A Thesis Presented in Part Fulfilment of the Requirements for the Degree of Master of Medicine in Pathology at the University of the Witwatersrand. November, 1963. / Modern pathology is forced to deal more and more with changes brought about by modern therapy on the one hand and by the abuse of drugs by the laity on the other ^ New diseases have been added, others altered. Skin allergies, collagen diseases, frightening foetal abnormalities due to thalidomide, peptic ulceration with cortison therapy are but a few of the end results of the abuse of the numerous pharmaceutical substances available with or without prescription to the public. To this list may be added the nephrotoxic effects of phena- cetin. / IT2018

No description available.

Estrogenic endocrine disrupting chemicals (EEDCs) are defined as chemicals that bind to estrogen receptors (ERs) and augment estrogenic functions, either through promoting or blocking estrogen receptor signaling. Recent reports highlight the growing concern surrounding environmental exposure to EEDCs and immune system modulation. A commonly prescribed EEDC, 17α-ethinyl estradiol, is a synthetic analog of 17β-estradiol (E2), and is also found in many environmental reservoirs of human and animal exposure. Little is known regarding the immunomodulatory effects of this EEDC. Autoimmune diseases, such as systemic lupus erythematosus (SLE), are characterized by a dysregulated immune system that has lost tolerance to self-antigens. The pathogenesis of SLE is still poorly understood. However, it is likely that genetics, epigenetics, hormones, and environmental factors, such as EEDC exposure, contribute to the pathogenesis and severity of SLE. The work presented in this dissertation focused on investigating the immunomodulatory effects of exogenous estrogens in mouse models of SLE. Chapter 1 describes an overview of environmental endocrine disruptors and autoimmune disease, with a particular emphasis on estrogens. Chapter 2 represents a review of the current and pertinent literature surrounding the contributions of sex differences, hormones, and EDCs to the induction of autoantibodies and development of autoimmunity, as well as the contributions of anti- microbial responses to SLE. We explored the contribution of dietary components to SLE disease severity. Mice fed a diet devoid of exogenous phytoestrogens developed significantly reduced glomerulonephritis and glomerular immune complex deposition compared to mice fed a diet containing soy isoflavones. Diet also influenced cytokine production and epigenetics of LPS-stimulated splenic leukocytes. We identified similar effects of E2 and EE implantation with regards to innate immunity, and distinct cellular subset, cytokine production profiles, gene expression, and epigenetic responses between E2 and EE treated NZB/WF1 mice. Oral exposure to a very low human relevant dose of EE promoted glomerulonephritis and augmented responses to viral and bacterial mimics in MRL/lpr mice. Overall, our findings suggest that chronic exposure to environmental EEDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals and have greatly expanded the current body of knowledge surrounding 17α-ethinyl estradiol. / Doctor of Philosophy / Chemicals that can disrupt the normal effects of hormones are termed endocrine disrupting chemicals (EDCs). Estrogenic EDCs promote or suppress the ability of estrogen receptors to carry out normal functions within the body. Normal immune system functions require a fine balance of inflammatory and anti-inflammatory cellular responses. This delicate balance is a prime target for dysregulation by EDC exposure. Autoimmune diseases, such as systemic lupus erythematosus, are characterized by a loss of immune tolerance to ones’ own cells and tissues. There is a lack of knowledge surrounding the immunomodulatory effects of a commonly prescribed EDC, 17α-ethinyl estradiol, especially as it pertains to autoimmune disease patients. The aim of this dissertation work is to investigate the immunomodulatory effects of exogenous EDC exposure in mouse models of SLE. We found that MRL/lpr mice fed a diet devoid of phytoestrogens had reduced kidney disease and immune-complex deposition and had augmented cytokine response and epigenetics in LPS-stimulated splenic leukocytes compared to mice fed a diet high in isoflavones. We next compared the immunomodulatory effects of chronic pharmacologic dose exposure to 17β-estradiol or EE, and found that while both estrogens have similar effects on innate immune cellular responses, EE has distinct effects on T cell population subsets, cytokine production, gene response and epigenetic alterations in female NZB/WF1 mice. Finally, chronic low-dose oral exposure to EE exacerbated clinical signs of kidney disease and suppressed the normal response of toll-like receptor 9 in MRL/lpr mice. Overall, we have found that chronic exposure to environmental estrogenic EDCs exacerbates lupus nephritis and alter an already dysregulated immune system in genetically susceptible individuals.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, characterized by the production of autoantibodies and immune-mediated injury to the kidney. Hyaluronan (HA) is a major component of the extracellular matrix, which is involved in immune-mediated renal injury. We have previously demonstrated that HA expression is increased in the glomerulus of patients with severe lupus nephritis, attributed in part to anti-dsDNA antibody-mediated induction of low molecular weight (LMW) HA and high molecular weight (HMW) HA synthesis in mesangial cells. The causal role of HA and its fragments in the pathogenesis of lupus nephritis has not been explored. In this project, two separate in vivo studies were undertaken to delineate the role of HA in disease progression in NZBWF1/J mice. In the first study, we determined the effect of 4-methylumbelliferone (MU), a specific inhibitor of HA synthesis, on renal function and histology in NZBWF1/J mice with active disease, with particular emphasis on inflammatory and fibrotic processes. In the second study, we investigated whether exogenous HA could exacerbate disease manifestations in pre-disease NZBWF1/J mice. The mechanisms underlying the effects of MU and exogenous LMW HA and HMW HA were investigated in mesangial cells isolated from NZBWF1/J mice. Female NZBWF1/J mice with established nephritis were randomized into 3 groups and treated with (1) PBS, (2) Arabic gum (Gum) or (3) MU (3g/kg/day) for 2, 4, 8 and 12 weeks. Treatment of mice with MU for 12 weeks reduced serum HA levels and abrogated intra-renal expression of HA compared to PBS and Gum treated mice. Inhibition of HA synthesis in the kidney resulted in decreased IgG and C3 deposition, reduced B cell, T cell and macrophage infiltration, matrix accumulation and TNF-, IL-6 and MCP-1 expression, which was associated with improved renal functions. To confirm that HA influenced pathogenesis of lupus nephritis, pre-disease NZBWF1/J mice were randomized to receive (1) PBS, (2) LMW HA or (3) HMW HA for periods up to 24 weeks. Administration of LMW HA and HMW HA into NZBWF1/J mice by tail-vein injection induced intra-glomerular deposition of IgG and C3, B cell infiltration, glomerular hypercellularity and tubular atrophy, which was accompanied by induction of MAPK signaling pathways, enhanced MCP-1 expression, and increased matrix deposition in the glomerular and tubular basement membranes. In vitro studies showed that exogenous IL-6, IL-1, TGF-1 and TNF- induced HA synthesis in murine mesangial cells (MMC), with over 80% secreted into the conditioned medium. This was accompanied by an increase in pro-inflammatory cytokine secretion and synthesis of fibronectin and laminin. Inhibition of HA synthesis with MU significantly decreased cytokine secretion and fibronectin synthesis. The ability of HA to induce inflammatory and fibrotic processes in mesangial cells was confirmed in separate studies in which MMC were incubated with exogenous LMW HA and HMW HA. In summary, these original findings provide evidence of a direct effect of HA on intra-renal inflammation and fibrosis in lupus nephritis. Approaches to inhibit HA synthesis may offer novel therapeutic strategies to delay disease progression. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

■Genetic diversity of feline morbillivirus isolated in Japan 著者最終稿版の公開のみ可能（The final version of Recordは不可）。出版社ウェブサイトへのリンクを以下の通り記載すること「The final version of record is available at http;//jgv.microbiologyresearch.org/」。■In vitro host range of feline morbillivirus 最終版はhttp://jsvetsci.jp/jvms/から入手可能である / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19581号 / 医博第4088号 / 新制||医||1013(附属図書館) / 32617 / 京都大学大学院医学研究科医学専攻 / (主査)教授 西渕 光昭, 教授 一山 智, 教授 木原 正博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lupus nephritis is characterized by the production of anti-dsDNA antibodies, deposition of immune complexes within the kidney parenchyma, proliferation of resident renal cells and induction of inflammatory and fibrotic processes. Approximately 70% of patients with lupus nephritis show immune aggregates along the tubular basement membrane, which is accompanied by an influx of infiltrating cells and increased intra-renal expression of IL-6. Much attention has focused on the inflammatory processes in the kidney during pathogenesis of lupus nephritis whereas mechanisms of fibrogenesis are less well characterized. Tubulo-interstitial injury is a key indicator of poor prognosis of renal function. Given that the tubulo-interstitium occupies over 80% of the kidney volume, injury to this compartment will have a major impact on renal function. There is evidence to show that proximal tubular epithelial cells (PTEC) undergo epithelial-to-mesenchymal transition (EMT) during pathological disorders and adopt a fibroblastic morphology with increased fibrogenic potential. We have previously demonstrated that anti-dsDNA antibodies bound directly to the surface of PTEC through cross-reactive proteins, which were subsequently internalized and translocated to the nucleus where they induced functional changes. Using a proteomic approach, this study identified the cross-reactive antigens that mediated anti-dsDNA antibody binding and intracellular localization in PTEC and the functional consequences thereafter, focusing on EMT and fibrogenic events. Human polyclonal anti-dsDNA antibodies isolated from patients with lupus nephritis bound to Ku70 in plasma membrane extracts isolated from PTEC, and to Ku70, Ku80 and major vault protein in cytosolic and nuclear fractions. Anti-dsDNA antibodies increased synthesis of Ku70, Ku80 and major vault protein in PTEC in a time-dependent manner. Expression of these proteins was localized to proximal tubules especially those undergoing atrophy, and staining was more prominent in renal biopsies from patients with lupus nephritis compared to non-lupus renal disease or control specimens. Binding of anti-dsDNA antibodies to PTEC increased phosphorylation of MAPK and PKC signaling pathways that was accompanied by a concomitant increase in IL-6, IL-8 and TGF-1 secretion and synthesis of β-catenin, fibroblast specific protein-1, fibronectin and laminin. Inhibition of MAPK and PKC signaling pathways with specific inhibitors revealed differential regulation of inflammatory and fibrotic processes by these signaling pathways. In this respect, increased ERK, p38 MAPK, JNK and PKC phosphorylation in PTEC following anti-dsDNA antibody stimulation enhanced IL-6, IL-8 and fibronectin synthesis, whereas increased ERK and JNK phosphorylation upregulated TGF-β1 secretion. Increased β-catenin synthesis was mediated through JNK and PKC phosphorylation. Taken together, our data suggest that PTEC contribute to the pathogenesis of renal inflammation and fibrosis in lupus nephritis. We hypothesize that anti-dsDNA antibodies bind to Ku70 on the plasma membrane of PTEC to mediate inflammation, cell activation and increased fibrogenesis. Although synthesis of EMT markers was increased in PTEC after anti-dsDNA antibody stimulation, transition to a fibroblastic morphology was not observed under our experimental setting suggesting that induction of the EMT cascade is an early event before phenotypic alterations. / published_or_final_version / Medicine / Master / Master of Philosophy

Thesis (doctoral)--Rijksuniversiteit te Groningen.

Abstract Type IV collagen is only found in basement membranes, where it is the major structural component, providing a framework for the binding of other basement membrane components and a substratum for cells. The type IV collagen molecule is triple-helical and composed of three a chains which exist as six distinct forms (α1 - α6). Abnormalities in this basement membrane collagen structure and function are connected to both inherited and acquired diseases. Alport syndrome is a hereditary kidney disease associated with extrarenal complications, such as sensorineural deafness and eye abnormalities. The disease is caused by mutations in the COL4A3, COL4A4 and COL4A5 genes, coding for the type IV collagen α3, α4 and α5 chain genes, respectively. About 85% of the Alport syndrome cases are X-linked dominant, caused by mutations in the COL4A5 gene. In order to develop a basis for automated mutation analysis of the COL4A5 gene, previously unknown intron sequences flanking exons 2 and 37 were determined. Intron sequences flanking the other 49 exons were expanded from 35 to 190, and additionally, two novel 9 bp exons (exons 41A and 41B) were characterized in the large intron 41. In addition to optimization of the PCR amplification and sequencing conditions for all 51 exons and exon flanking sequences, optimization for the 820 bp promoter region and for the two novel exons was performed as well. Mutations were found in 79 unrelated patients of the 107 studied. This gives a high mutation detection rate of almost 75% in comparison with 50%, at its best, in other extensive mutation analyses of the COL4A5 gene using SSCP analysis. None of the mutations involved the promoter region or exons 41A and 41B. Circulating antibodies against basement membrane components have been recognized in some autoimmune diseases. Goodpasture syndrome is a rare autoimmune disease characterized by progressive glomerulonephritis and pulmonary hemorrhage. The target of the antibodies in this disease has been shown to be the noncollagenous NC1 domain of type IV collagen α3 chain. For unknown reasons, a minority of Alport syndrome patients also develops antibodies against α3 and α5 chains after renal transplantation with manifestation of severe anti-GBM disease. In order to investigate the antibodies both in Goodpasture and Alport syndrome, the NC1 domains of all six type IV collagen chains were produced as recombinant proteins in bacterial and mammalian expression systems, and an ELISA method was developed for antibody detection. Antibodies were found in both syndromes, interestingly also in Alport syndrome patients without the anti-GBM disease. The results of this work have a significant clinical value by providing for the first time complete, effective DNA-based analysis of all exon/intron and promoter regions of the COL4A5 gene in Alport syndrome.