Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20–4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies

Metabolomic profile of systemic sclerosis patients

Systemic sclerosis (SSc) is an autoimmune disease of unknown aetiology characterized by vascular lesions, immunological alterations and diffuse fibrosis of the skin and internal organs. Since recent evidence suggests that there is a link between metabolomics and immune mediated disease, serum metabolic profile of SSc patients and healthy controls was investigated by1H-NMR and GC-MS techniques. The results indicated a lower level of aspartate, alanine, choline, glutamate, and glutarate in SSc patients compared with healthy controls. Moreover, comparing patients affected by limited SSc (lcSSc) and diffuse SSc (dcSSc), 6 discriminant metabolites were identified. The multivariate analysis performed using all the metabolites significantly different revealed glycolysis, gluconeogenesis, energetic pathways, glutamate metabolism, degradation of ketone bodies and pyruvate metabolism as the most important networks. Aspartate, alanine and citrate yielded a high area under receiver-operating characteristic (ROC) curves (AUC of 0.81; CI 0.726-0.93) for discriminating SSc patients from controls, whereas ROC curve generated with acetate, fructose, glutamate, glutamine, glycerol and glutarate (AUC of 0.84; CI 0.7-0.98) discriminated between lcSSc and dcSSc. These results indicated that serum NMR-based metabolomics profiling method is sensitive and specific enough to distinguish SSc from healthy controls and provided a feasible diagnostic tool for the diagnosis and classification of the disease

In vitro photostability and photoprotection studies of a novel 'multi-active' UV-absorber.

This paper reports on the synthesis and properties of a new UV-absorber (OC-NO) based on the most popular UV filter worldwide, ethylhexyl methoxycinnamate (OMC) in which the methoxy group has been replaced with a pyrrolidine nitroxide bearing antioxidant activity. This sunscreen active has therefore both UV-absorbing and antioxidant properties which could ideally address both the UV-B and UV-A skin photo-damage. For broad-spectrum coverage, the combinations of OC-NO with two commonly used UV-A absorbers (BMDBM and DHHB) were also studied. The results obtained reveal that OC-NO: (a) is as photostable as OMC after UV-A exposure; (b) acts as free radical scavenger as demonstrated by EPR and chemical studies; (c) reduces UV-A and UV-A+BMDBM induced lipid peroxidation in liposomes and cells, measured as reduced TBARS levels and increased C11-BODIPY red fluorescence, respectively; (d) has comparable antioxidant activity to that of vitamin E and BHT commonly used in skin care formulations; (e) is non-cytotoxic to human skin fibroblasts as assessed with the MTT assay when exposed to increasing doses of UV-A; and (f) OC-NO+DHHB is a promising, photostable broad spectrum UV-filter combination that concomitantly reduces UV-induced free radical damage. These results suggest that nitroxide/antioxidant-based UV-absorbers may pave the way for the utilization of 'multi-active' ingredients in sunscreens thereby reducing the number of ingredients in these formulations

WIF1, A WNT PATHWAY INHIBITOR, IS SILENCED IN SYSTEMIC SCLEROSIS BY DNA DAMAGE: A MECHANISM LINKING DNA DAMAGE TO WNT AND FIBROSIS

Dysregulation of Wnt signaling is common in a variety of human malignancies, carcinogenesis, aging and fibrosis. Wnt signaling is tightly controlled by several negative regulators, such as WIF1 (Wnt inhibitor factor 1). Activation of canonical Wnt signaling has been recently found in fibrotic diseases included Systemic Sclerosis (SSc). Objectives The objective of the present work is to identify the mechanism responsible for the silencing of WIF1 in SSc. Methods Skin fibroblasts from SSc patients and normal controls were treated with bleomycin or ATM-HDAC inhibitors. Cells were transiently transfected with the siRNA against c-jun and ATF-3 with Lipofectamine (Invitrogen). Total RNA was isolated and reverse-transcribed, according to the manufacturer’s instructions (Bio-Rad). Quantitative real-time PCR reactions were performed using SYBR-Green PCR Master Mix (Bio-Rad). The relative expression levels were calculated using the 2-ΔΔCT method. To analyzed protein expression, cells were lysed with RIPA buffer and subjected to western blot with specific antibodies. Results Our data indicate that WIF1 is silenced by DNA damage and the check point kinase, ATM. Cell derived from SSc patients reactivate WIF1 expression if exposed to ATM or HDACI-III inhibitors. ROS and SSc immunoglobulins silence WIF1 expression via PDGF receptor, stimulate b-catenin accumulation by inducing ROS-dependent DNA damage. Bleomycin, a drug widely used to induce local skin fibrosis in vivo, silences WIF1 and stimulates Wnt signaling and its effects are suppressed by ATM or HDAC inhibitors. Silencing of WIF1 in normal cells amplifies Wnt signaling and increases collagen expression. As molecular actors that silence WIF1 in DNA damaged cells, we report that the knocking down of the expression of transcription factors ATF-3 and c-jun relieves WIF1 inhibition and dowregulates collagen expression in SSc cells. Bleomycin profibrotic phenotype is caused by activation of ATF-3 which silences WIF1 and amplifies Wnt signaling and by c-jun which cooperates with Wnt and stimulates collagen expression. Conclusions These results explain Wnt signaling hypertrophy in fibrotic disease, unveil a direct link between DNA damage and Wnt, and pave a novel route to treat fibrosis

Agonistic anti-PDGF receptor autoantibodies from patients with systemic sclerosis impact human pulmonary artery smooth muscle cells function in vitro

One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia

Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis

Gut epithelial impairment, microbial translocation and immune system activation in inflammatory bowel disease-associated spondyloarthritis.

Objectives. Gut microbiota has been widely reported to be involved in systemic inflammation through microbial translocation and T cell activation in several diseases. In this work we aimed to investigate bacterial infiltration and epithelial impairment in the gut of patients with IBD-associated SpA (SpA-IBD), as well as the relationship of microbial translocation with immune system activation and their putative role in the pathogenesis of joint inflammation in IBD patients. Methods. Tight-junction proteins (TJPs) occludin and claudin-1/-4 and bacteria were assessed by real-time PCR analysis and immunohistochemical staining of the ileum. Intestinal fatty acid binding protein (I-FABP), lipopolysaccharides (LPS), soluble CD14 (sCD14), sclerostin and anti-sclerostin antibodies (anti-sclerostin-IgG) were assayed with ELISAs and peripheral mononuclear blood cells with flow cytometry. LPS and sCD14 were used in vitro to stimulate a human osteoblast cell line. Results. Compared with IBD, ileal samples from SpA-IBD patients showed bacterial infiltration, epithelial damage and downregulation of TJPs. In sera, they showed higher serum levels of I-FABP, LPS, sCD14 (the latter correlating with sclerostin and anti-sclerostin-IgG) and higher CD80þ/CD163þ and lower CD14þ mononuclear cells. In vitro experiments demonstrated that only the LPS and sCD14 synergic action downregulates sclerostin expression in osteoblast cells. Conclusion. SpA-IBD patients are characterized by gut epithelium impairment with consequent translocation of microbial products into the bloodstream, immune system activation and an increase of specific soluble biomarkers. These findings suggest that gut dysbiosis could be involved in the pathogenesis of SpA-IBD and it could hopefully prompt the use of these biomarkers in the follow-up and management of IBD patients