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Although there is still an emotional debate over the existence of mixed connective tissue disease, the evidence from animal models suggests that anti-U1RNP antibodies, similar to other autoantibodies in other connective tissue diseases (such as antisynthetase, anticentromere, and antitopoisomerase), play a pathophysiological role in this disease. Despite an antiendothelial effect of anti-U1RNP antibodies, which is reminiscent of anticentromere antibodies, patients with high-titer autoantibodies to U1RNP in the absence of anti-Sm antibodies do not usually have or develop typical systemic sclerosis. Instead, their severe Raynaud's syndrome is commonly accompanied by arthritis, which can be erosive, and by swollen/puffy hands and myositis. Pulmonary arterial hypertension is the major life-threatening complication in these patients and regular screening for this condition is essential.

Mixed connective tissue disease (MCTD) is a systemic disease seen in a group of patients with overlapping clinical features of lupus, scleroderma, polymyositis, and rheumatoid arthritis. A defining feature of MCTD is the presence of antibodies against the U1-ribonucleoprotein (U1-RNP) complex. Pulmonary hypertension is the major cause of death in MCTD. We report an autopsy case of MCTD with pulmonary hypertension. The U1-RNP antibody of this patient was 171.9 U (normal < 25.0 U). The immediate cause of death was attributed to acute pulmonary embolism at left lower lobe. A severe vasculopathy characterized by fibrotic occlusion of small veins and venules, associated with prominent capillary congestion, was consistent with pulmonary veno-occlusive disease (PVOD). This is the first case reported in which PVOD is the primary cause of pulmonary hypertension in MCTD.

Background: Anti-Sm antibodies and anti-RNP antibodies are considered to be diagnostic markers of systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). However, cross-reactivity between the antibodies diminishes their discriminating specificity between these diagnoses., Objective: We examined whether we could achieve better differentiation between these two disease entities using recombinant antigens to RNP70 and SmD and quantitative immunoassays., Patients/methods: Sera from 51 patients with SLE and 10 patients with MCTD and from a control group of 59 patients were used in a cross-sectional setting. Semiquantitative ELISAs for the detection of antibodies to RNP-70, RNP-A, RNP-C, SmBB' and SmD were used and the results were compared to conventional ELISA tests using U(1)-snRNP and a mixture of SmBB' and SmD as antigenic substrates., Results: Sera from MCTD patients showed higher levels of anti-RNP-70 antibodies than sera from SLE patients. Levels of anti-SmBB' or anti-SmD antibodies were not significantly different between SLE and MCTD sera. However, the presence of antibodies directed against SmD was more frequent in SLE., Conclusions: Our results suggest that the use of RNP-70 and SmD antigens may increase the practical value of immunoassays used to confirm a diagnosis of SLE or MCTD in patients with connective tissue disease.

Objective: To evaluate the presence of anti-endothelial cell antibodies (AECA) in patients with mixed connective tissue disease (MCTD) compared to those with systemic sclerosis (SSc) and to determine the candidates for the endothelial auto-antigen that reacts with AECA in patients with MCTD using a molecular cloning strategy., Methods: AECA were measured by a cellular enzyme-linked immunosorbent assay (ELISA) using fixed human umbilical vein endothelial cells (HUVEC) in 47 MCTD patients, 68 SSc patients, and 52 normal controls. A HUVEC cDNA expression library was immunoscreened with pooled sera from 6 patients with high AECA levels determined by cellular ELISA to explore the endothelial autoantigens in MCTD. An ELISA assay for anti-ribosomal protein P0 antibodies was used to assess the correlation with AECA levels., Results: The candidate target proteins recognized by AECA in MCTD included: (i) ribosomal protein P0; (ii) a putative oncogene derived from dek mRNA; (iii) SS-B/La protein; (iv) U1 RNA-associated 70K protein; and (v) DNA-binding protein B. A significant correlation between the levels of AECA and anti-ribosomal protein P0 antibodies was demon-strated in MCTD, but not in systemic sclerosis. The sera containing high levels of AECA from patients with MCTD frequently cross-reacted with ribosomal protein P0. On the other hand, sera without AECA activity from patients with MCTD never reacted with ribosomal protein P0., Conclusion: AECA were more frequently seen in patients with MCTD than in patients with SSc. Ribosomal protein P0 may be one of the major target antigens of AECA in patients with MCTD.

We report an unusual case of connective tissue disease characterized by the coexistence of signs, symptoms and immunological features of 4 defined autoimmune diseases: systemic lupus erythematosus (SLE), systemic sclerosis (SSc), polymyositis (PM) and rheumatoid arthritis (RA). A 53-year-old female was admitted in our clinic with massive polyserositis (pretamponade) as well as skin, joint, muscular lesions and altered general status. The problem we found was the difficulty of including this case in a known clinical entity; SSc/SLE/PM//RA overlap syndrome and mixed connective tissue disease were the two most plausible diagnoses. We discuss the particularities of these clinical and immunological associations and the appropriate therapeutic options used in this kind of patients.

Objectives: Although it has been reported that the numbers of both CD4(-)CD8(-) and CD4(+) natural killer T (NKT) cells are selectively decreased in the peripheral blood of patients with rheumatic diseases, there have been no reports concerning a novel subpopulation of CD8(+) NKT cells. To examine whether CD161(+)CD8(+) T cells, which are closely related to CD8(+) NKT cells, are also decreased in patients with rheumatic diseases, we have investigated the expression of CD161, together with that of CD28, CD25 and CD62L, on T cells in the peripheral blood of these patients., Methods: The rheumatic diseases evaluated in this study were systemic lupus erythematosus (SLE) (n= 54), mixed connective-tissue disease (MCTD) (n= 15), systemic sclerosis (SSc) (n= 14), polymyositis/dermatomyositis (PM/DM) (n= 13) and rheumatoid arthritis (RA) (n= 24). Healthy donors were examined as controls (n= 18). The expression of CD161, CD28, CD25 and CD62L on T cells was analysed by flow cytometry., Results: Both the frequency of CD161 expression on CD8(+) cells and the absolute number of CD161(+)CD8(+) cells were significantly decreased in patients with SLE, MCTD, SSc and PM/DM. Only the absolute number of CD161(+)CD8(+) T cells was significantly decreased in RA. CD161 expression on CD28(-)CD8(+) T cells was significantly decreased in SLE, MCTD and SSc. The absolute number of CD161(+)CD8(+)CD62L(-) T cells was significantly decreased in SLE, MCTD and SSc., Conclusions: Both the frequency and the absolute number of CD161(+)CD8(+) T cells were decreased in the peripheral blood of patients suffering from SLE, MCTD, SSc and PM/DM. This result suggests that there is also an abnormality of NKT cells in the CD8(+) population.

Mixed connective tissue disease (MCTD) is a connective tissue disorder that is often accompanied by various immunological abnormalities. In this study, we analyzed serum levels of rheumatoid factor (RF) isotypes in patients with MCTD and in normal controls to determine if any of these isotypes reflects the severity of the disease. IgM-RF, IgG-RF, and IgA-RF were positive in 48, 38, and 33% of the patients, respectively. The frequency of positive anti-SS-A antibody and decrease in white blood cell counts were significantly greater in patients with elevated IgA-RF levels than that in those with normal levels. These results suggest that the presence of RF isotypes can be regarded as one of the various immunological abnormalities of MCTD.

We investigated the clinical characteristics and immunoserological alterations in patients with mixed connective tissue disease (MCTD) associated with pulmonary arterial hypertension (PAH). Anti-U1RNP autoantibodies, anti-endothelial cell antibodies (AECA) and serum thrombomodulin (TM) as well as von Willebrand factor antigen (vWFAg) concentrations were measured in 25 patients with MCTD associated with PAH and in 154 MCTD patients without PAH. The results showed that the probability of survival was lower in MCTD patients with PAH than in the 154 MCTD-non-PAH patients (5-year survival rate in MCTD with PAH: 73%, versus 96% in MCTD-non-PAH; P < 0.01). AECA were more frequently present in the sera of MCTD patients with PAH than in MCTD-non-PAH (P < 0.001). Serum TM and vWFAg levels were higher in MCTD-PAH patients than in MCTD-non-PAH patients (TM: P < 0.001; vWFAg: P < 0.001). Significant correlation was noticed between the quantity of AECA and TM level (r = 0.466) as well as the quantity of AECA and vWFAg level (r = 0.550). In conclusion, our results suggest that in MCTD the presence of AECA and endothelial cell activation may play a role in the development of PAH and in the maintenance of obliterative vascular processes.

Objective: Extrahepatic autoimmune features of HCV infection include autoantibody production and the development of mixed cryoglobulinemia. Anti-Clq antibody, detected with high frequency in systemic lupus erythematosus and hypocomplementemic urticarial vasculitis, may have a direct pathogenic role in complement mediated autoimmune diseases. In this study, we investigate the prevalence of anti-Clq antibody in a population of patients with chronic HCV infection., Methods: Serum was obtained from a group of 50 patients with chronic HCV infection and control groups comprised of patients with SLE, rheumatoid arthritis (RA), scleroderma (PSS), Sjögren's syndrome (SS), mixed connective tissue disease (MCTD), and healthy individuals., Results: Anti-Clq antibody was detected in 38% of HCV patients compared with 2% of healthy controls (p < 0.0001). Levels were also significantly elevated in patients with SLE (61%), RA (20%), PSS (15%), SS (15%) and MCTD (15%)., Conclusion: In addition to numerous other autoantibodies, patients with chronic HCV infection exhibit increased production of anti-Clq IgG antibodies. This observation may have implications for the pathogenesis of the mixed cryoglobulinemic vasculitis syndrome.

Objective: To test whether immunizing mice with autoantigens closely linked to mixed connective tissue disease (MCTD) could induce an MCTD-like clinical syndrome distinguishable from systemic lupus erythematosus (SLE)., Methods: Transgenic and knockout C57BL/6-derived mice were immunized subcutaneously at age 8-12 weeks with U1-70-kd small nuclear RNP (70K) fusion protein along with either Freund's complete adjuvant (CFA) or U1 RNA. After 2 months, mice were killed and analyzed histologically and serologically., Results: Immunization of C57BL/6-derived mice transgenic for human HLA-DR4 with 70K and either CFA or U1 RNA led to anti-70K antibodies in 62% of mice (21 of 34), and diversified anti-RNP immune responses. MCTD-like lung disease also developed in 50% of immunized mice (17 of 34), and anti-70K antibodies were strongly correlated with lung disease. CFA and U1 RNA were comparably able to induce this syndrome. Mice deficient in Toll-like receptor 3 (TLR-3) also developed this same syndrome when immunized with 70K and CFA. However, TLR-3(-/-) mice failed to develop MCTD-like lung disease when treated with 70K and U1 RNA. Rather, TLR-3(-/-) mice immunized with 70K and U1 RNA developed an autoimmune syndrome characterized by glomerulonephritis typical of SLE., Conclusion: Exposure to 70K in an appropriate context is sufficient to induce autoimmunity and target organ injury consistent with MCTD. This system represents a new model of autoimmune interstitial lung disease, and establishes a closer link between anti-70K immunity and MCTD-like lung disease. Of note, changes in innate immune signaling can cause the same trigger to lead to the development of SLE-like nephritis rather than MCTD-like lung disease.

The patient was a 42-year-old woman diagnosed as having MCTD and Sjögren's syndrome in 1989, and who was taking oral prednisolone. Proteinuria and microscopic hematuria were pointed out for the first time in December 2004. She was referred to our hospital because of massive hemoptysis. Advanced renal failure, anemia and pulmonary alveolar hemorrhage were diagnosed on admission. She was positive for serum MPO-ANCA. The patient was started on a therapy that included steroids, cyclophosphamide and plasmapheresis. However, her respiratory condition was untreatable and she died on the 16th day of hospitalization. The autopsy revealed alveolar hemorrhage in the lungs and crescentic glomerulonephritis. This patient was considered as a rare case of MCTD associated with MPO-ANCA-positive microscopic polyangiitis.

Congenital heart block occurs in fetuses of Ro/SSA and La/SSB positive women. To investigate the stability of maternal autoantibody levels during pregnancy, we followed Ro52, Ro60 and La autoantibody IgG level variation and Ro52 subclass profiles longitudinally in selected congenital heart block risk pregnancies. Serum samples were obtained from 12 Ro/La positive women diagnosed with a systemic rheumatic disease and followed on average 60 months (range two to 84) which included 13 pregnancies. Seven children were affected by neonatal lupus, whereof four developed complete congenital heart block. Serum was also collected from the babies at birth. Ro52, Ro60 and La IgG as well as subclass antibodies were analysed by ELISA using recombinant antigens. Six Ro/La negative rheumatic patients were included as controls for antibody levels during pregnancy. Ro52, Ro60 and La IgG levels decreased progressively from early to late pregnancy, significantly for Ro52 and Ro60 (P < 0.01). No peaks or persistent elevation of antibody levels were noted in any of the CHB risk pregnancies. Ro52 IgG1 antibody levels were significantly higher than IgG2 (P < 0.01), IgG3 (P < 0.01) and IgG4 (P < 0.05) levels in the mothers during pregnancy. Ro52 IgG1 and IgG4 levels decreased significantly from early to late pregnancy (P = 0.02), while levels of IgG2 and IgG3 were low and the decrease was not significant. All IgG subclasses were transferred to the children. We conclude that maternal levels of Ro52, Ro60 and La autoantibodies tended rather to decrease than to increase during pregnancy.

A 66-year-old woman who was positive for human T-lymphotropic virus type I (HTLV-I) antibody developed mixed connective tissue disease (MCTD) with interstitial pneumonia, and was successfully treated with corticosteroid. One year later, under maintenance treatment of prednisolone (PSL), she contracted acute type adult Tcell leukemia/lymphoma (ATLL) without flaring of MCTD. MCTD is considered to be as one of the HTL-V-I-related inflammatory diseases, however the development of ATLL during the treatment of HTL-V-I-related MCTD has not been well studied. Here, we review the literature and raise the issue of the mutual interactions between MCTD-causative anti-HTLV-I immune response and anti-ATLL immune response.

Sjögren's Syndrome (SS) is the second most common autoimmune disorder after rheumatoid arthritis (RA). It can be found as a lone condition (primary Sjögren's Syndrome) or may accompany other autoimmune rheumatic diseases (secondary Sjögren's Syndrome). Despite such frequent occurrence, accurate diagnosis of Sjögren Syndrome is difficult. These difficulties result from highly variable symptoms of SS as well as from common presence of other autoimmune disorders. In the following article authors present current knowledge concerning clinical symptoms, diagnostic methods and latest clinical guidelines on the diagnosis of SS. Differential diagnosis of SS and systemic lupus erythematosus (SLE) is also discussed.

We studied the presence of secondary Sjögren's syndrome (SS) and the composition of saliva, prevalence of oral pathogens, periodontitis, mouth mucosa, and teeth in patients with various rheumatic diseases and in healthy controls. The hypothesis was that different rheumatic diseases might cause differences in oral health characteristics because of the liability of secondary SS in the patients. The study involved 77 patients and 77 age-matched and sex-matched controls. Twenty patients were suffering from spondylarthropathy (SPA), 18 from ankylosing spondylitis (AS), 24 from rheumatoid arthritis (RA), and 15 from mixed connective tissue disease (MCTD). Clinical and radiographic oral health status was recorded and salivary flow rates were measured. Selected salivary proteins and immunoglobulins were analysed by routine methods. Minor salivary gland biopsy samples were taken from the patients for assessment of inflammatory focus scores. Differences between patients and controls and in between the different rheumatic diseases were analysed statistically. Secondary SS was diagnosed in 39% (30/77) of the patients. A severe periodontal condition (community periodontal index of treatment needs score 3 or 4) occurred in 58% (45/77) of the rheumatic patients compared with only 26% (20/77) of the controls (p < 0.0001). The severity of focal sialadenitis (focus score) correlated significant with salivary IgA, IgG, and IgM concentrations. Salivary albumin, total protein, IgG, and IgM concentrations were higher in all patient groups than in the controls. The number of patients with low salivary flow rates was higher in all patient groups compared to controls. Oral yeast counts were significantly higher in the patients than in the controls (p < 0.001). In a subgroup analysis, patients with SS had higher values for salivary IgA and IgM than patients without SS. Dental caries and oral lactobacilli were more frequent in patients with SS, but SS was not associated with periodontitis. No major differences were noted in other salivary biochemical parameters between these two groups. Patients with rheumatic diseases, irrespective of specific diagnosis, thus had various alterations in salivary flow and composition and oral health. The findings may reflect the autoimmune inflammation of the salivary glands frequently observed in these patients.

Antibodies to U1-RNP are part of the clinical definition of mixed connective tissue disease (MCTD). These antibodies and other well-defined antibodies tend to arise together in affected patients. Although still speculative, hypotheses that link U1-RNP antibodies to the development of autoimmunity in MCTD and that associate U1-RNP antibodies with mechanisms of tissue injury in MCTD have emerged and are being tested. Salient features of these hypotheses include: (1) an antigen-driven response that is due to impaired clearance of potentially immunogenic self-antigens, (2) inadequate B- and T-cell tolerance to RNP autoantigens, and (3) immunogenic properties of the RNA component of targeted ribonucleoproteins. Further studies are needed to establish whether anti-RNP antibodies have prognostic importance that is relevant to practicing clinicians.

This article describes other manifestations of mixed connective tissue disease (MCTD). We focus on inflammatory arthritis, the gastrointestinal tract (the esophagus in particular), the kidney, skin, and hematologic changes such as thrombocytopenia. Due to the complexity of potential organ involvements in MCTD, vigilance with appropriate diagnosis and treatment is warranted.

After a short description of the mechanism of the development of polysystemic autoimmune diseases, this paper presents the in vitro laboratory methods suitable for the characterization of the humoral and cellular elements of immune system, furthermore, summarizes the characteristic autoantibodies and other laboratory alterations in these diseases.

Objectives: To examine whether autoantibodies against bone morphogenetic protein receptor-II (BMPR-II) or activin receptor-like kinase 1 (ALK-1) are associated with pulmonary arterial hypertension (PAH) in patients with mixed connective tissue disease (MCTD)., Methods: We studied sera from 37 MCTD patients with or without PAH, six patients with idiopathic PAH, and 30 healthy controls. Circulating anti-BMPR-II and anti-ALK-1 antibodies were detected using immunoprecipitation of recombinant antigens generated by in vitro transcription/translation and indirect immunofluorescence of cultured cells that were induced to express these antigens by gene transfer. Anti-BMPR-II antibodies were further examined by immunoprecipitation and immunoblotting using a recombinant fragment of the extracellular domain of BMPR-II., Results: Serum anti-BMPR-II and anti-ALK-1 autoantibodies were not detected in MCTD patients irrespective of the presence or absence of PAH, or in patients with idiopathic PAH., Conclusions: Our finding does not support the hypothesis that autoantibody-mediated dysregulation of signals through BMPR-II or ALK-1 contributes to the development of PAH in patients with connective tissue diseases.

Aim: To evaluate prevalence of endocrine pathology in patients with circumscribed scleroderma (CS), baseline immunopathological alterations in CS patients., Material and Methods: Circulating immune complexes were studied by the Digeon method in a total of 70 CS patients (female 52, 74.3%; male 18, 25.7%; age 16 to 70 years)., Results: Circulating immune complexes assay by Digeon detected evident immune imbalance characteristic for metabolic disturbances of the connective tissue and affection of the endocrine system., Conclusion: CS patients need a combined examination including endocrinological and immunological tests.

Autoantibodies to the Sm antigens are specifically found in 5 to 30% of patients with systemic lupus erythematosus (SLE) depending on the detection system and the patient group. Several immunoassays designed for research and diagnostic laboratory use have been developed. The autoantigens employed in these tests include purified native proteins, recombinant polypeptides, and synthetic peptides. In the present study, we compared the clinical accuracy of anti-Sm autoantibody assays from commercial suppliers including different conventional enzyme-linked immunosorbent assay (ELISA) systems based on purified Sm antigens, an addressable laser bead assay and a newly developed anti-Sm peptide assay. Although the clinical sensitivity of all assays under investigation was comparable, relatively poor correlations and significant differences in specificity were found with a patient cohort of 150 patients. The sensitivity and specificity were 10 and 94%, respectively, for the anti-Sm ELISA from Euroimmun, 10 and 90%, respectively, for the QuantaLite Sm (INOVA), 12 and 88%, respectively, for the Sm assay in the Varelisa ReCombi ANA profile (Pharmacia Diagnostics), 10 and 94%, respectively, for the QuantaPlex Sm (INOVA), and 12 and 100%, respectively, for the new SmD3 peptide-based ELISA (Varelisa Sm Antibodies). The majority of positive test results within the control groups were found in patients with mixed connective tissue disease. Based on the results, we conclude that the detection of anti-Sm antibodies strongly depends both on the nature of the antigen and on the detection system. Finally, we conclude that the recently identified SmD peptide containing a symmetrical dimethylarginine at position 112 of D3 represents a promising tool for the detection of a highly specific subpopulation of anti-Sm antibodies.

The clinical cardiac manifestations most frequently reported in idiopathic inflammatory myopathies, myositis, are congestive heart failure, conduction abnormalities, that may lead to complete heart block and coronary artery disease. Although clinically overt cardiac involvement is rarely reported in myositis patients, subclinical manifestations are frequently observed and are predominated by conduction abnormalities and arrhythmias detected by ECG. Furthermore, cardiovascular manifestations constitute a major cause of death in myositis, thus cardiac involvement maybe overlooked in these patients. Also children with juvenile dermatomyositis may develop cardiac involvement although the frequency seems to be low. The underlying pathophysiologic mechanisms that may cause cardiac manifestations could involve myocarditis and coronary artery disease as well as involvement of the small vessels of the myocardium. In patients with mixed connective tissue disease (MCTD) clinically significant cardiac involvement is also rare, the most frequently reported manifestations being pericarditis and pulmonary hypertension, the latter often attributable to small vessel disease, and often a prognostic unfavourable manifestation.

Anti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.

Objective: To characterize the inflammatory cells, the expression pattern of adhesion molecules (ICAM-1 and VCAM-1), membrane attack complex (C5b-9), and major histocompatibility complex (MHC) antigens in muscle biopsy of mixed connective tissue disease (MCTD)., Method: We studied 14 patients with MCTD, and compared to 8 polimyositis (PM) patients, 5 dermatomyositis (DM) and 4 dystrophies. Inflammatory cells were examined for CD4+, CD8+, memory and naive T cells, natural killer cells, and macrophages. Expression of MHC-I and -II, ICAM-1, VCAM-1 and C5b -9 were characterized on muscle fibers and vessels., Results: Morphological analysis displayed a pattern of PM. Immunohistochemical study revealed a decreased number of capillaries, predominance of CD4+ and B cells in perivascular regions and predominance of CD8+ and CD45RO+ in endomysial regions. The expression of MHC-I on vessels and on degenerated muscle fibers, MHC-II expression on vessels and perifascicular muscle fibers, and the expression of ICAM-1 / VCAM-1 on endothelial cells indicated both vascular and cellular-immune mediated processes causing the muscular lesion., Conclusion: Our findings revealed a mixed mechanism in MCTD, both vascular involvement as DM, and cell-mediated like PM.

We report on a carrier of human T-lymphotropic virus type 1 (HTLV-1) who developed mixed connective tissue disease (MCTD). This patient suddenly manifested clinical symptoms and interstitial pneumonia ascribable to MCTD following long-term infection with HTLV-1. After initiation of oral prednisolone all manifestations quickly improved in parallel with a decrease in inflammatory reactions. In this patient HTLV-1 infection might have played an important role in the pathogenesis of MCTD. Since HTLV-1 can cause adult T-cell leukemia and HTLV-1-associated myelopathy, and also collagen diseases including MCTD, careful observation is necessary even in a carrier, particularly when autoantibodies are detectable in serum.

Background: HEp-2 cells that overexpress the human 60-kDa SSA antigen have been used to improve sensitivity and specificity for the detection of anti-SSA antibodies by indirect immunofluorescence. We describe a survey on the detection of anti-SSA antibodies using a commercial substrate that overexpresses SSA., Methods: The evaluation was done on 18 371 consecutive samples submitted to the laboratory for detection of anti-nuclear antibodies, from which 188 anti-SSA antibody-containing and clinically documented samples were obtained. The presence of anti-SSA antibodies produced a distinct bright speckled pattern with nucleolar staining in 10-20% of interphase cells. The identity of all anti-SSA antibodies was confirmed by dot-blot analysis., Results: Samples containing anti-SSA antibodies were separated into three main groups: group I, distinctive SSA pattern and other nuclear staining (50%); group II, only the distinctive SSA pattern (29%); group III, nuclear staining but without the distinctive SSA pattern (21%). Anti-SSA antibodies with concurrent SSB antibodies were associated with group I, whereas anti-SSA antibodies with concurrent U(1)-RNP antibodies were associated with group III. Group I included mainly patients with Sjogren syndrome and systemic lupus erythematosus (SLE), whereas group III included patients with mixed connective tissue disease and SLE. Diseases not classically associated with the presence of anti-SSA antibodies were found in group II in >50% of the cases., Conclusions: SSA-positive individuals were identified in a population selected on the basis of HEp-2000 positivity. Our study highlights diseases associated with anti-SSA antibodies and associations between the presence of the distinctive SSA pattern on HEp-2000 and some clinical conditions.

Mixed connective tissue disease (MCTD), undifferentiated connective tissue disease and overlap syndromes are autoimmune systemic diseases that must be differentiated. Antibodies against the U1-ribonucleoprotein complex --spliceosome-- allows the diagnosis of mixed connective tissue disease. Links between the immunologic and clinical phenomena are emerging. Longitudinal studies of patients with MCTD highlight the impact of pulmonary hypertension and contribute to define the disease. Immunogenetic studies hold MCTD as an independent disease.

A 47-year-old woman presented with facial spasm, swollen fingers and Raynaud's phenomenon due to cerebrovascular disorder and mixed connective tissue disease (MCTD). Although she was positive for both antineutrophil cytoplasmic antibodies against proteinase-3 (PR3-ANCA) and anti-U1 RNP antibodies, she did not meet the American College of Rheumatology classification criteria for Wegener's granulomatosis (WG). Physical and histopathological examinations revealed severe systemic atherosclerosis without any of the traditional risk factors. Elevated levels of malondialdehyde-modified LDL and antioxidized LDL autoantibodies, which are considered to be key factors in the pathogenesis of atherosclerosis, were also detected in the serum of this patient. In this case, systemic atherosclerosis might have been linked to these autoimmune reactions.

Background: Antinuclear antibody (ANA) tests are frequently used to screen children for chronic inflammatory diseases such as systemic lupus erythematosus (SLE). However, the diagnostic utility of this test is limited because of the large number of healthy children who have low-titer positive tests. We sought to determine the clinical utility of ANA tests in screening children for rheumatic disease and to determine whether there are specific signs or symptoms that enhance the clinical utility of ANA tests in children., Methods: We undertook a retrospective analysis of 509 new patient referrals. Charts of patients referred because of results of ANA testing were selected for further analysis. Children with JRA, SLE, and other conditions were compared using demographic data, chief complaints at the time of presentation, and ANA titers., Results: One hundred ten patients were referred because of an ANA test interpreted as positive. Ten patients were subsequently diagnosed with SLE. In addition, we identified one patient with mixed connective tissue disease, and an additional child with idiopathic Raynaud's phenomenon. Eighteen children of the children referred for a positive ANA test had juvenile rheumatoid arthritis (JRA). Another 80 children with positive ANA tests were identified, the majority of whom (n = 39, 49%) had musculoskeletal pain syndromes. Neither the presence nor the titer of ANA served to distinguish children with JRA from children with other musculoskeletal conditions. Children with JRA were readily identified on the basis of the history and physical examination. Children with SLE were therefore compared with children with positive ANA tests who did not have JRA, designated the "comparison group." Non-urticarial rash was more common in children with SLE than in children without chronic inflammatory disease (p = 0.007). Children with SLE were also older (mean +/- sd = 14.2 +/- 2.5 years) than the comparison group (11.0 +/- 3.6 years; p = 0.001). ANA titer was also a significant discriminator between children with SLE and children without chronic inflammatory disease. The median ANA titer in children with SLE was 1: 1,080 compared with 1:160 for other children (p < 0.0001). ANA titers of >/=1,080 had a positive predictive value for SLE of 1.0 while titers of

Objective: Present study addresses the issue whether apoptosis and necrosis increases the antigenicity of proteins recognized by antinuclear antibodies., Material and Methods: HEp-2 cells were cultured in standard conditions; apoptosis was induced by camptothecin and necrosis by mercuric chloride. Protein antigenicity of cell extracts was tested onto nitrocellulose membranes and probed with positive or negative sera for antinuclear antibodies by a luminescent-dot-ELISA system., Results: Apoptotic changes in HEp-2 cells appeared by 24 hours of camptothecin exposure, meanwhile the necrotic features become visible earlier. Luminescence was significantly superior in ANA positive sera than in ANA negative controls. Antinuclear antibody sera recognized better the antigens from the apoptotic and necrotic cells than controls without chemical treatments., Conclusions: Apoptosis and necrosis increase the ANA binding by better availability of intracellular antigens, or by disclosing cryptic epitopes.

Searching for a mechanism underlying autoimmunity in autism, we postulated that gliadin peptides, heat shock protein 60 (HSP-60), and streptokinase (SK) bind to different peptidases resulting in autoantibody production against these components. We assessed this hypothesis in patients with autism and in those with mixed connective tissue diseases. Associated with antigliadin and anti-HSP antibodies, children with autism and patients with autoimmune disease developed anti-dipeptidylpeptidase I (DPP I), anti-dipeptidylpeptidase IV (DPP IV [or CD26]) and anti-aminopeptidase N (CD13) autoantibodies. A significant percentage of autoimmune and autistic sera were associated with elevated immunoglobulin G (IgG), IgM, or IgA antibodies against three peptidases, gliadin, and HSP-60. These antibodies are specific, since immune absorption demonstrated that only specific antigens (e.g., DPP IV absorption of anti-DPP IV), significantly reduced IgG, IgM, and IgA antibody levels. For direct demonstration of SK, HSP-60, and gliadin peptide binding to DPP IV, microtiter wells coated with DPP IV were reacted with SK, HSP-60, and gliadin. They were then reacted with anti-DPP IV or anti-SK, anti-HSP, and antigliadin antibodies. Adding SK, HSP-60, and gliadin peptides to DPP IV resulted in 27 to 43% inhibition of the DPP IV-anti-DPP IV reaction, but DPP IV-positive peptides caused 18 to 20% enhancement of antigen-antibody reactions. We propose that (i) superantigens (e.g., SK and HSP-60) and dietary proteins (e.g., gliadin peptides) in individuals with predisposing HLA molecules bind to aminopeptidases and (ii) they induce autoantibodies to peptides and tissue antigens. Dysfunctional membrane peptidases and autoantibody production may result in neuroimmune dysregulation and autoimmunity.

Objective: To investigate the clinical significance of anti-TS1-RNA antibodies in patients with mixed connective tissue disease (MCTD)., Methods: Anti-TS1-RNA antibodies were detected by immunoprecipitation using 32P-UTP labeled TS1-RNA as the antigen source. In total, 104 patients with MCTD, 30 with Sjögren's syndrome, 30 with systemic lupus erythematosus (SLE), 25 with systemic sclerosis, 23 with polymyositis or dermatomyositis, and 10 with rheumatoid arthritis were examined. Specificity of anti-TS1-RNA antibodies was analyzed by immunoprecipitation using HeLa cell extracts., Results: The frequency of anti-TS1-RNA antibodies was 31.7% in patients with MCTD, significantly higher than in SLE (p < 0.05). In anti-TS1-RNA positive patients, the incidence of hypertension and proteinuria and the frequency of anti-Sm and anti-dsDNA antibodies associated with SLE were higher than those of anti-TS1-RNA negative patients. Clinical features of SS such as sicca complex, the serum level of IgA, and anti-SSA antibodies were also elevated. The frequency of anti-TS1-RNA antibodies was significantly higher in SLE patients with anti-U1-RNP antibodies (p < 0.01); however, anti-TS1-RNA positive sera did not precipitate the specific RNA including U1 RNA in immunoprecipitation using HeLa cell extracts. In longitudinal studies, the level of anti-TS1-RNA antibodies changed in parallel with disease activity., Conclusion: We found that the level of anti-TS1-RNA antibodies was possibly correlated with the disease activity of lupus-like clinical features in patients with MCTD.

Systemic autoimmune diseases form a diverse group which includes: systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), scleroderma, dermato-polymyositis, Wegener's granulomatosis, Sjögren syndrome. Although multisystem involvement is the hallmark of these diseases, the heart seems to be less affected than other organ systems. The aim of the study was to study possible cardiac abnormalities in patients with documented systemic autoimmune diseases and to assess whether there was any relation between antiphospholipid, anti-dsDNA antibodies and myocardial dysfunction findings. 76 patients (53 with SLE, 9 with MCTD, 8 with scleroderma, 6 with Wegener's granulomatosis) were subjected to our study, 69% of these patients manifested cardiac involvement, based on two-dimentional echocardiografic examination (36%--post-inflammatory valvular thickening, 20%--pericardial effusions, 15%--valvular regurgitation, 7%--left atrial enlargement, 5%--left ventricular hypertrophy, 4%--left ventricular dysfunction). None of the patients showed characteristic, acute Libman-Sacks endocarditis, which probably can be explained by chronic corticosteroid-treatment. Clinical evidence of cardiac abnormalities has been observed, in as many as 58% of cases with positive echocardiographic findings. The frequency and extend of cardiac pathology positively correlated with the detection of antiphospholipid antibodies. No such relationship was observed in patients with the presence of very high titers of antinuclear antibodies (anti-dsDNA). In conclusion, our results indicate that echocardiography is a useful method for assessment and monitoring cardiac involvement in the systemic autoimmune diseases.

Mixed connective tissue disease is an overlap syndrome characterized by features of different systemic autoimmune diseases and a high titer of U1-snRNP antibodies. We examine here the autoantibodies to nucleoporin p62 in a severe case of mixed connective tissue disease in a young male patient. Thus far, p62 antibodies have mainly been described in cases of primary biliary cirrhosis. We speculate that the presence of p62 antibodies is an indication of a poor prognosis in connective tissue disorders.

We studied the prevalence and clinical significance of anti-U1 RNA antibodies in patients with systemic sclerosis. The presence of anti-U1 RNA antibodies was determined using immunoprecipitation in systemic sclerosis patients with anti-U1 RNP antibodies (n=36), antitopoisomerase I antibodies (n=20), or anticentromere antibodies (n=20), mixed connective tissue disease patients (n=23), systemic lupus erythematosus patients with anti-U1 RNP antibodies (n=26), and normal controls (n=20). Moreover, antigen specificities for anti-U1 RNP antibodies were examined in patients with systemic sclerosis by immunoblotting and enzyme-linked immunosorbent assay. Anti-U1 RNA antibodies was detected in 22 of 36 systemic sclerosis patients (61%) with anti-U1 RNP antibodies, 14 of 23 patients (61%) with mixed connective tissue disease, and eight of 26 systemic lupus erythematosus patients (31%) with anti-U1 RNP antibodies. Anti-U1 RNA antibodies were not detected in other groups. As for systemic sclerosis patients, the frequencies of pulmonary fibrosis and reduced percentage diffusion capacity for carbon monoxide were significantly greater in patients with anti-U1 RNA antibodies than in those without (76%vs 18%, p<0.005; 82%vs 27%, p<0.005, respectively). Moreover, patients with anti-U1 RNA antibodies had significantly lower percentage diffusion capacity for carbon monoxide and percentage vital capacity values than those without (51.9+/-16.8 vs 79.4+/-16.4, p<0.01; 83.8+/-21.4 vs 101.4+/-12.9, p<0.05, respectively). Regarding the antigen specificities of anti-U1 RNP antibodies in systemic sclerosis patients, the frequency of anti-70 kDa antibodies determined by immunoblotting was significantly higher in patients with anti-U1 RNA antibodies than in those without (77%vs 43%, p<0.05). This finding was also confirmed by enzyme-linked immunosorbent assay for anti-70 kDa antibodies (86%vs 43%, p<0.05). These results indicate that anti-U1 RNA antibodies may be a serologic indicator for pulmonary fibrosis in systemic sclerosis patients with anti-U1 RNP antibodies.

We retrospectively analysed the data of 1519 antiphospholipid antibody (APLA) positive patients between 1986 and 1999. Among them 637 were considered to have antiphospholipid syndrome (APS) based on the 1999 preliminary classification criteria, while 704 patients had no clinical signs of the syndrome. Our aim was to compare the autoantibody profile and clinical characteristics of primary and secondary APS, moreover to evaluate the associations between different APLA and specific symptoms attributable to APS. In our results, the APLA profiles for primary and SLE-associated secondary APS were similar. Among the evaluated clinical symptoms, cerebrovascular thrombosis was found to be more frequent in the SLE-associated, than in the primary APS group (P = 0.04). We identified important differences in the clinical profile of patient populations with various types of APLA. Venous thrombosis occurred more frequently in subjects withlupus anticoagulant (LA), than in those with IgG or IgM type ACLA (P < 0.0001), while coronary, carotid and peripheral artery thrombosis occurred more often in subjects with IgG or IgM ACLA (P < 0.0001). These findings may support the role of antibodies to cardiolipin or its cofactor, beta2glycoprotein I (beta2-GPI) in the initiation and progression of atherosclerosis. Cerebrovascular thrombosis was detected in larger proportion of LA or IgG ACLA-positive patients compared with to IgM ACLA-positive subjects, while the occurrence of foetal loss was similar in all three groups.

Interleukin-18 (IL-18) and its inducer IL-12 have multiple biological activities that are important in generating Th1 responses and inflammatory tissue damage. We investigated serum concentration of the novel proinflammatory Th1 cytokine; IL-18, and its inducer IL-12 in patients with immune rheumatic diseases. Group I comprised32 patients of systemic lupus erythmatosus (SLE), Group II comprised 36 patients of rheumatoid arthritis (RA). Group III comprised 9 patients (2 patients of Behcet, 2 patients of Dermatomyositis, 2 patients of Sicca syndrome, one patient of Scleroderma, and 2 patients of Mixed connective tissue disease). Group IV is a control group consists of 21 sex and age matched healthy subjects and correlated their levels with autoantibody concentration (ANA and ds-DNA), clinical grades and SLE disease activity index (SLEDAI). Serum IL-18, IL-12, ANA and ds-DNA were measured by enzyme immuno sorbent assay. IL-18, IL-12 and ANA were significantly higher in the three studied groups than in the control group (IL-18; P < 0.001 in the three groups, IL-12; P = 0.019, P = 0.002, and P = 0.006, and ANA; P < 0.001, P = 0.002,and P = 0.006, respectively).ds-DNA was significantly higher in SLE patients than in control group (P < 0.001). There were significant positive correlations between; A) levels of IL-18,and both ANA and ds-DNA in SLE patient (r = 0.41,P = 0.001, r = 0.58 and P=0.001 respectively); and B) IL-18 and ANA in both RA and group III patients (r = 0.32, P = 0.005, r = 0.61and P = 0.022 respectively). Also, there were significant positive correlation between the levels of IL-18 and clinical grades of the three groups (r = 0.60,P = 0.001, r = 0.79,P = 0.001, r = 0.78 and P= 0.001 respectively). In SLE patients , IL-18 concentration shows significant positive correlation with SLEDAI score (r = 0.76, P = 0.001). In conclusion, the elevation of proinflammatory cytokines (IL-18 and IL-12 ) may trigger the inflammatory process in immune rheumatic diseases and IL-18 is correlated with disease activity

Objectives: Pulmonary hypertension (PH) is one of the major fatal causes in patients with mixed connective tissue disease(MCTD), which showed remarkable angiopathy from large to small vessels in the lungs. However, the etiology of PH in MCTD is still unknown. Even the lung tissues of MCTD patients without overt clinical PH represent minor vascular damages such as microthrombus or slight intimal thickening. These findings suggest some serum factors cause endothelial cell damage especially to pulmonary micro vessels, which leads to PH in MCTD. To elucidate the mechanisms of PH in MCTD we studied the anti-endothelial cell antibodies (AECA) in the sera of MCTD patients, which are considered to correlate with activity in some collagen diseases, and compared the three kinds of endothelial cells, especially the effects on pulmonary endothelial cells., Materials and Methods: Sera from 14 MCTD patients who satisfied the Kasukawa's criteria in Japan including 4 cases of PH, 8 cases of non-PH and 3 untreated cases, and 5 healthy controls were analyzed as follows: (1) AECA to human pulmonary arterial endothelial cells (HPAEC), pulmonary microvascular endothelial cells (HMVE-L) and human aortic endothelial cells(HAEC) were analyzed by an indirect immunofluorescence method using flow cytometry. (2) Effects of MCTD patients' and healthy controls' sera on cell proliferation and induction of apoptosis on cultured HPAEC were investigated by methods of MTS and TUNEL. (3) A cytotoxic effect of patients' sera in combination with activated NK cells on HPAEC were studied by a method of LDH concentration., Results: (1) Patients' sera from MCTD have IgG type AECA, and sera from MCTD patients with PH showed a higher intensity of AECA compared with non-PH and control cases (P < 0.01). (2) Only patient's sera revealed no potency of cell proliferation and induction of apoptosis in every kinds of endothelial cells compared with controls. (3) Sera from MCTD patients with PH, and from untreatment patients were high intensity of AECA, which shows cytotoxicity by addition of activated NK cells., Conclusions: Apoptosis of pulmonary arterial endothelial cells induced by AECA in combination with activated NK cells may be the fist step of vascular damage associated with pulmonary hypertension in patients with MCTD.

Objective: To establish an enzyme-linked immunosorbent assay (ELISA) using a complex of in vitro-transcribed U1 RNA and recombinant 70-kd, A, and C proteins (C-ELISA) to detect anti-U1 RNP antibodies reactive in double immunodiffusion (DID), but not in ELISA using the proteins alone (P-ELISA)., Methods: Sera from 196 patients with mixed connective tissue disease were used to test reactivity in P- and C-ELISAs, and the specificity of the sera was also tested by DID and immunoprecipitation (IP)., Results: In P-ELISA, 15 of 196 sera positive for anti-U1 RNP in DID did not react, while all sera reacted in C-ELISA. The reactivity of 15 sera to the U1 RNA was tested by IP and ELISA, and only 3 sera reacted with the U1 RNA. These results indicated that the increased reactivity in C-ELISA was not due to the U1 RNA itself. We confirmed that the 70-kd and A proteins were bound directly to the U1 RNA by IP using antibodies to His-tag, and we tested the reactivity of the sera to the U1 RNA-70-kd protein complex and the U1 RNA-A protein complex by IP. All sera reacted with the U1 RNA-70-kd protein complex, and 1 sample reacted with the U1 RNA-A protein complex., Conclusion: These results suggest that some anti-U1 RNP-positive sera specifically recognize the conformational structure altered by the binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is as useful as the DID method for detecting anti-U1 RNP antibodies.

Introduction: 179 patients with mixed connective tissue disease (MCTD) were follow-up, and the cause of death was analyzed in 12 died patients., Patients and Methods: The survival of 179 patients with MCTD was evaluated by using Kaplan-Meier's method. Clinically and immunological data of the patients were analyzed between 1 and 25 years follow-up period (mean: 13.1 +/- 5.5 years)., Results: The five-year survival rate was 96.4%, 10-year survival rate was 93.9%, and the 15-year survival rate was 89.6%. The cause of death was pulmonary hypertension in 5 patients, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in 3 cases, infection in 3 cases (hepatitis C virus induced hepatic coma in 2 patients, Staphylococcus sepsis in one patient), and on one patient myocarditis. The pulmonary hypertension was the most serious prognostic factor., Conclusion: In patients with MCTD the pulmonary hypertension with endothelial cells proliferation and microangiopathy developed very quickly, and there was progressive and therapy resistant statement. The secondary virus and bacterial infections may develop in the patients who were followed-up the long term period. Their survival rate was better than the data in the literature. This fact may cause genetic-demographic factors, and the sequential follow-up of the patients.

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-x(L), and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro-translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

Scleroderma is a connective tissue disease that causes fibrosis and vascular abnormalities, but that also has an autoimmune component. Many patients with scleroderma have a positive antinuclear antibody, and there can be family histories of other connective tissue diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. Some patients have features of scleroderma and other autoimmune conditions. This article will review recent literature to help in the understanding of scleroderma with overlap features. Recent reports of scleroderma overlap with rheumatoid arthritis suggest distinct features of diffuse scleroderma with positive Scl-70, pulmonary fibrosis, and later seropositive erosive rheumatoid arthritis. SLE rarely occurs with scleroderma. Sjögren syndrome symptoms are common in scleroderma. In primary Sjögren syndrome, anticentromere antibody positive patients have more Raynaud phenomenon. Antibodies that occur in scleroderma that are thought to be specific are present in other connective tissue diseases. For instance, Scl-70 antibody is reported in as many as 35% of patients with scleroderma but can be present in 25% of patients with SLE. Myositis or myopathy can be features of scleroderma. Scleroderma overlap with polymyositis is frequently anti-PM Scl antibody positive, whereas anti-Jo-1 does not normally occur in the overlap of scleroderma and polymyositis but is usually exclusively positive in polymyositis with arthritis and alveolitis. A better prognosis is found with PM Scl antibody in myositis. Vasculitis is not a typical feature of scleroderma, but has been reported. Eosinophilic fasciitis is rare, and the onset could be associated with simvastatin.