Skin gene expression correlates of severity of interstitial lung disease in systemic sclerosis.

OBJECTIVE: We undertook this hypothesis-generating study to identify skin transcripts correlating with severity of interstitial lung disease (ILD) in systemic sclerosis (SSc). METHODS: Skin biopsy samples from 59 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort or an open-label imatinib study (baseline visit) were examined by global gene expression analysis using Illumina HT-12 arrays. Skin transcripts correlating with concomitantly obtained forced vital capacity (FVC) values and the modified Rodnan skin thickness score (MRSS) were identified by quantitative trait analysis. Also, immunofluorescence staining for selected transcripts was performed in affected skin and lung tissue. Plasma levels of CCL2, soluble SELP, and soluble P-selectin glycoprotein ligand 1 (sPSGL-1) were examined in all patients enrolled in the GENISOS cohort (n = 266). RESULTS: Eighty-two skin transcripts correlated significantly with FVC. This gene list distinguished patients with more severe ILD (FVC <70% predicted) in unsupervised hierarchical clustering analysis (P < 0.001). These genes included SELP, CCL2, and matrix metalloproteinase 3, which are involved in extravasation and adhesion of inflammatory cells. Among the FVC correlates, 8 genes (CCL2, HAPLN3, GPR4, ADCYAP1, WARS, CDC25B, PLP1, and STXBP6) also correlated with the MRSS. Immunofluorescence staining revealed that SELP and CCL2 were also overexpressed in affected skin and lung tissue from SSc patients compared to those from controls. Plasma levels of CCL2 and sPSGL-1 correlated with concomitantly obtained FVC values (r = -0.22, P = 0.001 and r = 0.17, P = 0.015, respectively). This relationship was independent of potential confounders (age, sex, ethnicity, smoking status, anti-topoisomerase I positivity, treatment with immunosuppressive agents, MRSS, disease type, and disease duration). CONCLUSION: A limited number of skin transcripts including genes involved in extravasation and adhesion of inflammatory cells correlate with severity of ILD.

Post-menopause is the main risk factor for developing isolated pulmonary hypertension in systemic sclerosis.

In scleroderma patients, isolated pulmonary hypertension (PHT) has been associated with selected HLA haplotypes, severe impairment of the diffusing capacity for carbon monoxide and the diagnosis of CREST. Most patients with CREST have a late-age onset of the disease, corresponding to the perimenopausal or postmenopausal period. We conducted a retrospective cohort study to determine the role of post-menopause and of the other known clinical and biological markers in the development of isolated pulmonary hypertension in Italian patients with systemic sclerosis. 189 female patients with scleroderma who had no ecographic signs of pulmonary hypertension (PHT) and radiographic signs of lung fibrosis at the first visit and did not develop significant pulmonary fibrosis during the observation time were included. Sixty-three out of 189 patients (33.3%) presented isolated pulmonary hypertension. A severe impairment of diffusing capacity for carbon monoxide at admission was found to be an early predictive element for its development. An increased risk was associated with postmenopausal condition (RR = 5.2, p = 0.000), CREST syndrome (RR = 2.8, p = 0.001) and haplotype HLA-B35 (RR = 2.8; p = 0.002). A significant positive interaction between postmenopausal condition and either HLA-B35 (RR = 15.2; p = 0.000) or the diagnosis of CREST (RR = 14.1; p = 0.000) was found. Postmenopausal condition alone or in combination with HLA-B35 and CREST syndrome is the main risk-factor for developing primary pulmonary hypertension in scleroderma patients. This suggests that hormonal replacement therapy could play a role in preventing isolated PHT in patients with systemic sclerosis.

Protease nexin-1 messenger RNA levels are not affected by serum or interferon beta in cultured systemic sclerosis fibroblasts.

The aim of the present study was to analyze the effect of serum and human recombinant beta interferon (rIFNbeta) treatment on PN-1 mRNA levels in cultured dermal fibroblasts obtained from the skin of healthy donors and from lesional skin of systemic sclerosis (SSc) patients with the limited (CREST syndrome) or the diffuse form of SSc. Total RNA was isolated from fibroblasts derived from the skin of healthy individuals and from lesional skin of patients with CREST syndrome and the diffuse form of SSc cultured under different conditions (1% or 10% serum-supplemented medium) and treated with 500 IU/ml of rIFNbeta. PN-1 gene expression was assessed by Northern blot analysis. We detected variable PN-1 mRNA levels in normal control fibroblasts as well as in SSc fibroblasts under the different culture conditions (1% or 10% serum-supplemented medium). Accumulated PN-1 mRNA levels found in normal cultured fibroblasts were similar to or even higher than in SSc fibroblasts. PN-1 messenger levels were not significantly altered by IFNbeta treatment in normal or SSc cultured fibroblasts despite the presence of an IFN-stimulated responsive element (ISRE) in the promoter of the PN-1 gene. Our findings suggest that PN-1 expression in SSc fibroblasts at the mRNA level requires further investigation in a large number of SSc patients to better characterize the role of this serpin in the pathogenesis of SSc. We conclude that the transcriptional regulation of PN-1 is not associated with IFNbeta, an antifibrotic cytokine naturally produced by fibroblasts.

Clinical, serological and genetic study in patients with CREST syndrome.

OBJECTIVE: To assess the clinical, serological and genetic features of Japanese patients with CREST syndrome. PATIENTS AND METHODS: Clinical features, autoantibodies and human histocompatibility leukocyte antigen (HLA) typing were studied in thirty patients with CREST syndrome, including 29 females and one male, with a mean age of 59.0 years (ranging from 40 to 76 years). RESULTS: Interstitial pneumonia on chest X-ray and renal involvement were rare. Mitral regurgitation and tricuspid regurgitation were present in 56.7% and 76.7%, respectively. Sjören's syndrome (SS) and primary biliary cirrhosis (PBC) were highly associated, however the positivity of the marker antibodies to those syndromes, such as anti-SSA, anti-SSB, anti-mitochondrial (AMA) and anti-smooth muscle autoantibodies were less frequent than that of primary SS and PBC without the other autoimmune diseases. The histological findings of PBC were all early stages in Scheuer's classification. HLA-Cw6 were associated with CREST-PBC overlap syndrome (p<0.05). However the HLA antigen was not correlated with CREST syndrome, and the frequency of HLA-DR2 between CREST syndrome with or without PBC was significantly different (p<0.01). CONCLUSION: It was suggested that there was a genetic difference between CREST syndrome alone and CREST-PBC overlap syndrome and there were differences (the positivity of AMA and the severity of bile duct lesion) between PBC and CREST-PBC overlap syndrome.

Association of clonally expanded T cells with the syndrome of primary biliary cirrhosis and limited scleroderma.

Clinical features of the CREST (calcinosis cutis, Raynaud's syndrome, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome are sometimes exhibited in patients with primary biliary cirrhosis (PBC), but the postulated autoimmune mechanisms behind these conditions are poorly understood. Clonally expanded T cells may play an important role in disease pathogenesis. In this study, overrepresentation of one T-cell receptor beta chain variable region, TCRBV3, was documented in patients with PBC and/or CREST. Overrepresentation of the TCRBV3 gene mRNA was demonstrated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). T cells expressing TCRBV3 were analyzed by flow cytometry, were primarily CD8(+), and contained activated cells as assessed by expression of CD69. Clonally expanded T cells within this population were documented by both complementarity determining region 3 (CDR3) length polymorphism analysis and sequencing of T-cell receptor CDR3 cDNA. TCRBV3(+) clonal expansions were stable when followed for up to 5 years. The results of this study demonstrate that the T-cell repertoire of patients with PBC and CREST is characterized by expanded clonal populations of CD8(+) TCRBV3(+) T cells. These clonal expansions provide evidence that stimulation of clonal populations of CD8(+) T cells is associated with the clinical syndrome of PBC with CREST.

[Primary biliary cirrhosis (PBC)-CREST overlap syndrome complicated with Sjögren's syndrome].

We reported two sisters of primary biliary cirrhosis (PBC)-CREST overlap syndrome complicated with Sjögren's syndrome (SS). Both patients had Raynaud's phenomenon, sclerodactylia, telangiectasia, chronic sialoadenitis, chronic nonsuppurative destructive cholangitis by liver biopsy and were positive for anti-centromere antibodies. This is the first report of two sisters of PBC-CREST overlap syndrome complicated with SS.

Familial CREST syndrome.

The familial occurrence of scleroderma is uncommon particularly the limited (CREST) form. We describe 2 families in which such an association occurred. Family pedigree 1 consists of 2 of 3 sisters with CREST scleroderma. Both affected sisters shared HLA types and C4 allotypes including DR5, found more frequently in patients with scleroderma. The unaffected sister did not share this MHC allele. Family pedigree 2 includes a grandmother and grandson with CREST scleroderma as well as a family member with Raynaud's phenomena alone. We conclude that familial occurrence of scleroderma may be associated with shared class II MHC antigens.