Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage

MOLECULAR HETEROGENEITY OF THE 4TH COMPONENT OF COMPLEMENT (C4) AND ITS GENES IN VITILIGO

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*QO allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo