Detection of anti-SS-A/Ro and anti-SS-B/La antibodies of IgA and IgG isotypes in saliva and sera of patients with Sj?gren's syndrome.

To assess the frequency and the possibility of local production of autoantibodies against SS-A/Ro and SS-B/La in patients with primary Sj?gren's syndrome (SS), serum and saliva samples were obtained from 42 patients with SS, 10 with rheumatoid arthritis without sicca syndrome, and 12 healthy volunteers. Autoantibodies were detected using enzyme-linked immunosorbent assay and immunoblotting. The frequencies of IgA anti-SS-A antibody, IgA anti-SS-B antibody, IgG anti-SS-A antibody and IgG anti-SS-B antibody in serum from SS patients were 45%, 50%, 43% and 21%, respectively. The frequencies of IgA anti-SS-A antibody, IgA anti-SS-B antibody, IgG anti-SS-A antibody and IgG anti-SS-B antibody in saliva from SS patients were 31%, 33%, 40%, and 19%, respectively. We also found secretory IgA anti-SS-A and anti-SS-B antibodies accompanying secretory components in saliva and sera in representative SS patients. Significant correlations were found between serum and salivary levels of IgA anti-SS-A antibodies, and between serum and salivary levels of IgA anti-SS-B antibodies in SS patients. Significant correlations were also found between serum and salivary levels of IgG anti-SS-A antibodies, and between serum and salivary levels of IgG anti-SS-B antibodies in SS patients. Immunoblot analysis confirmed the presence of IgA-class autoantibodies against SS-A and SS-B in saliva and serum from representative patients. The presence of IgA- and IgG-class autoantibodies against SS-A and SS-B and those accompanying secretory components in saliva from SS patients suggests the local production of these antibodies and the relationship between local and systemic antibody responses.     

High prevalence of a variety of autoantibodies in a population of hepatitis C virus‐infected individuals

Hepatitis C virus (HCV) infection has been related to self‐reactivity, extrahepatic manifestations and autoimmune diseases. The main goals of this work were to study the prevalence of autoantibodies and their relationship with viral titers and biochemical markers of hepatic damage in patients infected with HCV. Autoantibodies (ANA, AMA, SMA, APC, LKM, DNAds, ANCA, ATG and RF) were determined in 73 individuals with chronic HCV infection and 44 healthy volunteers. The presence of these antibodies was related to demographic variables, viral titers and biochemical parameters. A high prevalence of autoantibodies, particularly for RF, that was associated with female gender was observed in HCV‐infected patients. In addition, SMA, ANA and ATG showed increased frequencies in HCV infection. Interestingly, the concurrent detection of SMA and more than one autoantibody was associated with high gGT levels. Notably, concurrent higher gGT, HCV and SMA levels were observed in male patients as compared to their female counterparts. These results indicate a relationship between HCV infection and the concurrent detection of various autoantibodies in the absence of symptoms of autoimmune diseases. They also suggest a link among the presence of a variety of autoantibodies simultaneously with SMA, increased gGT levels and HCV titers in a population of male patients.     

Anti-endothelial cell antibodies (AECA) in patients with propylthiouracil (PTU)-induced ANCA positive vasculitis are associated with disease activity

Increasing evidence has demonstrated that propylthiouracil (PTU) could induce ANCA positive vasculitis. However, our previous work has suggested that only one-fifth of the PTU-induced ANCA positive patients had clinical vasculitis and so the mechanism is not clear. Anti-endothelial cell antibodies (AECA) have been implicated in the pathogenesis of various vasculitides, including primary ANCA positive systemic vasculitis. The purpose of this study is to investigate the prevalence of AECA and their possible role in the pathogenesis of patients with PTU-induced ANCA positive vasculitis. Sera from 11 patients with PTU-induced ANCA positive vasculitis at both active and quiescent phases, and sera from 10 patients with PTU-induced ANCA but without clinical vasculitis, were studied. Sera from 30 healthy blood donors were collected as normal controls. Soluble proteins from 1% Triton-100 extracted in vitro cultured human umbilical vein endothelial cells were used as antigens and an immunoblotting technique was performed to determine the presence of AECA, and their specific target antigens were identified. In patients with PTU-induced ANCA positive vasculitis, 10 of the 11 patients in an active phase of disease were serum IgG-AECA positive and six protein bands of endothelial antigens could be blotted (61 kD, 69 kD, 77 kD, 85 kD, 91 kD and 97 kD). However, in the quiescent phase, seven of the 10 positive sera turned negative. None of the ANCA positive but vasculitis negative patients or normal controls were AECA positive. In conclusion, AECA could be found in sera from patients with PTU-induced ANCA positive vasculitis and were associated more closely with vasculitic disease activity.     

Keratinocytes from patients with lupus erythematosus show enhanced cytotoxicity to ultraviolet radiation and to antibody-mediated cytotoxicity.

Keratinocyte cytotoxicity is an important component of the immunopathology of photosensitive lupus erythematosus, and antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be an important mechanism by which autoantibodies, especially those specific for SS-A/Ro, can induce keratinocyte damage in models of photosensitive lupus. We provide further evidence that keratinocytes from patients with photosensitive lupus show significantly greater ultraviolet radiation (UVR)-induced cytotoxicity, and that ADCC of these targets is especially enhanced by autologous patient's serum or by anti-SS-A/Ro+ sera. Keratinocytes from normal uninvolved skin of 29 patients with cutaneous lupus erythematosus (LE) were grown in cell culture and tested as targets in cytotoxicity experiments in vitro. Cultured keratinocytes from patients with systemic lupus erythematosus (SLE) and subacute cutaneous lupus erythematosus (SCLE) showed significantly greater cytotoxicity following UVR treatment than did keratinocytes from normal adult controls or from neonatal foreskins (P < 0.01). The same cultures also showed greater UVR-induced binding of IgG from fractionated anti-SS-A/Ro+ preparations. ADCC experiments were also performed using keratinocytes cultured from patients with SLE, SCLE, discoid lupus erythematosus (DLE), and normal controls. When keratinocytes were incubated in autologous serum plus a standard mononuclear cell effector population, the percentage of ADCC observed was significantly greater in cultures containing keratinocytes and sera from the SLE and SCLE patients (P < 0.001). When cultured keratinocytes were added to different IgG antibody probes, plus standard mononuclear effector populations, greater ADCC was seen using the anti-SS-A/Ro probe and keratinocytes from patients with SLE or SCLE. With normal human neonatal keratinocyte targets, the anti-SS-A/Ro probe induced greater ADCC than that seen with anti-ssDNA or normal human serum. We have shown that keratinocytes from patients with some forms of lupus erythematosus (SLE and SCLE) show greater cytotoxicity in vitro when irradiated with UVR, and greater susceptibility to ADCC whether the antibody source is their own serum or an anti-SS-A/Ro probe.     

Clinical and immunological features of Sj?gren's syndrome in patients with primary biliary cirrhosis with emphasis on focal sialadenitis

Serological and pathological findings in 21 patients with primary Sj?gren's syndrome (primary SS) were compared with those in 32 patients with primary biliary cirrhosis (PBC). In ELISA, anti-SS-B/La antibodies were detected in sera from 14 (67%) of the patients with primary SS, but only from 12 (38%) of those with PBC. With the Ouchterlony test, anti-SS-A/Ro antibodies were found in sera from 15 (71%) of the primary SS patients, but in no PBC patient. Of those PBC patients investigated prospectively with objective tests, four of 11 (36%) had keratoconjunctivitis sicca, and five of 15 (33%) had pathological sialometry results. In contrast, all PBC patients but one (i.e., 14 of 15 or 93%) showed evidence of focal sialadenitis. In immunochemical study of PBC patients, IgM immunoreactivity was found in the stroma, particularly adjacent to excretory ducts and acini in salivary glands (5 of 5), whereas no such IgM deposits were observed in patients with primary SS (3 of 3), nor in healthy controls (n = 20). We conclude that the frequency of anti-SS-A/Ro and anti-SS-B/La antibodies in serum is lower in PBC patients than in patients with primary SS. The incidence of focal sialadenitis is high in PBC, though only one third of the PBC patients studied here showed clinical evidence of glandular dysfunction. With immunochemical techniques, sialadenitis associated with PBC is distinguishable by its significant IgM reaction from sialadenitis in primary SS.     

A major autoepitope is present on the amino terminus of a human SS-A/Ro polypeptide

A human SS-A/Ro antigen is present on the polypeptide component of a particle composed of hyRNA and a 60 kD protein. We have now purified the Wil-2 cell 60 kilo dalton (kD) SS-A/Ro protein and determined its amino-terminal amino acid sequence. A synthetic peptide corresponding to residues 7 to 24 of this sequence (RoSP7-24) exhibited enzyme-linked immunosorbent assay (ELISA) binding activity with immunodiffusion-defined, monospecific anti-SS-A/Ro sera. In addition, ELISA binding of monospecific anti-SS-A/Ro sera to native SS-A/Ro antigen was partially inhibited (35%) by KLH-RoSP7-24. Sera from patients known to frequently produce precipitating anti-SS-A/Ro antibody (subacute cutaneous lupus erythematosus [SCLE], 56 patients; Sj?gren's syndrome [SS], 41 patients; mothers of infants with neonatal LE [NLE], 10 individuals; infants with congenital heart block [CHB], 5 patients) were tested for reactivity to RoSP7-24 in ELISA. Overall, 38% of SCLE sera, 36% of SS sera, 50% of maternal NLE sera and 20% of CHB infant sera had anti-RoSP7-24 binding levels greater than 2 standard deviations above the mean of that of normal individuals. Of the sera which had anti-SS-A/Ro detected by double immunodiffusion and/or counterimmunoelectrophoresis, 68% of SCLE patients, 71% of SS patients, 55% of NLE mothers and 20% of CHB infants had significantly elevated RoSP7-24 ELISA binding levels. These findings strongly suggest that a major autoepitope of native human SS-A/Ro resides on the amino terminal portion of the Wil-2 SS-A/Ro 60 kD polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The prevalence of autoantibody and its relationship with genotypes of hepatitis C virus in patients with chronic hepatitis C virus infection

The prevalence of autoantibody in the patients with chronic hepatitis C infection, and the relationship between the autoantibodies and HCV genotypes were investigated in this study. One hundred and eight anti‐HCV positive and 86 anti‐HCV negative patients were included in the study. Anti‐HCV were studied by enzyme immunassay (EIA). HCV RNA was determined by real time polymerase chain reaction (PCR) and HCV genotypes were determined by a reverse‐line blot hybridization. Anti‐nuclear antibodies (ANA), anti‐smooth muscle antibodies (ASMA), Anti‐mitochondrial antibodies (AMA), liver kidney microsomal antibodies (LKM) were detected by indirect immunofluorescence assay. Among patients, 13 (12.03%) of 108 were positive for at least one autoantibody. The positivity was not observed in control group. The most prevalent autoantibody in anti‐HCV positive group was ANA. ANA was positive in six HCV patients with genotype 1. In HCV patients with genotype 1, the frequencies of ANA, ASMA, AMA and LKM1 were six, two, three and one, respectively. In HCV patients with genotype 2, ANA was positive one patient and ASMA, AMA and LKM1 were not detected in HCV patients with genotype 2. In conclusion, the autoantibodies in patients with chronic hepatitis C in the study were low as compared to those reported in previous studies.     

Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis.

Fifty-five serum samples from patients with reactive arthritis (ReA), 40 from patients with ankylosing spondylitis (AS) and three from patients with chronic sacroiliac joint arthritis were analysed for the presence of ANCA of IgG class by means of enzyme immunosorbent assay using lactoferrin (Lf), myeloperoxidase (MPO) and antigen extracted from azurophil granules ('alpha-antigen') containing proteinase 3 (PR3) as substrate. IgG-ANCA were found in 31 (56%) patients with ReA. Twenty-three (42%) had anti-Lf antibodies, nine (16%) had anti-MPO and eight (15%) had anti-alpha-antigen antibodies, none of which reacted with PR3. Only six (14%) AS or sacroiliac joint arthritis patients had ANCA (P < 0.001). Three (7%) had anti-Lf, two (5%) anti-MPO and two (5%) anti-alpha-antigen antibodies. Yersinia and Salmonella bacteria were separated by SDS-PAGE and blots were incubated with serum from rabbits immunized with human Lf. The hyperimmune serum recognized a band of 78 kD from both bacteria which was not seen when preimmune serum was used. The reaction to the 78-kD antigen could be completely inhibited when anti-Lf antibodies were absorbed on Lf coupled to cyanogen bromide-activated Sepharose, possibly indicating cross-reacting epitopes in Lf and enterobacterial antigen.     

Anti-neutrophil cytoplasmic antibody (ANCA) in malaria is directed against cathepsin G

Autoantibodies of diverse specificities are detected in sera of patients with acute malaria. The clinical relevance of these autoantibodies is not clear, though there are reports associating some autoantibodies with specific disease manifestations. We have investigated the occurrence of ANCA in the sera of 93 patients during episodes of acute malaria. Sera were tested by indirect immunofluorescence (IIF) and by ELISA for antibodies to neutrophil cytoplasmic components proteinase 3 (PR3), myeloperoxidase (MPO), cathepsin G (CG), human leucocyte elastase (HLE), and lactoferrin (LF). Forty-seven sera samples (50.5%) were positive by IIF, all except one with the atypical ANCA pattern (a-ANCA). When screened by ELISA, anti-CG antibodies were detected in 52 samples (56%), while anti-PR3 and anti-MPO antibodies were detected in three and one samples, respectively. Antibody binding to HLE and LF was not significant. Anti-CG antibodies were detected in 93% of the IIF-positive sera. A combination of anti-CG and anti-PR3 antibodies was noted in three samples. Our study demonstrates the presence of ANCA in sera from patients with acute malaria, almost all with the a-ANCA pattern on IIF. The antibody specificity, noted for the first time in our study, appears to be predominantly directed against CG. The significance of CG and CG-ANCA in the pathogenesis and clinical manifestations of malaria has yet to be elucidated.     

High prevalence of anti-cardiolipin and other autoantibodies in a healthy elderly population.

Serum samples from 64 apparently healthy individuals (32 men and 32 women, mean age 81.0 years) were examined for the prevalence of several autoantibodies, including rheumatoid factor (RF), antinuclear antibodies (ANA), antibodies to extracted cellular antigens Ro (SSA), La (SSB), Sm, U1nRNP and Scl-70. IgG and IgM isotype-specific ELISA methods were applied for the detection of antibodies to ssDNA (anti-ssDNA), to dsDNA (anti-dsDNA) and to cardiolipin (anti-CL). The sera of this elderly population were found to contain a plethora of autoantibodies; RF was detected in 14.1%, ANA in 31.3% and anti-Ro (SSA) in 1.6% of the individuals. Precipitating antibodies to La (SSB), Sm, U1nRNP and Scl-70 were absent, while 15.6% of the sera displayed precipitating antibodies to a common undefined human spleen antigen. ELISA methods revealed anti-ssDNA in 17.2% of the individuals, anti-dsDNA in 14.1% and anti-CL in an extremely high incidence (51.6%). Notably, the above autoantibodies were exclusively of IgG isotype. Tests of 261 sera from healthy non-elderly individuals disclosed only anti-CL (IgG and IgM isotypes) in 2.3% of them. The levels of IgA and IgG immunoglobulins were increased in 23.4% and 29.7% of the elderly subjects, respectively. IgM was elevated in 3.1%, but it was also found decreased in 9.4%. This study documents the high incidence of autoantibodies in the healthy elderly, including for the first time, anti-CL antibodies. Furthermore, the relative impairment in IgM autoantibody production observed, possibly indicates the involution of the senescent immune system.     

Autoantibody prevalence in children with liver disease due to chronic hepatitis C virus (HCV) infection

HCV infection and interferon-alpha (IFN-α) therapy have been associated with autoimmunity. To assess whether chronic liver disease (CLD) due to HCV infection or its treatment with IFN-α cause autoimmune manifestations, the prevalence of tissue autoantibodies in 51 children with chronic HCV infection and 84 with other CLD was analysed by standard techniques. Sixty-five percent of patients with chronic HCV infection, 66% with chronic hepatitis B infection and 60% with Wilson's disease were positive for at least one autoantibody. In the 51 subjects with chronic HCV infection (29 treated with IFN-α, 22 untreated), tested on 165 occasions over a median of 9 months (range 5–42 months), autoantibodies to nuclei (ANA), smooth muscle (SMA), gastric parietal cell (GPC) and/or liver kidney microsomal type 1 (LKM-1) were similarly prevalent in treated and untreated patients (90% versus 68%, P = 0.12). Positivity for SMA was present in 67%, GPC in 32%, ANA in 10%, LKM-1 in 8% of cases. Treatment with IFN-α had to be suspended due to transaminase elevation in one SMA-positive, one ANA-positive but in three of four LKM-1-positive patients. Our results show that: (i) autoantibodies are common in viral-induced hepatitis and Wilson's disease; (ii) positivity for SMA, GPC, ANA is part of the natural course of chronic HCV infection, their prevalence being unaffected by IFN-α; and (iii) IFN-α should be used cautiously in the treatment of LKM-1/HCV-positive patients.     

Epitope specificity of myeloperoxidase antibodies: identification of candidate human immunodominant epitopes

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are a common feature of systemic vasculitides and have been classified as autoimmune conditions based, in part, on these autoantibodies. ANCA are subdivided further based on their primary target: cytoplasm (c-ANCA) or perinuclear region (p-ANCA). p-ANCAs commonly target myeloperoxidase (MPO), an enzyme with microbicidal and degradative activity. MPO antibodies are non-specific for any single disease and found in a variety of vasculitides, most commonly microscopic polyangiitis. Despite their prevalence, their role in human disease pathogenesis remains undefined. We sought to characterize the sequential antigenic determinants of MPO in vasculitis patients with p-ANCA. Of 68 patients with significant levels of p-ANCA, 12 have significant levels of MPO antibodies and were selected for fine specificity epitope mapping. Sequential antigenic targets, including those containing amino acids (aa) 213-222 (WTPGVKRNGF) and aa 511-522 (RLDNRYQPMEPN), were commonly targeted with a prevalence ranging from 33% to 58%. Subsequent analysis of autoantibody binding to the RLDNRYQPMEPN peptide was assessed using a confirmatory enzyme-linked immunosorbent assay format, with six patients displaying significant binding using this method. Antibodies against this epitope, along with four others (aa 393-402, aa 437-446, aa 479-488 and aa 717-726), were reactive to the heavy chain structure of the MPO protein. One epitope, GSASPMELLS (aa 91-100), was within the pro-peptide structure of MPO. B cell epitope prediction algorithms identified all or part of the seven epitopes defined. These results provide major common human anti-MPO immunodominant antigenic targets which can be used to examine further the potential pathogenic mechanisms for these autoantibodies.     

Rheumatic manifestations of hepatitis C virus infection are associated with autoantibodies but not viremia

BackgroundTo investigate the associations between extrahepatic manifestations, autoantibodies, and viremia in patients with hepatitis C virus (HCV) infection.MethodsThis cross-sectional study recruited patients with HCV infection from the outpatient department of a tertiary medical center in Northern Taiwan between January 2017 and August 2019. Autoantibody profiles and the clinical parameters of HCV infection were evaluated using laboratory tests, and a questionnaire was used to record extrahepatic manifestations. HCV infection status, including inactive HCV infection, active hepatitis, and cirrhosis, was defined according to abdominal ultrasonography findings and alanine transaminase levels.ResultsA total of 77 patients with HCV were recruited, with 19.5% and 16.9% of patients, respectively, presenting with arthritis and dry eyes. Autoantibody screening revealed rheumatoid factor (RF), antinuclear antibody (ANA), anti-Ro antibody, and anti-La antibody positivity in 20.8%, 23.4%, 13.0%, and 2.6% of the patients, respectively. The presence of RF was associated with arthritis, whereas the presence of ANA was associated with dry eyes but not dry mouth. Active hepatitis and HCV-related cirrhosis were associated with viremia, but not autoantibody profiles.ConclusionIn this single-center study, the prevalence of extrahepatic manifestations and autoantibodies did not differ in patients stratified by the HCV infection status. Rheumatic manifestations were associated with the presence of autoantibodies but not with viremia.     

Prevalence in myositis of antibodies recognizing anti-U3 RNA probably in a novel complex with 22/25 kD protein and not fibrillarin.

New antibodies against a U3 snRNP, which were named anti-Myo 22/25 antibodies, were detected in four (8%) of 53 serum samples from patients with polymyositis/dermatomyositis (PM/DM) by RNA immunoprecipitation. In the protein immunoprecipitation analysis, all four serum samples precipitated 22 kDa and 25 kDa proteins, which were not precipitated by normal serum or serum positive for antifibrillarin antibodies. Three of the four PM/DM patients had other identified autoantibodies including anti-PL-12 antibodies, antihistone antibodies (AHA), anti-SS-A antibodies and anti-SS-B antibodies defined by double immunodiffusion, ELISA or RNA immunoprecipitation, although there were no significant correlations between anti-Myo 22/25 antibodies and clinical or laboratory findings. There may be a subgroup of PM/DM patients whose sera are positive for anti-Myo 22/25 antibodies.     

Mechanisms of autoantibody production and the relationship between autoantibodies and the clinical manifestations in Sjögren's syndrome

The major target organs of Sj?gren's syndrome (SS) are lacrimal glands and salivary glands where prominent lymphocytic infiltration occurs, which may induce varying levels of autoantibody production. Multiple factors, including environmental stress, viral infection, hormonal imbalance, and apoptosis, are thought to be involved in the pathogenesis of SS. Production of anti-SS-A/Ro and anti-SS-B/La antibodies is thought to be regulated by the presentation of autoantigens in context with an aberrant expression pattern of human leukocyte antigen (HLA) in situ. Molecular mimicry with some viral sequences is also hypothesized. The apoptosis-resistance phenotype of B cells in labial salivary glands (LSGs) of SS is important in autoantibody production. CD40/CD40L (CD40 ligand) and Bcl-2 family proteins, in tandem with B cell-activating factor (BAFF), are supposed to protect infiltrating lymphocytes from apoptosis. Anti-muscarinic3 receptor antibody plays an important role in cholinergic hyperresponsiveness in SS. Fragmentation of autoantigens such as SS-B/La or alfa-fodrin during the process of apoptosis causes the redistribution of these autoantigens, leading to the production of autoantibodies in SS. In this review, the role of autoantibodies found in SS, corresponding to clinical aspects of each antibody as well as the mechanisms of production, is discussed.     

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.     

慢性丙型肝炎患者血清ANA、anti-LKM1的检测及其产生机制探讨

目的 观察慢性丙型肝炎(CHC)患者中抗核抗体(ANA)、抗肝肾微粒体抗体(anti-LKM1)的检出情况,并深入探讨其产生机制.方法 通过多因素分析探讨自身抗体产生与年龄、性别、HCV RNA含量、HCV基因型、生化指标及临床特征等指标的关系.结果 360例CHC患者中,ANA阳性率为12.5%(451360),anti-LKMi的阳性率为2.5%(91360).CHC患者的自身抗体检出率高于慢性乙型肝炎(CHB)患者(15%vs2.9%,P=0.006)而低于自身免疫性肝炎(AIH)患者(15%vs47.9%,P<0.001);女性患者的自身抗体检出率高于男性(P<0.05);自身抗体阳性组HCV RNA含量低于自身抗体阴性组(1.23×107 vs 7.2× 107拷贝/L,P<0.05).自身抗体阳性组和阴性组患者的年龄、HCV基因型、生化指标、肝硬化发生率的差异均无统计学意义.接受干扰素治疗组和未接受干扰紊治疗组患者的自身抗体检出率差异无统计学意义(P>0.05).结论 CHC患者血清中可检测出AIH相关自身抗体;自身抗体可能并非由干扰素治疗所诱发;很可能是HCV引发自身免疫,导致自身抗体的出现.     

Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.     

Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen,enhancing recognition by anti-Ro60 kD autoantibodies

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide–calreticulin is recognized specifically by anti‐Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi‐step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175–184aa(10p) and 216–232aa(17p). The interaction of the peptide–calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty‐eight anti‐Ro60 KD positive and 23 anti‐Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti‐Ro60 kD positive sera bound the complex calreticulin‐17p, while 95% of the same sera had activity against the complex calreticulin ? 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti‐Ro60 kD positive sera. Anti‐Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10ρ and 17ρ or with the complexes calreticulin ? 10p and calreticulin‐17p (<5%). These results suggest that calreticulin can induce conformation‐dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.