杉浦 一充

Background. The myositis-specific autoantibodies that characterize certain forms of inflammatory myopathy are useful in diagnosing dermatomyositis (DM) / polymyositis and predicting its prognosis. Autoantibodies to small ubiquitin-like modifier activating enzyme (SAE) have been identified as a DM-marker antibody in European Caucasians. Objective and methods. This study investigates the frequency and clinical characteristics of anti-SAE autoantibodies in Japanese patients with DM. Sera from 110 Japanese patients, including 13 with juvenile DM, were screened for anti-SAE antibodies by enzyme-linked immunosorbent assays. Positive sera were further examined by immunoblotting of the immunoprecipitates. Results. Only two patients (1.8%) were confirmed to have anti-SAE antibodies, and neither of these two patients had amyopathic or juvenile DM. One patient with anti-SAE had DM complicated with pulmonary arterial hypertension, and the other had cancer-associated DM. Both had hallmark cutaneous manifestations of DM. Conclusion. This is the first report of anti-SAE antibodies from an Asian single center cohort. Although Japanese patients with anti-SAE antibodies have a clinical phenotype similar to that of Caucasian patients, their frequency was lower in the Japanese patients than in the previously reported Caucasian patients. © Informa UK, Ltd.

Autoantibodies against melanoma differentiation-associated gene 5 (MDA5) are important serological markers in dermatomyositis (DM) with rapidly progressive interstitial lung disease (ILD). Recent studies noted that anti-MDA5 antibody (anti-MDA5ab), ferritin, and IL-18 are useful biomarkers for evaluating the responses to treatment and the status of ILD in anti-MDA5ab-positive DM. In this study, we further studied the importance of anti-MDA5ab levels and of ferritin and IL-18 concentrations in our patients. These biomarkers could be sometimes useful for evaluating ILD status and/or predicting the prognosis in patients with anti-MDA5ab-positive DM with several exceptional cases. A single-point evaluation of anti-MDA5ab levels and of ferritin and IL-18 concentrations has limitations in predicting the prognosis of ILD with DM. We consider that the timing of initial therapy and the anti-MDA5ab isotype, in addition to the patient's age, are also crucial factors for predicting the prognosis.

Objective: The constituents of the centromere region, centromere protein (CENP)-A, -B, and -C, are mainly targeted by anticentromere antibodies (ACA). Many other proteins also assemble around CENP-A nucleosomes in interphase nuclei to form the interphase centromere complex (ICEN). CENP-H, -I, -K, -L, -M, -N, -T, and -U have been reported as the constitutive components of ICEN. In this study, we examined the reactivities of ACA to the 8 CENPs for the purpose of investigating their autoantigenicity. Methods: Sera from 95 patients with ACA were tested by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) with the recombinant C-terminal of CENP-B (Ct-CENP-B). Next, the sera were examined for autoantibodies against the 8 CENPs by WB with each recombinant protein. Furthermore, the coiled-coil motifs and granzyme B (GB) cleavage for various CENPs were analyzed with computer tools. Results: Out of 95 ACA-positive sera, 85 and 93 sera were positive for anti-Ct-CENP-B antibodies in WB and in ELISA, respectively. In WB using the 8 CENPs, no sera reacted to any other 7 CENPs, except 1 serum, which reacted weakly to CENP-T. We were unable to find any obvious relationships between the autoantigenicity of CENPs and coiled-coil-forming probabilities or potential substrates for GB. Conclusion: This study demonstrates that ACA rarely target the 8 CENPs, in contrast to CENP-B. Protein structures might not contribute in a major way to the autoantigenicity of CENPs.

Linear cutaneous lupus erythematosus (LCLE) is a rare subtype of cutaneous lupus erythematosus. We describe a 22-year-old Japanese man who had an 11-year history of asymptomatic linear erythema from the right upper back to the dorsum of the right hand. The lesions followed the lines of Blaschko and spread over three large joints. Histological findings were compatible with discoid lupus erythematosus. Although the most common site for LCLE is the face, a few cases of LCLE on the extremities have been reported. To our knowledge, this is the first reported case of an extraordinarily long, continuous LCLE skin lesion. © 2013 by the article author(s).

Anti-Th/To autoantibodies have been recognized as serological markers of systemic sclerosis (SSc) for more than 20 years. However, validated immunoassay kits to test this specificity have not been commercially available. SSc autoantibodies are basically mutually exclusive and are associated with a certain subset of the disease and/or with organ involvement. Anti-Th/To are generally considered to be markers of the limited cutaneous type of SSc with the involvement of certain internal organs. The excellent correlation between anti-Rpp25 as detected by their novel chemiluminescent method and anti-Th/To as detected by immunoprecipitation suggest that the new assays may become widely available tests for clinicians in future and could help to clarify the clinical significance of anti-Th/To in SSc as well as other conditions over different races or countries.

Hailey-Hailey disease (HHD, MIM 16960) is an autosomal dominant disease characterized by suprabasal cell separation (acantholysis) of the epidermis. The clinical features vary and include crusted erosions with vesicular pustules, and erythematous scaly plaques at sites of friction and flexures. The skin lesions are often exacerbated by heat, sweating, mechanical trauma, infection and exposure in ultraviolet B (UVB) (1). Patients have a defect in ATP2C1 encoding the ATPase, Ca2+-transporting, type 2C, member 1; (ATP2C1) on the Golgi apparatus (2). We performed mutation analysis of ATP2C1 in a Japanese patient with HHD and identified the heterozygous novel mutation c.212delT (p.Leu71ArgfsX26). This is a very early truncating mutation, which clearly suggests that haploinsufficiency is an underlying pathomechanism of HHD. © 2013 Acta Dermato-Venereologica.

Advances in immunology, biochemistry, and molecular biology have enabled the development of a number of assays for measuring autoantibodies. ELISA has been widely used, because it can deal with relatively large numbers of serum samples more quickly than other immunologic methods, such as immunoblotting and immunoprecipitation. Recombinant autoantigens, which are generally produced in E. coli using the relevant cloned cDNA, are necessary for ELISA. Conventional clinical ELISA tests are limited in their ability to purify proteins free of bacterial contaminants, and the process is labor intensive. We recently developed new ELISA tests that utilize simple in vitro transcription and translation labeling of autoantigens in order to measure dermatomyositis- (DM-) specific autoantibodies, including autoantibodies to Mi-2, MDA5, NXP-2, TIF1-alpha, and TIF1-gamma. This method may allow for the rapid conversion of cDNAs to a chemiluminescent ELISA to detect autoantibodies that are found not only in DM but also in other autoimmune diseases.

Objective. Autoantibodies against melanoma differentiation-associated gene 5 (MDA-5) are one of the serological markers for DM. Anti-MDA-5 antibodies are especially associated with rapidly progressive interstitial lung disease (ILD) in amyopathic DM (ADM). It is known that the antibody status of anti-ENAs does not generally change significantly with disease course. For anti-MDA-5 antibodies, however, few longitudinal studies have investigated such changes. This study aimed to establish a quantitative assay for anti-MDA-5 antibodies towards assessing the long-term outcome of ADM patients who had anti-MDA-5 antibodies. Methods. We established ELISA for measuring anti-MDA-5 antibody levels using in vitro transcription and translation recombinant protein. The antibody levels were measured at different time points in 11 clinically ADM patients who tested positive for the anti-MDA-5 antibody on their first visit (range of follow-up 3 months to 16 years). Results. At the stage of clinical remission, six patients received no medication and the four others received low-dose CS. ELISA showed that anti-MDA-5 antibodies disappeared in nine of the patients and fell to just above the cut-off in one patient; in the patient who died, the antibodies remained. Conclusion. Our results suggest that anti-MDA-5 antibodies may be useful as a marker for monitoring disease activity in ILD complicated with ADM. Serial monitoring at short intervals is required to evaluate whether anti-MDA-5 antibody levels correlate with ADM disease activity.

Lens epithelium-derived growth factor (LEDGF)/dense fine speckles 70 kDa protein (DFS70) is a transcription cofactor that enhances growth and is overexpressed in various cancers. In the epidermis, LEDGF/DFS70 localizes to the nucleus of keratinocytes (KCs) in the basal layers and to the cytoplasm of cells in the upper layers. However, the biological and pathological relevance of LEDGF/DFS70 in the epidermis is virtually unknown. Compared with normal epidermis, we detected strong nuclear staining of LEDGF/DFS70 in both the spinous and basal layers of the epidermis of psoriatic skin. To investigate the roles of LEDGF/DFS70 in the epidermis of psoriatic skin, we generated HaCaT cells that constitutively express enhanced green fluorescence protein (EGFP)-LEDGF (EGFP-LEDGF-HaCaT) or EGFP alone (EGFP-HaCaT) as a control. EGFP-LEDGF-HaCaT cells had increased expression of IL-6, which was attenuated by LEDGF-specific RNA interference and the p38-specific inhibitors SB-239063 and SB-203580. Furthermore, EGFP-LEDGF-HaCaT cells had increased expression of S100A7 and S100A9 and decreased expression of filaggrin. These findings are compatible with the expression pattern in psoriatic tissues. Taken together, these results strongly suggest that ectopic expression of LEDGF/DFS70 in KCs could be involved in the pathology of psoriasis vulgaris.

Objectives. Myositis-specific autoantibodies are useful for diagnosing PM/DM. Recently, two new myositis-specific autoantibodies against melanoma differentiation-associated gene 5 (MDA5) and transcriptional intermediary factor 1-γ (TIF1-γ) were identified in DM. Here, we detected these autoantibodies in patient sera using new assays with recombinant MDA5 and TIF1-γ, and associated clinical features with the presence of anti-MDA5 or anti-TIF1-γ antibodies. Methods. We screened 135 Japanese patients with various CTDs, including 82 with DM. DM patients were classified as clinically amyopathic DM (CADM), cancer-associated DM or classical DM without cancer. Anti-MDA5 and anti-TIF1-γ antibodies were detected by their ability to immunoprecipitate biotinylated recombinant proteins.Results. Sera from 21 (26%) of 82 DM patients immunoprecipitated MDA5, and every anti-MDA5-positive patient had DM (except one patient with SSc). Sera from 20 (65%) of 31 CADM patients reacted with MDA5. Notably, anti-MDA5-positive DM patients had significantly more interstitial lung disease than anti-MDA5-negative DM patients (95 vs 32%, P < 0.001). Sera from 12 (15%) of 82 DM patients immunoprecipitated TIF1-γ, and anti-TIF1-γ antibodies were only detected in DM patients. Strikingly, 7 (58%) of 12 patients with cancer-associated DM had sera that reacted with TIF1-γ. Anti-TIF1-γ-positive DM patients had significantly more internal malignancies than anti-TIF1-γ-negative DM patients (58 vs 9%, P < 0.001).Conclusions. Anti-MDA5 and anti-TIF1-γ antibodies were confirmed to be serological DM subset markers. Anti-MDA5 and anti-TIF1-γ antibodies were detected based on their ability to immunoprecipitate biotinylated recombinant MDA5 and TIF1-γ, and were closely associated with life-threatening complications in DM. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) is a transducer of mitogenic signals, and is involved in the pathogenesis and progression of a number of cancers, including non-small cell lung cancer (NSCLC). Gefitinib is an EGFR-TK inhibitor that is clinically used to treat NSCLC; however, this drug frequently causes adverse effects, including skin eruptions. The mechanism underlying these skin reactions is elusive, although it is assumed that they are caused by the inhibition of EGFR-TK signalling in epidermal and adnexal cells. In this article, we demonstrate by immunocytochemistry that the skin lesions of patients treated with oral gefitinib had higher expression of CCL2 and CCL5 compared to normal human epidermis. Further, PD153035, a gefitinib prototype, induced CCL2 and CCL5 mRNA and protein expression in HaCaT and HSC-1 keratinocyte cell lines with or without interleukin-1 (IL-1) treatment in vitro. PD153035 also reduced the levels of interleukin-1 receptor 2 (IL-1R2), an IL-1 decoy receptor. Moreover, we demonstrate that reduction in IL-1R2 by RNA interference increased IL-1-mediated CCL2 and CCL5 mRNA and protein expression. Taken together, our data strongly suggest that IL-1-mediated signalling is activated to induce the high expression of CCL2 and CCL5 via reduction in IL-1R2 in the skin lesions caused by gefitinib.

The unfolded protein response (UPR), which is induced by stress to the endoplasmic reticulum (ER), is involved in the functional alteration of certain cells, such as the differentiation of B cells to plasma cells. The aim of this study is to determine whether the UPR is activated during epidermal keratinocyte (KC) differentiation. Here, we show that the expression of the UPR-induced proteins Bip/GRP78 and HRD1 was increased in cells in the suprabasal layers of normal human epidermis that contain KCs undergoing differentiation as well as in skin-equivalent cultured KCs. However, Bip/GRP78 and HRD1 were poorly expressed in proliferating KCs in squamous cell carcinoma and psoriasis vulgaris tissues. The epidermal growth factor receptor tyrosine kinase inhibitor, PD153035, which induces KC differentiation, upregulated UPR-induced marker mRNAs and proteins. Furthermore, microarray analyses and quantitative PCR revealed that ER stress-inducing reagents, tunicamycin (TU), thapsigargin, and brefeldin A, altered the expression of genes essential for human epidermal KC differentiation, including C/EBP beta, KLF4, and ABCA12 in vitro. However, ABCA12 and KLF4 mRNA did not increase with TU treatment after siRNA-mediated knockdown of XBP-1. Taken together, our findings strongly suggest that the UPR is activated during normal epidermal KC differentiation and induces C/EBP beta, KLF4, and ABCA12 mRNAs.

Objective. The ankle and toe brachial indices (ABI and TBI) are calculated as the ankle and toe systolic blood pressures divided by the highest brachial systolic pressure, respectively. We sought to evaluate the efficacy of ABI and TBI as an objective, non-invasive assessment of vascular involvement in patients with systemic sclerosis (SSc) and to investigate the clinical significance of TBI in SSc. Methods. ABI and TBI were measured using an oscillometric method in 136 outpatients, including 77 with SSc, 29 with systemic lupus erythematosus (SLE), 16 with primary Sjögren's syndrome (SjS), and 14 with dermatomyositis (DM). We also analyzed 21 healthy controls. Results. The mean ABI and frequency of reduced ABI values (&lt 1.0) did not differ significantly between disease groups. TBI values in patients with SSc and lSSc were significantly lower than in those with SjS and DM, respectively (p&lt 0.01). Patients with SSc and lSSc had significantly lower TBI values than healthy controls (p&lt 0.05). Reduced TBI values (&lt 0.6) were significantly more common in patients with SSc, including both dSSc and lSSc, than in those with SLE (p&lt 0.05). Similarly, the frequency of decreased TBI was higher in patients with SSc and dSSc than in those with SjS or healthy controls (p&lt 0.05). Skin ulcers (p=0.041) or overlap with rheumatoid arthritis (p=0.018) were associated with reduced TBI values by logistic regression analysis. Conclusion. The TBI value is a useful, non-invasive tool to evaluate vascular involvement in SSc. © Copyright Clinical and Experimental Rheumatology 2009.

Objectives: To investigate the prevalence of anti-ribosomal P protein (anti-P) antibodies in Japanese patients with connective tissue diseases (CTDs) using enzyme-linked immunosorbent assays (ELISAs) and western blotting (WB) and to evaluate the indirect immunofluorescence (IIF) staining patterns of anti-P-positive sera. Methods: Anti-P antibodies were measured by two different commercially available ELISA kits and WB in 239 outpatients, 99 with systemic sclerosis (SSc), 73 with systemic lupus erythematosus (SLE), 45 with dermatomyositis (DM), and 22 with Sjogren's syndrome (SjS). Sera positive for anti-P antibodies by WB were analysed by IIF. Results: The frequency of positive WB findings in SLE (18/73, 25%) was higher than in other diseases. ELISA kits A and B for anti-P antibodies showed 21% and 43% sensitivity, and 93% and 88% specificity, respectively, for diagnosing SLE, based on the manufacturer's cut-off values. Receiver operating characteristic (ROC) curve analysis, based on positive WB findings, determined a new cut-off threshold but revealed that both ELISA kits still had good diagnostic characteristics. In IIF assays on anti-P antibody positive sera, typical anti-P antibody cytoplasmic staining patterns (n = 8) were seen less frequently than other staining patterns (n = 17). Conclusions: Routine screening for anti-P antibodies by IIF has low sensitivity. ELISAs using cut-off values established by individual facilities are suitable for detecting anti-P antibodies and provide a tool with good diagnostic characteristics, on a parity with WB.

We evaluated the utility of HEp-2 cells transfected with the 60 kDa SS-A/Ro as a substrate for indirect immunofluorescence (IIF-HEp-2000) to compare several methods for screening Japanese serum samples for anti-SSA/Ro antibodies. Serum samples from 243 Japanese patients were analyzed by IIF for anti-nuclear antibodies (ANAs), using HEp-2 cells (IIF-HEp-2), and for anti-SS-A/Ro 60 kDa antibodies, using IIF-HEp-2000 and enzyme-linked immunosorbent assay (ELISA). We performed double immunodiffusion and immunoprecipitation experiments, using the products of in vitro transcription and translation, to analyze sera for which there were discrepancies in the results of the IIF-HEp-2000 assay and ELISA. A total of 93 of the 243 serum samples showed findings positive for anti-SS-A/Ro antibodies. Notably, eight of the 93 sera gave positive findings for anti-SS-A/Ro antibodies by IIF-HEp-2000 but ANA-negative by IIF-HEp-2 analysis. Seven sera possessing anti-SS-A/Ro antibodies gave false negative results by IIF-HEp-2000; however, those samples were all ANA positive. ELISA for anti-SS-A/Ro antibodies showed that five and two samples gave false positive and negative results, respectively. Analysis by IIF-HEp-2000 was useful for primary screening of patients for ANAs, especially for anti-SS-A/Ro antibodies; the test could detect anti-SS-A/Ro antibodies not identified on standard substrates in samples obtained from the Japanese population, as reported for the Caucasian population.

Objectives: To determine if anti-cyclic citrullinated peptide (anti-CCP) antibody titers can distinguish the overlap syndrome of systemic sclerosis and rheumatoid arthritis (SSc-RA) in patients with systemic sclerosis (SSc) and to investigate the clinical significance of anti-CCP antibodies in SSc. Methods: Serum levels of anti-CCP antibodies were measured by enzyme-linked immunosorbent assay in 159 outpatients: 114 with SSc, 14 with rheumatoid arthritis, 7 with SSc-RA overlap syndrome, and 24 with Sjögren's syndrome. In patients with SSc and SSc-RA, we also measured serum levels of matrix metalloproteinase-3 and anti-agalactosyl IgG antibody. Results: Elevated serum levels of anti-CCP antibodies were observed in 3 of 114 patients (2.6%) with SSc, 9 of 14 patients (64%) with RA, 6 of 7 patients (86%) with SSc-RA, and only 1 of 24 patients (4.2%) with SjS. In patients with SSc-RA, serum anti-CCP antibody levels were significantly higher than those seen in SSc (p&lt 0.001). The sensitivity, specificity, and predictive values of elevated anti-CCP titers for SSc-RA were higher than either matrix metalloproteinase-3 and anti-agalactosyl IgG antibodies as markers. In addition, almost all SSc-RA and SSc patients with elevated serum levels of anti-CCP antibodies exhibited arthralgias and interstitial pneumonia. Conclusions: Anti-CCP antibody titers are a reliable marker of SSc-RA facilitating its distinction from SSc alone. © Copyright Clinical and Experimental Rheumatology 2008.

Autoantibodies against dense fine speckles 70 (DFS70) are found in 10% of healthy individuals, but only in a tiny population of patients with autoimmune rheumatic disease. The antibody may thus be a marker of autoimmune rheumatic disease negativity. To investigate this possibility, we examined the presence of various disease-marker autoantibodies in anti-DFS70 antibody-positive patients with autoimmune rheumatic disease. Serum samples from 500 patients with various types of autoimmune rheumatic disease were examined for anti-DFS70 antibodies by indirect immunofluorescence and immunoblotting. Various disease-marker autoantibodies were measured by enzyme-linked immunosorbent assay. Twenty-two patients were positive for anti-DFS70 antibodies. Eighteen patients also had disease-marker autoantibodies including anti-double stranded DNA, anti-cardiolipin, anti-SS-A, or other antibodies. In one patient with Sjogren syndrome and two patients with dermatomyositis, no disease-marker antibodies were found; however, one patient with dermatomyositis had a concomitant anti-cytoplasmic antibody. All seven systemic lupus erythematosus patients fulfilled the classification criteria for this disease even if anti-nuclear antibody-positive findings were excluded. One patient with morphea had high-titer anti-single stranded DNA antibody. According to this and previous studies, patients with only anti-DFS70 antibody are rarely diagnosed as having autoimmune rheumatic disease. Recognizing dense fine speckle patterns in anti-nuclear antibodies tests is, thus, very important for analysis of laboratory results in rheumatology clinics.

Objective To evaluate the physical functional impairment inpatients with systemic sclerosis (SSc) using the Health Assessment Questionnaire (HAQ) and to estimate the correlation of HAQ scores with the severity of SSc. Method One hundred and twenty-four outpatients with connective tissue disease, including 50 patients with SSc, were evaluated using the HAQ. Twelve patients were classified as having diffuse cutaneous SSc (dSSc) and 38 limited cutaneous SSc (ISSc). The severity classification and the guidelines for treatment (2004) were applied to Japanese SSc patients in order to evaluate the relationship between HAQ scores and disease activity in patients with multiple organ involvement. Results In dSSc the HAQ category scores for eating, walking, grip, activity and the HAQ-disability index (HAQ-DI)showed the greatest deficits in all disease groups. The severity of disease activity correlated significantly with the scores for walking, reach, and the HAQ-DI. The severity of joint, heart, and pulmonary hypertension were correlated independently with the HAQ-DI score by multiple linear regression analysis. Conclusion Patients with dSSc suffer greater functional impairment than patients with other connective tissue diseases, and improvements in hand use and walking represent very important targets for both drug development and rehabilitation. As improvement in organ involvement (joints, heart as well as pulmonary hypertension) can lead to reduced functional impairment, they constitute an important target for therapy in SSc.

p80 coilin is a nuclear autoantigen that strongly accumulates in Cajal bodies (CB) and is considered a marker for CBs. No clear clinical features have been associated with anti-p80 autoantibodies, and the epitopes recognized by these antibodies are entirely unknown. We determined the epitope specificity for 34 anti-p80 coilin-positive sera from Japanese patients to better understand its clinical significance. Western blotting and immunoprecipitation analysis revealed that the autoepitope of p80 coilin was restricted to a single region encompassing amino acid residues 421-576 from the C-terminus of p80 coilin. Additionally, sera from 30 of 34 (88%) patients with anti-p80 coilin antibody titers were also positive for anti-DPS70/LEDGF antibody. This autoantibody is sometimes found in patients with some allergic diseases but rarely in patients with connective tissue disease (CTD). Other similar characteristics between these two autoantibodies were also noted. In contrast, few anti-p80 coilin positive patients were positive for CTD-marker antibodies, such as anti-Sm, anti-dsDNA, and anti-SS-A. We conclude that the presence of anti-p80 coilin autoantibodies is not associated with any particular clinical syndromes nor is it diagnostic of any conditions. (c) 2005 Elsevier Ltd. All rights reserved.

Circulating prolactin levels in a woman with systemic lupus erythematosus (SLE) were measured over a period of 4 years, and her hyperprolactinemia remained associated with disease activity throughout. Circulating prolactin could thus be a better practical marker than standard parameters such as anti-double-stranded DNA antibodies and serum complement in some patients with SLE. © Japan College of Rheumatology and Springer-Verlag Tokyo 2005.

Alopecia areata (AA) has been suspected to be an autoimmune disease, although there is no distinct evidence, we investigated the relationship between AA and autoantibodies against dense fine speckles 70, kDa (DFS70) in 111 patients with alopecia and 105 healthy controls. The sera from 59 out of 111 (53%) Japanese alopecia patients were positive for anti-nuclear antibody (ANA), as compared to the sera of 16 out of 1:05 (15%) healthy controls (p < 0.001). Twenty percent (22/111) of the alopecia patients were shown to be positive for the prevalence of anti-DFS70 antibodies, as compared to 8% (8/105) of the healthy controls (P < 0.01). IgG subclass analysis by ELISA showed that IgG1 and IgG2-anti-DFS70 antibodies were dominant in alopecia patients. The DFS70 gene expression in the hair structures was clearly detected in both those with and those without the anti-DFS70 antibody by RTPCR. immunohistochemical techniques showed that the DFS70 was localized predominantly in the outer root sheath (ORS) cells. The elevated anti-DFS70 antibodies in alopecia patients and the localization of the DFS70 in the ORS suggest that autoantibodies against the DFS70 are related to the etiology in a certain population of AA. (C) 2004 Elsevier Ltd. All rights reserved.

Autoantibodies against DFS70 (Dense Fine Speckles 70) are found in 30% of Japanese atopic dermatitis patients, and less frequently in patients with other diseases. We have recently reported that they are also seen in 11% of hospital workers, but in only similar to2% of patients with systemic rheumatic disease. In this study, in order to investigate the possible pathological role of anti-DFS70 antibodies, fine epitope mapping was carried out using 93 anti-DFS70 autoantibody-positive sera. Immunoblotting using overlapping peptides failed to reveal major linear epitopes. Western blotting using various truncated proteins showed a strikingly uniform epitope distribution on a suspected tertiary structure expressed by DFS70(349-435). Some sera showed reactivity only in an immunoprecipitation assay using an in vitro translated DFS70. Circular dichroism analysis revealed that DFS349-435 contains an approximately 40% alpha-helical conformation, while an overlapping, non-antigenic peptide is composed of random coiled structures. The skewed single major epitope enabled us to establish a highly quantitative ELISA for the epitope region. Antibody titers showed no significant differences between the diseased group and healthy individuals. We propose that anti-DFS70 antibody may be a natural autoantibody, which might modify or reflect the inflammatory process of various disorders. (C) 2004 Elsevier Ltd. All rights reserved.

We report the case of a child with unilateral linear scleroderma disturbing the right lower limb. It took three-quarters of a year to be diagnosed as linear scleroderma and another 8 months to be treated with oral steroids. Although functional disabilities of the right knee and foot were improved with the steroid therapy, the limb asymmetry has remained. We believe that early treatment with steroids is essential for childhood linear scleroderma with lesions over any joints. © Japan College of Rheumatology and Springer-Verlag Tokyo 2004.

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.

The 1994 case definition of chronic fatigue syndrome is widely used not only for diagnosis but also for clinical and laboratory-based observations of this clinical entity. The criteria for the 1994 case definition are based primarily on symptoms and not on physical signs or chemical or immunological tests. This situation has resulted in conflicting clinical and laboratory observations that in all likelihood is due to different populations of patients being studied in different centers. Based on some of the recent publications, there appears to be an emerging picture of this disease entity that we propose could be used to subgroup chronic fatigue syndrome into four different subclasses. These subclasses would consist of chronic fatigue with primarily nervous system disorders such as impaired memory or concentration and headache, chronic fatigue with primarily endocrine system disorders such as unrefreshing sleep and postexertional malaise, chronic fatigue with musculoskeletal system disorders such as muscle pain and joint pain, and chronic fatigue with immune system/infectious disorders such as sore throat and tender lymph nodes. It is suggested that if clinical and laboratory-based studies on chronic fatigue syndrome were conducted on more homogeneous subgroups of patients, the data from one center to the other might not be as conflicting and more insights can be shed on the nature of this clinical condition.

Objective. To elucidate the frequency and clinical significance of anti-annexin V antibodies in patients with scleroderma. Methods. The study population consisted of 66 patients with scleroderma. Their sera were examined for IgG anti-annexin V antibodies by ELISA and immunoblotting. Results. IgG anti-annexin V antibodies were detected by ELISA in 12 patients (18.2%) with scleroderma. Anti-annexin V antibodies were associated with digital ischemia in the patients with scleroderma. Only one patient of the 12 had anticardiolipin antibodies. Although 6 patients with anti-annexin V antibodies were also examined for activated partial thromboplastin time (APTT), none showed prolonged APTT. No IgG anti-annexin V antibodies were detected by immunoblotting. Conclusion. Anti-annexin V antibodies in scleroderma are related to digital ischemia. These antibodies may be associated with the pathogenesis of vascular involvement in scleroderma.

We isolated a cDNA encoding a human homologue of ZF5 (hZF5), which has five Kruppel-like C2H2 type zinc fingers at carboxyl terminus and the BTB/POZ (poxvirus and zinc finger) at the amino terminus, using autoimmune sera from a patient with overlap syndrome (dermatomyositis and scleroderma). Sequencing of the entire cDNA revealed an open reading frame (ORF) of 1349 bp with a deduced protein sequence of 449 amino acid residues and a calculated molecular weight of 51.3 kDa. The deduced amino acid sequence of hZF5 is highly homologous to mouse ZF5 (99.3% identity). Immunofluorescence studies revealed that HA-tagged hZF5 transiently expressed in COS-7 cells showed the nuclear dot pattern in the BTB/POZ domain-dependent manner.

The Ki-67 antigen, which is recognized by the monoclonal antibody Ki- 67, is a marker of cell proliferation. During cDNA cloning using sera from a patient with systemic lupus erythematosus, we obtained a positive clone encoding part of Ki-67. We determined the frequency of autoantibodies against Ki-67 in patients with systemic autoimmune diseases. Serum samples from 252 patients with rheumatic diseases were examined by immunoblotting with HeLa nuclear extract and the recombinant N-terminus of the Ki-67 antigen. Autoantibodies against Ki-67 antigen were detected in two out of 76 patients with systemic lupus erythematosus and one out of 90 patients with scleroderma. While, in a previous report, anti-Ki-67 antibodies were frequently targeted by a certain strain of autoimmune mice, our results indicated that Ki-67 was a minor target of autoantibodies among Japanese patients with systemic autoimmune diseases.