Coxsackievirus B4-Induced Development of Antibodies to 64,000-Mr Islet Autoantigen and Hyperglycemia in Mice

Diabetogenic Coxsackievirus B4 infection produces a diabetes syndrome in susceptible mice resembling insulin-dependent diabetes mellitus. We reported a two- to threefold increased expression of a 64,000-M_r (64 K) islet autoantigen in the infected mice preceding the development of hyperglycemia, suggesting a possible role of the virus in autoimmunity. To assess if the virus could be a trigger of autoimmunity, 64 K autoantibody development was correlated with hyperglycemia and virus replication in islets during early and late infection. Additionally, the effects of blood removal from these mice on the incidence of hyperglycemia and antibody production were evaluated. Noninfected control mice were essentially 64 K antibody negative, the infected consistently positive. Approximately 30% of the animals developed antibodies by 72 h postinfection (pi.) and 90% by 4-6 wk p.i. Virus-induced hyperglycemia appeared bimodal: hyperglycemia in 50% of the mice by 1 wk p.i. which decreased to 30% by 3 wk and then increased to 80-100% by 6 wk p.i. Infectious virus was abundant in the islets at 72 h but undetectable later. Hyperglycemia seen at 6 wk decreased dramatically (67-73%) if the mice were bled once between 72 h and 2 wk p.i. Only 50-60% of the mice bled once were 64 K positive compared to 90% positive nonblood mice. Coxsackievirus may initiate or enhance the autoimmune response.     

Alpha-fodrin as a putative autoantigen in Graves' ophthalmopathy

Alpha-fodrin, an intracellular organ-specific cytoskeleton protein is a recently identified autoantigen associated with Sicca- and Sjogren's syndrome (SS). SS frequently affects patients with Graves' ophthalmopathy (GO). We have therefore cloned and expressed the human recombinant 120-kDa fodrin-fragment. A sequential purification procedure was applied to isolate the recombinant protein. Using sera from patients with SS, the antigenicity of the purified fodrin fragment was demonstrated by immunoblotting. Sera from 144 patients with GO and 1200 blood donors were screened for the presence of anti-alpha-fodrin IgA and IgG antibodies by a newly developed ELISA using the human alpha-fodrin fragment as an autoantigen. In contrast to controls (<1% IgA only, P < 0.001) and to subjects with various autoimmune diseases (P < 0.001), alpha-fodrin antibodies were detected in 22% of patients with GO (n = 32). IgA and IgG antibodies were present in 21 (15%) and 14 (10%) GO subjects, respectively. A total of 45 patients with GO (31%) had at least one fodrin- or SS-antibody. GO patients with SS showed SS- and high titres of alpha-fodrin-antibodies. In GO patients, fodrin antibodies correlated with TPO- (P < 0.05) and SS-A (P = 0.002) antibodies. Thus, for the first time, antibodies reactive with fodrin are reported in patients with GO.     

Limkain b1, a novel human autoantigen localized to a subset of ABCD3 and PXF marked peroxisomes

Detection of self-reactive antibodies has an established role in the diagnosis and monitoring of many human autoimmune diseases. Autoantibodies with restricted reactivity to cytoplasmic compartments and structures are an occasional incidental finding following routine examination of serum for antinuclear antibody reactivity. A prerequisite for rational exploitation of self-reactive antibodies, in either clinical or research settings, is the establishment of the molecular identity of the target autoantigen(s). Here we report on the identification of a novel autoantigen that co-localizes with a subset of cytoplasmic microbodies marked by ABCD3 (PMP-70) and/or PXF (PEX19). Immunoscreening a HeLa cell cDNA expression library with a human autoimmune serum identified two clones that encode fragments of limkain b1 (LKAP). We demonstrate that mouse polyclonal antibodies raised against a bacterially expressed fragment of limkain b1 mark the same cytoplasmic structures as human serum, as does an EGFP:LKAPCT429 fusion protein expressed in HeLa cells. An immunoblot screen against a bacterially expressed MBP:LKAPCT429 fusion protein substrate, using a cohort of 16 additional human sera that display Hep 2 cell cytoplasmic staining patterns similar to the prototype serum, identified three additional sera reactive to limkain b1. This is the first report establishing the molecular identity of a peroxisomal autoantigen. Preliminary results suggest that limkain b1 may be a relatively common target of human autoantibodies reactive to cytoplasmic vesicle-like structures.     

体外培养人胰岛素瘤细胞的特性

目的分离培养人胰岛素瘤细胞,并研究它的功能特性。方法原代分离人胰岛素瘤细胞,经多次筛选培养进行纯化。通过荧光显微镜观察细胞特有的自发荧光;RT-PCR方法检测胰岛素mRNA的表达和胰岛细胞自身抗原IA-2、IAR和ICAp69的表达;应用MTT方法检测抗人IA-2抗体对胰岛素瘤细胞的杀伤。结果分离的人胰岛素瘤细胞经传40代,在悬浮培养条件下可自发聚集成胰岛样结构,并有自发荧光。该细胞表达胰岛素mRNA并合成胰岛素,但明显低于大鼠胰岛素瘤细胞INS-1。该细胞也表达人胰岛细胞自身抗原IA-2、IAR和ICAp69,并且用抗人IA-2抗体与之孵育后可出现抗体剂量依赖性的细胞杀伤。结论此分离培养的人胰岛素瘤细胞表达人胰岛素和人胰岛细胞自身抗原,因此可以作为研究人Ⅰ型糖尿病发病机制的细胞模型。     

Identification of antigenic domains on the human sodium-iodide symporter which are recognized by autoantibodies from patients with autoimmune thyroid disease

The sodium-iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease. In the present study we have characterized the antigenic domains on the human symporter which are recognized by autoantibodies from patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH). Deletion derivatives of complementary DNA (cDNA) encoding the Na(+)/I(-) symporter were constructed using polymerase chain reaction (PCR) amplification. These deletion constructs were translated in vitro with the concomitant incorporation of [(35)S]methionine into the protein products. The reactivity of seven GD and six AH sera, which were known to contain symporter-binding antibodies, to each of the radiolabelled modified symporters was then determined in immunoprecipitation experiments. Analyses of the results obtained in the radiobinding assays suggest the existence of multiple antibody binding sites on human NIS (hNIS), including regions between amino acids (aa) 1--134, 191--286, 290--411, 411--520 and 520--588. Computer prediction of the potential B cell epitopes on the symporter revealed that, apart from aa 134--191, all the epitope domains identified overlapped, at least in part, with areas predicted to be highly antigenic. Interestingly, the antigenic domains represented by aa 191--286, 290--411 and 411--520 include regions of the polypeptide which form putative extracellular domains in the secondary structure model of the rat symporter. No correlation between the recognition of specific epitopes on the human symporter and the type of autoimmune thyroid disease was demonstrated.     

Anti‐p200‐Pemphigoid – Eine neue blasenbildende Autoimmundermatose

Anti‐p200 pemphigoid is an autoimmune skin disease characterized by tense blisters, subepidermal split formation, and mainly neutrophilic inflammatory infiltration of the dermal‐epidermal junction (DEJ). Direct immunofluorescence microscopy of perilesional skin biopsies demonstrates linear deposits of IgG and C3 along the DEJ, while by indirect immunofluorescence microscopy on NaCl‐split human skin, patients' IgG labels the dermal side. The antigenic target of the autoantibodies is a 200 kD protein (p200) of the lower lamina lucida that can be detected in human dermal extracts by immunoblotting. While p200 is thought to be important for cell‐matrix adhesion, its exact identity is unknown. To date, the p200 autoantigen has been demonstrated to be distinct from bullous pemphigoid antigens 180 und 230, laminin 1, 5, and 6, α6β4 integrin, and type VII collagen. Biochemical characterization of the p200 molecule revealed a noncollagenous N‐glycosylated acidic protein with an isoelectric point of approximately 5.5. We provide an overview on pathogenesis, clinical features, diagnosis, and treatment of this unique autoimmune dermatosis.     

Presence of rheumatoid arthritis (RA) synovial autoantigen recognized by T cells in RA joints

Abstract

The disease-specific antigen in rheumatoid arthritis (RA) is still unknown. Analyses of the T cell receptor (TCR) repertoire suggested that certain antigen-driven T cells infiltrate into the legion. Efforts to establish T cell lines or clones which recognize RA-specific antigen showed that multiple antigen-reactive T cells were detected in inflamed joints. These antigens fall in two categories, one is foreign antigens which induce molecular mimicry and the other is autoantigens. To date, few etiological studies have shown convincing relationships between RA and infectious diseases. Further, lymphocytes reactive to candidate RA autoantigens were often observed in other autoimmune patients. To determine the primary and RA-specific autoantigen, we focused on T cells specific for RA synovial nurse cells. In the present review, we summarize previous studies on tissue-infiltrating T cells in RA and our recent studies on RA synovial nurse cell-specific autoantigen recognized by T cell clones established from RA joints.     

Vitiligo and alopecia areata: apples and oranges?

Vitiligo and alopecia areata are common autoimmune diseases of the skin. Vitiligo is caused by the destruction of melanocytes and results in the appearance of white patches on any part of the body, while alopecia areata is characterized by patchy hair loss primarily on the scalp, but may also involve other areas as well. At first glance, the two diseases appear to be quite different, targeting different cell types and managed using different treatment approaches. However, the immune cell populations and cytokines that drive each disease are similar, they are closely associated within patients and their family members, and vitiligo and alopecia areata have common genetic risk factors, suggesting that they share a similar pathogenesis. Like apples and oranges, vitiligo and alopecia areata have some obvious differences, but similarities abound. Recognizing both similarities and differences will promote research into the pathogenesis of each disease, as well as the development of new treatments.     

利用重组抗原检测抗SSB自身抗体及其临床意义

目的：利用重组抗原检测自身免疫疾患者血清中抗ＳＳＢ自身抗体并探讨其临床意义。方法：应用ＤＮＡ重组技术构建ＳＳＢ基因工程菌，并表达ＳＳＢ融合蛋白。经ＧＳＴ柱层析法化后，分别经蛋白质印迹（ＷＢＴ）法和酶联免疫吸附（ＥＬＩＳＡ）法检测２０坐干燥综合征患者、６０份系统性红斑狼疮患者和３０份正常人血清。结果：经常规试剂盒检测为ＳＳＢ（＋）的２４份患者血清，利用重组抗原检测均阳性，经常剂剂盒检测为ＳＳＢ（－）