BACTERIAL CONTRIBUTIONS TO SYSTEMIC LUPUS PATHOGENESIS

系统性红斑狼疮(SLE)是一种自身免疫性关节炎。 病因不明。明确地域的系统性红斑狼疮发病率增加 地区研究表明，这种自身免疫性疾病可能有环境因素 基础。由于免疫系统在很大程度上是微生物的对手， 有人提出，自身免疫可能作为一种疾病状态而出现。 由微生物分子模仿免疫耐受的人类 组件。与系统性红斑狼疮相关的抗原成分，即Sm 与核糖核蛋白小颗粒相关的抗原 (SNRNPs)，是高度保守的细胞内分子 真核生物，但被认为不存在于原核生物中。 分子序列重合的证据表明 SM和原核组分缺失。然而，最近的证据表明 核糖核蛋白颗粒与人类SNRNPs高度同源 确实存在于两组真细菌中，尽管这两组都不存在 众所周知，细菌在人类中具有传染性。短链RNPs的存在 在感染性原核生物中可以提供一种抗原性刺激， 可能会导致易感人群的自身免疫力。为了测试这一点 假设，这项提议打算使用聚合酶链式反应 (聚合酶链式反应)以确定常见的感染性真细菌是否也 拥有与SNRNPs相关的基因。U1和U6SnRNA特异性 引物将与来自各种原核生物的基因组DNA反应 微生物努力发现SnRNA基因的范围 在真细菌中。DNA片段的Southern杂交 SNRNP阳性的真细菌也将被用来筛选 基因组DNA中的SnRNA基因。为了解决这个问题， 原核生物单链RNA代表了某种形式的自发分子模仿 或者它们是否在原核生物、RNA剪接等方面具有功能作用 将开发体外和体内的检测方法，使用带有 SV4O内含子以及真核和原核两种启动子位点。这个 质粒将在体外转录，并在人或 假定含有剪接成分的细菌提取物；核糖核酸 剪接反应的产物将通过反向扩增 转录酶-聚合酶链式反应(RT-PCR)，并在电泳法上进行可视化分离。 序列确定的内含子探针的Southern杂交 (未拼接)或已拼接外显子(已拼接)应验证是否存在任何拼接 活动。反义杂交法去除人U1SnRNA的提取液 将在不同数量的U1SnRNA提取液中孵育- 以阳性原核细胞判断是否存在“剪接” 原核生物成分与细胞之间可发生互补作用 真核剪接系统。此外，还将对 含有内含子的原核细胞将被用作 体内检测系统。从这些转化株中提取RNA 细菌和RT-PCR扩增可用于体内评估 拼接性能。最后，含有SnRNP的原核表达载体的能力 刺激SLE的细胞提取物将通过SLE免疫进行评估- 模型小鼠，MRL/LPR。自发发展成系统性红斑狼疮的MRL/LPR小鼠 在五个月龄左右，将接种不同数量的 含SnRNP的原核提取液及其发展的时间进程 抗Sm抗体的数量将通过与遗传的比较来确定 不易产生系统性红斑狼疮或抗Sm抗体的相似小鼠， 即MRL/+。老鼠。接种不含以下物质的细菌提取物 也可以对SNRNPs，即大肠杆菌进行评估。抗Sm遗嘱的制作 用ELISA法和免疫荧光法进行监测。这些措施的结果 实验应该提供一个更清楚的迹象：SNRNPs的范围 在原核细胞中；原核生物的SNRNPs是否是“偶然的” 分子模拟或功能剪接组件；以及 原核单核细胞核糖核酸核糖核酸酶的免疫与致病作用 SLE。

Systemic Lupus Erythematosus (SLE) is an autoimmune arthritic disease of unknown etiology. Increased incidence of SLE in defined geographic regions suggests that this autoimmune disease may have an environmental basis. As the immune system functions largely as a microbial adversary, it has been suggested that autoimmunity may arise as a disease state prompted by microbial molecular mimicry of immune-tolerable, human components. The antigenic components associated with SLE, namely the Sm antigen associated with small nuclear ribonucleoprotein particles (snRNPs), are highly conserved, intracellular molecules within eukaryotes, but are not thought to exist in prokaryotic organisms. Evidence for coincident molecular sequences suggesting mimicry between Sm and prokaryotic components is absent. However recent evidence suggests that ribonucleoprotein particles displaying high homology to human snRNPs do exist among two groups of eubacteria, although neither of these bacteria are known to be infectious in humans. The existence of snRNPs among infectious prokaryotes could provide an antigenic stimulus which might lead to autoimmunity in susceptible individuals. To test this hypothesis, this proposal intends to use the polymerase chain reaction (PCR) to determine whether commonly infectious eubacteriaI organisms also possess genes associated with snRNPs.. Both U1 and U6snRNA-specific primers will be reacted with genomic DNA from a variety of prokaryotic microorganisms in an effort to discover the extent of the snRNA genes among the eubacteria. Southern hybridization with PcR probes from the snRNP-positive eubacterial organisms will also be used to screen for snRNA genes in genomic DNA. To address the question of whether the prokaryotics snRNAs represent some form of spontaneous molecular mimicry or whether they have a functional role in prokaryotes, RNA splicing assays will be developed, both in vitro and in vivo, using a plasmid with an SV4O intron and both eukaryotic and prokaryotic promoter sites. The plasmid will be transcribed in vitro and incubated in either human or bacterial extracts presumed to contain the splicing components; the RNA products of the splicing reaction will be amplified by reverse transcriptase-PCR (RT-PCR) and visualized on electrophoretic separations. Southern hybridizations with sequence-defined probes for the intron (unspliced) or spliced exons (spliced) should verify any splicing activity. Human extracts depleted of U1snRNA by antisense hybridization will be incubated in varying quantities of extracts derived from U1snRNA- positive prokaryotic cells to determine whether "splicing complementation" can occur between prokaryotic components and the eukaryotic splicing system. In addition, transformation of the prokaryotic cells with the intron-containing plasmid will be used as the in vivo assay system. Extraction of the RNA from these transformed bacteria and amplification with RT-PCR should allow assessment of in vivo splicing performance. Finally, the ability of snRNP-containing prokayotic cell extracts to stimulate SLE will be assessed by immunization of SLE- model mice, MRL/lpr. The MRL/lpr mice, which spontaneously develop SLE around five months of age, will be inoculated with varying amounts of snRNP-containing prokaryotic extract, and the time course of development of anti-Sm antibodies will be determined in comparison to genetically similar mice which are not prone to develop SLE or anti-Sm antibodies, i.e. MRL/+. Mice. inoculated with extracts from bacteria not containing snRNPs, i.e. E. coli, may also be evaluated. Production of anti-Sm will be monitored by ELISA and immunofluorescence assays. The outcome of these experiments should provide a clearer indication of: the extent of snRNPs in prokaryotic cells; whether the prokaryotic snRNPs are "fortuitous" molecular mimics or functional splicing components; and whether inoculation with prokaryotic snRNPs contributes to the pathogenesis of SLE.