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1.  Echocardiographic follow-up of patients with systemic sclerosis by 2D speckle tracking echocardiography of the left ventricle 
Background
Subclinical myocardial involvement is common in systemic sclerosis (SSc) and associated with poor prognosis. Early detection, particularly during follow-up, is important. Two-dimensional speckle tracking echocardiography (STE) has already been shown to detect early left ventricular systolic impairment in SSc patients with advanced disease. The aim of this study was to assess the ability of STE to diagnose changes in left ventricular function in patients with SSc with preserved LV ejection fraction (LVEF) and normal pulmonary pressure over time.
Methods
This single-center pilot study included nineteen SSc patients without pulmonary hypertension and preserved LVEF (55.2 ± 10.8 years, 13 women, mean modified Rodnan Skin Score of 8.2 ± 6.5, median disease duration 6 ± 4.5 years). We performed STE at baseline and after two years (mean 756.6 ± 8.8 days). Pulmonary hypertension was ruled out in all patients by right heart catheterization (average mean PAP 17.7 ± 3.5 mmHg).
Results
The LVEF remained unchanged (63.3 ± 4.2% vs. 63.2 ± 5.0%, P = ns), but the global longitudinal peak systolic strain of the left ventricle was significantly lower: baseline -22.0 ± 2.3% vs. follow-up -20.8 ± 2.1% (P = 0.04). The regional analysis showed a heterogeneous distribution of segmental systolic dysfunction that did not match any particular coronary artery distribution. In contrast, the LV diastolic function remained stable during follow-up.
Conclusion
STE might be a sensititive and valuable method to detect early LV systolic impairment in patients with SSc and preserved LVEF during two years. Prospective evaluations are needed for prognostic implications of these changes.
doi:10.1186/1476-7120-12-13
PMCID: PMC3986623  PMID: 24678809
Systemic sclerosis; Speckle tracking; 2D strain; Myocardial involvement
2.  Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients 
Introduction
Agonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.
Methods
Protein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.
Results
Both AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.
Conclusion
In our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.
doi:10.1186/ar4503
PMCID: PMC4060229  PMID: 24612997
3.  Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis 
Introduction
Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT1R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed.
Methods
Anti-AT1R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT1R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy.
Results
Anti-AT1R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs.
Conclusions
We conclude that angiotensin and endothelin-receptor activation via anti-AT1R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT1R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.
doi:10.1186/ar4457
PMCID: PMC3978438  PMID: 24472528
4.  A genome-wide association study follow-up suggests a possible role for PPARG in systemic sclerosis susceptibility 
Introduction
A recent genome-wide association study (GWAS) comprising a French cohort of systemic sclerosis (SSc) reported several non-HLA single-nucleotide polymorphisms (SNPs) showing a nominal association in the discovery phase. We aimed to identify previously overlooked susceptibility variants by using a follow-up strategy.
Methods
Sixty-six non-HLA SNPs showing a P value <10-4 in the discovery phase of the French SSc GWAS were analyzed in the first step of this study, performing a meta-analysis that combined data from the two published SSc GWASs. A total of 2,921 SSc patients and 6,963 healthy controls were included in this first phase. Two SNPs, PPARG rs310746 and CHRNA9 rs6832151, were selected for genotyping in the replication cohort (1,068 SSc patients and 6,762 healthy controls) based on the results of the first step. Genotyping was performed by using TaqMan SNP genotyping assays.
Results
We observed nominal associations for both PPARG rs310746 (PMH = 1.90 × 10-6, OR, 1.28) and CHRNA9 rs6832151 (PMH = 4.30 × 10-6, OR, 1.17) genetic variants with SSc in the first step of our study. In the replication phase, we observed a trend of association for PPARG rs310746 (P value = 0.066; OR, 1.17). The combined overall Mantel-Haenszel meta-analysis of all the cohorts included in the present study revealed that PPARG rs310746 remained associated with SSc with a nominal non-genome-wide significant P value (PMH = 5.00 × 10-7; OR, 1.25). No evidence of association was observed for CHRNA9 rs6832151 either in the replication phase or in the overall pooled analysis.
Conclusion
Our results suggest a role of PPARG gene in the development of SSc.
doi:10.1186/ar4432
PMCID: PMC3978735  PMID: 24401602
5.  Angiotensin II Type 1 Receptor Antibodies and Increased Angiotensin II Sensitivity in Pregnant Rats 
Hypertension  2011;58(1):77-84.
Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-α and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia- inducible factor 1α was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an “autoimmune-activating receptor” that could contribute to Ang II sensitivity and possible to preeclampsia.
doi:10.1161/HYPERTENSIONAHA.111.171348
PMCID: PMC3597080  PMID: 21576625
preeclampsia; angiotensin II; immunology; autoimmune disease
6.  Items for developing revised classification criteria in systemic sclerosis: results of a consensus exercise with the ACR/EULAR working committee for classification criteria in systemic sclerosis 
Arthritis care & research  2012;64(3):351-357.
Background
Classification criteria for systemic sclerosis (SSc) are being updated.
Objective
To select a set of items potentially useful for the classification of SSc using consensus procedures including the Delphi and nominal group techniques (NGT).
Methods
Items were identified through two independent consensus exercises performed by the Scleroderma Clinical Trials Consortium (SCTC) and the EULAR Scleroderma Trials and Research Group (EUSTAR). The first-round items from both exercises were collated and redundancies were removed leaving 168 items. A 3-round Delphi exercise was performed using a 1–9 scale (1=completely inappropriate and 9=completely appropriate) and a consensus meeting using NGT. During the last Delphi, the items were ranked on a 1–10 scale.
Results
Round 1: 106 experts rated the 168 items. Those with a median score <4 were removed, resulting in a list of 102 items. Round 2: The items were again rated for appropriateness and subjected to a consensus meeting using NGT by European and North American SSc experts (n=16), resulting in 23 items. Round 3: SSc experts (n=26) then individually scored each of the 23 items in a last Delphi round, using an appropriateness score (1–9) and ranking their 10 most appropriate items for classification of SSc. Presence of skin thickening, SSc-specific autoantibodies, abnormal nailfold capillary pattern and Raynaud’s phenomenon ranked highest in the final list that also included items indicating internal organ involvement.
Conclusion
The Delphi exercise and NGT resulted in a set of 23 items for classification of SSc which will be assessed for their discriminative properties in a prospective study.
doi:10.1002/acr.20679
PMCID: PMC3288452  PMID: 22052558
Delphi technique; nominal group technique; systemic sclerosis; scleroderma; classification; classification criteria
7.  Validation of potential classification criteria for systemic sclerosis 
Arthritis care & research  2012;64(3):358-367.
Background
Classification criteria for systemic sclerosis (SSc) are being updated jointly by ACR and EULAR. Potential items for classification were reduced to 23 using Delphi and Nominal Group Techniques. We evaluated the face, discriminant and construct validity of the items to be further studied as potential criteria.
Methods
Face validity was evaluated using the frequency of items in patients sampled from the Canadian Scleroderma Research Group, 1000 Faces of Lupus, the Pittsburgh, Toronto, Madrid and Berlin CTD databases. SSc (n=783) were compared to 1071 patients with diseases similar to SSc (mimickers): SLE (n=499), myositis (n=171), Sjögren’s syndrome (n=95), Raynaud’s phenomenon (RP) (n=228), MCTD (n=29), and idiopathic PAH (n=49). Discriminant validity was evaluated using odds ratios (OR). For construct validity, empiric ranking was compared to expert ranking.
Results
Compared to mimickers, SSc are more likely to have skin thickening (OR=427), telangiectasias (OR=91), anti-RNA polymerase III antibody (OR=75), puffy fingers (OR=35), finger flexion contractures (OR=29), tendon/bursal friction rubs (OR=27), anti-topoisomerase-I antibody (OR=25), RP (OR=24), finger tip ulcers/pitting scars (OR=19), anti-centromere antibody(OR=14), abnormal nailfold capillaries (OR=10), GERD symptoms (OR=8), and ANA, calcinosis, dysphagia, esophageal dilation (all OR=6), interstitial lung disease/pulmonary fibrosis (OR=5) and anti-PM-Scl antibody (OR=2). Reduced DLCO, PAH, and reduced FVC had OR<2. Renal crisis and digital pulp loss/acro-osteolysis did not occur in SSc mimickers (OR not estimated). Empiric and expert ranking were correlated (Spearman rho 0.53, p=0.01).
Conclusion
The candidate items have good face, discriminant and construct validity. Further item reduction will be evaluated in prospective SSc and mimicker cases.
doi:10.1002/acr.20684
PMCID: PMC3376721  PMID: 22052658
Systemic Sclerosis; Scleroderma; Classification Criteria; Validity; Bayesian
8.  CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice 
Introduction
The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).
Methods
Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.
Results
Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5 × 106 purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.
Conclusions
Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.
doi:10.1186/ar4188
PMCID: PMC3672693  PMID: 23446139
9.  A GWAS follow-up study reveals the association of the IL12RB2 gene with systemic sclerosis in Caucasian populations 
Human Molecular Genetics  2011;21(4):926-933.
A single-nucleotide polymorphism (SNP) at the IL12RB2 locus showed a suggestive association signal in a previously published genome-wide association study (GWAS) in systemic sclerosis (SSc). Aiming to reveal the possible implication of the IL12RB2 gene in SSc, we conducted a follow-up study of this locus in different Caucasian cohorts. We analyzed 10 GWAS-genotyped SNPs in the IL12RB2 region (2309 SSc patients and 5161 controls). We then selected three SNPs (rs3790567, rs3790566 and rs924080) based on their significance level in the GWAS, for follow-up in an independent European cohort comprising 3344 SSc and 3848 controls. The most-associated SNP (rs3790567) was further tested in an independent cohort comprising 597 SSc patients and 1139 controls from the USA. After conditional logistic regression analysis of the GWAS data, we selected rs3790567 [PMH= 1.92 × 10−5 odds ratio (OR) = 1.19] as the genetic variant with the firmest independent association observed in the analyzed GWAS peak of association. After the first follow-up phase, only the association of rs3790567 was consistent (PMH= 4.84 × 10−3 OR = 1.12). The second follow-up phase confirmed this finding (Pχ2 = 2.82 × 10−4 OR = 1.34). After performing overall pooled-analysis of all the cohorts included in the present study, the association found for the rs3790567 SNP in the IL12RB2 gene region reached GWAS-level significant association (PMH= 2.82 × 10−9 OR = 1.17). Our data clearly support the IL12RB2 genetic association with SSc, and suggest a relevant role of the interleukin 12 signaling pathway in SSc pathogenesis.
doi:10.1093/hmg/ddr522
PMCID: PMC3298110  PMID: 22076442
10.  The Systemic Lupus Erythematosus IRF5 Risk Haplotype Is Associated with Systemic Sclerosis 
PLoS ONE  2013;8(1):e54419.
Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10−8, OR  = 1.22, CI 95%  = 1.14–1.30; rs2004640: P  = 4.60×10−7, OR  = 0.84, CI 95%  = 0.78–0.90; rs10488631: P  = 7.53×10−20, OR  = 1.63, CI 95%  = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10−22, OR  = 1.75, CI 95%  = 1.56–1.97) better explained the observed association (likelihood P-value  = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
doi:10.1371/journal.pone.0054419
PMCID: PMC3553151  PMID: 23372721
11.  Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis 
Arthritis Research & Therapy  2012;14(3):R154.
Introduction
The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc).
Methods
In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes.
Results
No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis.
Conclusions
Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.
doi:10.1186/ar3890
PMCID: PMC3446540  PMID: 22731751
12.  Frequency of disease-associated and other nuclear autoantibodies in patients of the German network for systemic scleroderma: correlation with characteristic clinical features 
Arthritis Research & Therapy  2011;13(5):R172.
Introduction
In the present study, we analysed in detail nuclear autoantibodies and their associations in systemic sclerosis (SSc) patients included in the German Network for Systemic Scleroderma Registry.
Methods
Sera of 863 patients were analysed according to a standardised protocol including immunofluorescence, immunoprecipitation, line immunoassay and immunodiffusion.
Results
Antinuclear antibodies (ANA) were detected in 94.2% of patients. In 81.6%, at least one of the autoantibodies highly associated with SSc or with overlap syndromes with scleroderma features was detected, that is, anti-centromere (35.9%) or anti-topoisomerase I (30.1%), followed in markedly lower frequency by antibodies to PM-Scl (4.9%), U1-ribonucleoprotein (U1-RNP) (4.8%), RNA polymerases (RNAPs) (3.8%), fibrillarin (1.4%), Ku (1.2%), aminoacyl-transfer RNA synthetases (0.5%), To (0.2%) and U11-RNP (0.1%). We found that the simultaneous presence of SSc-associated autoantibodies was rare (1.6%). Furthermore, additional autoantibodies were detected in 55.4% of the patients with SSc, of which anti-Ro/anti-La, anti-mitochondrial and anti-p25/p23 antibodies were most frequent. The coexistence of SSc-associated and other autoantibodies was common (43% of patients). SSc-associated autoantibodies disclosed characteristic associations with clinical features of patients, some of which were previously not acknowledged.
Conclusions
This study shows that five autoantigens (that is, centromere, topoisomerase I, PM-Scl, U1-RNP and RNAP) detected more than 95% of the known SSc-associated antibody responses in ANA-positive SSc patients and characterise around 79% of all SSc patients in a central European cohort. These data confirm and extend previous data underlining the central role of the determination of ANAs in defining the diagnosis, subset allocation and prognosis of SSc patients.
doi:10.1186/ar3495
PMCID: PMC3308107  PMID: 22018289
systemic sclerosis; scleroderma; autoantibodies; antinuclear antibodies
13.  Erectile dysfunction is frequent in systemic sclerosis and associated with severe disease: a study of the EULAR Scleroderma Trial and Research group 
Introduction
Erectile dysfunction (ED) is common in men with systemic sclerosis (SSc) but the demographics, risk factors and treatment coverage for ED are not well known.
Method
This study was carried out prospectively in the multinational EULAR Scleroderma Trial and Research database by amending the electronic data-entry system with the International Index of Erectile Function-5 and items related to ED risk factors and treatment. Centres participating in this EULAR Scleroderma Trial and Research substudy were asked to recruit patients consecutively.
Results
Of the 130 men studied, only 23 (17.7%) had a normal International Index of Erectile Function-5 score. Thirty-eight per cent of all participants had severe ED (International Index of Erectile Function-5 score ≤ 7). Men with ED were significantly older than subjects without ED (54.8 years vs. 43.3 years, P < 0.001) and more frequently had simultaneous non-SSc-related risk factors such as alcohol consumption. In 82% of SSc patients, the onset of ED was after the manifestation of the first non-Raynaud's symptom (median delay 4.1 years). ED was associated with severe cutaneous, muscular or renal involvement of SSc, elevated pulmonary pressures and restrictive lung disease. ED was treated in only 27.8% of men. The most common treatment was sildenafil, whose efficacy is not established in ED of SSc patients.
Conclusions
Severe ED is a common and early problem in men with SSc. Physicians should address modifiable risk factors actively. More research into the pathophysiology, longitudinal development, treatment and psychosocial impact of ED is needed.
doi:10.1186/ar3748
PMCID: PMC3392836  PMID: 22348608
14.  Genome-wide association study of systemic sclerosis identifies CD247 as a novel susceptibility locus 
Nature genetics  2010;42(5):426-429.
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify novel SSc susceptibility loci we conducted the first genome wide association study (GWAS) in a population of Caucasian ancestry including a total of 2296 SSc patients and 5171 controls. Analysis of 279,621 autosomal single nucleotide polymorphisms (SNPs) followed by replication testing in an independent case-control set of European ancestry (2,753 SSc patients / 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22-23; rs2056626, P = 2.09 × 10−7 in the discovery samples, P = 3.39 × 10−9 in the combined analysis). Additionally, we confirm and firmly establish the role of MHC (2.31 × 10−18), IRF5 (P =1.86 × 10−13) and STAT4 (P =3.37 × 10−9) gene regions as SSc genetic risk factors.
doi:10.1038/ng.565
PMCID: PMC2861917  PMID: 20383147
15.  Effect of sildenafil on digital ulcers in systemic sclerosis: analysis from a single centre pilot study 
Annals of the Rheumatic Diseases  2010;69(8):1475-1478.
Objective
In this pilot study, the effect of sildenafil on digital ulcer (DU) healing and related clinical symptoms was analysed.
Methods
A total of 19 patients with systemic sclerosis (SSc) were treated with maximally tolerated sildenafil doses up to 6 months. Primary outcome was the healing of DUs. Changes in other clinical symptoms were also evaluated.
Results
In all, 49 DUs were present at baseline; this decreased to 17 ulcers (p<0.001) at the end of sildenafil treatment. Furthermore, the visual analogue scale (VAS) score for Raynaud's phenomenon (RP), pain and activity improved (p=0.003, p=0.002 and p=0.05, respectively). A total of 9 patients developed 12 new DUs during sildenafil treatment.
Conclusions
This study indicates an effect of sildenafil on DU healing in patients with SSc and an improvement of RP and associated symptoms that should be validated in controlled studies.
doi:10.1136/ard.2009.116475
PMCID: PMC2938897  PMID: 19900936
16.  The EUSTAR model for teaching and implementing the modified Rodnan skin score in systemic sclerosis 
Annals of the Rheumatic Diseases  2007;66(7):966-969.
Objective
To evaluate the ability to teach scleroderma experts and young rheumatologists to perform the modified Rodnan skin score test.
Methods
Three international “teaching courses for teachers” were conducted with 6–9 experts who performed 3–9 skin score tests each. In addition, an international course for 90 young rheumatologists, in which 18 patients with systemic sclerosis (SSc) participated, was also organised. Finally, a local repeated training course for 5–9 rheumatologists was performed, in which 6–7 patients with SSc participated.
Results
When 6–9 scleroderma specialists investigated the patients, the intraclass correlation coefficient (ICC) showed “good” to “excellent” values (0.865 and 0.710, respectively). When 90 young rheumatologists were involved in one teaching course, the coefficient of variation (CV) was relatively satisfactory (35%) owing to the high number of investigators, and with a considerable within‐patient SD value of 5.4.
Repeated teaching of 5–9 young rheumatologists in local courses clearly improved the consistency. The ICC increased from 0.496 to a “good” level of 0.722. The within‐patient SD values for intraobserver variability ranged between 2.5 and 2.9. The intraobserver CV was about 20%.
Conclusions
This study strongly supports the need for standardisation among different centres when using skin scoring for clinical trials. The intraobserver variability and within‐patient SD values can be significantly reduced by repeated teaching. For inexperienced rheumatologists, at least one repeated teaching course is needed.
doi:10.1136/ard.2006.066530
PMCID: PMC1955103  PMID: 17234649
17.  Clinical and serological evaluation of a novel CENP-A peptide based ELISA 
Introduction
Anti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA.
Methods
Sera collected from SSc patients (n = 334) and various other diseases (n = 619) and from healthy controls (n = 175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA® CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies.
Results
The CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA® CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho = 0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n = 131) and various controls (n = 134) was significantly better using the CENP-A as compared to CENP-B ELISA (P < 0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P = 0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P = 0.0103), specific joint involvement (Jaccoud) (P = 0.0006) and anti-phospholipid syndrome (P = 0.0157) between ACA positive SLE patients and the entire SLE cohort were observed.
Conclusions
Anti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.
doi:10.1186/ar3029
PMCID: PMC2911886  PMID: 20487535
18.  Early right ventricular systolic dysfunction in patients with systemic sclerosis without pulmonary hypertension: a Doppler Tissue and Speckle Tracking echocardiography study 
Background
Isovolumetric acceleration (IVA) is a novel tissue Doppler parameter for the assessment of systolic function. The aim of this study was to evaluate IVA as an early parameter for the detection of right ventricular (RV) systolic dysfunction in patients with systemic sclerosis (SSc) without pulmonary hypertension.
Methods
22 patients and 22 gender- and age-matched healthy subjects underwent standard echocardiography with tissue Doppler imaging (TDI) and speckle tracking strain to assess RV function.
Results
Tricuspid annular plane systolic excursion (TAPSE) (23.2 ± 4.1 mm vs. 26.5 ± 2.9 mm, p < 0.006), peak myocardial systolic velocity (Sm) (11.6 ± 2.3 cm/s vs. 13.9 ± 2.7 cm/s, p = 0.005), isovolumetric contraction velocity (IVV) (10.3 ± 3 cm/s vs. 14.8 ± 3 cm/s, p < 0.001) and IVA (2.3 ± 0.4 m/s2 vs. 4.1 ± 0.8 m/s2, p < 0.001) were significant lower in the patient group. IVA was the best parameter to predict early systolic dysfunction with an area under the curve of 0.988.
Conclusion
IVA is a useful tool with high-predictive power to detect early right ventricular systolic impairment in patients with SSc and without pulmonary hypertension.
doi:10.1186/1476-7120-8-3
PMCID: PMC2822748  PMID: 20096122
19.  Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients 
Arthritis Research & Therapy  2009;11(4):R111.
Introduction
Interstitial lung disease (ILD) is a frequent manifestation of systemic sclerosis (SSc), and cytokines can contribute to the disease pathology. The aim of the current study was to identify specific changes in cytokine levels that may serve as disease markers and possible targets for therapy.
Methods
Cytokines were measured with bioplex analysis in 38 bronchoalveolar fluids (BALFs) from 32 SSc patients (27 with alveolitis and 11 without alveolitis) and 26 control patients. In the case of SSc patients, cytokines were correlated with the respective bronchoalveolar lavage (BAL) cell differentiation, lung function, and thoracic HR-CT score. For 35 BALF samples derived from 29 SSc patients, follow-up investigations of clinical data, lung-function parameter, or thoracic HR-CT scans were available to evaluate the predictive capacity of BALF cytokines and chemokines.
Results
High IL-7 levels were characteristic of SSc-associated interstitial lung disease (ILD) and, in addition, when compared with ILD-negative SSc patients, ILD-positive SSc patients revealed higher IL-4, IL-6, IL-8, and CCL2 (MCP-1) BALF levels. High CCL2 and IL-8 BALF concentrations were associated with neutrophilic and mixed alveolitis. Cytokine levels of IL-4, IL-8, and CCL2 correlated negatively with lung-function parameters; CCL2 concentrations also correlated with HR-CT scores. High concentrations of several cytokines were associated with the progress of ILD and end-stage ILD. Univariate analyses revealed high IL-2 and tumor necrosis factor-alpha (TNF-α) levels as the best predictors for progressive disease, together with lung-function parameters, young age, and neutrophilic alveolitis. Multivariate analyses partially confirmed these results but did not sufficiently converge because of the limited number of patients.
Conclusions
The association of BALF cytokines with lung fibrosis and its progress suggests that cytokines contribute to the pathogenesis of ILD and hence could be regarded as potential therapeutic targets.
doi:10.1186/ar2766
PMCID: PMC2745793  PMID: 19615053
20.  High frequency of corticosteroid and immunosuppressive therapy in patients with systemic sclerosis despite limited evidence for efficacy 
Introduction
In systemic sclerosis (SSc) little evidence for the effectiveness of anti-inflammatory and immunosuppressive therapy exists. The objective of this study was to determine the extent to which SSc patients are treated with corticosteroids and immunosuppressive agents.
Methods
Data on duration and dosage of corticosteroids and on the type of immunosuppressive agent were analyzed from 1,729 patients who were registered in the German Network for Systemic Scleroderma (DNSS).
Results
A total 41.3% of all registered SSc patients was treated with corticosteroids. Corticosteroid use was reported in 49.1% of patients with diffuse cutaneous SSc and 31.3% of patients with limited cutaneous SSc (P < 0.0001). Among patients with overlap disease characteristics, 63.5% received corticosteroids (P < 0.0001 vs. limited cutaneous SSc). A total 16.1% of the patients received corticosteroids with a daily dose ≥ 15 mg prednisone equivalent. Immunosuppressive therapy was prescribed in 35.8% of patients. Again, among those patients with overlap symptoms, a much higher proportion (64.1%) was treated with immunosuppressive agents, compared with 46.4% of those with diffuse cutaneous SSc sclerosis and 22.2% of those with limited cutaneous SSc (P < 0.0001). The most commonly prescribed drugs were methotrexate (30.5%), cyclophosphamide (22.2%), azathioprine (21.8%) and (hydroxy)chloroquine (7.2%). The use of these compounds varied significantly between medical subspecialties.
Conclusions
Despite limited evidence for the effectiveness of corticosteroids and immunosuppressive agents in SSc, these potentially harmful drugs are frequently prescribed to patients with all forms of SSc. Therefore, this study indicates the need to develop and communicate adequate treatment recommendations.
doi:10.1186/ar2634
PMCID: PMC2688174  PMID: 19261182
21.  Diagnostic value of anti-topoisomerase I antibodies in a large monocentric cohort 
Introduction
In the present study, the detection of anti-topoisomerase I (anti-topo I) autoantibodies was evaluated for diagnosis and risk assessment of systemic sclerosis (SSc) patients in a well characterized large monocentric cohort.
Methods
Sera from patients with SSc (diffuse n = 96, limited n = 113), from patients with overlap syndromes (n = 51), from patients with other diseases associated with SSc (n = 20), as well as from disease controls (n = 487) were analysed for the presence of anti-topo I antibodies by line immunoblot assay and ELISA. Assessment of organ manifestations was performed as proposed by the European Scleroderma Trial and Research network.
Results
The applied test systems for the detection of anti-topo I antibodies revealed a diagnostic sensitivity for SSc of approximately 24% and a diagnostic specificity of at least 99.6%. The sensitivity to identify patients with diffuse SSc amounted to 60%. Patients with anti-topo I antibodies showed a higher burden of skin and lung fibrosis, contractures, electrocardiogram changes, as well as digital ulcers and had more active disease than antibody-negative patients. Signal strengths correlated only weakly with disease activity, with modified Rodnan skin score, with predicted forced vital capacity, and with predicted diffusion capacity levels (P = 0.01, ρ = 0.234, ρ = 0.413, ρ = -0.215, ρ = -0.219). High signal intensities were associated with an increased mortality in diffuse SSc patients (P = 0.003).
Conclusions
Diagnosis and risk assessment of SSc patients can be supported by the detection of anti-topo I antibodies. Signal intensities as obtained by line immunoblot assay or ELISA can be used as a surrogate marker for fibrosis, active disease and worse prognosis.
doi:10.1186/ar2622
PMCID: PMC2688262  PMID: 19232127
22.  Antibodies against PM/Scl-75 and PM/Scl-100 are independent markers for different subsets of systemic sclerosis patients 
Introduction
Anti-PM/Scl antibodies are present in sera from patients with polymyositis (PM), systemic sclerosis (SSc), and PM/SSc overlap syndromes. The prevalence of antibodies against the 75- and 100-kDa PM/Scl proteins and their clinical associations have not been studied in SSc patients in detail so far but could provide a valuable tool for risk assessment in these patients. Furthermore, it remains speculative whether commercially available test systems detecting only anti-PM/Scl-100 antibodies are sufficient in SSc patients.
Methods
Two hundred eighty sera from SSc patients, patients with other connective tissue diseases (n = 209), and healthy blood donors (n = 50) were analyzed for the presence of anti-PM/Scl-75 and anti-PM/Scl-100 antibodies by means of line immunoblot assay. For the SSc patients, possible associations between both subsets of anti-PM/Scl antibodies with clinical and laboratory findings were studied.
Results
The determination of anti-PM/Scl reactivity revealed a diagnostic sensitivity of 12.5% and a specificity of 96.9% for SSc. Among anti-PM/Scl-positive SSc patients, 10.4% and 7.1% were positive for anti-PM/Scl-75 and anti-PM/Scl-100 antibodies, respectively. The highest prevalences of reactivity to PM/Scl were detected in diffuse SSc (19.8%) and overlap syndromes (17.6%). Patients with diffuse SSc showed mainly an anti-PM/Scl-75 response, whereas most cases of overlap syndromes were characterized by reactivity to both PM/Scl antigens. The presence of anti-PM/Scl-75/100 antibodies was associated with muscular and lung involvements as well as with digital ulcers; pulmonary arterial hypertension was found less frequently. Anti-PM/Scl-75 antibodies were detected more frequently in younger and more active patients with joint contractures. Anti-PM/Scl-100 antibodies were associated with creatine kinase elevation; however, gastrointestinal involvements were observed less frequently.
Conclusions
Anti-PM/Scl antibodies are common in distinct SSc subsets and are associated with several clinical symptoms. They are directed mainly to the PM/Scl-75 antigen. Consequently, the detection of anti-PM/Scl antibodies by tests based only on PM/Scl-100 as an antigen source may miss a relevant number of SSc patients positive for these antibodies.
doi:10.1186/ar2614
PMCID: PMC2688254  PMID: 19220911

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