Secondary Sjögren''s syndrome and chromosome six markers

Patients with Sj?gren's syndrome (SS) in association with either Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) or Sclerodactyly were tested for HLA-A, B and Dr antigens and the phenotypes of the 4th component of complement (CA). Significant associations were identified between HLA-Bw62, Dr4, C4B2.9, C4A8 and females with SS-RA. The MHC antigen frequencies noted for secondary Sj?gren's syndrome differed from those reported for their respective primary diseases and the implications of these observations are discussed.     

The significance of autoantibodies coexisting with anti-centromere antibodies in sera of patients with primary biliary cirrhosis

Anti-centromere antibody (ACA) has been believed to be specific to patients with CREST syndrome, a variant of scleroderma (PSS). This study was undertaken to clarify the distribution of ACA in various diseases and the significance of autoantibodies coexisting with it. The sera of patients with primary biliary cirrhosis (PBC) along with collagen diseases and aged subjects were examined for ACA by immunofluorescence method (IF) using cultured HEp-2 cells and chromosomes prepared from K 562 cells. ACA were found in sera of 10 patients with PBC, one with scleroderma, one with cerebral infarction and one with chronic renal failure respectively. ACA positive sera were examined for antibodies against other nuclear antigens including nRNP, Sm, Scl-70, SS-A and SS-B and cytoplasmic antigens by double immunodiffusion methods using rabbit thymus extract etc. as the antigens and by IF method using cryostat sections of rat kidney and stomach. In 13 sera with ACA, antimitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA) and anti SS-A antibody were found in 10, 4 and one sera respectively. In 10 PBC patients with ACA, various collagen disease-related disorders were found to coexist; CREST syndrome in one, CRST syndrome in one, Raynaud's phenomenon in two and Sj?gren's syndrome in 5. These results would indicate that ACA may be one of the common serological abnormalities among patients with PBC, CREST syndrome and Sj?gren's syndrome.     

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

An antibody reacting with splenic red pulp macrophages in the sera of patients with rheumatic diseases

An antibody was detected in the sera of patients with certain rheumatic diseases that reacted with the cytoplasm of the splenic red pulp (SRP) cells of adult mice. This antibody was detected in the sera of all patients with mixed connective tissue disease (MCTD), 53% of patients with systemic lupus erythematosus (SLE), 42% with Sj?gren's syndrome (SS), and 10% with rheumatoid arthritis (RA). However, this antibody was found neither in the sera from patients with other types of rheumatic diseases nor in healthy volunteers. The screening of this antibody may be useful in diagnoses of MCTD, SLE, and SS. In the present study, we also performed the characterization of the cells reacting with this antibody. The cells proved to be acid phosphatase positive phagocytes in the SRP, that is, red pulp macrophages. Moreover, a histochemical analysis of the reacting antigen in these cells has demonstrated that its antigenic activity is NaIO4 and RNase sensitive, suggesting that the antigen may be associated with RNA.     

An experimental protocol for the fractionation and 2DE separation of HeLa and A-253 cell lysates suitable for the identification of the individual antigenic proteome in Sjögren's syndrome

Sj?gren's syndrome (SS) is an autoimmune disease affecting exocrine glands, especially the salivary and lacrimal glands. Although most of the SS patients' sera have autoantibodies that can target a variety of antigens, it is not clear what determines which proteins will become autoantigens. The muscarinic receptor M3, an integral plasma membrane protein, has been proposed as a possible autoantigen in SS, and is endogenous in HeLa cells. The aim of this study was to develop a method that is able to separate and identify antigens recognised by sera from SS patients using lysates of HeLa and A-253 cells in 2D Western Blot (2DWB). The HeLa and A-253 cell lysates were fractionated in soluble and membrane-bound proteins, and the membrane-bound proteins were enriched for integral proteins. The fractions were tested using WB, confirming the presence of the main cell compartments. The rehydration solution containing ASB-14 performed better than the others in all three steps (active rehydration, focus and transfer), and efficiently separated the muscarinic receptor M3. The M3 receptor was also detected in lysates from A-253 cells. The presence of this receptor in this cell line has not been proven earlier. This work develops a suitable protocol to perform a mapping of the autoantibodies present in the sera of single SS patients, using lysates from epithelial cell lines that represent the main cell compartments as an antigen source. It is our future aim to use this protocol to perform a mapping of the antibodies present in the sera of individual SS patients.     

The presence of immunosuppressive ''p15E-like'' factors in the serum and urine of patients suffering from malign and benign breast tumours.

Autoantibodies in sera from patients with systemic lupus erythematosus (SLE) and onchocerciasis recognize calreticulin (CaR), a calcium-binding protein, as antigen. In this study we present the immunological properties of two synthetic peptides prepared to correspond to the 1-24 and 7-24 amino acid sequence of CaR. In contrast to information previously reported for the recombinant protein, the CaR-peptide analogues appeared immunoreactive to anti-Ro/SSA autoimmune sera. Human sera from patients with SLE, Sjögren's syndrome (SS), rheumatoid arthritis (RA), as well as mixed connective tissue disease (MCTD), demonstrated a positive autoimmune response (binding of antibodies), to the CaR-peptide analogues. These findings suggest that anti-calreticulin autoantibodies are not restricted to any disease specificity.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

Preparative isolation of p67, A, B, B' and D from nRNP/Sm and Sm antigens by reverse-phase chromatography. Use in a polypeptide-specific ELISA for independent quantitation of anti-nRNP and anti-Sm antibodies

The p67 (67 kDa) and A (33 kDa) polypeptides of nRNP/Sm antigen and the B, B' (28 and 29 kda) and D (16 kDa) polypeptides of 'free' Sm antigen were isolated and used in enzyme-linked immunoadsorbent assays (ELISA) for human autoantibodies. ELISA specificity was demonstrated using monoclonal antibodies. The ELISA using HPLC-purified polypeptides was found to be more sensitive than immunoblotting for detecting antibody. 86% of sera with precipitating anti-nRNP antibodies were positive in the ELISA, as were all sera with precipitating anti-Sm antibodies. Patients with rheumatoid arthritis (RA), Sj?grens syndrome (SS) and undifferentiated connective tissue disease (UCTD) had low levels of anti-p67 with a prevalence 11.6% and 18%, respectively, whilst patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) had high levels and prevalence rates of 55.2% and 80%, respectively. Anti-B or anti-D antibodies were detected at high levels in SLE (prevalence 30%) but were found rarely in UCTD and MCTD (prevalence 7% and 10%) and not at all in RA or SS sera.     

Immunoserological area--with special reference to the current topics on antinuclear antibodies

In 1948, Hargraves described the phenomenon of the LE cells and equated it with systemic lupus erythematosus (SLE). Kunkel and his colleagues elucidated the immunochemical basis of this phenomenon, showing that it was related to the circulating autoantibodies to deoxyribonucleoprotein (DNP). Their pioneering studies led to the continuing discoveries of antinuclear antibodies (ANA) and today's considerable knowledge concerning the molecular identity of antigens and further consolidation of ANA. In addition to the role of diagnostic markers, spontaneously occurring autoantibodies, including anti-PCNA (proliferating cell nuclear antigen) antibody and anticentromere antibody (ACA), turned out to be useful as powerful probes for cell biology. In this study, the sera of patients with primary biliary cirrhosis (PBC) along with collagen diseases were examined for the presence of antinuclear antibodies. In using HEp-2 cells and chromosomes derived from K 562 as the substrates for the immunofluorescence method, the frequency of ANA and ACA in PBC sera were 84% and 44% respectively. Anti-DNA, antiSS-A and antiSS-B antibodies were found in one, 4 and one sera respectively. On the other hand, antibodies to nRNP, Sm and Scl-70 were not found in PBC sera. In some PBC patients with ACA, various complications related to collagen diseases were found to coexist. Our results indicated that there might be common serological abnormalities among patients with PBC, CREST syndrome and Sj?gren's syndrome.     

Monoclonal anti-la antibody derived from a mouse with experimental SLE is similar to human anti-la antibodies

The La antigen is a highly conserved protein, originally defined by sera of patients with Sj?gren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.     

Identification of antigenic regions of the human Ro 60 kDa protein using recombinant antigen and synthetic peptides.

Autoantibodies reacting with the human Ro 60 kDa protein are present in anti-Ro/SS-A positive sera from patients with several different connective tissue diseases including Sj?gren's syndrome and systemic lupus erythematosus. To investigate the humoral immune response to this protein, the pattern of antibody recognition of recombinant Ro 60 kDa proteins encoded by both full-length and deletion clones was analysed by immunoblotting. An antigenic region recognized by nearly all anti-Ro 60 kDa positive sera was found to reside in the middle part of the protein. In addition, some sera reacted with two other antigenic regions located in the amino-terminal and carboxyl-terminal part of the protein. For further mapping, overlapping peptides covering the most frequently recognized region of the protein were synthesized by solid-phase methods and used as antigens in ELISA. Three major patterns of reactivity to Ro 60 kDa peptides were found. These results not only indicate the presence of an immunodominant region but also heterogeneity in the autoimmune human response to the Ro 60 kDa antigen.     

Cytoplasmic staining pattern characteristic for anti Jo-1 antibody in HEp-2 cells]

We wished to confirm the staining pattern of HEp-2 cells by the anti Jo-1 antibody, because we found antibodies in serum with positive anti Jo-1 antibody which showed either a fibrilar cytoplasmic staining or a nuclear speckled staining pattern in indirect immuno fluorescence+ examinations using HEp-2 cells. Sera available from eight patients with PM DM (polymyositis and/or dermatomyositis) showed positive anti Jo-1 antibody in the double immuno-diffusion technique but had various staining patterns of HEp-2 cells in the immunofluorescent examination. We examined these eight sera with the immuno-blotting method utilizing whole cell extract of HeLa cells, and found the 50 kDa band from all sera tested, to which Jo-1 antigen had been reported to move. We eluted the antibody which formed the 50 kDa band from the nitrocellulose membrane and applied it on HEp-2 cells. This maneuver gave us the fine granular cytoplasmic staining of anti Jo-1 antibody on HEp-2 cells. We therefore concluded that the anti Jo-1 antibody should have cytoplasmic staining on HEp-2 cells although observers might miss it due to other types of associated antinuclear antibodies.     

Anti-Golgi Antibody in Rheumatoid Arthritis Patients Recognizes a Novel Antigen of 79 kDa (Doublet) by Western Blot

We have detected cytoplasmic anti-Golgi antibody (AGA) during a routine immunofluorescence test for detecting autoantibodies. Two sera from patients with rheumatoid arthritis (RA) reacted to the Golgi complex by an indirect immunofluorescence technique on HEp-2 cells. Localization of AGA in the Golgi complex was confirmed by double-staining with antibodies to beta-COP. The effect of monensin on the integrity and morphology of the Golgi complex was also studied. To confirm the presence of AGA further, we performed immuno-electron microscopy. Both sera reacted with cytoplasmic antigen located in the Golgi complex of various animal tissues. Furthermore, by using the Western blot technique, both sera reacted to a relative molecular weight (MW) of 79 kDa (doublet) Golgi antigen purified from rat liver. To our knowledge, this study may be the first to identify the relative MW of Golgi antigen by the Western blot method. Identification of this antibody could provide better understanding of protein synthesis and secretion. The presence of AGA in RA patients further substantiates the diversified nature of autoantibody production seen in this disease.     

Ro (SSA) and La (SSB) antibodies

Summary This review traces the historical development of information regarding the Ro (SSA) and La (SSB) autoantibody systems over the past twenty years. Clinical and serologic findings are integrated with fundamental observations in this rapidly expanding area of research. Retrospective analysis of the physicochemical properties of the antigens and the cellular staining characteristics of antibodies to these antigens suggest that SjD and Ro and SSA, as well as SjT and La, SSB, and Ha antigens probably are similar macromolecules. The immunologic identity of Ro with SSA and La with SSB and Ha has been established previously. Antibodies to these antigens are directed against macromolecules containing small RNA nucleotides.Antibodies to the Ro (SSA)-La(SSB) antigen system commonly are detected in the sera of patients with systemic lupus erythematosus and Sjögren's syndrome and appear to be of diagnostic significance. These antibodies occur in up to one quarter of patients with systemic lupus erythematosus (SLE) without the sicca complex, but also in patients with ANA negative SLE who have a prominent photosensitive dermatitis and may have serious renal disease, subacute cutaneous SLE, and in infants and mothers of infants with neonatal SLE. Thus, these antibody systems form a serologic link between many unusual connective tissue diseases and systemic SLE.Antibodies to Ro (SSA)-La(SSB) are associated not only with Sjögren's syndrome occurring alone, but also with Sjögren's syndrome occurring in the setting of other connective tissue diseases including SLE and rheumatoid arthritis. Anemia, leukopenia, and thrombocytopenia, as well as hyperglobulinemia and the presence of rheumatoid factor, cryoglobulins, and antibodies to nuclear antigens are associated significantly with Ro positivity in Sjögren's syndrome patients. There is a striking association of vasculitis in the clinical setting of Sjögren's syndrome with the presence of antibodies to Ro (SSA). In addition to peripheral nerve involvement, unusual central nervous system manifestations as well as myositis occur in these Ro(SSA) positive Sjögren's syndrome patients. Deposition of immunoglobulin and complement within vessel walls of kidney and muscle from Ro positive patients with Sjögren's syndrome suggests a possible role for immune complex deposition in the pathogenesis of the vasculitis.Supported by National Institutes of Health grant 5ROI-AM-25650-03 and Research Career Development Award 5-KO-4-AM-00524-02     

Evaluation of commercial enzyme immunoassays compared to immunofluorescence and double diffusion for autoantibodies associated with autoimmune diseases.

A commercially available enzyme immunoassay system for detecting autoantibodies to double-stranded DNA, deoxyribonucleoprotein, Smith, ribonuclearprotein, Sj?gren's syndrome-associated antigens A and B, and scleroderma-associated antigen 70 was compared to the conventional immunofluorescence assay for double-stranded DNA and double diffusion assays for extractable nuclear antigens. There was excellent correlation between methods, but it appears that the enzyme immunoassays are more sensitive. Based on the results of this study, the authors recommend performing anti-nuclear antibody screening at two dilutions, with enzyme immunoassay follow-up of appropriate patient sera that are positive on anti-nuclear antibody testing. Nucleolar and centromere pattern anti-nuclear antibodies are diagnostic for variants of scleroderma and need no further evaluation. Negative anti-nuclear antibody tests performed using HEp-2 tissue culture cells require no further evaluation.     

Multicenter evaluation study on a new HEp2 ANA screening enzyme immune assay.

The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sj?gren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations.     

At least three distinct B cell epitopes reside in the C-terminal half of La protein, as determined by a recombinant DNA approach

The La antigen is a nuclear protein that is one of the major target antigens of autoantibodies found in the sera of patients with primary Sj?gren's syndrome. The formation of such autoantibodies is therefore likely to reflect the basic immunopathogenesis of this disorder. A recombinant DNA strategy has been used to examine the La protein for sequences that encode autoimmunizing B cell epitopes. We have isolated and characterized a 1.2-kb-long cDNA from a human liver cDNA library encoding a region of the La protein; this region contains 296 amino acids, including the C terminus. A sub-library of recombinant DNA in the expression vector pEX was made from portions of the La cDNA. Individual fusion proteins were tested by immunoblotting and enzyme-linked immunosorbent assay for their ability to react with anti-La autoantibodies contained in sera from patients with Sj?gren's syndrome. In this way, we have identified at least three distinct epitopes in the C-terminal half of the La protein. Every anti-La serum tested contained antibodies against all three of the antigenic regions identified. Furthermore, most of the sera display similar ratios between the titers of antibodies with the three kinds of specificity. Our data suggest that the production of anti-La autoantibodies may be antigen driven.     

Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.     

Clinical and serological features of patients with autoantibodies to GW/P bodies

GW bodies (GWBs) are unique cytoplasmic structures involved in messenger RNA (mRNA) processing and RNA interference (RNAi). GWBs contain mRNA, components of the RNA-induced silencing complex (RISC), microRNA (miRNA), Argonaute proteins, the Ge-1/Hedls protein and other enzymes involving mRNA degradation. The objective of this study was to identify the target GWB autoantigens reactive with 55 sera from patients with anti-GWB autoantibodies and to identify clinical features associated with these antibodies. Analysis by addressable laser bead immunoassay (ALBIA) and immunoprecipitation of recombinant proteins indicated that autoantibodies in this cohort of anti-GWB sera were directed against Ge-1/Hedls (58%), GW182 (40%) and Ago2 (16%). GWB autoantibodies targeted epitopes that included the N-terminus of Ago2 and the nuclear localization signal (NLS) containing region of Ge-1/Hedls. Clinical data were available on 42 patients of which 39 were female and the mean age was 61 years. The most common clinical presentations were neurological symptoms (i.e. ataxia, motor and sensory neuropathy) (33%), Sj?gren's syndrome (SjS) (31%) and the remainder had a variety of other diagnoses that included systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC). Moreover, 44% of patients with anti-GWB antibodies had reactivity to Ro52. These studies indicate that Ge-1 is a common target of anti-GWB sera and the majority of patients in a GWB cohort had SjS and neurological disease.     

毒蕈碱受体3抗原表位筛选及其在原发性干燥综合征诊断中的作用

运用计算机软件分析毒蕈碱受体3(M3R)蛋白的结构特点及抗原决定簇分布,根据结果设计合成3条多肽。比较合成多肽与抗M3受体抗体阳性干燥综合征(SS)患者血清IgG的反应强弱,获得一条抗原抗体反应最强的抗原表位多肽(N49)。进一步以多肽N49作为包被抗原,检测原发性SS(pSS)患者41例,继发SS(sSS)患者25例,系统性红斑狼疮(SLE)患者34例,类风湿关节炎(RA)患者80例和正常人174例血清中抗M3多肽IgG抗体,结果显示该多肽抗体在原发性SS患者血清中的阳性率为53.7%,明显高于sSS患者28%、SLE患者8.8%、RA患者25%和正常人12.1%(P<0.05)。抗N49多肽抗体在pSS诊断中的敏感性为53.7%,特异性为83.7%,而且该抗体的检测对于抗SSA抗体、抗SSB抗体或抗α-胞衬蛋白(-αfodrin)抗体阴性的pSS患者的诊断具有补充作用。表明,筛选获得的M3抗原表位多肽N49,其检测抗体在pSS临床血清诊断中具有很好的应用价值。