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Toll-like receptors (TLR) play an essential role in the activation of both innate and adaptive immune responses. Salivary gland epithelial cells (SGEC) may participate in the development of glandular inflammatory reactions that characterize primary Sjögren's syndrome (pSS). In this study we sought to assess the expression and function of several TLR molecules in cultured non-neoplastic SGEC obtained from pSS patients and disease controls. Long-term cultured non-neoplastic SGEC derived from pSS patients (SS-SGEC) and disease controls (control-SGEC), as well as the monocytic cell line THP-1 (positive control cell line), were examined by reverse transcription–polymerase chain reaction (RT–PCR) analysis and quantitative real-time PCR for mRNA expression of TLR1, -2, -3 and -4 molecules. TLR function was assessed by the induction of the expression (flow cytometry) of the immunoregulatory molecules CD54/intercellular adhesion molecule-1 (ICAM-1), CD40, CD86/B7·2, major histocompatibility complex (MHC) class I and MHC class II following treatment with the TLR ligands: Staphylococcus aureus peptidoglycan (TLR2), the synthetic dsRNA analogue polyinosinic:cytidylic acid (TLR3) and Escherichia coli lipopolysaccharide (TLR4). SGEC were found to express functional TLR2, -3 and -4 molecules, as attested by dose-dependent up-regulation of surface ICAM-1, CD40 and MHC-I expression (as well as of reciprocal TLR mRNA) following treatment with the respective TLR-ligands. SS-SGEC lines displayed significantly higher constitutive expression of TLR1 ( P = 0·0027), TLR2 ( P = 0·01) and TLR4 ( P = 0·03) mRNA compared to control-SGEC. This study demonstrates that cultured SGEC express functional TLR molecules; the high constitutive TLR expression by SS-SGEC is probably suggestive of the intrinsic activation of epithelial cells in pSS and further supports the role of this type of tissue in pathogenesis of the disorder. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration, destruction of the salivary and lacrimal glands and production of autoantibodies against a variety of cellular proteins. The aberrant immune response against these autoantigens may begin or extend to other proteins that are not yet defined. Several studies have shown that autoantibody production is taking place in the affected salivary glands. In the present study, using proteomic approaches, we aimed to: (a) identify new autoantigens in the salivary glands of primary SS (pSS) patients and (b) evaluate the epigenetic changes of known autoantigens. Total parotid gland extracts of pSS patients were analysed using two-dimensional gel electrophoresis, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with pSS patients' sera or purified autoantibodies and immunoprecipitation using homologous IgG. Identification of the unknown proteins was performed using mass spectrometry (MS). Immunoblot analysis on two-dimensional gels using purified anti-La/SSB antibodies revealed that pSS salivary glands contain high levels of post-translationally modified La/SSB autoantigen, while the native form of the protein is recognized faintly, in contrast to normal controls. Moreover, salivary glands of pSS patients contain post-translationally modified actin that becomes immunogenic in the microenviroment of the affected tissue. The alteration of the physicochemical properties of self-proteins could thus contribute to the break of immune tolerance against them. [ABSTRACT FROM AUTHOR]

Ro60 kDa is a member of the Ro/LaRNP ribonucleoprotein complex and its major linear B cell epitope, corresponding to the region 169–190aa, has been found to be the initial target of the autoimmune response in patients with systemic lupus erythematosus. This sequence contains one serine and two arginine amino acid residues, which can potentially be modified post-translationally by phosphorylation or citrullination, respectively. The aim of this study was to develop an immunoassay for anti-Ro60 kDa epitope antibody detection and to investigate the changes in the antigenicity of the Ro60 kDa epitope when it is post-translationally modified, by either citrullination or phosphorylation. Peptide analogues corresponding to the unmodified form of the epitope, its phosphorylated form, and a form with both arginine residues citrullinated were synthesized. The peptide coating conditions were investigated and it was found that the use of highly hydrophilic surfaces increase the efficiency of the coating, as well as the sensitivity of the method for anti-peptide antibody detection. All peptides were tested by the optimized enzyme-linked immunosorbent assay (ELISA) against 119 sera from patients with primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis with anti-Ro/SSA reactivity, 20 sera from patients with systemic diseases without anti-Ro/SSA immune reactivity, as well as against 65 sera from normal individuals. A large proportion of the tested sera reacted against all three peptide analogues, although with a preference for the unmodified form of the epitope. In conclusion, post-translational modifications of the major Ro60 kDa B cell epitope can alter the autoantibody binding. [ABSTRACT FROM AUTHOR]

Sera from patients with primary Sjögren Syndrome (pSS) or Systemic Lupus Erythematosus (SLE) often contain autoantibodies directed against La/SSB. The sequence 349–368aa represents the major B-cell epitope of La/SSB, also it contains, at position 366, a serine aminoacid residue which constitutes the main phosphorylation site of the protein. In this study we investigated the differential recognition of the 349–368aa epitope and its phosphorylated form by antibodies found in sera from patients with systemic autoimmune diseases. Peptides corresponding to the sequence of the unphosphorylated (pep349–368aa) and the phosphorylated form (pep349–368aaPh) of the La/SSB epitope 349–368aa, as well as to a truncated form spanning the sequence 349–364aa and lacking the phosphorylation site (pep349–364aa), were synthesized. Sera from 53 patients with pSS and SLE with anti-La/SSB specificity, 30 patients with pSS and SLE without anti-La/SSB antibodies, 25 patients with rheumatoid arthritis and 32 healthy individuals were investigated by ELISA experiments. Autoantibodies to pep349–368aaPh were detected in sera of anti-La/SSB positive patients with a higher prevalence compared to the pep349–368aa (66% versus 45%). Pep349–368aaPh inhibited the antibody binding almost completely (92%), while pep349–368aa inhibited the binding only partially (45%). Anti-La/SSB antibodies presented a higher relative avidity for the phosphorylated than the unphosphorylated peptide. Immunoadsorbent experiments using the truncated peptide pep349–364aa indicated that the flowthrough showed a selective specificity for pep349–368aaPh, while the eluted antibodies reacted with both peptide analogues of the La/SSB epitope. These data suggest that sera from pSS and SLE patients with anti-La/SSB reactivity possess autoantibodies that bind more frequently and with a higher avidity to the phosphorylated major B-cell epitope of the molecule. [ABSTRACT FROM AUTHOR]

Coxsackie virus RNA has recently been detected in biopsy specimens of minor salivary glands from patients with primary Sjögren's Syndrome (pSS). A peptide derived from Coxsackie virus 2B protein (pepCoxs) presents 87% sequence homology with the 222–229 region of the major linear B-cell epitope of Ro60 kD autoantigen (pep216–232). Synthetic peptides corresponding to pep216–232:216KALSVETEKLLKYLEAV232 and pepCoxs:31MVTSTITEKL LKNLVKI47, were prepared. Sera from 42 patients with pSS and 43 patients with systemic lupus erythematosus (SLE) as well as sera from 27 healthy individuals (normal controls) and sera from 30 patients with rheumatoid arthritis (disease controls) were tested against the two homologous peptides. Twenty-five percent of SLE sera and 33·3% of pSS sera reacted against pep216–232, whereas 28% of SLE sera and 37% of pSS sera recognized the pepCoxs. The sera reacting with pep216–232 were apparently the same as those reacting with pepCoxs. Normal sera and disease control sera presented only a limited reactivity against both peptides (ranging from 3·7% to 10%). Both peptides reacted more prominently with anti-Ro/La (+) sera from pSS patients. Thus, pep216–232 was recognized by 17% of the anti-Ro (+) sera and by 42% of the anti-Ro/La (+) sera, whereas pepCoxs was recognized by 28·5% and 38% of the a-Ro(+) and a-Ro/La(+) sera, respectively. Purified anti-pep216–232 antibodies readily reacted with both peptides while inhibition experiments revealed the specificity of this reaction. These results suggest a possible cross-reaction between antibodies to the major linear B-cell epitope of Ro60 kD autoantigen and the homologous pepCoxs in pSS patients. This cross-reaction might potentially play a role in autoantibody formation and the perpetuation of the autoimmune response against Ro/SSA and La/SSB. [ABSTRACT FROM AUTHOR]

Immunotherapies against autoimmune diseases have been of limited success. Preventive vaccines could be developed on the basis to abrogate unwanted immune responses to defined autodeterminants. In this study it is shown that immunization of BALB/c mice with two linear T and B cell epitopes of the human La/SSB autoantigen (spanning the regions 289–308aa and 349–364aa) and their complementary forms specified by the complementary mRNA, results in characteristic B and T cell responses. Mice immunized with the 289–308aa epitope or its complementary peptide elicited specific antibodies against both epitopes. In contrast, mice immunized with the 349–364aa epitope or its complementary peptide mounted antibody titres against the immunizing peptide only. According to these data, the 289–308aa epitope and its complementary form were capable to generate an idiotypic–anti-idiotypic response, which were cross-regulated. Peptide-specific T cell proliferation and cytokine production in vitro revealed the induction of a two-stage T helper response (Th1→Th2 type) after immunization with either the epitope 289–308 or its complementary peptide. IgG1 was the predominant subclass after immunization with the two forms of epitopes 289–308 and 349–364, while a response of the IgG2b > IgG2a was obtained after the immunization with the complementary form of 349–364 epitope reflecting the TH2/TH1 polarization, respectively. Our data suggest that the complementary peptides of two immunodominant epitopes of human LaSSB can mimic the autoantibodies against these epitopes and establish an active idiotypic–anti-idiotypic network. [ABSTRACT FROM AUTHOR]

SUMMARY 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. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated a possible antigenic relation between the carboxyl terminal portion of anti-Ro60kD autoantigen and a nucleocapsid protein (N) of vesicular stomatitis virus (VSV). In order to investigate whether anti-Ro60kD autoantibodies react with the VSV homologous region of the Ro60kD protein we synthesized, according to Merrifield's method, the EYRKKMDI octapeptide (8p) sharing a common sequence with the N protein of VSV. Sera from 61 patients with autoimmune rheumatic diseases (34 systemic lupus erythematosus (SLE), 21 Sjögren's syndrome (SS) and six rheumatoid arthritis (RA)) as well as 59 from normal blood donors were tested for the presence of anti-Ro60kD autoantibodies by ELISA and immunoblot (IB) and anti- 8p antibodies by ELISA. Antibodies to 8p were found in 9/31 of anti-Ro60kD IB-positive sera, 5/30 of anti-Ro60kD-negative sera and 2/59 of normal control sera. The concordance between the anti-8p ELISA and the anti-Ro60kD IB was very poor (x2 = 0.71, P = 0.4) in contrast to the anti- Ro60kD ELISA and the anti-Ro IB(x2 = 27.6, P = 10-7). Subsequent affinity purification of the anti-8p antibodies from a strong positive anti-8p and anti-Ro60kD SLE serum yielded 95% depletion of the anti-8p activity and 37% reduction of the anti-Ro60kD activity. Inhibition assays with the affinity-purified anti-8p antibodies demonstrated that the octapeptide gave 94.5% inhibition of the anti-Ro60kD activity, while Ro60kD protein led to 42.3% inhibition of the anti-8p, Preincubation of the serum with the octapeptide produced 4% inhibition of anti-Ro60kD ELISA, These results indicate that the anti-8p antibodies account only for a minority of the anti- Ro60kD autoantibodies. [ABSTRACT FROM AUTHOR]

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 lo 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), lo the CaR-peptide analogues. These findings suggest that anti-calreticulin autoantibodies are not restricted to any disease specificity. [ABSTRACT FROM AUTHOR]

The aetiology of sustained autoantibody production in human autoimmune diseases is unknown. Evidence for structural similarities and common clonal origin among autoantibodies have been demonstrated through the expression of cross-reactive idiotype (CRI). In the present study we use four monoclonal antibodies (MoAbs) with specificity for non-overlapping CRI on human rheumatoid factor (RF) autoantibodies to define the structural features of polyclonal RF characteristic of patients with autoimmune rheumatic diseases. The pattern of CRI expression in the serum of 12 patients with rheumatoid arthritis (RA), eight with systemic lupus erythematosus (SLE) and 20 with primary Sjögren's syndrome and 34 normal individuals were determined in parallel with the level of IgM RF, IgA RF and autoantibodies to the cellular antigens SS-A, SS-B, Sm, nRNP and dsDNA and cryoglobulins. The results demonstrate significant elevation in the level of IgM and IgA expressing VnI (G6 and G8) and VnIII (B6 and DI2) associated CRI in the serum of patients with autoimmune rheumatic diseases compared with normal individuals. These increases paralleled, but did not equal the increase in the level of immunoglobulins and RF. However, when expressed as proportion of immunoglobulin, only the Vnl-associated CRI were significantly elevated in patients compared with normal individuals. The proportion of IgM RF expressing the VnI-associated CRI was higher in patients with Sjögren's syndrome compared with SLE and RA. Furthermore, the proportion of IgA RF expressing the G6 CRI was higher than G6+ IgM RF. These findings imply that different mechanisms contribute to RF production in autoimmune diseases. It is suggested that polyclonal B cell activation is likely to be a contributing mechanism. However, such polyclonal activation is unlikely to be random since a selective elevation in the level of specific autoantibodies and VH I-associated CRI is observed. Furthermore, the data demonstrate that a proportion of auto-antibodies in autoimmune diseases are immunoglobulin germline gene encoded. This is more evident in some patients with primary Sjögren's syndrome, where RF is likely to be oligoclonal or monoclonal in individuals with lymphoproliferation. [ABSTRACT FROM AUTHOR]

Monoclonal or oligoclonal B cell products have been described in the sera and urine of patients with primary Sjögren's syndrome (PSS). In addition, monoclonal expansion of plasma cells has been found in the exocrine glands of PSS patients with circulating monoclonal B cell products. The goal of this study was to raise an anti-idiotype to a cryoprecipitable monoclonal IgM kappa rheumatoid factor (RF) from it PSS patient. Using the F(ab')2 fragments of the rabbit IgG anti-idiotype an idiotype-specific ELISA was developed and sera from 32 patients with PSS (13 with monoclonal IgMκ), 33 with rheumatoid arthritis, three with rheumatoid arthritis-t-Sjögren's syndrome (SS), 30 with systemic lupus erythematosus, six with Waldenström's macroglobulinaemia, and 20 healthy controls were tested. The idiotype was primarily found in PSS patients with circulating monoclonal IgMκ- and more often in those who had a ratio of κ:λ intracytoplasmically positive plasma cells greater than 3:1 in the lymphocytic infiltrates of minor salivary glands, and systemic manifestations. The idiotype was also found in PSS and rheumatoid arthritis patients without circulating monoclonal cryoglobulins as well as in two of the six patients with Waidenstrom's macroglobulinacmia. Our results suggest that the monoclonal process observed in PSS could involve restricted idiotypic clones that are susceptible to malignant transformation. [ABSTRACT FROM AUTHOR]

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 141% and anti-CL in an extremely high incidence (51.6%). Notably, the above autoantibodies were exclusively of lgG 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. [ABSTRACT FROM AUTHOR]