6 cytokine family and has been shown to produce several positive effects when supplemented during oocyte in vitro maturation. IL6 has predominantly been studied as being supplemented post-fertilization. However, published transcriptomic work described receptors for IL6, IL11, and LIF as present in cumulus cells at the time COCs are removed from their follicles. Consequently, we wanted to investigate if supplementing 25 ng/ml of IL6, IL11, or LIF would improve IVM bovine oocyte competency. Several experiments were completed (4replicates/experiment; 30-60 cumulus-oocyte complexes (COCs)/replicate). In Experiment 1, COCs were in vitro matured for 16 or 22 hours, then meiotic stage was assessed after denuding, fixation, and DNA staining. No cytokine treatment influenced the percentage of oocytes that achieved metaphase II at either time point. In Experiment 2, COCs were in vitro matured for 4 hours before snap freezing. and processing to examine changes in five cumulus-expressing transcripts associated with oocyte competency (CX43, CX37, AREG, TNFAIP6, HAS2). Our chosen housekeeping gene, HPRT1, served as the internal control. An increased abundance of AREG occurred following exposure to LIF but not with the other treatments. Supplementation with IL6 and IL11 but not LIF tended to increase TNFAIP6 abundance (P<0.10). No other transcript differences were detected. In Experiment 3, we examined whether supplementing these cytokines during IVM affects subsequent fertilization and blastocyst rates. No effects were detected on cleavage rates but at day 8 increases in blastocyst yield were detected for LIF and IL11, but not IL6. LIF showed a tendency to increase hatching rates. In Experiment 4, we aimed to assess how the cytokine treatments affected cryosurvivability. Blastocysts (5-10/replicate, 9 replicate studies) were frozen at a rate of -0.6 degrees C/min until reaching -32 degrees C, then were stored in liquid nitrogen for 4-8 weeks before being thawed and incubated in conventional embryo culture medium (SOF-BE1) for 3 days. No treatment effects were noted for re-expansion, hatching, and overall survivability. In summary, these results implicate IL11 and LIF as potential mediators of oocyte competency. However, the evidence presented here suggest that IL6 and IL11 may function differently than LIF when provided during COC maturation. / Master of Science / The numerous similarities in the regulation of early embryonic development between human and cow has made bovine embryos an excellent model for exploring how to optimize assisted reproductive techniques (ARTs) and other methods for improving and preserving fertility in humans. Pregnancy loss is also very similar in both cattle and humans. In beef cattle, more than 50 percent of reproductive failures occur before day 16 of gestation. In women, approximately 15 percent of all clinically recognized pregnancies result in spontaneous loss, however, several more pregnancies fail prior to ever being clinically recognized. Various ARTs are used to treat sub-fertile conditions in cattle, and these technologies are generally deemed as a viable way to improve fertility. However, IVP embryos are inferior in their ability to properly fertilize and develop to the blastocyst stage, the stage when embryos are normally transferred. Furthermore, IVP-generated embryos are inferior at maintaining pregnancies. There are two primary restraints to the IVP process: a low percentage of oocytes that become fertilized and produce transferable embryos and transferred IVP embryos have decreased chances of maintaining a viable embryo than embryos produced in vivo. Very little is known about the various hormone and molecular factors that promote oocyte and embryo development. Therefore, a primary objective for bovine oocyte and embryo research is to classify these factors and implement them into their maturation and culture media to improve overall IVP efficiency. My lab studies members of the IL6 cytokine family as potential factors that might play a role in the development of oocytes and embryos. The aim of this work is to assess the capacity of three molecules within this family, IL6, IL11, and Leukemia inhibitory factor (LIF) to improve oocyte development, fertilization rate, and blastocyst yield when supplemented during in vitro maturation (IVM). This work revealed that both IL11 and LIF improved IVP bovine blastocyst development at day 8. Unfortunately, none of the treatments had any effect on fertilization rates. LIF increased the expression of a cumulus-specific transcript known to aid in cumulus expansion. Cumulus cells are the somatic cells immediately surrounding the oocyte. Cumulus expansion is a key indicator of proper oocyte maturation. We did not observe any treatment effect on post-thaw survival of cryopreserved bovine embryos. This indicates that our treatments did not help the embryos maintain viability after undergoing a slow-freeze cryopreservation protocol followed by thawing and culture. In summary, this work showed that IL11 and LIF have potential benefits to the in vitro production of bovine embryos when supplemented at IVM. However, future work is needed to assess how these molecules are causing these improvements. Our results indicate that IL11 and LIF may function differently from IL6 when supplemented during IVM.

No description available.