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Objectives SSc is an autoimmune disease with chronic and persistent inflammation in its pathogenesis. To examine the expression pattern of IL-16 in SSc lesions, the serum concentration of IL-16 in SSc patients and the relationship between serum IL-16 levels and the clinical symptoms of SSc were investigated. Methods Using immunohistochemical analysis, we examined the quantity and localization of IL-16 in affected skin obtained from SSc patients. We also measured serum levels of IL-16 in SSc patients using an ELISA. We then validated the correlation between serum IL-16 levels and clinical symptoms in patients with SSc. Results In the skin, IL-16 was expressed on the lymphocytes around the capillaries. Furthermore, the proportion of IL-16-positive cells was statistically higher in patients with dcSSc than in those with lcSSc patients (43.9 vs 29.1%, P < 0.05). The serum IL-16 levels in SSc patients were statistically significant elevated compared with healthy controls (297.0 vs 194.9 pg/ml, P < 0.05). Increased serum IL-16 levels in SSc patients were correlated with the proportion classified as dcSSc, skin score and the presence of cutaneous symptoms of erythema and pigmentation. Conclusion The regional up-regulation of IL-16 in the skin is not only associated with skin sclerosis, but also with systemic IL-16 activation. IL-16 may play a role in the pathogenesis of SSc. Moreover, serum IL-16 levels may be useful as a biomarker for determining the severity of the skin sclerosis. Inhibiting IL-16 activation may be effective in treating SSc. [ABSTRACT FROM AUTHOR]

Abstract: Several effective drugs have been identified for the treatment of systemic sclerosis (SSc). However, in advanced cases, not only their effectiveness is reduced but they may be also harmful due to their side‐effects. Therefore, early diagnosis and early treatment is most important for the treatment of SSc. We established diagnostic criteria for SSc in 2003 and early diagnostic criteria for SSc in 2011, for the purpose of developing evaluation of each organ in SSc. Moreover, in November 2013, the American College of Rheumatology and the European Rheumatology Association jointly developed new diagnostic criteria for increasing their sensitivity and specificity, so we revised our diagnostic criteria and severity classification of SSc. Furthermore, we have revised the clinical guideline based on the newest evidence. In particular, the clinical guideline was established by clinical questions based on evidence‐based medicine according to the New Minds Clinical Practice Guideline Creation Manual (version 1.0). We aimed to make the guideline easy to use and reliable based on the newest evidence, and to present guidance as specific as possible for various clinical problems in treatment of SSc. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that various nucleic acids are present in human sera, and attracted attention for their potential as novel disease markers in many human diseases. In this study, we tried to evaluate the possibility that DNA and RNA of collagens exist in human sera, and determined whether their serum levels can be useful biomarkers in scleroderma patients. The RNA or DNA of collagens were purified from sera, and detected by polymerase chain reaction or quantitated by real-time polymerase chain reaction. Among approximately 18 360 bases of full-length α1(I) collagen DNA, various regions were detected by polymerase chain reaction in human sera. However, α2(I) collagen DNA, α1(I) collagen RNA or α2(I) collagen RNA were not detectable. α1(I) Collagen DNA in sera was quantitative using our method. The levels of serum α1(I) collagen DNA were significantly increased in scleroderma patients compared with healthy control subjects or systemic lupus erythematosus patients. According to the receiver-operator curve analysis, serum α1(I) collagen DNA levels were shown to be effective as a diagnostic marker of scleroderma. Furthermore, when we determined the association of serum α1(I) collagen DNA levels with clinical/laboratory features in scleroderma patients, those with elevated α1(I) collagen DNA levels showed significantly higher prevalence of pitting scars/ulcers. In summary, elevation of serum α1(I) collagen DNA levels in scleroderma patients may be useful as the diagnostic marker, reflecting the presence of vasculopathy. [ABSTRACT FROM AUTHOR]

A multicenter, open-label study was performed to investigate the safety and tolerability of bosentan in Japanese patients with systemic sclerosis ( SSc) and secondary digital ulcers. Twenty-eight patients were enrolled. The safety and tolerability of bosentan was monitored over 52 weeks of study treatment (primary end-point), while incidence and healing of digital ulcers were also assessed up to week 16. The following adverse events occurred in 5% or more of patients during the 52-week treatment period: upper respiratory tract infection (50.0%), abnormal liver function tests (42.9%), digital ulcers (25.0%), anemia (17.9%), peripheral edema (14.3%), diarrhea (10.7%), urinary tract infection (7.1%), arthralgia (7.1%), constipation (7.1%) and herpes zoster (7.1%). Eight patients experienced at least one serious adverse event, including drug-related serious adverse events in two patients, which were abnormal liver function tests and fluid retention (pericardial effusion) in one patient each. During the 16-week observation period, seven out of 28 patients (25%) developed new digital ulcers. In this study, adverse events were comparable with those previously reported with bosentan. Approximately half of the patients had adverse events associated with abnormal liver function tests, thus we conclude that liver function should be monitored regularly during treatment with bosentan. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lnc RNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lnc RNAs in patients with systemic sclerosis ( SSc) and determined whether lnc RNAs controls collagen expression in dermal fibroblasts. lnc RNA expression was determined by real-time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real-time PCR. We found TSIX, one of the lnc RNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor ( TGF)-β1 si RNA. TSIX si RNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease-related cytokines. In addition, TSIX si RNA significantly reduced type I collagen mRNA stability, but not protein half-lives. Furthermore, we first investigated serum lnc RNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF-β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lnc RNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs. [ABSTRACT FROM AUTHOR]

Objective. To clarify the clinical course of SSc in Japanese patients with early-onset disease. It is well known that ethnic variations exist in the clinical features and severity of SSc. However, neither the clinical course nor prognostic factors have been thoroughly investigated in the Japanese population.Methods. Ninety-three Japanese patients of early-onset SSc (disease duration: <3 years) with diffuse skin sclerosis and/or interstitial lung disease were registered in a multi-centre observational study. All patients had a physical examination with laboratory tests at their first visit and at each of the three subsequent years. Factors that could predict the severity of skin sclerosis and lung involvement were examined statistically by multiple regression analysis.Results. Two patients died from SSc-related myocardial involvement and four patients died from other complications during the 3-year study. Among various clinical data assessed, the initial modified Rodnan total skin thickness score (MRSS) and maximal oral aperture were associated positively and negatively with MRSS at Year 3, respectively. Additionally, initial ESR tended to be associated with final MRSS. Pulmonary vital capacity (VC) in the third year was significantly associated with initial %VC. Furthermore, patients with anti-topo I antibody tended to show reduced %VC at Year 3.Conclusions. Several possible prognostic factors for skin sclerosis and lung function were detected in Japanese patients with early SSc. Further longitudinal studies of larger populations will be needed to confirm these findings. [ABSTRACT FROM PUBLISHER]

Jinnin M, Ihn H, Yazawa N, Asano Y, Yamane K, Tamaki K. Serum levels of manganese superoxide dismutase in patients with localized scleroderma. The objective was to determine the serum levels of manganese superoxide dismutase (Mn SOD) in patients with localized scleroderma and investigate their clinical significance in this disease. Serum samples from 15 patients with localized scleroderma and 20 healthy volunteers were examined by a specific enzyme-linked immunosorbent assay. Serum levels of Mn SOD were significantly higher in patients with generalized morphea than those in healthy individuals. And the patients with elevated serum Mn SOD levels had significantly larger number of sclerotic lesions and significantly higher serum levels of soluble interleukin-2 receptor than those without it. These results suggested that the serum levels of this enzyme may be a serological marker for the disease activity and the extent of skin involvement in this disease. [ABSTRACT FROM AUTHOR]

Serum containing anti-U1RNP antibodies reacts with the nuclear matrix, the relatively insoluble component of the cell nucleus, in addition to U1RNP. In this study, we determine the serum titer and clinical correlations of antinuclear matrix antibodies in samples from patients with anti-U1RNP antibodies. The patients with anti-U1RNP antibodies were classified as having mixed connective tissue disease (MCTD, 15 patients), systemic sclerosis (SSc, 12 patients), systemic lupus erythematosus (SLE, 7 patients), and undifferentiated CTD (UCTD, 9 patients). Antinuclear matrix antibodies were detected using indirect immunofluorescence staining on HCl-treated HEp-2 cells. The antinuclear matrix antibody titer was significantly higher in serum from patients with MCTD or SSc than in serum from patients with SLE or UCTD. The antinuclear matrix antibody titer was significantly increased in serum from patients with sclerodactyly, pitting scars, contracture of the phalanges, and decreased carbon monoxide diffusion capacity. Thus, a higher titer of antinuclear matrix antibodies in serum from patients with anti-U1RNP antibodies may be associated with a clinical diagnosis of MCTD or SSc rather than a diagnosis of SLE or UCTD. [ABSTRACT FROM AUTHOR]

• CXCL17 reduced type I collagen expression via MMP1 and miR-29 in skin fibroblasts. • CXCL17 levels in SSc skin were lower than those in healthy controls. • CXCL17 attenuated the skin fibrosis induced by bleomycin in mice. Systemic sclerosis (SSc) is characterized by excessive deposition of collagen in the skin and internal organs. Recent studies have shown that chemokine (C-X-C motif) ligands (CXCLs) are involved in the pathogenesis of SSc. Our aim was to examine the anti-fibrotic potential of CXCL17, a newly discovered chemokine, in cultured skin fibroblasts and in a bleomycin-induced SSc mouse model. Moreover, we examined serum level of CXCL17 in patients with SSc. Type I collagen expression was evaluated in SSc skin and cultured fibroblasts treated with CXCL17 using immunoblotting and quantitative reverse transcription-PCR. Serum CXCL17 levels were determined using enzyme-linked immunosorbent assay in 63 patients with SSc and 17 healthy subjects. A bleomycin-induced SSc mouse model was used to evaluate the effect of CXCL17 on skin fibrosis. CXCL17 reduced the expression of type I collagen in healthy control fibroblasts. CXCL17 also induced matrix metalloproteinase 1 (MMP1) and miR-29 expression in fibroblasts, indicating that CXCL17 regulates type I collagen expression in part via post-transcriptional mechanisms through MMP1 and miR-29. We found that local injection of CXCL17 attenuated bleomycin-induced skin fibrosis in mice. CXCL17 levels in SSc skin were lower than those in healthy controls, in contrast to the high serum CXCL17 levels in patients with SSc. The low expression of CXCL17 in SSc skin possibly affects type I collagen accumulation in this disease. Our data indicate that understanding CXCL17 signaling may lead to a better therapeutic approach for SSc. [ABSTRACT FROM AUTHOR]

Background Various cytokines have been indicated to be involved in the pathogenesis of systemic sclerosis (SSc). IL-22 is one of the member of IL-10 cytokine family, and several studies have implicated IL-22 signaling in the pathogenesis of autoimmune diseases. Objectives To clarify the role of IL-22 in the regulatory mechanism of ECM expression and to determine the contribution of IL-22 to the phenotype of SSc. Methods The effect of IL-22 on ECM expression in normal fibroblasts was determined by using PCR array, real-time PCR and immunoblotting. microRNA expression was evaluated by real-time PCR. The expression levels of IL-22 in the skin and sera were determined by using immunohistochemical staining and ELISA. Results IL-22 significantly increased the expression of type I collagen protein without changing its mRNA levels in cultured normal human dermal fibroblast. The expression of let-7a, one of the microRNAs which have negative effect on type I collagen expression, was significantly decreased by the treatment with IL-22 in dermal fibroblasts. There was no significant difference in the serum levels of IL-22 between SSc patients and control subjects. However, the expression of IL-22 was detected in the infiltrated lymphocytes in the SSc dermis, but not in normal dermis. IL-22 receptors were expressed in both normal and SSc dermal fibroblasts to the similar extent. Conclusion IL-22 expressed in infiltrated lymphocytes may stimulate the up-regulation of type I collagen protein in dermal fibroblasts via let-7a down-regulation in SSc skin. [ABSTRACT FROM AUTHOR]

Background We recently generated induced pluripotent stem cells (iPSCs) from cultured dermal fibroblasts of systemic sclerosis (SSc-iPSC) to study the disease mechanisms. Objective In the present study, we have performed gene expression analysis using cultured SSc dermal fibroblasts, SSc-iPSC, and fibroblasts re-differentiated from SSc-iPSC (SSc-iPSC-FB). Methods mRNA and protein levels of collagen and integrins were analyzed using PCR array, PCR, immunoblotting, and immunofluorescence. Results We compared expression pattern of TGF-β-related genes between normal iPSC (NS-iPSC) and SSc-iPSC by PCR array, and found constitutive and significant down-regulation of S100A8, Smad6, and TGF-β2 in SSc-iPSC. The expression of these genes was not altered in cultured SSc fibroblasts or SSc-iPSC-FB compared to NS fibroblasts or NS-iPSC-FB, respectively. On the other hand, the expression of collagen, integrin α and β was up-regulated in SSc fibroblasts, while SSc-iPSC-FB showed normalized levels of collagen and integrin β. Conclusions So far, there have been no reports investigating disease-derived iPSCs of SSc. Our results suggest that S100A8, Smad6, and TGF-β2 may be the key molecules of this disease. On the other hand, the normalization of collagen and integrins by iPSC reprogramming suggests that epigenetic modifications of genes may play a role in the mechanism of collagen accumulation seen in SSc fibroblasts, and that gene reprogramming may become novel therapeutic approach. As the limitation of this study, we established only one iPSC line from each patient, which may not be enough to discuss disease-specific phenotypes. Larger studies including increased number of iPSC lines are needed in the future. [ABSTRACT FROM AUTHOR]

Background Exosomes are small vesicles shed from various cells. They contain proteins, lipids, and nucleic acids, and are regarded as a tool of cell-cell communication. Objectives To reveal the putative role of exosomes in systemic sclerosis (SSc), and to elucidate the effect of exosomes on wound healing. Methods The expression of common markers for exosomes (CD63, CD9, and CD81) and type I collagen were examined with real-time PCR, immunohistochemical analysis, ELISA, immunoblotting, and flow cytometry. The effect of serum-derived exosomes on wound healing was tested on full-thickness wounds in the mid-dorsal skin of BALB/c mice. Results The expression levels of CD63 as well as CD9 and CD81 tended to be increased in SSc dermal fibroblasts compared to normal fibroblasts. Increased exosomes in a cultured media of SSc fibroblasts stimulated the expression levels of type I collagen in normal fibroblasts. As the mechanism, collagen-related microRNA levels in SSc fibroblast-derived exosomes were dysregulated, indicating that both the amount and the content of exosomes were altered in SSc. On the other hand, SSc sera showed significantly decreased exosome levels compared to normal sera. The frequencies of vascular involvements, including skin ulcers or pitting scars, were significantly increased in patients with decreased serum exosome levels. The healing of mice wounds was accelerated by treatment with serum-derived exosomes. Conclusions Vascular abnormalities in SSc may account for the decreased serum exosome levels by the disturbed transfer of exosomes from the skin tissue to the blood stream. Our study suggests the possibility that SSc patients with vascular involvements have decreased serum exosome levels, which causes the delay of wound healing due to down-regulation of collagen, resulting in higher susceptibility to pitting scars and/or ulcers. Exosome research will lead to a detailed understanding of SSc pathogenesis and new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Hypoxia may play an important role in the pathogenesis of systemic sclerosis (SSc). Carbonic anhydrase IX (CA IX) is one of the hypoxia markers and its extracellular domain can be released into the serum. However, the clinical significance of serum CA IX levels in SSc is still unknown. The aim of this study is to evaluate the possibility that serum CA IX levels can be a specific disease marker of SSc. Serum samples were obtained from SSc patients and healthy controls. Patients diagnosed as scleroderma spectrum disorder (SSD), who did not fulfill the ACR criteria of SSc but were thought that they might develop SSc in the future, were also included in this study. Serum CA IX levels were measured with specific enzyme-linked immunosorbent assays. SSD patients had significantly lower CA IX levels than diffuse cutaneous SSc (dcSSc), limited cuntaneous SSc (lcSSc) and healthy control groups. Also, we found a significant decrease in the values in dcSSc patients compared to those of lcSSc patients. Serum levels of CA IX may be useful for the differentiation of lcSSc from SSD. Decreased serum CA IX levels in spite of the presence of hypoxia in SSc may indicate an impaired response to hypoxia, which leads to the persistent hypoxic condition. Our results suggest that the abnormal response to hypoxia may already exist in SSD patients, and may be involved in its pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Background: Rapamycin has been shown to exert an anti-fibrotic effect on skin fibrosis in a certain subset of patients with systemic sclerosis (SSc) and in bleomycin-treated animal models. Objectives: To investigate the mechanism responsible for the anti-fibrotic effect of rapamycin especially by focusing on human α2(I) collagen (COL1A2) and matrix metalloproteinase 1 (MMP1) genes in normal and systemic sclerosis (SSc) dermal fibroblasts. Methods: The expression levels of type I procollagen and MMP1 proteins were analyzed by immunoblotting and the mRNA levels of COL1A2 and MMP1 genes were evaluated by quantitative real-time RT-PCR. The activities of COL1A2 and MMP1 promoters were determined by reporter analysis. Results: Rapamycin significantly decreased the levels of type I procollagen protein and COL1A2 mRNA, while significantly increasing the levels of MMP1 protein and mRNA in normal dermal fibroblasts. Similar effects of rapamycin were also observed in SSc dermal fibroblasts. Importantly, the inhibitory and stimulatory effects of rapamycin on the mRNA levels of COL1A2 and MMP1 genes, respectively, were significantly greater in SSc dermal fibroblasts than in normal dermal fibroblasts. In SSc dermal fibroblasts, rapamycin affected the expression of COL1A2 gene at the post-transcriptional level. In contrast, rapamycin altered the expression of MMP1 gene at the transcriptional level through the JNK/c-Jun signaling pathway in those cells. Conclusion: Rapamycin has a potential to directly regulate the deposition of type I collagen in extracellular matrix through inhibiting type I collagen synthesis and promoting its degradation by MMP1, suggesting that this drug is useful for the treatment of SSc. [Copyright &y& Elsevier]

Abstract: Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. There is an urgent need to develop new therapeutic approaches against skin fibrosis. Although intravenous immunoglobulin (IVIG) may be one of the promising treatments, the mechanisms by which IVIG improves the fibrosis of SSc remain unknown. Objectives: To compare the cytokine profile in the sera and skin of SSc patients before and after IVIG administration, and try to clarify the mechanism of the effect of IVIG. Methods: Each three patients received 5-day administration of IVIG, or the same dose of physiologic saline for placebo. Cytokine levels were determined by ELISA array, immunostaining, and real-time PCR. Results: Cytokine array revealed that the serum levels of IFN-γ and IL-12, representative Th1 cytokines, were increased by IVIG treatment, but not by placebo. The percentage of IFN-γ- and IL-12-positive cell number/CD4-positive T lymphocyte cell number was also significantly increased by IVIG in SSc skin. Furthermore, mRNA expression of IFN-γ and IL-12 in SSc skin tissue was significantly up-regulated after IVIG treatment. Conclusion: The expression of Th1 cytokine is reported to be decreased in SSc. Our study suggested IVIG recovered the suppressed levels of Th1 cytokines, and that the treatment improves skin fibrosis by correcting the Th1/Th2 balance. In order to facilitate the clinical use of IVIG for SSc, it is necessary to perform a larger study in the future. [Copyright &y& Elsevier]