Occurrence of IgA and IgG autoantibodies to calreticulin in coeliac disease and various autoimmune diseases

Calreticulin (CRT), a high-affintiy calcium binding protein and chaperone, was recently identified as one of the targets of autoantibodies in coeliac disease. We evaluated the level of IgA and IgG antibodies to CRT in sera from patients with coeliac disease and various autoimmune diseases. The level of antibodies to gliadin (shown previously to cross-react with CTR), isolated enterocytes and tissue transglutaminase were determined for comparison. The mean level of IgA antibodies to CRT was significantly higher (P< 0.001) in sera from coeliac patients with active disease (139.9+/-11.2 AU/+/-SE) than in healthy controls (20.9+/-1.7 AU). In sera of patients with systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), multiple sclerosis (MS) and autoimmune thyroiditis (AT) or inflammatory bowel disease (IBD) the mean level (25.8+/-3.7 to 38.1+/-5.6 AU) did not exceed the cut-off value. A low level of these antibodies, however, was detected in some sera of patients with MS and IBD. The level of IgG anti-CRT antibodies was increased in coeliac patients (mean 125.4+/-8.0 AU, P< 0.001) when compared to that in healthy controls (33.9+/-2.3 AU). The IgG anti-CRT antibodies were also detected in about 30% of SLE patients sera (54.1+/-3.6 AU, P< 0.001), but the mean level reached only half that detected in coeliac patients.     

Anti-C1q receptor/calreticulin autoantibodies in patients with systemic lupus erythematosus (SLE)

SLE is a disease characterized by the presence of multiple autoantibodies and high levels of circulating immune complexes. We studied the presence and functional relevance of autoantibodies directed against a receptor for the collagen-like stalks of the first subcomponent of complement, also known as calreticulin (cC1qR/CaR), in patients with SLE. In a cross-sectional study it was found that higher titres of antibodies against cC1qR/CaR are present in sera of SLE patients compared with normal donors. No association between anti-cC1qR/CaR titres and SLE disease activity was found. Following gel filtration of SLE serum it was found that anti-cC1qR/CaR reactivity is associated with the peak of monomeric IgG. Purified IgG from patients was able to specifically immunoprecipitate cC1qR/CaR. Since we have shown previously that cC1qR/CaR is able to inhibit the haemolytic activity of C1q, we determined a possible pathogenic role for anti-cC1qR/CaR on complement regulation. IgG derived from SLE serum reversed the inhibitory capacity of cC1qR/CaR in a dose-dependent fashion up to 63%, whereas IgG from normal donors had no significant effect. With respect to the capacity of anti-cC1qR/CaR antibodies to activate neutrophils, it was found that incubation of normal neutrophils with F(ab′)₂ anti-cC1qR/CaR resulted in a very limited oxidative burst. However, cross-linking of F(ab′)₂ anti-cC1qR/CaR on the neutrophils clearly induced neutrophil activation. Pre-incubation of the SLE-derived F(ab′)₂ with cC1qR/CaR prevented activation of neutrophils up to 81 ± 5%. These results suggest that the presence of anti-cC1qR/CaR antibodies in patients with SLE may modulate complement and neutrophil activation.     

Onset and regulation of anti-lamin B autoantibody production is independent of the level of polyclonal activation

Anti-lamin B autoantibodies are associated with both systemic lupus erythematosus (SLE) and autoimmune liver disease. We examined the possibility that the underlying clinical feature in patients with anti-lamin B autoantibodies might be chronic autoimmune liver disease, and whether the hypergammaglobulinemia present in both disorders is involved in generating anti-lamin B autoantibodies. A lamin B fusion protein (MLB1), consisting of amino acids 77-533 of lamin B fused to TrpE, was used to screen sera from 84 patients with SLE for anti-lamin B autoantibodies. 3/4 prototype human lamin B antisera, 5/84 SLE sera (6%), and 0/30 sera from healthy individuals reacted with MLB1 on immunoblots at a 1:500 dilution. Of the 9 anti-lamin B autoantibody positive patients studied, all but 1 fulfilled at least four ARA criteria for SLE. None of the patients displayed evidence of chronic autoimmune liver disease, suggesting that autoimmune liver disease is not strongly associated with anti-lamin B antibodies in SLE. In SLE, as in "lupoid hepatitis", anti-lamin B autoantibodies are often produced transiently during periods of increased disease activity. Although polyclonal hypergammaglobulinemia is also associated with increased activity of both diseases, anti-lamin B autoantibody production in 2 patients was independent of total immunoglobulin levels, antibodies to irrelevant proteins, and production of some other autoantibodies. Thus, polyclonal activation is insufficient to explain either the initiation or regulation of anti-lamin B autoantibody production, supporting the hypothesis that antinuclear antibodies are antigen-selective.     

Autoantibodies to major and minor nuclear lamins are not restricted to autoimmune diseases.

Autoantibodies to lamins, the major polypeptide components of the nuclear lamina, have been reported in selected sera from patients with autoimmune diseases, including anti-lamin B in systemic lupus erythematosus (SLE) and anti-lamins AC in autoimmune chronic active hepatitis (CAH). We have studied the frequency, specificity, and isotypy of autoantibodies to major and minor lamins by immunoblotting on purified rat liver lamins in 190 sera from normal controls (n = 62), rheumatic disease controls (n = 42), and autoimmune disease patients (n = 86). The frequency of anti-lamin in normal controls was 85.5%, and ranged from 77 to 100% in the other groups. Anti-lamin frequency was not related to age, sex, or disease duration. Reactivity with lamin A or with minor lamins only was observed with 7 various sera and 2 normal sera, respectively. Between groups, the proportions of reactive sera were not different for lamins AC (18-47%) and for lamin B (22-36%). In particular, anti-lamin B and anti-lamins AC were not more common in SLE or CAH than in normal sera. The most frequent lamin specificity of SLE sera was anti-lamins ABC. Anti-lamin isotypes were IgG and/or IgM. Titers of IgM antibodies were not higher in any group. However, IgG anti-lamin titers were higher in CAH than in normal, ankylosing spondylitis, or SLE sera. The highest end point titers (greater than or equal to 1:3200) were observed with CAH, SLE, and rheumatoid arthritis (RA) sera with IgG anti-lamins AC, B, or ABC, or with IgM anti-lamins ABC. None of these SLE and RA patients had evidence of liver disease. Reactivity with minor lamins was more frequent in CAH. We conclude that anti-lamin autoantibodies are present in sera from most individuals and that the highest titers are found in sera from patients with autoimmune diseases.     

Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization

Extracellular calreticulin (CRT) as well as anti‐CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in ‘epitope spreading’ to other autoantigens such as the Ro/SS‐A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti‐CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjögren’s syndrome. Approximately 40% of all SLE patients were positive for anti‐CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1–289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N‐terminal half of the protein in 69% of the SLE sera from active disease patients, while the C‐domain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P‐domains. Sera from both healthy and disease controls and primary Sjögren’s syndrome patients were non‐reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N‐terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera.

The ability of synthetic peptides encompassing almost the entire sequence of snRNP U1A polypeptide to be recognized in ELISA by sera of autoimmune patients was investigated. Sera from 18 patients with mixed connective tissue disease (MCTD), 145 with systemic lupus erythematosus (SLE) and 120 with other rheumatic autoimmune diseases were tested with 13 overlapping peptides. Among them, peptide 257-282 and, to a lower extent, peptide 1-11 were recognized by MCTD, SLE and Sjögren''s syndrome sera. In contrast, peptide 35-58 was recognized by 94% of MCTD and only 19% of SLE sera. It did not react with any of the other patient sera. The ELISA results were compared with the pattern of reactivity observed in immunoblotting. The results indicate that peptide 35-58 probably contains a major epitope recognized by MCTD autoantibodies. It is noteworthy that in snRNP particles, this region of U1A interacts with RNA and presents only limited homology with the corresponding sequence 32-50 of U2B''''.     

Antiribonuclease H2 antibodies are an immune biomarker for systemic lupus erythematosus

We previously reported that autoantibodies against the proliferating cell nuclear antigen protein (PCNA)-binding protein chromatin assembly factor-1 (CAF-1) are specifically found in patients with systemic lupus erythematosus (SLE). PCNA and its complex constituents elicit autoimmune responses in patients with SLE, suggesting that autoantibody diversification likely occurs owing to epitope spreading. Therefore, we sought to clarify whether patients with SLE exhibit an autoimmune response to Ribonuclease H2 (RNase H2), another PCNA-binding protein that regulates cell division. As results, RNase H2 autoantibodies were detected in the sera of 33.9% (19/56) of SLE patients, which was significantly higher than that observed in sera from other patients with systemic autoimmune diseases (polymyositis/dermatomyositis, systemic sclerosis, Sjogren’s syndrome, mixed connective tissue disease and rheumatoid arthritis) and healthy controls. Regression analysis also showed that serum anti-RNase H2 levels were strongly correlated to that of CAF-1 in SLE patients. Our data support the use of RNase H2 autoantibodies as a serum biomarker for SLE diagnosis. Moreover, the strong correlation observed between RNase H2 and CAF-1 suggests that intermolecular epitope spreading may play a critical role in autoantibody production and diversification in SLE.     

High circulating levels of free interleukin-18 in patients with active SLE in the presence of elevated levels of interleukin-18 binding protein

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies particularly to nuclear antigens and by an abnormal production of proinflammatory cytokines. In the present study, we measured the levels of the proinflammatory cytokine IL-18 and its natural inhibitor, the IL-18 binding protein (IL-18BP), in sera of SLE patients at various stages of the disease. This is the first study to present IL-18BP levels in sera of SLE patients as well as the calculated, biologically active, free IL-18 concentrations that are most probably more relevant to the pathology of SLE. Sera from 48 unselective SLE patients (total of 195 samples) were obtained longitudinally with a mean follow-up period of 11.1 ± 8.9 years and were compared to sera from 100 healthy volunteers. Circulating levels of IL-18, IL-18BP and free IL-18 in the SLE patients were significantly higher than the levels of healthy controls (5 fold, 6 fold and 3 fold for IL-18, IL-18BP and free IL-18, respectively) and correlated with disease activity as scored by SLEDAI-2K. Furthermore, these levels during active disease (SLEDAI-2K ≥ 6) were higher compared to the levels measured in the sera of the same patients during remission or during mild disease (SLEDAI-2K 0–5). The high levels of IL-18 and IL-18BP in sera of active SLE patients suggest their possible role in the pathogenesis and course of the disease. However, despite the elevated levels of IL-18BP during active disease, free IL-18 remained more than 2 fold higher than the levels in healthy controls suggesting a potential benefit of administration of exogenous IL-18BP as a novel therapeutic approach for active SLE.     

系统性红斑狼疮患者血清脱氧核糖核酸酶活性检测的临床意义

检测系统性红斑狼疮(SLE)患者血清脱氧核糖核酸酶(DNase)的活性,探讨其在判断SLE疾病活动及狼疮肾炎(LN)中的临床意义.采集116例SLE患者、30例疾病对照者和30名健康体检者(对照组)的血清,用ELISA法定量检测血清DNase活性.结果表明:SLE患者血清DNase活性明显低于疾病对照组和健康对照组(P<0.01),且疾病活动期低于缓解期(P<0.01);DNase活性与SLE疾病活动程度呈负相关(P<0.01);狼疮肾炎组DNase活性低于SLE非狼疮肾炎组,两组比较有统计学意义(P<0.05).DNase活性下降可能是促进SLE及LN发生、发展的重要因素之一.血清DNase活性检测可用于判断SLE及其疾病活动程度的指标之一,同时也可作为判断LN的危险因素之一.     

Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. The SLE Study Group.

Recent studies have shown that sera of patients suffering from systemic autoimmune diseases contain autoantibodies directed against the eukaryotic ribosomal protein L7 [1]. In the present study we screened a large panel of sera from patients with systemic lupus erythematosus (SLE) for the presence of anti-L7 autoantibodies and their relationship to clinical, serological and genetic parameters of SLE. By means of an ELISA employing recombinant protein L7 as antigen we detected anti-L7 autoantobodies in 172 of 506 SLE sera (34%). Negative correlations were observed between the presence of anti-L7 autoantibodies, serum IgG levels and proteinuria; a potentially positive relationship existed with lung fibrosis. In order to analyse further this possibility we screened sera of 129 patients suffering from progressive systemic sclerosis (PSS) for anti-L7 reactivity; 45 of these patients had lung fibrosis. Of the PSS patients, 41% exhibited anti-L7 autoantibodies, but positive reactions were evenly distributed among patients with and without lung fibrosis. Protein L7 thus represents a major autoantigen of systemic autoimmune diseases, but does not so far define a distinct subpopulation of patients.     

IgG antibodies from patients with primary Sjögren''s syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.

Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (≤7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21–41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21–41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.     

Evaluation of the Reactivity of Sera from Patients with Systemic Lupus Erythematosus Against the Human MCP1

This study evaluates metaphase chromosome protein 1 (MCP1), a nuclear antigen, as a diagnostic marker for systemic lupus erythematosus (SLE). Reactivity of sera from 114 Portuguese patients with autoimmune rheumatic disease or from healthy blood donors (HBD), against MCP1, produced in bacteria (bact-MCP1) or in its native form (native-MCP1), was determined by immunoblotting. Predictive and discriminative power of MCP1 reactivity for SLE diagnosis in disease-control groups was evaluated by logistic regression, its diagnostic value determined by receiver-operating characteristic analysis and compared with similar analysis of antinuclear antibody and double-stranded DNA (dsDNA). We demonstrated that native-MCP1, in contrast to bact-MCP1, reacts with SLE sera with significant predictive and discriminative power versus other autoimmune diseases (odds ratio [OR] ≤3.537 and ≥3.265; area under the receiver-operating characteristic curve [AUC] ≤0.643 and ≥0.636) or versus HBD (OR?=?5.006; AUC?=?0.671), showing a good diagnostic power with high specificity (82.1% versus HBD) and low sensitivity for SLE, similar to those of dsDNA. The reactivity of SLE sera with native-MCP1 was shown to be dependent on the presence of phosphorylated residues. Native-MCP1 was shown to have diagnostic value as a specific marker for SLE diagnosis and, therefore, is a suitable substrate for a new antibody test. The widely reported importance of phosphorylated epitopes as targets for autoantibodies in SLE could also be confirmed for native-MCP1.     

Induction of apoptosis in monocytes and lymphocytes by serum from patients with systemic lupus erythematosus − an additional mechanism to increased autoantigen load?

The most likely source of autoantigens in systemic lupus erythematosus (SLE) is apoptotic material. Because increased levels of circulating apoptotic cells are found in SLE we wanted to investigate the capacity of serum from patients with SLE or other autoimmune or infectious diseases and normal healthy donors (NHD) to induce apoptosis in normal monocytes, lymphocytes and corresponding cell lines, in relation to clinical and immunological data. Monocytes and lymphocytes from healthy donors were incubated with sera from 37 SLE patients, 37 sex- and age-matched NHD and sera from patients with rheumatoid arthritis, vasculitis, sepsis and mononucleosis. Sera from SLE patients were sampled at both active and inactive disease. The apoptosis-inducing effect (AIE) of these sera was monitored with flow cytometry using annexin V and propidium iodide (PI) binding. The AIE in monocytes and lymphocytes was significantly higher in sera from SLE patients than in other patient groups and NHD (P < 0.001) and was also higher when cell lines were used. Level of C5a in cell culture supernatant correlated with AIE in monocytes (r = 0.451, P = 0.005), suggesting involvement of complement. Heat-inactivation of sera did not affect the AIE, nor did depletion of IgG by protein G absorption of serum. Kinetic analyses showed a peak in apoptosis induction at 12-16 h, with a delayed PI positivity. AIE was equally high using sera from active and inactive SLE cases, and did not correlate with the SLE Disease Activity Index (SLEDAI). Thus, SLE serum has a strong and apparently disease-specific apoptosis-inducing capacity, which could contribute to a high load of potential autoantigen.     

Linear epitopes of two different autoantigens-La/SSB and myelin basic protein--with a high degree of molecular similarity,cause different humoral immune responses

The region 147-154aa of La/SSB presents 83% sequence similarity with the 139-146aa region of the human myelin basic protein (MBP). The aim of this study was to investigate the prevalence and significance of antibodies against both epitopes in sera from patients with systemic autoimmune diseases, and to compare the humoral responses produced after rabbit immunization. Peptides 147-154aa of La/SSB and 139-146aa of the MBP were attached on tetrameric sequential oligopeptide carriers and used for immunizations of New Zealand White rabbits. Antibodies to immunizing peptides, as well as to the peptides corresponding to other previously defined La/SSB epitopes (289-308aa, 349-364aa), to the intact human MBP (hMBP) and to the recombinant human La/SSB (rechLa) proteins, were identified using specific ELISA assays. Sera from 45 patients with Sjogren's syndrome (pSS), 49 with Systemic Lupus Erythematosus (SLE), and 18 with Rheumatoid Arthritis (RA) were tested against the two peptides and the hMBP. Twenty-two per cent of sera from pSS patients, 27% of SLE patients, and none from RA sera reacted with the La epitope; 27% from pSS sera, 22% of SLE sera, and 17% of RA sera gave a positive reaction against the MBP peptide. Finally, 19% of pSS, 30% of SLE, and 38% of RA sera reacted with the hMBP. Thirty-five days after immunization of rabbits with the La epitope, antibodies were produced against all three La/SSB peptides, the MBP peptide, and the hMBP and rechLa proteins. Rabbits immunized with the MBP peptide produced antibodies against the immunizing peptide and the mimicking peptide of La shortly after immunization, whilst antibodies against the other La epitopes and the two intact proteins were produced later. Inhibition experiments in rabbit sera with high reactivity against hMBP, using the MBP peptide as inhibitor, revealed that 80% of serum reactivity was abolished. In conclusion, a significant proportion of human autoimmune sera reacted with both La and MBP derived peptides, as well as with hMBP. La 147-154aa peptide, when used for animal immunizations, induces a fast epitope spreading involving both La and MBP. In contrast, the mimicking MBP epitope induces a delayed response against the other La epitopes. Thus, despite the fact that these peptides present molecular similarity, they induce different immune responses.     

Enhanced recognition of reactive oxygen species damaged human serum albumin by circulating systemic lupus erythematosus autoantibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore, we hypothesized that oxidative by-products, such as hydroxyl radical (*OH), could lead to neoantigens like *OH damaged human serum albumin (HSA), which could in turn initiate autoimmunity in SLE. In the present study, the binding characteristics of SLE autoantibodies with native and *OH damaged HSA were assessed. SLE patients (n = 74) were examined by direct binding ELISA and the results were compared with healthy age- and sex-matched controls (n = 44). High degree of specific binding by 52.7% of patients sera towards *OH damaged HSA, in comparison to its native analogue (p < 0.05) was observed. Normal human sera showed negligible binding with either antigen. Competitive ELISA and gel retardation assays reiterate the direct binding results. The increase in total serum protein carbonyl levels in the SLE patients was largely due to an increase in oxidized albumin. HSA of SLE patients (SLE-HSA) and normal subjects (normal-HSA) were purified. Spectroscopic analysis confirmed that the SLE-HSA samples contained higher levels of carbonyls than normal-HSA (p < 0.01). SLE-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in SLE patients.     

Enhanced recognition of reactive oxygen species damaged human serum albumin by circulating systemic lupus erythematosus autoantibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore, we hypothesized that oxidative by-products, such as hydroxyl radical (√OH), could lead to neoantigens like √OH damaged human serum albumin (HSA), which could in turn initiate autoimmunity in SLE. In the present study, the binding characteristics of SLE autoantibodies with native and √OH damaged HSA were assessed. SLE patients (n = 74) were examined by direct binding ELISA and the results were compared with healthy age- and sex-matched controls (n = 44). High degree of specific binding by 52.7% of patients sera towards √OH damaged HSA, in comparison to its native analogue (p < 0.05) was observed. Normal human sera showed negligible binding with either antigen. Competitive ELISA and gel retardation assays reiterate the direct binding results. The increase in total serum protein carbonyl levels in the SLE patients was largely due to an increase in oxidized albumin. HSA of SLE patients (SLE-HSA) and normal subjects (normal-HSA) were purified. Spectroscopic analysis confirmed that the SLE-HSA samples contained higher levels of carbonyls than normal-HSA (p < 0.01). SLE-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in SLE patients.     

Presence of antibodies to replication protein A in some patients with systemic lupus erythematosus (SLE)

Presence of antibodies directed against replication protein A (RPA), a DNA binding protein complex composed of three subunits (RPA-70, RPA-32 and RPA-14) was investigated among patients with SLE and other autoimmune diseases using immunoblot analysis to RPA-70 and RPA-32 recombinant proteins. Anti-RPA antibodies were found in two out of 108 sera from SLE patients, one of them showing reactivity against RPA-32 and RPA-70 and the other reacting only against RPA-32. Sera from 108 patients with other autoimmune disorders as well as from 42 healthy control individuals were negative. Thus, the frequency of these antibodies in SLE is estimated to be 2–3%. The study demonstrates that RPA is one target more of the wide array of autoantigens that elicit an immune response in SLE. The presence of anti-RPA autoantibodies seems to be circumscribed to a small number of patients with SLE.     

Elevated levels of soluble CD40 ligand (sCD40L) in serum of patients with systemic autoimmune diseases

The CD40–CD40L costimulatory pathway is involved in the evolution of many autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Increased levels of sCD40L in the serum have been associated with disease activity in SLE. The aim of this study was to investigate the role of sCD40L in the development of lupus nephritis and examine its possible association with cryoglobulinemia in Sjögren's syndrome. We used a 2-site sandwich ELISA to measure the levels of sCD40L in sera, from 64 patients with SLE, RA and SS and 17 healthy blood donors. Biological specimens from the affected tissues such as urine from patients with lupus nephritis and saliva from patients with SS were also tested. In this regard, paired sera and first morning urine samples from 6 SLE patients (3 with active lupus nephritis and 3 with inactive lupus nephritis) were tested with the sCD40L ELISA protocol as well as paired sera and salivary samples from 5 patients with SS and cryoglobulinemia, 5 patients with SS and anti-Ro or anti-La autoantibodies and 5 age-matched healthy control donors. We also examined possible correlations of sCD40L levels with several laboratory and clinical parameters in SS and SLE. We found that sera from SLE and SS patients had significantly higher levels of sCD40L compared to sera from healthy control donors. No sCD40L was detected, in urine samples of patients with either active or inactive nephritis and in salivary samples from SS patients or normal subjects. Soluble CD40L is elevated in sera of SS and SLE patients but further investigation is needed to determine its possible role in SLE nephritis and Sjögren's syndrome.     

Major Trypanosoma cruzi antigenic determinant in Chagas'' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease.