Racial variability in immune responses only partially explains differential systemic sclerosis disease severity.

OBJECTIVE: To understand if autoantibodies account for racial variation in disease severity, we compared autoantibody distribution and associated phenotype between self-identified black and white systemic sclerosis (SSc) patients. METHODS: 803 black and 2178 white SSc patients had systematic testing for autoantibodies using Euroimmun (centromere (ACA), RNA-polymerase III (POLR3), Scl70, PM/Scl, NOR90, Th/To, Ku, U3RNP and Ro52) and commercial ELISA (U1RNP). In this observational study, logistic regression was performed to assess the association between self-identified race and outcomes, adjusting for autoantibodies. To estimate whether the effect of race was mediated by autoantibody status, race coefficients from multivariate models including and excluding autoantibodies were compared. RESULTS: Anti-Scl70, anti-U1RNP, anti-U3RNP, anti-Th/To, anti-Ku and anti-NOR90 were more common in the black cohort than in the white cohort, which was enriched for ACA, anti-POLR3 and anti-PM/Scl. Black individuals had a higher prevalence of severe Raynaud's, skin, lung, gastrointestinal and renal disease whereas white individuals had a higher prevalence of severe heart and muscle disease. Adjusting for autoantibodies decreased the effect of race on outcome for telangiectasias, forced vital capacity <70%, pulmonary hypertension and severe lung, heart, muscle and gastrointestinal disease by 11%-44% and increased the association between race and renal crisis and severe kidney disease by 37%-52%. CONCLUSIONS: This study is the largest systematic analysis of autoantibody responses in a geographically diverse population of black SSc patients. Black and white individuals with SSc have distinct autoantibody profiles. Autoantibodies explain only a fraction of the effect of race on clinical outcomes, suggesting other factors contribute to disparate outcomes between these groups.

A PD-1highCD4+ T Cell Population With a Cytotoxic Phenotype is Associated With Interstitial Lung Disease in Systemic Sclerosis.

OBJECTIVE: T cells contribute to tissue injury in systemic sclerosis (SSc), yet the specific T cell subsets expanded in patients with SSc remain incompletely defined. Here we evaluated specific phenotypes and functions of peripheral helper T (Tph) and follicular helper T (Tfh) cells, which have been implicated in autoantibody production, and assessed their associations with clinical features in a well-characterized cohort of patients with SSc. METHODS: Mass cytometry of T cells from peripheral blood mononuclear cells of patients with SSc and controls were evaluated using t-distributed stochastic neighbor embedding visualization, biaxial gating, and marker expression levels. Findings were validated with flow cytometry and in vitro assays. RESULTS: The frequencies of PD-1highCXCR5+ Tfh cells and PD-1highCXCR5- Tph cells were similar in patients with SSc and controls. t-distributed stochastic neighbor embedding visualization (tSNE) revealed distinct populations within the PD-1highCXCR5- cells distinguished by expression of HLA-DR and inducible costimulator (ICOS). Among PD-1highCXCR5- cells, only the HLA-DR+ICOS- cell population was expanded in patients with SSc. Cytometric and RNA sequencing analyses indicated that these cells expressed cytotoxic rather than B cell helper features. HLA-DR+ICOS- PD-1highCXCR5- cells were less potent in inducing B cell plasmablast differentiation and antibody production than comparator T helper cell populations. HLA-DR+ICOS-PD-1highCXCR5- cells were significantly associated with the presence and severity of interstitial lung disease among patients with SSc. CONCLUSION: Among PD-1highCXCR5- T cells, a subset of HLA-DR+ICOS- cells with cytotoxic features is specifically expanded in patients with SSc and is significantly associated with interstitial lung disease severity. This potential cytotoxicity appearing in the CD4 T cell population can be evaluated as a prognostic disease biomarker in patients with SSc.

Protein biomarkers of disease progression in patients with systemic sclerosis associated interstitial lung disease.

Systemic sclerosis is a rare connective tissue disease; and interstitial lung disease (SSc-ILD) is associated with significant morbidity and mortality. There are no clinical, radiologic features, nor biomarkers that identify the specific time when patients are at risk for progression at which the benefits from treatment outweigh the risks. Our study aimed to identify blood protein biomarkers associated with progression of interstitial lung disease in patients with SSc-ILD using an unbiased, high-throughput approach. We classified SSc-ILD as progressive or stable based on change in forced vital capacity over 12 months or less. We profiled serum proteins by quantitative mass spectrometry and analyzed the association between protein levels and progression of SSc-ILD via logistic regression. The proteins associated with at a p value of < 0.1 were queried in the ingenuity pathway analysis (IPA) software to identify interaction networks, signaling, and metabolic pathways. Through principal component analysis, the relationship between the top 10 principal components and progression was evaluated. Unsupervised hierarchical clustering with heatmapping was done to define unique groups. The cohort consisted of 72 patients, 32 with progressive SSc-ILD and 40 with stable disease with similar baseline characteristics. Of a total of 794 proteins, 29 were associated with disease progression. After adjusting for multiple testing, these associations did not remain significant. IPA identified five upstream regulators that targeted proteins associated with progression, as well as a canonical pathway with a higher signal in the progression group. Principal component analysis showed that the ten components with the highest Eigenvalues represented 41% of the variability of the sample. Unsupervised clustering analysis revealed no significant heterogeneity between the subjects. We identified 29 proteins associated with progressive SSc-ILD. While these associations did not remain significant after accounting for multiple testing, some of these proteins are part of pathways relevant to autoimmunity and fibrogenesis. Limitations included a small sample size and a proportion of immunosuppressant use in the cohort, which could have altered the expression of inflammatory and immunologic proteins. Future directions include a targeted evaluation of these proteins in another SSc-ILD cohort or application of this study design to a treatment naïve population.

Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.

Peripheral blood leucocyte telomere length is associated with progression of interstitial lung disease in systemic sclerosis.

BACKGROUND: Peripheral blood leucocyte telomere length (PBL-TL) is associated with outcomes in patients with idiopathic pulmonary fibrosis. Whether PBL-TL is associated with progression of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is unknown. METHODS: A retrospective observational cohort study was performed using prospectively collected data from 213 patients with SSc followed at the University of California San Francisco (UCSF) Scleroderma Center. PBL-TL was measured by quantitative PCR of DNA isolated from peripheral blood. Associations between PBL-TL and pulmonary function test trends in patients with SSc-ILD were assessed by longitudinal analysis using Generalised Linear Mixed Models. Findings were validated in a cohort of 61 patients with SSc-ILD enrolled in the Stanford University Scleroderma Center database. RESULTS: Patients with UCSF SSc with ILD were found to have shorter PBL-TL compared with those without ILD (6554±671 base pairs (bp) vs 6782±698 bp, p=0.01). Shorter PBL-TL was associated with the presence of ILD (adjusted OR 2.1 per 1000 bp TL decrease, 95% CI [1.25 to 3.70], p=0.006). PBL-TL was shorter in patients with SSc-ILD lacking SSc-specific autoantibodies compared with seropositive subjects (6237±647 bp vs 6651±653 bp, p=0.004). Shorter PBL-TL was associated with increased risk for lung function deterioration with an average of 67 mL greater loss in per year for every 1000 bp decrease in PBL-TL in the combined SSc-ILD cohorts (longitudinal analysis, adjusted model: 95% CI -104 mL to -33 mL, p<0.001). CONCLUSIONS: These findings suggest that telomere dysfunction may be associated with SSc-ILD progression and that PBL-TL measurement may be useful for stratifying risk for SSc-ILD progression.

Autoantibodies targeting telomere-associated proteins in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune fibrotic disease affecting multiple tissues including the lung. A subset of patients with SSc with lung disease exhibit short telomeres in circulating lymphocytes, but the mechanisms underlying this observation are unclear. METHODS: Sera from the Johns Hopkins and University of California, San Francisco (UCSF) Scleroderma Centers were screened for autoantibodies targeting telomerase and the shelterin proteins using immunoprecipitation and ELISA. We determined the relationship between autoantibodies targeting the shelterin protein TERF1 and telomere length in peripheral leucocytes measured by qPCR and flow cytometry and fluorescent in situ hybridisation (Flow-FISH). We also explored clinical associations of these autoantibodies. RESULTS: In a subset of patients with SSc, we identified autoantibodies targeting telomerase and the shelterin proteins that were rarely present in rheumatoid arthritis, myositis and healthy controls. TERF1 autoantibodies were present in 40/442 (9.0%) patients with SSc and were associated with severe lung disease (OR 2.4, p=0.04, Fisher's exact test) and short lymphocyte telomere length. 6/6 (100%) patients with TERF1 autoantibodies in the Hopkins cohort and 14/18 (78%) patients in the UCSF cohort had a shorter telomere length in lymphocytes or leukocytes, respectively, relative to the expected age-adjusted telomere length. TERF1 autoantibodies were present in 11/152 (7.2%) patients with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disease believed to be mediated by telomere dysfunction. CONCLUSIONS: Autoantibodies targeting telomere-associated proteins in a subset of patients with SSc are associated with short lymphocyte telomere length and lung disease. The specificity of these autoantibodies for SSc and IPF suggests that telomere dysfunction may have a distinct role in the pathogenesis of SSc and pulmonary fibrosis.

The Utility of Plasma Vascular Biomarkers in Systemic Sclerosis: A Prospective Longitudinal Analysis.

OBJECTIVE: In cross-sectional studies, pulmonary hypertension (PH) and ischemic digital lesions are 2 scleroderma vascular outcomes associated with abnormalities in biomarkers of angiogenesis. The clinical usefulness of these biomarkers is unknown, in part due to lack of data on longitudinal measurement. This prospective longitudinal study was undertaken to evaluate vascular biomarker measurements in patients with systemic sclerosis (SSc) over time. METHODS: We conducted a prospective cohort study of 300 patients with SSc who were followed up for at least a 5-year period and lacked evidence of PH and/or active ischemic digital lesions at enrollment. Levels of hepatocyte growth factor (HGF), soluble Flt-1 (sFlt-1), soluble endoglin, endostatin, and placental growth factor (PLGF) were obtained at multiple time points and assessed for their ability to predict the development of PH/ischemic digital lesions. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) were calculated. RESULTS: Forty-six patients (15%) developed PH and 69 patients (23%) developed an ischemic digital lesion. In time-to-event analyses, the following 3 biomarkers measured at cohort entry were found to be significantly associated with the development of PH: HGF (HR 1.99 [95% CI 1.24-3.17], P = 0.004), sFlt-1 (HR 3.04 [95% CI 1.29-7.14], P = 0.011), and PLGF (HR 2.74 [95% CI 1.32-5.69], P = 0.007). As time approaching PH diagnosis decreased, there was no corresponding increase in any biomarker level. Upon converting each continuous vascular biomarker into a binary variable, a dose-response relationship was observed for the number of elevated biomarkers at cohort entry and the risk of developing PH. With each additional elevated biomarker at cohort entry, there was a 78% increase in the risk of developing PH (HR 1.78 [95% CI 1.2-2.6], P = 0.004). CONCLUSION: These data suggest that molecules involved in angiogenesis reflect vascular perturbation, and that elevations in these biomarkers at first encounter can indicate patients who are at risk of PH development.

Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

OBJECTIVE: Autoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied. METHODS: We developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients. RESULTS: We found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRß chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025). CONCLUSION: These findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma.

Longitudinal assessment of interstitial lung disease in single lung transplant recipients with scleroderma.

OBJECTIVE: To investigate the natural history of fibrotic lung disease in recipients of a single lung transplant for scleroderma-associated interstitial lung disease (ILD). METHODS: Global ILD (including ground glass, nodular opacities and fibrosis) was categorized into severity quintiles on first and last post-transplant CT scans, and percent fibrosis by manual contouring was also determined, in nine single lung transplant recipients. Quantitative mean lung densities and volumes for the native and allograft lungs were also acquired. RESULTS: In the native lung, global ILD severity quintile worsened in two cases and percent fibrosis worsened in four cases (range 5-28%). In the lung allograft, one case each developed mild, moderate and severe ILD; of these, new fibrotic ILD (involving <10% of lung) occurred in two cases and acute cellular rejection occurred in one. The average change in native lung density over time was +2.2 Hounsfield Units per year and lung volume +1.4 ml per year, whereas the allograft lung density changed by -5.5 Hounsfield Units per year and total volume +27 ml per year (P = 0.011 and P = 0.039 for native vs allograft density and volume comparisons, respectively). CONCLUSIONS: While the course of ILD in the native and transplanted lungs varied in this series, these cases illustrate that disease progression is common in the native lung, suggesting that either the immune process continues to target autoantigens or ongoing fibrotic pathways are active in the native lung. Mild lung disease may occur in the allograft after several years due to either allograft rejection or recurrent mild ILD.

Clinical and Molecular Phenotyping in Scleromyxedema Pretreatment and Posttreatment With Intravenous Immunoglobulin.

OBJECTIVE: Scleromyxedema (SMX) is a rare systemic sclerosis mimic that often responds to intravenous immunoglobulin (IVIG) therapy, yet the resulting clinical and biochemical changes have not been well characterized. To better understand the pathogenesis of the disease and the efficacy of IVIG, we sought to explore whether IVIG would introduce a measurable biologic effect corresponding with clinical improvement. METHODS: Fifteen patients with SMX were recruited for the study. Clinical information and peripheral blood mononuclear cells for flow cytometry were obtained immediately before and again 1-2 weeks after patients received IVIG therapy. Ten patients also underwent skin biopsies for gene expression analysis both before and after IVIG therapy. Clinical data included measures of skin involvement (modification of the modified Rodnan skin thickness score [MMRSS] and percentage of body surface area) and several patient-reported outcome measures assessing patients' skin. RESULTS: Posttreatment, the average MMRSS score decreased from mean ± SD 13.6 ± 2.6 to 10.3 ± 1.9; P = 0.003. There were also significant improvements in skin flexibility (mean ± SD 5.4 ± 0.8 to 3.2 ± 0.7; P = 0.003) and softening (mean ± SD 4.9 ± 0.9 to 2.6 ± 0.6; P = 0.022). Baseline levels of Tc17 cells (CD8+CCR6+CXCR3+CCR4-) correlated with the extent of skin involvement as measured by MMRSS pretreatment (r = 0.69, P = 0.012) and decreased after IVIG therapy (mean ± SD 3.4% ± 3.2% to 1.3% ± 1.7%; P = 0.008). Posttreatment analysis of RNA in skin tissue revealed a decrease in gene expression of transforming growth factor ß (TGFß) cytokines as well as several interferon-inducible proteins. CONCLUSION: This open-label study further supports the evidence that patients with SMX respond both objectively and subjectively to IVIG therapy. Biologic studies suggest a role for T lymphocytes in the pathogenesis of the disease and reveal the potential significance of TGFß and interferon pathways.

HLA and autoantibodies define scleroderma subtypes and risk in African and European Americans and suggest a role for molecular mimicry.

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

Heart-lung transplantation: A viable option for connective tissue diseases.

BACKGROUND: While lung transplantation (LTx) has been effective for connective tissue disease (CTD) patients with pulmonary involvement, outcomes for heart-lung transplantation (HLTx) are less defined. The aim of this study is to evaluate HLTx in CTD patients utilizing the UNOS database. METHODS: HLTx patients with CTD (HLTx-CTD) were compared to both LTx patients with CTD (LTx-CTD) and HLTx patients with all other indications (HLTx-OI) from 1999 to 2018. Primary outcome was 1- and 5-year graft survival. Secondary outcomes included freedom from first-year rejection and outcomes prior to transplant discharge. RESULTS: 1143/29 323 adults received first-time HLTx or LTx for CTD. Seventeen were HLTx-CTD (3.3% of total HLTx) and 1126 were LTx-CTD (3.9% of total LTx). There were 492 HLTx-OI. Transplant hemodynamic values including cardiac output, pulmonary capillary wedge pressure, and calculated pulmonary vascular resistance were significantly worse for HLTx-CTD vs LTx-CTD (4.2 vs 5.4 L/min, P = .005; 14 vs 10 mm Hg, P = .009; 439 vs 267 dynes, P = .007, respectively). Cardiac status 1 was more common for HLTx-CTD vs HLTx-OI (94% vs 56%, P < .001). HLTx-CTD 1 and 5-year graft survival was similar compared to LTx-CTD and HLTx-OI. CONCLUSION: HLTx-CTD is a valid option for carefully selected patients with CTD cardiac and pulmonary involvement with similar morbidity and mortality compared to LTx-CTD and HLTx-OI.

Digital ulcers: should debridement be a standard of care in systemic sclerosis?

Digital ulcers are a serious, recurrent complication in patients with systemic sclerosis. They are often slow to heal and exquisitely painful. Local wound care, such as debridement of the wound bed, is an essential component in the management of digital ulcers in systemic sclerosis. However, digital ulcer debridement is not a standard of care, and there is substantial international variation in the use of this approach. In this Viewpoint, we discuss the assessment of the wound bed and different methods of debridement using the model of tissue management, infection and inflammation, moisture control, and wound edge or epidermal advancement, known as TIME. We highlight the challenges in standard practice and the need for research into local wound care for this type of ulceration, before suggesting a potential roadmap to develop a standardised approach to support ulcer debridement in systemic sclerosis. Debridement might be the missing component in optimising the management of digital ulcers and we propose that the approach should be rigorously investigated as a standard of care in this common complication of systemic sclerosis.

Regulatory T cells in skin are uniquely poised to suppress profibrotic immune responses.

At the center of fibrosing diseases is the aberrant activation of tissue fibroblasts. The cellular and molecular mechanisms of how the immune system augments fibroblast activation have been described; however, little is known about how the immune system controls fibroblast function in tissues. Here, we identify regulatory T cells (Tregs) as important regulators of fibroblast activation in skin. Bulk cell and single-cell analysis of Tregs in murine skin and lungs revealed that Tregs in skin are transcriptionally distinct and skewed toward T helper 2 (TH2) differentiation. When compared with Tregs in lung, skin Tregs preferentially expressed high levels of GATA3, the master TH2 transcription factor. Genes regulated by GATA3 were highly enriched in skin "TH2 Treg" subsets. In functional experiments, Treg depletion resulted in a preferential increase in TH2 cytokine production in skin. Both acute depletion and chronic reduction of Tregs resulted in spontaneous skin fibroblast activation, profibrotic gene expression, and dermal fibrosis, all of which were exacerbated in a bleomycin-induced murine model of skin sclerosis. Lineage-specific deletion of Gata3 in Tregs resulted in an exacerbation of TH2 cytokine secretion that was preferential to skin, resulting in enhanced fibroblast activation and dermal fibrosis. Together, we demonstrate that Tregs play a critical role in regulating fibroblast activation in skin and do so by expressing a unique tissue-restricted transcriptional program that is mediated, at least in part, by GATA3.

Vascular complications in systemic sclerosis: a prospective cohort study.

Two major complications in scleroderma patients that cause substantial morbidity and mortality are ischemic digital lesions (DL) and pulmonary hypertension (PH). The clinician's ability to predict which patients will develop these complications is imperfect. We conducted a prospective observational cohort study of 300 patients with scleroderma who were followed for at least a 5-year period. At baseline, patients lacked evidence of PH and were without a current DL. At each 6-month visit, the patient was examined for signs/symptoms of PH and/or a DL. The primary outcomes were (1) PH defined as a mean pulmonary artery pressure ≥ 25 mmHg by right heart catheterization and (2) ≥ 1 DL defined as new onset of severe vascular compromise. Thirty patients (10%) developed PH (11 group 1/PAH, 4 group II, 15 group III) and 69 developed DL. The average time from enrollment until diagnosis of PH was 3.2 ± 2 years. In multivariable analyses, patients who developed PH were more likely to have diffuse disease (HR 3.2, p = 0.004), a forced vital capacity (FVC)/diffusing capacity of the lungs for carbon monoxide (DLCO) ratio > 1.6 (HR 1.7, p = 0.008), and elevated RVSP (HR = 1.07, p = 0.007). Patients who developed PAH were more likely to have a FVC/DLCO ratio > 1.6 (HR = 5.8, p = 0.014), and patients who developed group III PH were less likely to have an elevated FVC (HR = 0.92, p = 0.001). Patients were more likely to develop a DL if they had a history of prior DL (HR = 7.0, p < 0.001), or were men (HR = 2.3, p = 0.007). In a prevalent cohort of scleroderma patients, individuals who develop PH or DL have simple to measure clinical features that can predict these complications years before they occur.

Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans.

OBJECTIVE: Whole-exome sequencing (WES) studies in systemic sclerosis (SSc) patients of European American (EA) ancestry have identified variants in the ATP8B4 gene and enrichment of variants in genes in the extracellular matrix (ECM)-related pathway that increase SSc susceptibility. This study was undertaken to evaluate the association of the ATP8B4 gene and the ECM-related pathway with SSc in a cohort of African American (AA) patients. METHODS: SSc patients of AA ancestry were enrolled from 23 academic centers across the US under the Genome Research in African American Scleroderma Patients consortium. Unrelated AA individuals without serologic evidence of autoimmunity who were enrolled in the Howard University Family Study were used as unaffected controls. Functional variants in genes reported in the 2 WES studies in EA patients with SSc were selected for gene association testing using the optimized sequence kernel association test (SKAT-O) and pathway analysis by Ingenuity Pathway Analysis in 379 patients and 411 controls. RESULTS: Principal components analysis demonstrated that the patients and controls had similar ancestral backgrounds, with roughly equal proportions of mean European admixture. Using SKAT-O, we examined the association of individual genes that were previously reported in EA patients and none remained significant, including ATP8B4 (P = 0.98). However, we confirmed the previously reported association of the ECM-related pathway with enrichment of variants within the COL13A1, COL18A1, COL22A1, COL4A3, COL4A4, COL5A2, PROK1, and SERPINE1 genes (corrected P = 1.95 × 10-4 ). CONCLUSION: In the largest genetic study in AA patients with SSc to date, our findings corroborate the role of functional variants that aggregate in a fibrotic pathway and increase SSc susceptibility.

Patient-reported outcome instruments for assessing Raynaud's phenomenon in systemic sclerosis: A SCTC Vascular Working Group Report.

The episodic nature of Raynaud's phenomenon (RP) in systemic sclerosis (SSc) has led to a reliance on patient-reported outcome (PRO) instruments such as the Raynaud's Condition Score (RCS) diary. Little is known about the utilisation in routine clinical practice and health professional attitudes towards existing PRO instruments for assessing SSc-RP. Members of the Scleroderma Clinical Trials Consortium Vascular Working Group (SCTC-VWG, n=28) were invited to participate in a survey gauging attitudes towards the RCS diary and the perceived need for novel PRO instruments for assessing SSc-RP. Nineteen SCTC-VWG members (68% response rate) from academic units based in North America (n=9), Europe (n=8), South America (n=1) and Australasia (n=1) took part in the survey. There was broad consensus that RCS diary returns could be influenced by factors including seasonal variation in weather, efforts made by patients to avoid or ameliorate attacks of RP, habituation to RP symptoms, evolution of RP symptom characteristics with progressive obliterative microangiopathy, patient coping strategies, respondent burden and placebo effect. There was consensus that limitations of the RCS diary might be a barrier to drug development (79% of respondents agree/strongly agree) and that a novel PRO instrument for assessing SSc-RP should be developed with the input of both clinicians and patients (84% agree/strongly agree). Perceived potential limitations of the RCS diary have been identified along with concerns that such factors might impede drug development programs for SSc-RP. There is support within the systemic sclerosis community for the development of a novel PRO instrument for assessing SSc-RP.

Flow cytometric discrimination of seven lineage markers by using two fluorochromes.

Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping.

PTP4A1 promotes TGFß signaling and fibrosis in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFß signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFß signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFß-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.