Etiology and Pathogenesis of Pemphigus

描述(由申请者提供)：这笔赠款旨在发现巴西农村地区地方性叶状天疱疮(PF)的病原学，也被称为Fogo Selvan em(FS)。这种表皮特异性自身免疫性疾病是由致病的IgG4抗桥粒芯糖蛋白1(DSG1)自身抗体介导的。1994年，我们确定了巴西利毛维德(LV)的美洲印第安人保护区(LV)，那里有大约1,400人，FS的患病率约为3%。重要的是，在过去的18年里，我们对这一人群进行了临床和血清学跟踪，确定了12名个体从正常到FS的转变，并报告了暴露于供血节肢动物是FS的潜在危险因素。此外，利什曼病、恰加斯病和盘尾丝虫病患者的血清中含有抗DSG1抗体，这些昆虫是寄生虫的携带者。最近，我们发现抗DSG1的IgG4自身抗体与LJM11“黄色”蛋白的构象表位发生交叉反应，LJM11“黄色”蛋白的构象表位来自于利什曼病的媒介Lutzmyia long gipalpis。本研究的目的是确定DSG1如何成为FS免疫系统的靶标，并确定启动抗自身反应的环境抗原(S)。在以往研究的基础上，我们假设嗜血性昆虫的唾液抗原是FS致敏抗原的来源。这笔赠款的目的是确定DSG1和LJM11上与FS患者的IgG4抗体发生交叉反应的表位，并测试患者血清中存在的这些抗体是否是来自与外来或自身抗原结合的天然B细胞，或者两者兼而有之。在目标1中，我们将从FS患者的血清和B细胞中确定抗DSG1的多克隆抗体与DSG1和LJM11的构象表位。此外，我们将通过被动转移实验来测试结合人DGS1的小鼠抗LJM11抗体是否具有致病性。在目标2中，我们将通过噬菌体展示的方法从FS B细胞中制备IgG4单抗(ScFv)，并通过淘汰法筛选DSG1和LJM11特异性克隆。这些单链抗体将进行致病性测试，并用于更精确的表位定位。此外，将产生“回复”的单抗，并测试其与DSG1和LJM11的反应性，以努力揭示FS患者IgG4抗体反应的初始触发因素。目的3和4涉及在三个队列和利毛维德人口中进行的血清流行病学研究。我们将继续寻找可能诱发正常人的免疫球蛋白G和FS患者的免疫球蛋白G4的危险因素。从正常状态到疾病状态的转变可能会在队列的后续研究中发现。随着这些新研究的完成，我们将更好地了解环境抗原如何在某些遗传易感个体中触发导致皮肤病的自身免疫IgG4反应。同时，新的表位特异性免疫分析和潜在的新的治疗干预措施可能从这些研究中进化出来。

DESCRIPTION (provided by applicant): This grant is aimed at discovering the etiology of endemic pemphigus foliaceus (PF), also known as Fogo Selvagem (FS) in rural Brazil. This epidermal-specific autoimmune disease is mediated by pathogenic IgG4 anti-desmoglein 1 (Dsg1) autoantibodies. In 1994, we identified the Amerindian Reservation of Limao Verde (LV), Brazil composed of ~1,400 individuals where the prevalence of FS was ~3%. Importantly, we have followed this population clinically and serologically for the last 18 years, identifying transitions from normal to FS in 12 individuals, and reported that exposure to blood feeding arthropods is a potential risk factor for FS. Moreover, the sera of patients with leishmaniasis, Chagas disease and onchocerciasis, where these insects are involved as carriers of parasites, possess anti-Dsg1 antibodies. Recently, we have shown that IgG4 anti-Dsg1 autoantibodies cross-react with conformational epitopes of the LJM11 "yellow" protein from the saliva of Lutzomyia longipalpis, the vector of leishmaniasis. The objective of this proposal is to determine how Dsg1 becomes the target of the immune system in FS and to identify the environmental antigen(s) that initiate the anti-self response. Based on previous studies, we hypothesize that salivary antigens from hematophagous insects are the source of sensitizing antigen in FS. The aims of this grant will identify epitopes on Dsg1 and LJM11 that cross-react with IgG4 antibodies from FS patients and test whether these antibodies present in patient sera derive from na¿ve B cells that bind foreign or self-antigens or both. In Aim 1 we will determine the conformational epitopes bound by IgG4 anti-Dsg1 polyclonal antibodies from sera and B cells of FS patients on Dsg1(self) and LJM11(sand fly) antigens. Moreover, we shall test if murine anti-LJM11 antibodies, that bind human Dgs1, are pathogenic by passive transfer experiments. In Aim 2 we shall generate IgG4 monoclonal antibodies (scFv) from FS B cells by phage display methods and select Dsg1 and LJM11 specific clones by panning procedures. These scFv antibodies will be tested for pathogenicity and used for more precise epitope mapping. Further, "revertant" monoclonal antibodies will be generated and tested for reactivity with Dsg1 and LJM11 in an effort to disclose the initial trigger of the IgG4 antibody response in FS. Aim 3 and 4 are concerned with seroepidemiological studies in three cohorts and the population of Limao Verde. We shall continue searching for risk factors that may precipitate the IgG autoimmune response in normal subjects and the IgG4 in FS patients. The transition from normal to disease state will likely be uncovered on the follow up studies of the cohorts. At the completion of these novel studies we shall have a better understanding of how environmental antigens might trigger, in certain genetically predisposed individuals, an autoimmune IgG4 response that leads to skin diseases. In parallel, new epitope-specific immunoassays and potential novel therapeutic interventions may evolve from these studies.