Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen.

Herpes gestationis (HG) is a putative autoimmune bullous dermatosis of pregnancy which shares many findings with bullous pemphigoid (BP), a disease of the elderly. This study identifies for the first time the antigen detected by HG autoantibodies and compares it with that recognized by BP autoantibodies. Sera from 16 HG and 17 BP patients, and from normal pregnant women were evaluated by immunofluorescent (IF) studies and immunoblotted against human epidermal extracts. 89% of HG sera with circulating antibodies by IF recognized a 180-kD protein by immunoblotting. 71% of BP sera recognized a 240-kD band, but 47% detected a 180-kD protein that comigrated with the antigen detected by HG sera. None of the control sera recognized any specific bands. These findings suggest that the 180-kD epidermal protein may be the antigen detected by the HG factor and they also define immunologic similarities between HG and BP.     

A 52-kD protein is a novel component of the SS-A/Ro antigenic particle

Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti- 60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti- 52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.     

Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis.

Linear IgA bullous dermatosis (LABD) is a rare blistering skin disease characterized by basement membrane zone deposition of IgA. This study identifies a tissue antigen detected by patient serum and then isolates the autoantibody using epidermis and protein bands blotted on nitrocellulose as immunoabsorbents. Sera from 10 patients (9 with cutaneous disease and 1 with cicatrizing conjunctivitis) were evaluated. Indirect immunofluorescence revealed an IgA anti-basement membrane antibody in 6 of 10 sera with monkey esophagus substrate and 9 of 10 sera with human epidermal substrate. Immunoblotting was performed on epidermal and dermal extracts prepared from skin separated at the basement membrane zone with either sodium chloride or EDTA. Saline-separated skin expressed a 97-kD band in dermal extract alone that was recognized by 4 of 10 sera. EDTA-separated skin expressed the 97-kD band in both epidermal (4 of 10 sera) and dermal (6 of 10 sera) extract. Immunoabsorption of positive sera with epidermis purified an IgA antibody that reacted uniquely with the 97-kD band. In addition, IgA antibody bound to nitrocellulose was eluted from the 97-kD band and found to uniquely bind basement membrane zone. It is likely that the 97-kD protein identified by these techniques is responsible for basement membrane binding of IgA in LABD.     

Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.

Bullous pemphigoid (BP) is a blistering skin disease in which autoantibodies develop to hemidesmosomal components of the epidermal basement membrane zone, including two major antigenic proteins of the 230-kD antigen (BPAG1) and the 180-kD antigen (BPAG2). The present study demonstrated the precise ultrastructural localization of the epitopes for autoantibodies against BPAG1 and BPAG2 in normal skin. Autoantibodies against either BPAG1 or BPAG2 were affinity-purified using nitrocellulose membrane, which was blotted with SDS-PAGE-fractionated antigens from human epidermal extract as the immunoabsorbent. Postembedding, immunogold electron microscopy was performed after skin was processed by rapid freezing and freeze substitution fixation without chemical fixatives. Purified autoantibodies against BPAG1 bound only to the intracellular domain of the hemidesmosome, and 80% of the gold labeling was within 40-140 nm from the plasma membrane (mean distance 91 nm inside). In contrast, the autoantibodies against BPAG2 bound along the plasma membrane of the hemidesmosome, and 80% of the gold labeling was within 10 nm outside to 50 nm inside the cells (mean distance 12 nm inside). These results suggest that the autoantibodies against BPAG1 and BPAG2 react with the epitopes localizing in distinct regions of the hemidesmosome complex, and may play different roles in the blister formation in patients with BP.     

Human autoantibody to defensin: disease association with hyperreactive onchocerciasis (sowda)

Chronic hyperreactive onchodermatitis (sowda) is a severe form of onchocerciasis observed in a subset of individuals infected with the filarial nematode Onchocerca volvulus. SDS-PAGE and immunoblot analyses of O. volvulus adult worm extracts were used to characterize the antigens of the marked antibody response of sowda patients. One 2.5-kD antigen was recognized by sera from all 35(100%) sowda patients that were studied. In comparison, only 7 of 44 (16%) patients with generalized onchocerciasis and 11 of 21 (52%) of exposed individuals with no microfilariae in skin snips and no signs of disease showed reactivity to this antigen. Microfilaricidal treatment of sowda patients with improvement of the clinical status was associated with a decrease or disappearance of antibodies to the 2.5-kD antigen. Amino acid sequencing of the antigen indicated identity to human defensins 1- 3 of neutrophils. Defensin was demonstrated by immunohistochemical staining in onchocercal nodules on the surface of adult filariae and in the surrounding tissue. A similar staining pattern was observed for other proteins present in neutrophils such as myeloperoxidase, elastase, and the L-1 protein complex (MRP 8/MRP 14), indicating that neutrophils, macrophages, and their proteins predominate in the environment adjacent to the worms. These results demonstrate an association between the presence of autoantibodies to defensins and an infectious disease of known etiology. The association with a particular form of onchocerciasis, sowda, suggests a link between formation of autoantibodies to defensin and enhanced immune reactivity towards the parasite.     

A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.     

Autoantibodies to the constitutive 73-kD member of the hsp70 family of heat shock proteins in systemic lupus erythematosus

Serum from patients with systemic lupus erythematosus (SLE) frequently contain IgM and IgG autoantibodies to the constitutively expressed 73-kD/pI 5.5 member of the hsp70 family of heat shock proteins, as determined by one-dimensional (SDS-PAGE) and two-dimensional (IEF/SDS-PAGE) immunoblotting, and by solid-phase SLE Ig immunoprecipitation experiments using hsp70 protein-specific mAbs as probes. Autoantibodies to hsp70 also were detected in a minority of sera from patients with other rheumatic or viral diseases, but not in normal sera. These data may provide additional insight into etiologic and pathophysiologic mechanisms in this and related autoimmune disorders.     

The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128- kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS.     

Autoantibodies to the heat-shock protein hsp90 in systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) develop multiple autoantibodies to self-antigens. Analysis of autoantibody systems in this and related autoimmune disorders can provide information of etiologic and pathogenetic significance. We report here a previously unrecognized autoantibody to the 90,000-D heat-shock protein, hsp90, a molecule thought to have important functions in the cellular response to stress, virus-induced transformation, steroid hormone receptor action, and cellular activation. Autoantibodies to hsp90 were exclusively of the IgG class, and were detected in approximately 50% of unselected patients with SLE and 2/6 patients with idiopathic polymyositis. Anti-hsp90 antibodies were not detected in sera from 10 normal subjects, 10 patients with rheumatoid arthritis, or 7 patients with scleroderma. The identity of this major intracytoplasmic antigen was established by its specific removal from nonionic detergent cell lysates following immunoabsorption with monospecific rabbit anti-hsp90, and by demonstration of increased synthesis following a 10-min 45 degrees C heat shock. These data define the frequent occurrence of a novel autoantibody to a major heat-shock protein in patients with SLE.     

Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression.

Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.     

Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE.     

Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1.

Type 1, liver kidney microsomal autoantibodies (LKM-1) are associated with a subgroup of idiopathic autoimmune type, chronic active hepatitis (CAH). The antigenic specificity of LKM-1 autoantibodies from 13 patients was investigated by immunoblot analysis of human liver microsomal proteins. Polypeptides of 50, 55, and 64 kD were detected with these antisera. A high titer LKM-1 serum was selected to screen a human liver lambda gt11 cDNA expression library, resulting in the isolation of several complementary (c)DNA clones. Autoantibodies affinity purified from proteins expressed by two of the immunopositive cDNA clones, HLD8.2 and HLD13.2, specifically react with a 50-kD protein of human liver microsomes and display immunofluorescence staining of the proximal renal tubular epithelia characteristic of LKM-1 sera. Determination of the sequence of HLD8.2 revealed that it encodes a recently described cytochrome P450db1. A bacterial fusion protein constructed from HLD8.2 proved to be a specific and sensitive diagnostic reagent. All sera from patients with LKM-1 positive liver disease react with this fusion protein. No reaction was seen, however, for sera from patients with other types of autoimmune liver diseases, viral hepatitis, systemic immunological disorders, or healthy controls.     

免疫印迹检测抗眼肌抗体

用含ＴｒｉｔｏｎＸ－１００及脱氧胆酸钠的ＴＤＯＣ缓冲液自牛眼肌提取了眼肌细胞膜抗原，建立了抗眼肌多肽抗体的免疫印迹检测法。     

Distinct antibody specificities to a 64-kD islet cell antigen in type 1 diabetes as revealed by trypsin treatment

Type 1 diabetes is associated with antibodies that immunoprecipitate a 64-kD islet cell membrane protein from detergent extracts of pancreatic islets. In this study we have determined whether mild trypsin treatment of islet membranes can release fragments of the antigen that bind antibodies in the serum of Type 1 diabetic patients. Partial tryptic proteolysis of [35S]methionine-labeled 64-kD antigen immunoprecipitated from detergent extracts of rat islets resulted in the formation of 50-, 40-, and 37-kD fragments. Similar sized fragments were recovered when sera from diabetic patients were employed to immunoprecipitate polypeptides solubilized by mild trypsin treatment of a particulate fraction of radiolabeled rat islets. Of 27 diabetic patients, 22 possessed antibodies to the 50-kD polypeptide and 21 to the 40- and 37-kD polypeptides. A positive association was found between 64k antibodies and antibodies to the 50-kD fragment but not between 64k antibodies and antibodies to the 40- or 37-kD fragments. Some 64k antibody negative patients possessed antibodies that efficiently immunoprecipitated the latter fragments. Serum from 25 of 27 (93%) diabetic patients immunoprecipitated at least one of the three tryptic polypeptides. One of 20 nondiabetic controls immunoprecipitated a 50-kD polypeptide and all controls were negative for antibodies to 40- and 37-kD fragments. Thus, Type 1 diabetes is associated with the presence of at least two antibody reactivities to distinct determinants of the 64-kD antigen, and some patients may possess antibodies to a cryptic epitope on the detergent-solubilized molecule. These data suggest that the detection of antibodies (present in 93% of patients) to epitopes on tryptic polypeptides of the 64-kD antigen may be of even greater diagnostic value for the onset of Type 1 diabetes than analyses of antibodies reactive with the intact 64-kD antigen.     

Novel biosensor-based analytic device for the detection of anti-double-stranded DNA antibodies

BACKGROUND: Patients with systemic lupus erythematosus (SLE) develop a wide variety of serologic manifestations, including double-stranded DNA autoantibodies (anti-dsDNA). The determination of the potentially pathogenic autoantibodies is diagnostically relevant. METHODS: We developed a novel surface plasmon resonance (SPR) biosensor chip for studies of dsDNA and anti-dsDNA binding. A synthetic oligonucleotide was coupled to biotinylated human transferrin, hybridized with the complementary antistrand, and ligated with a human recombinant dsDNA fragment 233 bp in length. After surface immobilization of this antigenic construct, diluted sera from SLE patients and healthy donors were analyzed with the resulting SPR biosensor system. RESULTS: This SPR biosensor allowed specific detection of anti-dsDNA. In pilot experiments, sera from SLE patients were distinguished from control sera. We also confirmed the specificity of this biosensor by supplementing anti-dsDNA-positive sera with salmon sperm DNA, which blocked the surface binding of anti-dsDNA in a concentration-dependent manner. CONCLUSIONS: An SPR biosensor monitors interactions in real time under homogeneous conditions, providing information about binding kinetics and affinities. Its applicability critically depends on the design of the solid-state surface of the sensor chips. Covalently immobilizing dsDNA as the antigen to the surface in a flow-through cell assured maximal stability for multiple serum injections and regeneration cycles. This technique, which adds a new analytic quality to existing methods, may be beneficial in the diagnosis and clinical monitoring of SLE.     

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 M(r). Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.     

Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen.

Serum SS-A/Ro autoantibodies are commonly found in patients with Sjogren's syndrome, systemic lupus erythematosus, neonatal lupus, and subacute cutaneous lupus. Two proteins of 60 and 52 kD have been described as targets for these autoantibodies. To define the 52-kD component unambiguously, cDNA clones were isolated from human HepG2 and MOLT-4 cell cDNA libraries. The identity of cDNA was established by (a) the specificity of the antibody affinity purified from the recombinant protein, (b) the reactivity of the purified recombinant protein with prototype SS-A/Ro sera in immunoblot and ELISA, and (c) two-dimensional gel comigration of MOLT-4 cell 52-kD protein and the recombinant protein. A 1.9-kb cDNA encoded the complete 52-kD protein containing 475 amino acids (Mr 54,082). Putative zinc-finger domains and a leucine zipper motif were identified in the amino-terminal half of the 52-kD protein, implicating its possible association with DNA/RNA. Sequence homology detected between the 52-kD protein and human ret transforming protein, and mouse T cell gene expression down-regulatory protein rpt-1, may provide leads to the functional role of the 52-kD protein in addition to the possibility that these proteins might constitute members of a subfamily of finger proteins.     

Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus.

Homogenized tissue from the frontal cortex of normal human brains obtained at postmortem examination was used to absorb lymphocytotoxic antibody from the serum of six patients with systemic lupus erythematosus (SLE). Four absorptions of all of the SLE sera with equal volumes of homogenized brain tissue at 4 degrees C depleted their cytotoxic capacity more than 90%. Three of the six sera, however, retained some lymphocytotoxicity despite extensive brain absorption. Absorbed lymphocytotoxic antibodies were eluted from brain tissue absorbents at 37 degrees C. Cytotoxicity of the brain eluates was blocked by antibodies to human IgM (mu-chain specific) but not anti-IgG. The unabsorbed SLE sera, brain-absorbed sera, and brain eluates were equally cytotoxic to T (thymus-derived) and B (bone marrow-derived) cells fractionated from normal human peripheral blood lymphocytes. Thus, the lymphocytotoxic antibodies in SLE serum exhibit no preference for circulating human T cells. An analysis of the clinical records of 40 patients with SLE whose serum cytotoxic capacity had been determined revealed that circulating lymphocytotoxicity is greater in sera of patients with central nervous system (CNS) manifestations than in other SLE patients. This observation suggests a possible role for brain-reactive lymphocytotoxic antibodies in the development of CNS disease in SLE.     

The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies.

The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most specific serological markers of RA. Using indirect immunofluorescence, AKA label the stratum corneum of various cornified epithelia and APF the keratohyalin granules of human buccal mucosa epithelium. We recently demonstrated that AKA recognize human epidermal filaggrin. Here, we report the identification of the major APF antigen as a diffuse protein band of 200-400 kD. This protein is seen to be closely related to human epidermal (pro) filaggrin since it was recognized by four antifilaggrin mAbs specific for different epitopes, and since the APF titers of RA sera were found to be correlated to their AKA titers and to their immunoblotting reactivities to filaggrin. Immunoabsorption of RA sera on purified epidermal filaggrin abolished their reactivities to the granules of buccal epithelial cells and to the 200-400-kD antigen. Moreover, antifilaggrin autoantibodies, i.e., AKA, affinity purified from RA sera, were shown to immunodetect the 200-400-kD antigen and to stain these granules. These results indicate that AKA and APF are largely the same autoantibodies. They recognize human epidermal filaggrin and (pro) filaggrin-related proteins of buccal epithelial cells. Identification of the epitopes recognized by these autoantibodies, which we propose to name antifilaggrin autoantibodies, will certainly open new paths of research into the pathophysiology of RA.