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

Adeno-associated virus type 5 infection via PDGFRα is associated with Interstitial lung disease in systemic sclerosis and generates composite peptides and epitopes recognized by the agonistic immunoglobulins present in patients with systemic sclerosis

Objectives: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in SSc patients. Since PDGFRα is targeted of the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. Methods: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL). Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and Immunoaffinity-purified anti-PDGFRα antibodies from sera of SSc patients. Results: AAV5 was detected in the BAL of 41 out of 66 (62.1%) SSc patients with interstitial lung disease and in 17 of 66 controls (25.75 %; p<0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from SSc patients, not from healthy controls. Conclusions: These data link AVV5 with the immune reactivity to endogenous antigens in SSc, and provide a novel element in the pathogenesis of SSc

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

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

hUC-MSC do not induce any biological effects on control mice treated with endotracheal saline.

<p>Histology (<b>A-B</b>), collagen content (<b>C-D</b>) and macrophage infiltration (<b>E-H</b>) of mouse lungs 21 days after sterile saline endotracheal injection followed by hUC-MSC intravenous infusion. Lung sections obtained from C57BL/6 mice (n = 8 per group) that received endotracheal sterile saline only (saline) or endotracheal sterile saline followed by intravenous hUC-MSC (saline+hUC-MSC) were stained with H&E (<b>A-B</b>), Picrosirius Red (<b>C-D</b>), anti- galectin-3 (<b>E-F</b>) and anti-arginase I (<b>G-H</b>) antibodies. Representative microscopic images (10× or 40× magnification) of three independent experiments are shown. Quantitative real-time PCR gene expression analysis of Col1A1, TGFβ and αSMA (<b>I</b>), and IL-1β, IL-2, IL-6 and IL-10 (<b>J</b>) in whole lung mRNA obtained at day 21 from C57BL/6 mice receiving the aforementioned treatments. Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. (<b>K</b>) Quantification of galectin-3 and arginase I positive macrophages in C57BL/6 mouse lungs 21 days after saline injection with or without subsequent hUC-MSC infusion. Results are mean ± SD of positive immunostained cell count per sample (n = 8 per group) and are representative of three independent experiments.</p

Decrease of galectin-3 positive cells in bleomycin-injured lung tissue upon administration of hUC-MSC.

<p>Macrophage infiltration in mouse lungs at days 8 (<b>A-D</b>) and 21 (<b>E-H</b>) after endotracheal injection of sterile saline (saline) (<b>A, E</b>) or bleomycin (bleomycin) (<b>B, F</b>), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) (<b>D, H</b>) or sterile saline (bleomycin+saline) (<b>C, G</b>). Lung sections obtained from C57BL/6 mice (n = 8 per group) were immunostained with anti-galectin-3 antibody. At each time point, the number of immunoreactive macrophages infiltrating the lungs are significantly decreased by hUC-MSC infusion in comparison to bleomycin-treated mice. Representative microscopic images (40× magnification) of three independent experiments are shown. (<b>I)</b> Cell count of galectin-3 positive macrophages in C57BL/6 mouse lung sections, and (<b>J</b>) quantitative real-time PCR analysis of galectin-3 gene expression in whole lung mRNA, obtained at days 8 and 21. Results are expressed as mean ± SD of positively immunostained cell count per sample (n = 5 per group) and are representative of three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 compared to bleomycin treated mice.</p