Introduction
Systemic sclerosis (SSc) is a heterogenous disorder that appears to result from interplay between vascular pathologies, tissue fibrosis and immune processes, with evidence for ...deregulation of chemokines, which normally control immune trafficking. We recently identified altered levels of chemokine CCL21 in SSc associated pulmonary arterial hypertension (PAH). Here, we aimed to define target organ expression and biomarker characteristics of CCL21.
Materials and methods
To investigate target organ expression of CCL21, we performed immunohistochemistry (IHC) on explanted lung tissues from SSc-PAH patients. We assessed serum levels of CCL21 by ELISA and Luminex in two well-characterized SSc cohorts from Oslo (OUH, n=552) and Zurich (n=93) University hospitals and in 168 healthy controls. For detection of anti-CCl21 antibodies, we performed protein array analysis applying serum samples from SSc patients (n=300) and healthy controls. To characterize circulating CCL21 in SSc, we applied immunoprecipitation (IP) with antibodies detecting both full length and tailless and a custom-made antibody detecting only the C-terminal of CCL21. IP products were analyzed by SDS-PAGE/western blot and Mass spectrometry (MS).
Results
By IHC, we found that CCL21 was mainly expressed in the airway epithelial cells of SSc patients with PAH. In the analysis of serum levels of CCL21 we found weak correlation between Luminex and ELISA (r=0.515, p<0.001). Serum levels of anti-CCL21 antibodies were higher in SSc patients than in healthy controls (p<0.001), but only 5% of the SSc population were positive for anti-CCL21 antibodies in SSc, and we found no correlation between anti-CCl21 and serum levels of CCL21. By MS, we only identified peptides located within amino acid (aa) 23-102 of CCL21, indicating that CCL21 in SSc circulate as a truncated protein without the C-terminal tail.
Conclusion
This study demonstrates expression of CCL21 in epithelial lung tissue from SSc patients with PAH, and indicate that CCL21 in SSc circulates as a truncated protein. We extend previous observations indicating biomarker potential of CCL21, but find that Luminex is not suitable as platform for biomarker analyses. Finally,
in vivo
generated anti-CCL21 antibodies exist in SSc, but do not appear to modify serum CCL21 levels in patients with SSc-PAH.
Abstract
Objectives
In SSc, gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) ...represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling.
Methods
We analysed duodenal biopsies obtained pre-intervention (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving an intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling.
Results
In patients receiving FMT, the number of podoplanin- and CD64-expressing cells in the mucosa were lower at week 2 compared with baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T cell receptor complexes, and chemokine receptors, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16.
Conclusion
Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement.
Trial registration
ClinicalTrials.gov, http://clinicaltrials.gov, NCT03444220.
Abstract
Objectives
To evaluate the severity and evolution of patient-reported gastrointestinal tract (GIT) symptoms in systemic sclerosis (SSc) patients, assess predictive factors for progression ...and determine the impact of standard of care treatment.
Methods
SSc patients from the Leiden and Oslo cohorts were included. We assessed clinical data and patient-reported GIT symptoms measured by the validated University of California, Los-Angeles Gastrointestinal-tract (UCLA-GIT) score at baseline and annually. GIT severity and progression was determined. Logistic regression was applied to identify risk factors associated with baseline GIT symptom severity. Linear mixed-effect models were applied to assess progression in GIT symptom burden and to identify predictive factors. We repeated all analysis in patients with early disease (inception cohort) to exclude the effect of longstanding disease and increase insights in development of GIT symptom burden early in the disease course.
Results
We included 834 SSc patients with baseline UCLA GIT scores, 454 from Leiden and 380 from Oslo. In the total cohort, 28% reported moderate-severe GIT symptoms at baseline, with increased risk for severity conferred by ACA, smoking and corticosteroid use, while use of calcium channel blockers appeared protective. In the inception cohort, 23% reported moderate-severe GIT symptoms at baseline, with increased risk for females and with smoking. Over time, symptom burden increased mainly for reflux/bloating. Female sex and ACA predicted GIT symptom progression.
Conclusion
High GIT symptom burden is present early in SSc disease course. Both for prevalence and for progression of GIT symptom burden, female sex and smoking were identified as risk factors.
Objective
Systemic sclerosis (SSc) is a major cause of pulmonary arterial hypertension (PAH). Murine models indicate key roles for chemokines CCL19 and CCL21 and their receptor CCR7 in lung ...inflammation leading to PAH. The objective of this study was to assess the chemokine CCL19–CCL21 axis in patients with SSc‐related PAH.
Methods
Serum samples obtained from 2 independent prospective SSc cohorts (n = 326), patients with idiopathic PAH (n = 12), and healthy control subjects (n = 100) were analyzed for CCL19/CCL21 levels, by enzyme‐linked immunosorbent assay. The levels were defined as either high or low, using the mean + 2 SD value in controls as the cutoff value. Risk stratification at the time of PAH diagnosis and PAH‐related events were performed. Descriptive and Cox regression analyses were conducted.
Results
CCL21 levels were higher in patients with SSc compared with controls and were elevated prior to the diagnosis of PAH. PAH was more frequent in patients with high CCL21 levels (≥0.4 ng/ml) than in those with low CCL21 levels (33.3% versus 5.3% P < 0.001). In multivariate analyses, CCL21 was associated with PAH (hazard ratio HR 5.1, 95% CI 2.39–10.76 P < 0.001) and occurrence of PAH‐related events (HR 4.7, 95% CI 2.12–10.46, P < 0.001). Risk stratification at the time of PAH diagnosis alone did not predict PAH‐related events. However, when risk at diagnosis was combined with high or low CCL21 level, there was a significant predictive effect (HR 1.3, 95% CI 1.03–1.60 P = 0.027). A high CCL21 level was associated with decreased survival (P < 0.001).
Conclusion
CCL21 appears to be a promising marker for predicting the risk of SSc‐related PAH and PAH progression. CCL21 may be part of a dysregulated immune pathway linked to the development of lung vascular damage in SSc.
To determine survival and causes of death in an unselected and complete cohort of Norwegian patients with systemic sclerosis (SSc) compared to the background population.
Multiple methods were used to ...identify every patient with SSc living in southeast Norway, with a denominator population of 2,707,012, between 1999 and 2009. All patients who met either the American College of Rheumatology criteria or the Medsger and LeRoy criteria for SSc were included. Every patient was matched for sex and age with 15 healthy controls drawn from the national population registry. Vital status at January 1, 2010, was provided for patients and controls by the national population registry. Causes of death were obtained from death certificates and by chart review.
Forty-three (14%) of 312 patients with SSc died during the study period. The standardized mortality rate (SMR) was estimated to be 2.03 for the entire cohort and 5.33 for the subgroup with diffuse cutaneous (dc) SSc. The 5- and 10-year survival rates were 91% and 70%, respectively, for dcSSc and 98% and 93% for limited cutaneous (lc) SSc. Causes of death were related to SSc in 24/43 (56%) patients, mostly cardiopulmonary diseases (n = 13), including pulmonary hypertension (n = 8). Factors associated with fatal outcome included male sex, dcSSc, pulmonary hypertension, and interstitial lung disease.
Compared to the Norwegian background population, our cohort of 312 unselected patients with SSc had decreased survival. The survival rates observed were, however, better than those previously reported from SSc referral centers.
Objective
Pulmonary arterial hypertension (PAH) is a major complication in systemic sclerosis (SSc), a disease marked by vascular and lymphatic vessel abnormalities. This study was undertaken to ...assess the role of the lymphangiogenic factors vascular endothelial growth factor C (VEGF‐C) and angiopoietin 2 (Ang‐2) and the soluble forms of their respective cognate receptors, soluble VEGF receptor 3 (sVEGFR‐3) and soluble TIE‐2, in patients with SSc, and to evaluate their predictive ability as markers for PAH development in SSc.
Methods
In this cohort study, we used multiplex bead assays to assess serum levels of lymphangiogenic factors in 2 well‐characterized SSc cohorts: an unselected identification cohort of SSc patients from Oslo University Hospital (n = 371), and a PAH‐enriched validation cohort of SSc patients from Zurich University Hospital and Oslo University Hospital (n = 149). As controls for the identification and validation cohorts, we obtained serum samples from 100 healthy individuals and 68 healthy individuals, respectively. Patients in whom SSc‐related PAH was identified by right‐sided heart catheterization (RHC) in both cohorts were studied in prediction analyses. PAH was defined according to the European Society of Cardiology/European Respiratory Society 2015 guidelines for the diagnosis and treatment of PAH. Associations of serum levels of lymphangiogenic factors with the risk of PAH development were assessed in logistic regression and Cox regression analyses. Associations in Cox regression analyses were expressed as the hazard ratio (HR) with 95% confidence interval (95% CI).
Results
In the identification cohort, SSc patients had lower mean serum levels of VEGF‐C and higher mean serum levels of Ang‐2 compared to healthy controls (for VEGF‐C, mean ± SD 2.1 ± 0.5 ng/ml in patients versus 2.5 ± 0.4 ng/ml in controls; for Ang‐2, mean ± SD 6.1 ± 7.6 ng/ml in patients versus 2.8 ± 1.8 ng/ml in controls; each P < 0.001); these same trends were observed in SSc patients with PAH compared to those without PAH. The association of serum VEGF‐C levels with SSc‐PAH was confirmed in the PAH‐enriched RHC validation cohort. For prediction analyses, we assembled all 251 cases of SSc‐PAH identified by RHC from the identification and validation cohorts. In multivariable Cox regression analyses adjusted for age and sex, the mean serum levels of VEGF‐C and sVEGFR‐3 were predictive of PAH development in patients with SSc (for VEGF‐C, HR 0.53 95% CI 0.29–0.97, P = 0.04; for sVEGFR‐3, HR 1.21 95% CI 1.01–1.45, P = 0.042).
Conclusion
These findings support the notion that lymphangiogenesis is deregulated during PAH development in SSc, and indicate that VEGF‐C could be a promising marker for early PAH detection in patients with SSc.
IntroductionIn the multisystem inflammatory disorder systemic sclerosis (SSc), gastrointestinal tract (GIT) affliction is highly prevalent. There are no known disease modifying therapies and the ...negative impact is substantial. Aiming for a new therapeutic principle, and inspired by recent work showing associations between gut microbiota changes and GIT symptoms in SSc, we performed a pilot study on faecal microbiota transplantation (FMT) with the single-donor bacterial culture ‘Anaerobic Cultivated Human Intestinal Microbiome (ACHIM)’. Motivated by positive pilot study signals, we designed the ReSScue trial as a phase II multicentre, placebo-controlled, randomised 20-week trial to evaluate safety and efficacy on lower GIT symptoms of FMT by ACHIM in SSc.Methods and analysesWe aim to include 70 SSc participants with moderate to severe lower GIT symptoms, defined by the validated patient-reported University of California Los Angeles Scleroderma Clinical Trial Consortium GIT 2.0 2.0 questionnaire. The trial includes three parts. In part A1 (induction phase) lasting from week 0 to week 12, participants will be randomised 1:1 to repeat infusions of 30 mL ACHIM or placebo at week 0 and 2 by gastroduodenoscopy. In part A2, which is an 8-week subsequent maintenance phase, all study participants will receive 30 mL ACHIM at week 12 and followed until week 20 on continued blind. In part B, which will last until the last participant completes part A2, the participants will be followed through a maximum 16-week extended monitoring period, for longer-term data on safety and intervention effects. Primary endpoint is change from baseline to week 12 in UCLA GIT subscale scores of diarrhoea or bloating, depending on the worst symptom at baseline evaluated separately for each patient. Secondary endpoints are safety measures and changes in UCLA GIT scores (total, diarrhoea and bloating).Ethics and disseminationThis protocol was approved by the Northern Norwegian Committee for Medical Ethics. Study findings will be published.Trial registration numberNCT04300426; Pre-results.Protocol versionV.3.1.
Dysregulation of Fractalkine (CX3CL1) and its receptor CX3CR1 has been linked to the pathobiology of chronic inflammatory conditions. We explored CX3CL1 in systemic sclerosis (SSc) related ...progressive interstitial lung disease (ILD) and pulmonary hypertension (PH) in two different but complementary sources of biomaterial.
We collected lung tissue at the time of lung transplantation at UCLA from SSc-ILD patients (n = 12) and healthy donors (n = 12); and serum samples from the prospective Oslo University Hospital SSc cohort (n = 292) and healthy donors (n = 100). CX3CL1 was measured by ELISA. Cellular sources of CX3CL1/CX3CR1 in lung tissues were determined by immunohistochemistry and immunofluorescence. ILD progression and new onset PH endpoints were analysed.
CX3CL1 concentrations were increased in SSc in lung tissue as well as in sera. In the UCLA cohort, CX3CL1 was highly correlated with DLCO. In the SSc-ILD lungs, CX3CL1 was identified in reactive type II pneumocytes and airway epithelial cells. CX3CR1 stained infiltrating interstitial mononuclear cells, especially plasma cells. In the Oslo cohort, CX3CL1 correlated with anti-Topoisomerase-I-antibody and lung fibrosis. CX3CL1 was associated with ILD progression in multivariable regression analysis but not PH.
CX3CL1 is associated with progressive SSc-ILD but not SSc-PH. The CX3CR1/CX3CL1-biological axis may be involved in recruiting antibody secreting plasma cells to SSc lungs, thereby contributing to the immune-mediated pathobiology of SSc-ILD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Extensive skin disease and renal crisis are hallmarks of anti-RNA polymerase III (RNAP)-positive systemic sclerosis (SSc), while lung and heart involvement data are conflicting. Here, the aims were ...to perform time-course analyses of interstitial lung disease (ILD) and pulmonary hypertension (PH) in the RNAP subset of a prospective unselected SSc cohort and to use the other autoantibody subsets as comparators.
The study cohort included 279 patients with SSc from the observational Oslo University Hospital cohort with complete data on (1) SSc-related autoantibodies, (2) paired, serial analyses of lung function and fibrosis by computed tomography, and (3) PH verified by right heart catheterization.
RNAP was positive in 33 patients (12%), 79% of which had diffuse cutaneous SSc. Pulmonary findings were heterogeneous; 49% had no signs of fibrosis while 18% had > 20% fibrosis at followup. Forced vital capacity at followup was < 80% in 39% of the RNAP subset, comparable to the antitopoisomerase subset (ATA; 47%), but higher than anticentromere (ACA; 13%). Accumulated frequency of PH in the RNAP subset (12%) was lower than in ACA (18%). At 93% and 78%, the 5- and 10-year survival rates in RNAP were comparable to the ATA and ACA subsets.
In this cohort, the RNAP subset was marked by cardiopulmonary heterogeneity, ranging from mild ILD to development of severe ILD in 18%, and PH development in 12%. These data indicate that cardiopulmonary risk stratification early in the disease course is particularly important in RNAP-positive SSc.
To assess the prevalence of SSc in south-east Norway.
The survey was conducted in south-east Norway with a denominator population of 2,707,012, 56% of the total Norwegian population. All SSc patients ...living in the study area between 1 January 1999 and 31 December 2009 were included. Patients were identified by five overlapping acquisition routes, including all the rheumatology departments, private rheumatologists and the dermatology department in the study area. Only cases meeting the 1980 ACR and/or the Medsger and LeRoy classification criteria were included. The patients were assigned to three clinical subsets: limited SSc, lcSSc or dcSSc.
At the end of the study period, a total of 269 patients fulfilled the ACR and/or the Medsger and LeRoy SSc criteria, giving a point prevalence of 9.9/100,000 (95% CI 8.8, 11.2). The estimated prevalences of lSSc, lcSSc and dcSSc were 1.3/100,000, 6.9/100,000 and 1.8/100,000 (95% CIs 0.9, 1.8; 5.8, 7.8; 1.4, 2.5), respectively. The mean age at onset was 47 years and the female:male ratio was 3.8:1. The prevalence estimates of SSc in the 10 different counties in south-east Norway varied between 5.2 and 14.4/100,000 (95% CIs 2.8, 8.8; 10.3, 19.6).
This study establishes baseline estimates of the occurrence and disease characteristics in a large, unselected group of Norwegian SSc patients. Our data suggest that the prevalence of SSc in Norway is comparable with other northern European countries, supporting the notion of a north-south gradient of SSc in Europe with the lowest prevalence in northern Europe.
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