Mixed Connective Tissue Disease Patients Research Articles

Autoantibodies directed against labile epitopes on cell surface proteins in autoimmune disease patients: Proposal of a novel ELISA for the detection of anti-endothelial cell antibodies

This article describes a novel method for detecting anti-endothelial cell antibodies (AECAs). Sera from patients with systemic vasculitis or inflammatory conditions have been reported to contain antibodies (Abs) that bind to endothelial cells (EC), i.e., AECAs. AECAs are known to play immunogenic effects by triggering EC activation and vascular damage, but the immunopathological role of AECAs is not clear. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting have previously been used for detecting target antigens of AECAs. However, we assumed that these methods are not appropriate for searching genuine target antigens (Ags) on cell surface, and developed a novel solubilized cell surface protein-capture ELISA (CSP-ELISA). Ags were obtained as cell surface proteins from the plasma membrane of human umbilical vein endothelial cells (HUVECs); these cell surface proteins were biotinylated, solubilized with detergent, and captured on ELISA wells coated with NeutrAvidin™ biotin binding protein (NeuAvi). AECA titers in serum from 126 autoimmune disease patients and 122 healthy donors were tested. AECAs were detected in 28 of 36 (78%) of systemic lupus erythematosus (SLE) patients; in 13 of 16 (81%) of mixed connective tissue disease (MCTD) patients; and in 5 of 9 (56%) of systemic sclerosis (SSc) patients. Relatively weak denaturation of antigens on ELISA wells caused loss of binding of these autoantibodies (autoAbs). Thus, this newly developed CSP-ELISA method enables the detection of Abs to the labile epitopes of autoantigens (autoAgs) such as membrane proteins, and this method is generally applicable to various kinds of membrane proteins and the Abs against them. We propose CSP-ELISA for measuring AECAs in serum samples for routine laboratory testing.

Neuropsychological assessment in mixed connective tissue disease: comparison with systemic lupus erythematosus

The aims of the study were to assess cognitive functions (CF) in patients with mixed connective tissue disease (MCTD) and to compare MCTD patients with systemic lupus erythematosus patients with and without neuropsychiatric manifestations (NP-SLE and non-NP-SLE, respectively) in terms of CF. Neuropsychological examination was performed in 141 patients: 30 with MCTD (24 women, 6 men), mean age: 48.07 years, 37 with non-NP-SLE (36 women, 1 man), mean age: 40.76 years and 74 with NP-SLE (68 women, 6 men), mean age: 41.97 years. Neuropsychological tests and structured interview were used. Emotional state was assessed by Hospital Anxiety and Depression Scale and clinical review. We observed cognitive impairment in six MCTD patients (20%); in one (3%) the impairment was severe. MCTD patients achieved significantly higher results in seven out of 11 tests compared with patients with NP-SLE. MCTD and non-NP-SLE patients did not differ significantly. The differences were irrespective of premorbid IQ, education, disease duration and steroid treatment. In the majority of MCTD patients, CF were not impaired and severe impairment was unusual. Cognitive functioning was most disturbed in NP-SLE. The cognitive deficits observed in connective tissue diseases can be connected with nervous system involvement.

Identification of HnRNP-A2/B1 as a Target Antigen of Anti-Endothelial Cell IgA Antibody in Behçet's Disease

Behçet's disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease. We detected the target protein using western blotting and immunoprecipitation and determined the amino-acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/TOF). Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by ELISA. The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino-acid sequences of heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP-A2/B1). Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), 4 of 30 systemic lupus erythematosus patients (13.3%), 8 of 30 rheumatoid arthritis patients (26.7%), 9 of 30 Takayasu's arteritis patients (30%), 6 of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 were correlated with those against the recombinant streptococcal hsp-65.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

“To Be or Not To Be,” Ten Years After: Evidence for Mixed Connective Tissue Disease as a Distinct Entity

To determine if mixed connective tissue disease (MCTD) can be considered an independent clinical entity, to compare 3 different classification criteria for MCTD (Kasukawa, Alarcón-Segovia, and Sharp), and to define predictors (clinical features and autoantibodies) of potential evolution toward other connective tissue diseases (CTDs). One hundred sixty-one MCTD patients were evaluated retrospectively at the diagnosis and in 2008. They were classified, at the diagnosis, according to the 3 classification criteria of MCTD (Sharp, Alarcón-Segovia, and Kasukawa) and reclassified in 2008 according to their evolution. Statistical analyses were performed to find out predictors (clinical features and autoantibodies) of evolution into other CTDs. After a mean of 7.9 years of disease, 57.9% of patients still satisfied MCTD classification criteria of Kasukawa; 17.3% evolved into systemic sclerosis, 9.1% into systemic lupus erythematosus, 2.5% into rheumatoid arthritis, 11.5% was reclassified as affected by undifferentiated connective tissue disease, and 1.7% as suffering from overlap syndrome. Kasukawa's criteria were more sensitive (75%) in comparison to those of Alarcón-Segovia (73%) and Sharp (42%). The presence of anti-DNA antibodies (P = 0.012) was associated with evolution into systemic lupus erythematosus; hypomotility or dilation of esophagus (P < 0.001); and sclerodactyly (P = 0.034) with evolution into systemic sclerosis. MCTD is a distinct clinical entity but it is evident that a subgroup of patients may evolve into another CTD during disease progression. Initial clinical features and autoantibodies can be useful to predict disease evolution.

Histological analysis of esophageal muscular layers from 27 autopsy cases with mixed connective tissue disease (MCTD)

Esophageal symptoms in mixed connective tissue disease (MCTD) have been investigated radiologically. We investigated the esophageal lesions in MCTD histopathologically, and analyzed relationships between these lesions and autoantibodies extracted from the serum of MCTD patients. Esophageal tissues from 27 MCTD patients submitted to autopsy were examined. We compared histopathological features of the esophagus in different wall layers from the mucosa, submucosa, and muscular layer to the adventitia, and in the upper, middle, and lower portions of esophagus. The most striking change observed was severe atrophy and occasional loss of smooth muscle cells in the muscular layer, followed by fibrosis. These muscular changes were particularly prominent in the inner layer of the lower esophagus. Immunohistochemically, degenerated muscular tissues of the esophagus were positive for anti-IgG and anti-C3 antibodies, but not for anti-IgM antibodies. IgG fractions extracted from three MCTD patients were immunohistochemically used to examine whether some antibodies in MCTD patients showed reactivity for esophageal components. The IgG fractions isolated from MCTD patients reacted with smooth muscle from non-connective tissue disease cases, suggesting that some serum antibodies may trigger esophageal changes. These findings suggest that esophageal lesions associated with clinical dysphagia in MCTD may be related to autoantibodies.

Evaluation of Dot Immunoblot for identification of antibodies to Extractable nuclear antigen

This cross sectional study was done from January 2009 to June 2010 in Microbiology department of Dhaka Medical Collage. The study was designed to evaluate the advantages and reliability of dot immunoblot (DB) for identification of antibodies to extractable nuclear antigens (anti-ENA) in autoimmune connective tissue disorder (CTD). Sera from systemic lupus erythematosus patients (n = 37), systemic sclerosis patients (n = 13), rheumatoid arthritis patients(n=11), mixed connective tissue disease patients (n=6), polymyositis patients (n=7), and sjogren’s syndrome (n=2)were tested for antinuclear antibody (ANA) by Indirect Immunofluorescence (IIF) on HEp-2 cell and analyzed for the presence of antibodies to RNP, Sm, SSA, SSB, Scl-70, and Jo-1 (anti-ENAs) by dot immunoblot and ELISA. Out of 76sera of autoimmune connective tissue diseases 35 (46.05%) samples were found to be positive by DB and 33 (43.4%)were positive by ELISA. The dot blot assay exhibited 100% sensitivity for SSA, SSB, Scl-70 antibodies and for RNP the sensitivity was 90%; specificity was over 98% for these 4 ENA antibodies. Multiple reactivities were also found in a substantial number of patients sera. No serum was positive for anti-Sm and anti-Jo-1.Healthy controls also revealed no positive reactivity for anti-ENA by DB and ELISA. In conclusion, the dot immunoblot is a valid method for the detection of specific autoantibodies in autoimmune connective tissue disorder. Its most notable advantages are simultaneous detection of several autoantibodies in the same serum and its lower cost.DOI: http://dx.doi.org/10.3329/bjmm.v5i1.15815 Bangladesh J Med Microbiol 2011; 05 (01): 12-15

Pulmonary Arterial Hypertension in Connective Tissue Diseases

Pulmonary hypertension (PH) was found to be the primary cause of death in mixed connective tissue disease (MCTD). This led to investigation of the prevalence of PH in other connective tissue diseases (CTD). In 1998, the Ministry of Health and Welfare's MCTD Research Committee revealed complication of PH diagnosed by physicians in 5.02% MCTD patients, 0.90% systemic lupus erythematosus patients, 2.64% systemic sclerosis patients, and 0.56% polymyositis/dermatomyositis patients. These results have been supported by a similar survey performed in North America. As quite a few rheumatologists find right heart catheterization difficult to perform, doppler echocardiography is frequently used for screening and diagnosing PH. The MCTD Research Committee set the revised criteria for MCTD-PH, in which the threshold of estimated pulmonary arterial systolic pressure value for diagnosis of pulmonary arterial hypertension (PAH) is set at 36 mmHg, as proposed by the European Society of Cardiology. Right heart catheterization is strongly recommended for commencing the treatment. Since PH due to thromboembolism can potentially be cured surgically, lung perfusion scintigraphy should be performed for all patients diagnosed with PH. Most CTD-PH are PAH, and since idiopathic PAH (IPAH) patients sometimes have immune disorders, treatment for IPAH may be applicable to CTD-PH. The greatest difference between the treatment strategy for CTD-PH and IPAH is the usage of corticosteroids and other immunosuppressants. The MCTD Research Committee updated its therapeutic guidelines for MCTD-PH in 2011. Validation of these guidelines is also needed.

Clinical Significance of Anti-U1 RNP Antibodies Recognizing The Conformation Structure on U1 RNA/70-kd Protein Complex in Patients with Mixed Connective Tissue Disease

Objective. To study the clinical significance of anti-U1 ribonucleoprotein (RNP) antibodies recognizing the conformational structure of the U1 RNA/70-kd protein complex in patients with mixed connective tissue disease (MCTD). Methods. Antibodies recognizing the conformational structure of U1 RNP were detected in sera from 185 MCTD patients by comparing results obtained with enzyme-linked immunosorbent assays (ELISAs) using recombinant 70-kd, A and C proteins as the antigen sources (P-ELISA) with results obtained with ELISAs making use of those proteins plus in vitro-transcribed U1 RNA (C-ELISAs). Results. Fifteen of the 185 MCTD sera were positive in C-ELISAs but negative in P-ELISAs. Immunoprecipitation assays showed that, of those, 13 sera contained anti-U1 RNP antibodies that specifically recognized the conformational structure of the U1 RNA/70-kd protein complex. Comparison of the clinical characteristics revealed that 69% of the patients with anti-U1 RNA/70-kd protein complex antibodies also fulfilled the criteria for systemic lupus erythematosus (SLE). Moreover, the frequencies of almost all clinical features associated with SLE were higher in patients with antibodies against the U1 RNA/70-kd protein complex than in those without the antibodies; in particular, the frequencies of CNS lupus and positivity for anti-double stranded DNA antibodies were significantly higher in patients positive for anti-U1 RNA/70-kd protein complex antibodies. Conclusion. These results suggest that MCTD patients positive for anti-U1 RNA/70-kd complex antibodies that specifically recognize the conformational structure of the complex exhibit characteristic clinical features strongly associated with SLE and may represent a special subset of MCTD.

Sensorineural hearing loss as a common manifestation in patients with mixed connective tissue disease

Evaluation of: Hajas A, Szodoray P, Barath S et al.: Sensorineural hearing loss in patients with mixed connective tissue disease: immunological markers and cytokine levels. J. Rheumatol. 36, 1930–1936 (2009). In this study, Hajas et al. examined the prevalence of sensorineural hearing loss in patients with mixed connective tissue disease and its underlying mechanisms by measuring a series of immunological markers. Sensorineural hearing loss is a frequent organ manifestation affecting nearly half of patients with mixed connective tissue disease. Raynaud’s phenomenon, antiphospholipid and antiendothelial cell antibodies, increased serum levels of proinflammatory cytokines and a reduced number of regulatory T cells were found to be associated with sensorineural hearing loss in mixed connective tissue disease patients.

Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease.

IntroductionThe aim of the present study was to investigate the association between cardiovascular risk factors and endothelial dysfunction in patients with mixed connective tissue disease (MCTD) and to determine which biomarkers are associated with atherosclerotic complications, such as cardiovascular disease.MethodsFifty MCTD patients and 38 healthy age-matched and sex-matched controls were enrolled in this study. In order to describe endothelial dysfunction, we assessed flow-mediated dilation (FMD), nitrate-mediated dilation (NMD) and carotid artery intima-media thickness (IMT). We investigated FMD of the brachial artery after reactive hyperemia and NMD after sublingual nitroglycerin administration, while the IMT of the common carotid artery was determined by ultrasound. Anti-U1 ribonucleoprotein (anti-U1RNP) antibodies, anti-cardiolipin (anti-CL) antibodies, anti-endothelial cell antibody (AECA) and endothelial cell markers, such as soluble thrombomodulin (TM) and von Willebrand factor antigen (vWFAg), were assessed.ResultsThe endothelium-dependent vasodilation (FMD) was significantly impaired in patients with MCTD, as compared with controls (%FMD: 4.7 ± 4.2% vs. 8.7 ± 5.0%; P < 0.001), while the percentage NMD did not differ (%NMD: 14.3 ± 6.6% vs. 17.1 ± 6.7%; P = 0.073). Mean carotid IMT values were higher in patients than in controls (IMT: MCTD, 0.64 ± 0.13 mm vs. controls, 0.53 ± 0.14 mm; P < 0.001). FMD negatively correlated with disease duration, the levels of apolipoprotein A1, the paraoxonase-1 activity, and systolic blood pressure in MCTD patients. The percentage FMD was significantly lower in MCTD patients with cardiovascular diseases (CVD), than in those without CVD (%FMD: 3.5 ± 2.9 vs. 5.8 ± 4.8, P < 0.0002), while percentage NMD did not differ between patients with and without CVDs. Serum levels of autoantibodies (anti-U1RNP, AECA and anti-CL) were significantly higher in MCTD patients and differed between MCTD patients with and without CVD. Endothelial cell markers such as soluble TM (12.2 ± 8.1 ng/ml vs. 3.2 ± 1.3 ng/ml; P < 0.001) and vWFAg (224.1 ± 115% vs. 89.4 ± 27.1%, P < 0.001) were the highest in MCTD patients with CVD.ConclusionsFMD is a reliable sensitive marker of endothelial cell dysfunction in MCTD. Beside the traditional risk factors, anti-U1RNP, AECA and anti-CL antibodies may be important not only in the pathogenesis of MCTD but in the induction of endothelial cell activation, and may play crucial roles in the development of early atherosclerosis in MCTD.

Sensorineural Hearing Loss in Patients with Mixed Connective Tissue Disease: Immunological Markers and Cytokine Levels

To investigate the frequency of sensorineural hearing loss (SNHL) in patients with mixed connective tissue disease (MCTD). The study population consisted of 71 patients with MCTD (69 female; 2 male), with a mean age of 57.1 +/- 7.9 years and a mean disease duration of 14.5 +/- 8.0 years. All patients underwent audiological evaluation that included pure tone and speech audiometry. In addition, the systemic manifestations of the disease and drug therapy were recorded. All patients were tested for presence of autoantibodies. Fifty-one age-matched healthy subjects served as controls. SNHL was found in 33 (46.4%) of the 71 patients with MCTD. There was no correlation between SNHL and age and disease duration. An association was found between Raynaud's phenomenon (p < 0.03), secondary antiphospholipid syndrome (APS) (p < 0.05), and SNHL. MCTD patients with SNHL had higher serum levels of anti-U1RNP (p < 0.05), antiendothelial cell antibodies (p < 0.001), and IgG type anticardiolipin antibodies (p < 0.0001) than patients without SNHL. Serum levels of interferon-gamma and tumor necrosis factor-alpha were increased in MCTD patients with SNHL compared to patients without SNHL. The absolute number of natural (CD4+CD25(high)FoxP+) regulatory T cells (Treg) was lower compared to patients without SNHL. In MCTD, SNHL is a specific organ manifestation and appears frequently. We have found that pathogenic autoantibodies, decreased levels of regulatory T cells, and overexpression of proinflammatory cytokines may play a role in the pathogenesis of immune mediated inner ear disorders in MCTD.

The frequency of anti-β2-glycoprotein I antibodies is low and these antibodies are associated with pulmonary hypertension in mixed connective tissue disease

The objective of this study is to evaluate the prevalence of antiphospholipid antibodies, mainly anti-beta(2)-glycoprotein I (anti-beta(2)-GPI), and their possible clinical and laboratory relevance in mixed connective tissue disease (MCTD). This study included 39 consecutive patients with MCTD (Kasukawa's criteria) from January, 2005, to March, 2007, and compared them with 21 age- and sex-matched healthy controls. IgG and IgM anticardiolipin (aCL) and anti-beta(2)-GPI were measured by ELISA. Lupus anticoagulant (LA) was detected by functional coagulation tests. Medium to high titres of aCL and anti-beta(2)-GPI antibodies were found in sera from four (10.2%) MCTD patients. One of these patients was found to be positive for IgM aCL, IgM anti-beta(2)-GPI and LA antibodies simultaneously. Additionally, this patient had a previous history of foetal loss in the second trimester and new-onset pulmonary arterial hypertension (PAH). The other three patients had none of the manifestations of antiphospholipid syndrome (APS) or PAH. The mean value of IgG anti-beta(2)-GPI was higher among those MCTD patients with PAH than in the group without PAH (34.2 +/- 46.8 vs 12.3 +/- 9.1, P = 0.018). None of the controls were positive for antiphospholipid antibodies. High to moderate titres of anti-beta(2)-GPI as well as APS were rare in MCTD, and these antibodies may be correlated with the development of PAH in these patients.

Mixed Connective Tissue Disease, a Roundabout to Rheumatic Diseases?

Introduction: Mixed Connective Tissue Disease (MCTD) has originally been described as a syndrome that consisted of a combination of features typically found in patients with Systemic lupus erythematosus (SLE), Systemic Sclerosis (SSc), polymyositis/dermatomyositis (PM/DM), or rheumatoid arthritis (RA). Objective: The diagnosis of MCTD still remains controversial and it is critically dependent on the demonstration of high-titer anti-U1-RNP antibodies. When first described, MCTD, was believed to have a good prognosis. After longer follow-up studies this opinion has now been revisited: the prognosis in MCTD is in fact worse than that in LES, most of the deaths being due to pulmonary hypertension. The main question is whether or not MCTD patients have a distinctive clinical course, prognosis and treatment requirements. Conclusion: The importance of MCTD is not much in terms of diagnosis, but rather what antibody tells us about patients. The clinician must anticipate by treating the potential lethal complication of pulmonary hypertension, myositis and fibrosing alveolitis, and the tendency of the disease to evolve into one of its sister disease.

Chest abnormalities in juvenile-onset mixed connective tissue disease: Assessment with high-resolution computed tomography and pulmonary function tests

Mixed connective tissue disease (MCTD) is associated with several chest manifestations. Only a few studies have focused on chest manifestations in juvenile-onset MCTD (jMCTD), and the true prevalence of pulmonary abnormalities on high-resolution computed tomography (HRCT) in these patients is unknown. To investigate the occurrence of pulmonary abnormalities in jMCTD with particular reference to interstitial lung disease (ILD), and to evaluate a possible association between pulmonary findings and disease-related variables. Twenty-four childhood-onset MCTD patients with median disease duration of 10.5 years (range 1-21 years) were investigated in a cross-sectional study by means of HRCT, pulmonary function tests (PFT), and clinical assessment. Discrete ILD was identified in six patients (25%). Median extent of ILD was 2.0%, and all except one of the patients had very mild disease in which 5% or less of the parenchyma was affected. The CT features of fibrosis were mainly microcystic and fine intralobular. The most frequently abnormal PFT was carbon monoxide uptake from the lung, which was abnormal in 33% of the patients. PFT and disease duration were not significantly associated with HRCT findings of ILD. The prevalence of ILD in childhood-onset MCTD patients was lower than previously believed. In most of the patients with ILD, the findings were subtle and without clinical correlation. The results suggest a low extent of ILD in childhood-onset MCTD, even after long-term disease duration.

Human voltage-dependent anion selective channel 1 is a target antigen for antiglomerular endothelial cell antibody in mixed connective tissue disease

The purpose of this study was to identify the endothelial cell antigens that react with circulating antiendothelial antibody (AECA) in mixed connective tissue disease (MCTD). We screened serum AECA reactivity in 23 patients with MCTD using a human glomerular endothelial cell (HGEC) cellular ELISA. Proteomics, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the endothelial cell antigens of HGECs that reacted with serum antibodies from MCTD patients. Sera from 12 patients (52.0%) were positive for anti-HGEC antibody based on cellular ELISA. MALDI-TOF mass spectrometry used in combination with immunoblotting using serum antibody revealed one protein spot that represented a 36-kDa cell component of HGECs, with an isoelectric point (IP) of about 9, which had a high homology with the voltage-dependent anion-selective channel 1 (VDAC-1). This protein spot was confirmed to react with the antibody specific to VDAC-1. This is the first report of the presence of antibody to VDAC-1 from HGECs in the sera from MCTD patients. Although future studies will be needed to clarify the disease specificity of the a-VDAC-1 antibody in MCTD, the results show that modern proteomics technology is useful for identifying antigens that react with AECA in autoimmune diseases such as MCTD.

T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP).

In sera of patients with mixed connective tissue disease (MCTD) high titres of IgG autoantibodies to U1snRNP-specific proteins (70 kD, A, C) are found, suggesting an antigen-driven and T-cell-dependent process. In order to establish U1snRNP-specific T cell lines we cultured under various culture conditions mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells. Nine T cell lines were established by limiting dilution cloning from two MCTD patients and five T cell lines from a healthy individual. All T cell lines expressed the TCR alpha beta/CD3 complex. Surprisingly, most of the T cells lines exhibited the CD8 phenotype. Irrespective of this phenotype, all T cell lines showed a proliferative response to an N-terminal part (aa 51-195) of recombinant U1-specific 70-kD protein. One CD8+ T cell clone exhibited cytotoxic activity against an autologous B cell line pulsed with snRNP or recombinant fragments (aa 51-95 and aa 51-88). Interestingly, two T cell lines proliferated in response to four recombinant polypeptides representing different parts of the U1snRNP 70-kD protein. Since regions of sequence homology are distributed over the 70-kD molecule, it is suggested that conserved motifs may be recognized by the T cell lines.

Imbalance of CD4+ lymphocyte subsets in patients with mixed connective tissue disease.

CD4+ (helper/inducer) T lymphocyte subsets were studied in the peripheral blood from patients with mixed connective tissue disease (MCTD) by double-labelling immunofluorescence. The proportion of CD4+CD45RA+ cells was higher (P less than 0.01) when compared with controls, whereas CD4+CD29+ cells were markedly diminished (P less than 0.001). CD4+CD29+ cells were lower than in patients with progressive systemic sclerosis who were studied in parallel. Upon stimulation with phytohaemagglutinin, CD4+ cells from MCTD patients showed a strong reactivity to acquire the CD29+ phenotype. Expression of high levels of CD29 and other adhesion molecules might lead to facilitated localization of CD4+ cells to inflamed tissue. It is suggested that an increased responsiveness of CD4+ cells to activation signals in vivo and accumulation of CD4+CD29+ cells at tissue sites could result in depletion of this cell subset in the peripheral blood of patients with MCTD.

Human voltage-dependent anion selective channel 1 is a target antigen for antiglomerular endothelial cell antibody in mixed connective tissue disease

The purpose of this study was to identify the endothelial cell antigens that react with circulating antiendothelial antibody (AECA) in mixed connective tissue disease (MCTD). We screened serum AECA reactivity in 23 patients with MCTD using a human glomerular endothelial cell (HGEC) cellular ELISA. Proteomics, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the endothelial cell antigens of HGECs that reacted with serum antibodies from MCTD patients. Sera from 12 patients (52.0%) were positive for anti-HGEC antibody based on cellular ELISA. MALDI-TOF mass spectrometry used in combination with immunoblotting using serum antibody revealed one protein spot that represented a 36-kDa cell component of HGECs, with an isoelectric point (IP) of about 9, which had a high homology with the voltage-dependent anion-selective channel 1 (VDAC-1). This protein spot was confirmed to react with the antibody specific to VDAC-1. This is the first report of the presence of antibody to VDAC-1 from HGECs in the sera from MCTD patients. Although future studies will be needed to clarify the disease specificity of the a-VDAC-1 antibody in MCTD, the results show that modern proteomics technology is useful for identifying antigens that react with AECA in autoimmune diseases such as MCTD.

Successful Etanercept Therapy for Refractory Sacroiliitis in a Patient with Ankylosing Spondylitis and Mixed Connective Tissue Disease

The concurrence of ankylosing spondylitis (AS) in a patient with mixed connective tissue disease (MCTD) is rarely described in the literature. Significant and sustained efficacy with tumor necrosis factor (TNF)-α blockers has been demonstrated in AS patients. However, evidence to date has revealed associated side effects, including antinuclear antibody induction and development of a lupus-like syndrome. Several authors have reported lupus-like manifestations in MCTD patients treated with TNF-α blockers used to control peripheral polyarthritis. In our case report, we demonstrate a good response to etanercept therapy for refractory sacroiliitis in a patient with coexisting AS and MCTD, without development of a lupus-like syndrome. This demonstrates that etanercept therapy may be an appropriate therapeutic agent for sacroiliitis in MCTD patients, as it is in AS alone.

小児期発症全身性エリテマトーデスと混合性結合組織病の臨床的特徴の差異と抗U1-RNP抗体の意義

Mixed connective tissue disease (MCTD) includes clinical features of systemic lupus erythematosus (SLE), dermatomyositis/polymyositis (DM/PM), and systemic sclerosis (SSc) occurring in conjunction with a high anti-U1-RNP antibody titer. Childhood MCTD rarely manifests the symptoms and signs of DM/PM and SSc, and mostly does those of SLE. Thus, the diagnosis of childhood MCTD is inevitably based on the two major findings, Raynaud's phenomenon and a high titer of anti-U1-RNP antibody. However, in clinical setting there exist patients who have both anti-dsDNA antibody, a SLE disease-marker, and anti-U1-RNP antibody, a MCTD disease-marker, and thus it is hard to differentiate MCTD patients from SLE. Eighty children were enrolled in this study, and divided into 3 groups ; group A, those who are positive for anti-dsDNA antibody/negative for anti-U1-RNP antibody (48 cases, 60.0%), group B : those who are positive for both anti-dsDNA and anti-U1-RNP antibody (22 cases, 27.5%), group C; those who are negative for anti-dsDNA antibody/positive for anti-U1-RNP antibody (10 cases, 12.5%), and each of the clinical characteristics among these 3 groups was mutually examined. The results indicated that the frequency of hypocomplementemia in group B was close to group A rather than group C, and the frequencies of both hyper-gamma-globulinemia and Raynaud's phenomenon were very close to group C, but not to group A. On the contrary, the findings which seemed to be specific to MCTD, high titers of speckled type anti-nuclear antibody and rheumatoid factor, located at the middle between group A and group C. Thus, children in group B essentially carried characteristic symptoms and signs of both SLE and MCTD, and it will be difficult to differentiate these two diseases at the onset of the disease. Taken together, children with high titers of both anti-dsDNA antibody and anti-U1-RNP antibody as well as clinical symptoms and signs such as hyper-gamma-globulinemia, Raynaud's phenomenon, membranous nephritis, positive speckled type anti-nuclear antibody and rheumatoid factor should be followed and treated as children with MCTD along with SLE.