Efficacy and safety of tocilizumab in Japanese patients with systemic sclerosis and associated interstitial lung disease: A subgroup analysis of a global, randomised, controlled Phase 3 trial.

OBJECTIVES: The aim of this article is to investigate the efficacy and safety of tocilizumab in Japanese patients with systemic sclerosis. METHODS: Post hoc subgroup analysis of a global, randomised, controlled trial in patients treated with weekly tocilizumab 162 mg or placebo subcutaneously in a 48-week double-blind period (tocilizumab and placebo groups) followed by tocilizumab for 48 weeks in an open-label extension (continuous-tocilizumab and placebo-tocilizumab groups). RESULTS: Among 20 patients, 12 were randomised to tocilizumab (all had interstitial lung disease) and eight were randomised to placebo (six had interstitial lung disease). The modified Rodnan skin score improved in both treatment groups. The mean change in percent-predicted forced vital capacity was 3.3% [95% confidence interval (CI), -2.5 to 9.0] for tocilizumab and -3.8% (95% CI, -9.9 to 2.2) for placebo in the double-blind period and 2.0% (95% CI, -0.7 to 4.6) for continuous-tocilizumab and -1.4% (95% CI, -6.7 to 4.0) for placebo-tocilizumab in the open-label extension. Rates of serious adverse events per 100 patient-years were 19.3 for tocilizumab and 26.8 for placebo in the double-blind period and 0.0 for continuous-tocilizumab and 13.6 for placebo-tocilizumab in the open-label period. CONCLUSIONS: The efficacy and safety of tocilizumab in patients with systemic sclerosis were consistent between the Japanese subpopulation and the global trial population.

Nintedanib in patients with systemic sclerosis-associated interstitial lung disease: A Japanese population analysis of the SENSCIS trial.

OBJECTIVE: We examined the efficacy and safety of nintedanib in Japanese patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) in the global Safety and Efficacy of Nintedanib in Systemic Sclerosis (SENSCIS) trial. METHODS: Randomised patients received oral nintedanib 150 mg (N = 34) twice daily or placebo (N = 36) until the last patient reached 52 weeks of treatment (up to 100 weeks). Data were analysed using a subgroup analysis model with Japanese and non-Japanese patients as subgroup variables. RESULTS: In Japanese patients, the adjusted annual rate of forced vital capacity (FVC) decline over 52 weeks was -86.2 mL/year (nintedanib) and -90.9 mL/year (placebo); treatment difference, 4.67 mL/year (95% confidence interval, -103.28, 112.63). Treatment effect heterogeneity between Japanese and non-Japanese patients was not detected (treatment-by-visit-by-subgroup interaction; p = .49). FVC decline was smaller for nintedanib versus placebo through 100 weeks in Japanese patients. The most commonly reported adverse events with nintedanib were gastrointestinal and liver disorder events; most were mild-to-moderate in severity. CONCLUSION: In both Japanese and non-Japanese patients with SSc-ILD, nintedanib slowed the progression of ILD, with no heterogeneity detected between the subgroups. The safety profile for nintedanib in Japanese patients was similar to that observed in patients with idiopathic pulmonary fibrosis (ClinicalTrials.gov: NCT02597933).

Initial predictors of skin thickness progression in patients with diffuse cutaneous systemic sclerosis: Results from a multicentre prospective cohort in Japan.

OBJECTIVE: To identify initial parameters that predict worsening of skin thickening in patients with diffuse cutaneous systemic sclerosis (dcSSc) using a multicentre, prospective, observational cohort in Japan. METHODS: A total of 171 patients with dcSSc were selected from a prospective cohort database based on the following criteria: dcSSc, modified Rodnan total skin thickness score (mRSS) ≥7, disease duration <60 months, and valid mRSS data at one year. Worsening of skin thickness was defined as an increase in mRSS ≥3 points and an increase ≥25% from baseline to one year. Initial demographic and clinical parameters useful for predicting the progression of skin thickness were identified using univariate and multivariable analysis, and prediction models of skin thickening progression were built based on combinations of independent predictive parameters. RESULTS: Only 23 patients (13.5%) experienced worsening mRSSs at one year. Short disease duration, low mRSS, absence of nailfold bleeding, arthritis, and a high erythrocyte sedimentation rate at diagnosis were identified as predictors of subsequent worsening of the mRSS even after adjusting for the treatment. Assessment of the best predictive model revealed that patients with a disease duration ≤12 months and mRSS ≤19 had a risk of mRSS worsening within one year, with a sensitivity of 73.9% and specificity of 81.1%. CONCLUSION: Identification of predictors of subsequent worsening of skin thickness in dcSSc patients is useful for identifying patients who require intensive treatment with potential disease-modifying agents and for improving clinical trial design by characterizing eligible progressors in the Japanese population.

Clinical course of Japanese patients with early systemic sclerosis: A multicenter, prospective, observational study.

OBJECTIVES: To investigate the clinical course of Japanese patients with early diffuse cutaneous systemic sclerosis (dcSSc) and early SSc with interstitial lung disease (ILD). METHODS: We prospectively analyzed the clinical features of 207 Japanese patients with early dcSSc (n = 150) and limited cutaneous SSc (lcSSc) with ILD (n = 57) in 10 medical centers every year for 7 consecutive years. RESULTS: Mean modified Rodnan total skin thickness score (mRSS) was 18.3 and 67.4% of the cohort had ILD. Most patients started immunosuppressive therapy and vasodilators during 7 years (83.4% and 87.9%, respectively). Mean value of mRSS of total patients was significantly reduced from the initial registration after the first year. However, other parameters for physical function associated with skin sclerosis including fist closure, hand extension, and oral aperture were not so ameliorated during the study period. Health Assessment Questionnaire-disability index and serum KL-6 levels were constant throughout the course. Percent vital capacity and the presence of ILD, clinically suspected pulmonary arterial hypertension, and digital ulcers were gradually exacerbated during the period. CONCLUSION: In Japanese early dcSSc patients and SSc patients with ILD, mRSS was continuously reduced during 7 years of follow-up, but there was little improvement of physical disability and organ involvement.

Association of functional (GA)n microsatellite polymorphism in the FLI1 gene with susceptibility to human systemic sclerosis.

OBJECTIVE: Susceptibility genes that can account for characteristic features of SSc such as fibrosis, vasculopathy and autoimmunity remain to be determined. In mice, deficiency of Friend leukaemia integration 1 transcription factor (Fli1) causes SSc-like disease with these features. The human FLI1 gene contains (GA)n microsatellite, which has been shown to be associated with expression level. Because microsatellite polymorphisms are difficult to capture by genome-wide association studies, we directly genotyped FLI1 (GA)n microsatellite and examined its association with SSc. METHODS: Genomic DNA from 639 Japanese SSc patients and 851 healthy controls was genotyped for (GA)n microsatellite using the fragment assay. The cut-off repeat number for susceptibility to SSc was determined by receiver operating characteristics (ROC) analysis. Association with susceptibility and clinical characteristics was examined using logistic regression analysis. FLI1 mRNA levels were determined using quantitative RT-PCR. RESULTS: Based on the ROC analysis, (GA)n alleles with ≥22 repeats were collectively defined as L alleles and alleles with ≤21 repeats as S alleles. (GA)n L alleles were significantly associated with susceptibility to SSc (P = 5.0e-04, odds ratio 1.34, additive model). Significant association was observed both in diffuse cutaneous and limited cutaneous SSc. Among the SSc, (GA)n L alleles were significantly enriched in the patients with a modified Rodnan total skin thickness score ≥10 compared with those with a score <10. FLI1 mRNA levels were significantly decreased in healthy controls carrying (GA)n L alleles as compared with non-carriers. CONCLUSION: Extended repeat alleles of FLI1 (GA)n microsatellite may be associated with lower FLI1 mRNA levels and susceptibility to human SSc.

Availability of EuroQol-5-Dimensions-5-Level (EQ-5D-5L) as health-related QOL assessment for Japanese systemic sclerosis patients.

Objective: To assess the functional disability in Japanese patients with systemic sclerosis (SSc) using the EuroQol-5-Domain-5-Level health questionnaire (EQ-5D-5L), which was developed in Europe to demonstrate the cost utility of treatments for non-specific diseases.Methods: The EQ-5D-5L and Disability Index of the Health Assessment Questionnaire (HAQ-DI), which is a questionnaire for the quality of life for rheumatic diseases, were completed by 109 Japanese patients with SSc, and the clinical findings and laboratory data were collected at the same time.Results: There was a correlation between the EQ-5D-5L score and HAQ-DI score. The EQ-5D-5L index score was affected by the % of predicted vital capacity (%VC), pulmonary arterial hypertension, and renal crisis. The %VC and renal crisis were also indicated as factors reducing the quality of life in the HAQ-DI. There was no difference in the EQ-5D-5L score between the SSc subtypes or among autoantibodies.Conclusion: Our single-center study demonstrated the EQ-5D-5L to be a valuable assessment tool for functional disability in Japanese SSc patients, similarly to the disease specific HAQ-DI.

Human leukocyte antigen in Japanese patients with idiopathic inflammatory myopathy.

Objective: The human leukocyte antigen (HLA) is the strongest genetic risk factor for idiopathic inflammatory myopathy (IIM), and different HLA alleles have been reported to be associated with IIM susceptibility among different ethnic groups. In this study, we have investigated HLA alleles associated with IIM in Japanese patients.Methods: Genotyping of HLA-DRB1 and DPB1 were performed in 252 Japanese IIM patients (166 dermatomyositis [DM] and 86 polymyositis [PM] patients) and the association was analyzed with comparison to controls (n = 1026 for DRB1 and n = 413 for DPB1).Results: DRB1*08:03 was associated with IIM (p = 1.60 × 10-5, pc = .0005, odds ratio [OR] 2.11, 95% confidence interval [CI] 1.52-2.92) and DM (p = .0004, pc = .0128, OR 2.06, 95%CI 1.40-3.02). DPB1*05:01 was also associated with IIM (p = .0001, pc = .0021, OR 1.96, 95%CI 1.38-2.77) and DM (p = .0005, pc = .0075, OR 2.05, 95%CI 1.37-3.08). DRB1*09:01 (p = .0012, pc = .0368, OR 0.35, 95% CI 0.18-0.69) and DPB1*04:01(p = .0004, pc = .0057, OR 0.05, 95% CI 0.00-0.85) were protectively associated with PM. Two locus analyses suggested that DRB1*09:01 and DPB1*04:01 were independently associated with PM.Conclusion: Protective associations of HLA were detected in Japanese PM patients.

Increased expression levels of FcγRIIB on naïve and double-negative memory B cells in patients with systemic sclerosis.

OBJECTIVES: In systemic sclerosis (SSc), B cell hyperactivation and abnormality are considered to play an important role in the disease pathophysiology. We aimed to clarify if the abnormal activation of B cells involves inhibitory FcγRIIB on B cells in SSc patients. METHODS: Blood samples were collected from 76 SSc patients (38 limited cutaneous SSc and 38 diffuse cutaneous SSc) and 59 healthy controls. We evaluated the expression levels of FcγRIIB on different B cell subsets. B cells were classified into five subsets based on their surface phenotype as measured by flow cytometry: naïve B cells (CD19+IgD+CD27-), pre-switched memory B cells (CD19+IgD+CD27+), double-negative (DN) memory B cells (CD19+IgD-CD27-), switched memory B cells (CD19+IgD-CD27mid), and plasmablasts (CD19+IgD-CD27high). The expression levels of the activation markers CD80, CD86, and CD95 were also examined. RESULTS: The expression levels of FcγRIIB on SSc naïve and DN memory B cells were significantly increased compared to healthy controls (p<0.05 and p<0.001, respectively). CD80, CD86, and CD95 expression levels were significantly higher in all five B cell subsets, except for CD80 in switched memory B cells and plasmablasts. Increased FcγRIIB expression levels on DN memory B cells were associated with disease activity as assessed by the European Scleroderma Study Group Activity Index, presence of interstitial lung disease (ILD), and reduced lung function. Intravenous cyclophosphamide pulse therapy decreased FcγRIIB expression levels on memory B cell subsets. CONCLUSIONS: SSc B cells may exhibit compensatory elevation in the expression levels of FcγRIIB in order to suppress the abnormal activation of B cells. In addition, FcγRIIB expression levels may serve as a marker of severe complications, such as ILD, in SSc.

Long-term follow-up of finger passive range of motion in Japanese systemic sclerosis patients treated with self-administered stretching.

OBJECTIVE: Severe skin sclerosis in patients with systemic sclerosis (SSc) can result in a loss of hand function. The aim of this study is to examine the long-term changes of finger passive range of motion (ROM) in Japanese SSc patients treated with self-administered stretching. METHODS: This is a single-center, retrospective, observational cohort study. Forty-three Japanese patients with SSc were given instructions on self-administered stretching. ROM was assessed using a goniometer on their first visit and after 1 year, 3 years, 5 years and 9 years. Hand function was assessed by the Health Assessment Questionnaire disability index (HAQ-DI) at their first visit and after 9 years. RESULTS: Total passive ROM significantly improved in each finger after 3 years of finger stretching. Most patients (37 of 43 patients, 86%) improved or maintained total passive ROM and hand function within 9 years after their first visit. However, significant improvement of total passive ROM was lost in 6 of 43 SSc patients (14%) 9 years after their first visit. The HAQ-DI also was increased in these six patients. Multivariable analyses revealed that re-elevation of modified Rodnan total skin thickness score during the clinical course (OR = 5.260e + 7, 95% CI 1.52e + 150-uncalculated p = .0096) was the independent factor associated with deterioration of total passive ROM at 9 years. CONCLUSION: Patients with progressive skin sclerosis during the clinical course need multimodality therapy to maintain finger joint motion, since the effect of self-administered stretching is limited in these patients.

Blockade of p38 Mitogen-Activated Protein Kinase Inhibits Murine Sclerodermatous Chronic Graft-versus-Host Disease.

Bone marrow transplantation (BMT) of B10.D2 mice into sublethally irradiated BALB/c mice across minor histocompatibility loci is a well-established animal model for human sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) and systemic sclerosis (SSc). The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammation and cytokine production. Furthermore, the activation of p38 MAPK plays an important role in collagen production in SSc. We investigated the effects of p38 MAPK inhibitor, VX-702, on Scl-cGVHD mice. VX-702 was orally administered to Scl-cGVHD mice from day 7 to 35 after BMT. We compared skin fibrosis of Scl-cGVHD mice between the VX-702-treated group and control group. Allogeneic BMT increased the phosphorylation of p38 MAPK in the skin. The administration of VX-702 attenuated the skin fibrosis of Scl-cGVHD compared to the control mice. Immunohistochemical staining showed that VX-702 suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the dermis of Scl-cGVHD mice compared to the control mice. VX-702 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines, such as IL-6 and IL-13, in the skin of Scl-cGVHD mice. In addition, VX-702 directly inhibited collagen production from fibroblasts in vitro. VX-702 was shown to be a promising candidate for use in treating patients with Scl-cGVHD and SSc.

Transethnic meta-analysis identifies GSDMA and PRDM1 as susceptibility genes to systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by skin and systemic fibrosis culminating in organ damage. Previous genetic studies including genome-wide association studies (GWAS) have identified 12 susceptibility loci satisfying genome-wide significance. Transethnic meta-analyses have successfully expanded the list of susceptibility genes and deepened biological insights for other autoimmune diseases. METHODS: We performed transethnic meta-analysis of GWAS in the Japanese and European populations, followed by a two-staged replication study comprising a total of 4436 cases and 14 751 controls. Associations between significant single nuclear polymorphisms (SNPs) and neighbouring genes were evaluated. Enrichment analysis of H3K4Me3, a representative histone mark for active promoter was conducted with an expanded list of SSc susceptibility genes. RESULTS: We identified two significant SNP in two loci, GSDMA and PRDM1, both of which are related to immune functions and associated with other autoimmune diseases (p=1.4×10-10 and 6.6×10-10, respectively). GSDMA also showed a significant association with limited cutaneous SSc. We also replicated the associations of previously reported loci including a non-GWAS locus, TNFAIP3. PRDM1 encodes BLIMP1, a transcription factor regulating T-cell proliferation and plasma cell differentiation. The top SNP in GSDMA was a missense variant and correlated with gene expression of neighbouring genes, and this could explain the association in this locus. We found different human leukocyte antigen (HLA) association patterns between the two populations. Enrichment analysis suggested the importance of CD4-naïve primary T cell. CONCLUSIONS: GSDMA and PRDM1 are associated with SSc. These findings provide enhanced insight into the genetic and biological basis of SSc.

Autoantibody to scaffold attachment factor B (SAFB): A novel connective tissue disease-related autoantibody associated with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel connective tissue disease (CTD)-related autoantibody (autoAb) directed against scaffold attachment factor B (SAFB). METHODS: AutoAb specificity was analyzed using RNA and protein-immunoprecipitation assays. Autoimmune targets were affinity purified using patients' sera and subjected to liquid chromatography mass spectrometry. RESULTS: By immunoprecipitation assay, 10 sera reacted with a protein with a molecular weight of approximately 160 kDa. Liquid chromatography mass spectrometry of the partially purified autoantigen and additional immunoblot-based analyses revealed that the Ab specifically recognized SAFB. Anti-SAFB Abs were detected in 2 of 646 patients with systemic sclerosis (SSc) (0.3%), 1 of 1570 patients with polymyositis/dermatomyositis (0.06%), 4 of 270 patients with interstitial lung disease (ILD) (1.5%), 1 of 43 patients with overlap syndrome (2.3%) and 2 patients with other diseases including primary Raynaud's disease and eosinophilic pneumonia. Five patients with anti-SAFB Abs had Raynaud's phenomenon and 3 had nail fold punctate hemorrhage. Of note, 8 of the 10 patients (80%) suffered from ILD. None of the patients with anti-SAFB Abs had pulmonary arterial hypertension, heart disease, or renal involvement. CONCLUSIONS: Anti-SAFB Ab is a novel CTD-related autoAb possibly associated with ILD.

A novel splenic B1 regulatory cell subset suppresses allergic disease through phosphatidylinositol 3-kinase-Akt pathway activation.

BACKGROUND: IL-10-producing regulatory B (B10) cells potently suppress allergic diseases, such as contact hypersensitivity (CHS). Splenic B10 cells share overlapping phenotypic markers with CD5+ B1 B cells, CD1dhiCD21+CD23- marginal zone (MZ) B cells, and CD1dhiCD21+CD23+ T2-MZ precursor B cells but do not exclusively belong to either subset. OBJECTIVE: In this study we investigated the signaling mechanisms and a novel phenotypic parameter of B10 cells. METHOD: We performed microarray analysis comparing IL-10+ and IL-10- B cells. B cell-specific phosphatase and tensin homolog (PTEN)-deficient mice, which exhibit aberrant activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in B cells, were examined. RESULTS: Microarray analysis revealed that the PI3K-Akt pathway is important for IL-10 production in B cells. PI3K-Akt pathway inhibitors reduced B10 cell numbers in vitro. B10 cell numbers were significantly increased in B cell-specific PTEN-deficient mice. The CHS response was significantly diminished in PTEN-deficient mice. Unexpectedly, splenic B10 cells in these mice were found within the B1 B-cell subset but not within the MZ B-cell subset. In wild-type mice not only MZ B10 cells but also B1-B10 cells were identified in the spleen. In addition, these 2 B10 cell subsets were predominantly found within the CD9+CD80+ B-cell fraction. CONCLUSION: A novel splenic B1 regulatory cell subset (B1-B10 cells) was identified. Our findings show that the PI3K-Akt pathway in B cells is critical for B10 cell development and CHS response and that CD9/CD80 coexpression is a novel phenotypic parameter for both MZ-B10 and B1-B10 cells.

Classification of Japanese patients with mild/early systemic sclerosis (SSc) by the 2013 ACR/EULAR classification criteria for SSc.

OBJECTIVE: To classify Japanese patients with mild/early systemic sclerosis (SSc) by the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) Classification Criteria (new 2013 criteria). METHODS: We assessed 120 patients who visited Kanazawa University Hospital suspected of SSc and who did not meet the 1980 ACR preliminary classification criteria for SSc. We clinically diagnosed 16 patients with primary Raynaud's disease and 104 with mild/early SSc prior to being assessed by the new 2013 criteria. RESULTS: None of the 16 patients with primary Raynaud's disease met the new 2013 criteria. On the other hand, 94 out of the 104 patients (90.3%) with mild/early SSc by our clinical diagnosis met the new 2013 criteria. Among the 94 SSc patients, sclerodactyly was detected in 58 (62%), puffy fingers in 62 (66%), abnormal nailfold capillaries in 89 (95%), Raynaud's phenomenon in 93 (99%), and SSc-related autoantibodies (Abs) in 85 (90%). The median (range) score of these 94 patients was 12 (9-14). Ten mild/early SSc patients who did not meet the new 2013 criteria had the following clinical features: puffy fingers in 1 (10%), abnormal nailfold capillaries in 8 (80%), Raynaud's phenomenon in 9 (90%), and SSc-related autoAbs in 8 (80%). The median (range) score of these 10 patients was 7 (5-8). CONCLUSION: The new 2013 criteria can classify most mild/early Japanese SSc patients, which may contribute to early treatment interventions.

Decreased levels of regulatory B cells in patients with systemic sclerosis: association with autoantibody production and disease activity.

OBJECTIVE: B cell abnormalities characterized by autoantibody production and polyclonal B cell activation play an important role in the pathogenesis of SSc. IL-10 producing regulatory B (Breg) cells also play an important role in the negative immune response. We identified a human Breg cell subset that was predominantly found within the CD24(hi)CD27(+) B cell subpopulation. However, the role of Breg cells in SSc remains unknown. The aim of this study was to investigate the clinical association of Breg cells in SSc patients. METHODS: Blood IL-10 producing Breg cell levels were determined by FACS in 35 SSc patients and 30 healthy subjects. In a follow-up study, we analysed six individual dcSSc patients before and after treatment. RESULTS: The frequency of blood Breg cells was significantly lower in SSc patients than in healthy controls (P < 0.0001). Similarly, the frequency of CD24(hi)CD27(+) B cells was significantly lower in SSc patients than in healthy controls (P < 0.0001). SSc patients with decreased Breg cell levels often had interstitial lung disease (P < 0.05). Furthermore, Breg cell levels correlated negatively with the titre of anti-topo I antibody (Ab) and anticentromere Ab in SSc patients. For a follow-up study, Breg cell levels in dcSSc patients after treatment were found to be significantly increased compared with those before treatment (P < 0.05), accompanied by decreased disease activity. Thus, Breg cell levels were inversely correlated with disease activity of SSc. CONCLUSION: These results suggest that decreased Breg cell levels may contribute to the development of SSc.

B cells promote tumor immunity against B16F10 melanoma.

B cells are known to be critical mediators of tumor immunity; however, the mechanisms through which they exert this function remain unclear. B-cell linker protein (BLNK) is an essential component of the B-cell antigen receptor signaling machinery and is required for B-cell development, as evidenced by BLNK-deficient (BLNK(-/-)) mice, in which the development and function of B cells are severely impaired. Herein, we evaluated the role of B cells in the development of tumor immunity against B16F10 melanoma using BLNK(-/-) mice. B16F10 melanoma grew more aggressively in BLNK(-/-) mice, resulting in a twofold increase in tumor volume compared with wild-type mice. As predicted, tumor-infiltrating B-cell numbers were decreased in BLNK(-/-) mice. Paradoxically, tumor-infiltrating T-cell numbers were decreased in BLNK(-/-) mice, although inguinal lymph node T-cell numbers were increased. Adoptive transfer of B cells from wild-type mice into BLNK(-/-) mice attenuated B16F10 melanoma growth, with increasing numbers of B and T cells infiltrating into tumors. In addition, percentages of interferon-γ- and tumor necrosis factor-α-producing tumor-infiltrating T cells were restored. Taken together, our study supports the concept that B cells enhance tumor immunity against B16F10 melanoma by promoting T-cell infiltration into tumors and cytokine production within the tumor microenvironment.

Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon.

OBJECTIVE: The aim of this study was to identify cold-associated autoantibodies in patients with RP secondary to CTDs. METHODS: Indirect immunofluorescence staining was performed on non-permeabilized cold-stimulated normal human dermal microvascular endothelial cells (dHMVECs), using patients' sera. Cold-induced alterations in cell surface proteomes were analysed by isobaric tag for relative and absolute quantitation (iTRAQ) analysis. Serological proteome analysis (SERPA) was applied to screen cold-associated autoantigens. The prevalence of the candidate autoantibody was determined by ELISA in 290 patients with RP secondary to CTDs (SSc, SLE or MCTD), 10 patients with primary RP and 27 healthy controls. RESULTS: Enhanced cell surface immunoreactivity was detected in cold-stimulated dHMVECs when incubated with sera from patients with secondary RP. By iTRAQ analysis, many proteins, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), were found to be increased on the cell surface of dHMVECs after cold stimulation. By the SERPA approach, hnRNP-K was identified as a candidate autoantigen in patients with secondary RP. Cold-induced translocation of hnRNP-K to the cell surface was confirmed by immunoblotting and flow cytometry. By ELISA analysis, patients with secondary RP show a significantly higher prevalence of anti-hnRNP-K autoantibody (30.0%, 61/203) than patients without RP (9.2%, 8/87, P = 0.0001), patients with primary RP (0%, 0/10, P = 0.0314) or healthy controls (0%, 0/27, P = 0.0001). CONCLUSION: By comprehensive proteomics, we identified hnRNP-K as a novel cold-associated autoantigen in patients with secondary RP. Anti-hnRNP-K autoantibody may potentially serve as a biomarker for RP secondary to various CTDs.

Donor-derived regulatory B cells are important for suppression of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent cause of morbidity and mortality of allogeneic hematopoietic stem-cell transplantation. Sclerodermatous cGVHD (Scl-cGVHD) is characterized by fibrosis and autoimmune features resembling those of systemic sclerosis (SSc). Transplantation of B10.D2 bone marrow and splenocytes into irradiated BALB/c mice is an established model of human Scl-cGVHD. To examine the role of B cells in Scl-cGVHD, CD19-deficient (CD19(-/-)) mice were used as donors or recipients. CD19(-/-) donors induced more severe Scl-cGVHD than wild-type donors, but use of CD19(-/-) recipients resulted in no significant differences compared with wild-type recipients. Moreover, CD19 deficiency on donor B cells resulted in the expansion of splenic interleukin (IL) -6-producing monocytes/macrophages, cytotoxic CD8(+) T cells, and Th1 cells during the early stage of disease and increased the infiltration of T cells, TGF-ß-producing monocytes/macrophages, and Th2 cells into the skin in the later stage of Scl-cGVHD. IL-10-producing regulatory B cells (B10 cells) were not reconstituted by CD19(-/-) donor cells, and early adoptive transfer of B10 cells attenuated the augmented manifestations of CD19(-/-) donor-induced Scl-cGVHD. Therefore, donor-derived B10 cells have a suppressive role in Scl-cGVHD development, warranting future investigation of regulatory B-cell-based therapy for treatment of Scl-cGVHD and SSc.

High incidence of pulmonary arterial hypertension in systemic sclerosis patients with anti-centriole autoantibodies.

Systemic sclerosis (SSc)-related autoantibodies are useful tools in identifying clinically homogenous subsets of patients and predicting their prognosis. In this report, we described five SSc patients with anti-centriole antibodies. All five patients were females and had digital ulcers/gangrene. Four of five (80%) patients had pulmonary arterial hypertension (PAH). None of the five patients had active pulmonary fibrosis or developed renal crisis. Anti-centriole antibodies may be a marker for PAH and digital ulcers/gangrene.

FTY720 ameliorates murine sclerodermatous chronic graft-versus-host disease by promoting expansion of splenic regulatory cells and inhibiting immune cell infiltration into skin.

OBJECTIVE: Sphingosine 1-phosphate (S1P) exerts a variety of activities in immune, inflammatory, and vascular systems. S1P plays an important role in systemic sclerosis (SSc) pathogenesis. Regulation of S1P in fibrotic diseases as well as in SSc was recently reported. FTY720, an oral S1P receptor modulator, has been shown to be a useful agent for the prevention of transplant rejection and autoimmune diseases. Murine sclerodermatous chronic graft-versus-host disease (GVHD) is a model for human sclerodermatous chronic GVHD and SSc. We undertook this study to investigate the effects of FTY720 in murine sclerodermatous chronic GVHD. METHODS: FTY720 was orally administered to allogeneic recipient mice from day 0 to day 20 (short-term, early-treatment group), from day 0 to day 42 (full-term, early-treatment group), or from day 22 to day 42 (delayed-treatment group) after bone marrow transplantation. RESULTS: Delayed administration of FTY720 attenuated, and early administration of FTY720 inhibited, the severity and fibrosis in murine sclerodermatous chronic GVHD. With early treatment, FTY720 induced expansion of splenic myeloid-derived suppressor cells, Treg cells, and Breg cells. Vascular damage in chronic GVHD was inhibited by FTY720 through down-regulating serum levels of S1P and soluble E-selectin. FTY720 inhibited infiltration of immune cells into skin. Moreover, FTY720 diminished the expression of messenger RNA for monocyte chemotactic protein 1, macrophage inflammatory protein 1α, RANTES, tumor necrosis factor α, interferon-γ, interleukin-6 (IL-6), IL-10, IL-17A, and transforming growth factor ß1 in the skin. CONCLUSION: FTY720 suppressed the immune response by promoting the expansion of regulatory cells and reducing vascular damage and infiltration of immune cells into the skin. Taken together, these results have important implications for the potential use of FTY720 in the treatment of sclerodermatous chronic GVHD and SSc in humans.