New Animal Model for Studies of mucosal Immunity and IgA Nephropathy

研究粘膜免疫和 IgA 肾病的新动物模型

基本信息

批准号：
8098980
负责人：
JAN NOVAK
金额：
$ 21.76万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8098980
关键词：
Acetylgalactosamine Animal Model Antibodies Antigen-Antibody Complex Binding Biochemical Blood Circulation Bone Marrow Carbohydrates Cell Line Cells Characteristics Clinical Complex Defect Deposition Development Diagnosis End stage renal failure Enzyme-Linked Immunosorbent Assay Enzymes Epitopes Exoglycosidases Exons Experimental Animal Model Exposure to Foundations Future Galactose Galactosyltransferases Gas Chromatography Gene Targeting Generations Genes Genetic screening method Glomerular Mesangial Cell Glomerulonephritis Hematuria Human IgA1 Immune Immunoglobulin A Immunoglobulin G In Vitro Injection of therapeutic agent Injury Kidney Kidney Diseases Knock-in Mouse Laboratories Link Modeling Molecular Mouse Strains Mucosal Immunity Mucous Membrane Mus Nude Mice Pathogenesis Patients Physiological Play Polysaccharides Positioning Attribute Preparation Primates Production Proteinuria Renal Tissue Research Design Role Serum Site Small Interfering RNA Splenocyte Structure of glomerular mesangium Study models Testing Transgenic Mice Transgenic Organisms Urine Variant Western Blotting Wild Type Mouse base embryonic stem cell glycosylated IgA glycosylation glycosyltransferase human disease in vivo inhibitor/antagonist interest lymph nodes molecular mass novel public health relevance research study tool

项目摘要

DESCRIPTION (provided by applicant): is characterized by mesangial IgA1 immune deposits that originate from circulating immune complexes containing aberrantly-glycosylated IgA1, i.e., IgA1 with galactose (Gal)-deficient O-glycans. Several lines of evidence suggest a direct causal relationship between aberrant glycosylation, the formation of immune complexes containing aberrantly glycosylated IgA1, their deposition in the mesangium, and renal injury in IgAN. We determined that IgA1-producing cells of IgAN patients secrete IgA1 with Gal-deficient O- glycans and that this aberrancy is due to dysregulation in expression/activity of specific glycosyltransferases. These findings indicate that Gal-deficient IgA1 plays a pivotal role in the pathogenesis of IgAN. An understanding of the molecular basis for the variations in the carbohydrate content of IgA1 in IgAN is essential for the definition of the fundamental defects that result in the synthesis of the aberrant glycans in IgAN. Such studies have been frustrated, however, by the fact that it is not feasible to obtain sufficient cells of interest from human mucosal tissues, lymph nodes, or bone marrow and by the lack of animal models, as IgA1 is present exclusively in humans and hominoid primates. As a first step toward developing a murine model, we have now shown that immune complexes, prepared in vitro between human Gal-deficient IgA1 and anti-glycan IgG and i.v.-injected to nude mice, deposit in the mesangium and induce hematuria and proteinuria. Because mice do not have IgA with hinge-region O-glycans, we hypothesized that generating transgenic mice with IgA containing the human hinge region would result in murine IgA containing O-linked glycans that can be manipulated to resemble the aberrant human IgA1. Such a transgenic mouse strain would represent a new tool for studies of the role of the O-glycans in IgAN and in mucosal immunity. We propose to generate a knock-in transgenic mouse strain producing IgA with hinge region from human IgA1, assess its O-glycosylation, and, by inhibiting the key enzyme - 21,3-galactosyltransferase, to generate Gal-deficient O-glycans on the murine transgenic IgA. Furthermore, we will determine whether the immune complexes composed of murine transgenic Gal-deficient IgA and glycan-specific IgG deposit in renal mesangium of mice causing nephropathy. We will further develop and validate this first animal model truly reflecting the human disease by performing histological analysis of renal tissue and laboratory analysis of urine and serum. This transgenic mouse will open new possibilities for testing genetic and biochemical mechanisms involved in production of aberrantly O- glycosylated IgA and its mesangial deposition and clearance. Relevance: IgAN is the most common primary glomerulonephritis and leads to end-stage renal failure in 20% to 40% of patients. The current gaps in the understanding of the pathogenesis of IgAN represent a major barrier to the development of IgAN-specific treatments. The proposed studies will provide the foundation for the future development of a relevant physiological animal model of IgAN that will advance the understanding of the pathogenesis of human IgAN. PUBLIC HEALTH RELEVANCE: We propose to develop a murine model of IgAN. We will generate transgenic mice in which IgA will include the human hinge region containing O-linked glycans. We will further generate Gal-deficient O-glycans on the murine transgenic IgA and use anti-glycan IgG to form pathogenic complexes that will deposit in the kidneys.

描述（由申请人提供）：特征在于肾小球IgA1免疫沉积物，这些沉积物来自循环中含有异常糖基化IgA1的免疫复合物，即带有半乳糖（GAL）缺陷的O-糖质的IgA1。几条证据表明，异常糖基化，含有异常的糖基化IgA1的免疫复合物的形成之间存在直接因果关系，它们在中膜中的沉积和Igan的肾损伤。我们确定IGA患者产生的IGA1细胞用gal缺乏糖糖分泌Iga1，并且这种像差是由于特定糖基转移酶表达/活性失调引起的。这些发现表明，缺乏GAL IGA1在Igan发病机理中起关键作用。对IgA中IgA1碳水化合物含量变化的分子基础的理解对于定义导致Igan中异常聚糖的基本缺陷定义至关重要。然而，由于从人类和同型灵长类中，IGA1独家存在，因此从人类粘膜组织，淋巴结或骨髓缺乏感兴趣的细胞并缺乏动物模型是不可行的。作为开发鼠模型的第一步，我们现在表明，在人体缺乏的IgA1和抗糖基IgG和注射到裸鼠的静脉内，在体外制备的免疫复合物在体外制备，沉积在米松中并诱导血尿和蛋白尿。由于小鼠没有带有铰链区域O-聚糖的IgA，因此我们假设用含有人铰链区域的IgA产生转基因小鼠会导致含有鼠IgA的含有O连接的Glycans，这些聚糖可以被操纵以类似于异常的人IGA1。这种转基因小鼠菌株将代表研究O-聚糖在Igan和粘膜免疫中的作用的新工具。我们建议从人IGA1产生一种用铰链区域产生IgA的敲入转基因小鼠菌株，评估其O-糖基化，并通过抑制键酶-21,3-半乳糖基转移酶，从而在鼠类Transgenic IGA上产生gal缺乏的O-Glycans。此外，我们将确定是否由鼠类转基因gal缺乏的IgA和聚糖特异性IgG沉积物组成的免疫复合物中是否导致肾病的小鼠肾小肌中。我们将通过对肾脏组织和尿液和血清实验室分析进行组织学分析来进一步发展和验证真正反映人类疾病的第一个动物模型。这种转基因小鼠将开辟新的可能性，以测试与异常O-糖基化IgA产生的遗传和生化机制及其肾小球沉积和清除率。相关性：伊甘（Igan）是最常见的原发性肾小球肾炎，导致20％至40％的患者导致终末期肾衰竭。当前对Igan发病机理的理解的差距代表了Igan特异性治疗发展的主要障碍。拟议的研究将为Igan相关生理动物模型的未来发展提供基础，该模型将提高人们对人Igan发病机理的理解。公共卫生相关性：我们建议开发一种igan的鼠模型。我们将生成转基因小鼠，其中IgA将包括包含O连锁聚糖的人铰链区域。我们将进一步在鼠类转基因IgA上产生缺乏的gal o-glycans，并使用抗甘油can IgG形成将沉积在肾脏中的致病复合物。