Objective. To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor a (PDGFRa) in systemic sclerosis (SSc) and develop novel assays for detection of serum antiPDGFRa autoantibodies.Methods. Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRa and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRa IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRa recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRa extracellular domains, and expression analyses of alanine-scanned PDGFRa mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRa autoantibodies or, selectively, the agonistic ones.Results. Three types of anti-PDGFRa recombinant human mAb, with the same V H but distinct V L chains, were generated. Nonagonistic V H PAM-V k 13B8 recognized 1 linear epitope, whereas agonistic V H PAM-V l 16F4 and V H PAM-V k 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRa antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V H PAM-V k 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V H PAM-V k 16F4 epitope inhibited PDGFRa signaling triggered by serum IgG from SSc patients.Conclusion. Agonistic anti-PDGFRa autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRa signaling in patients with SSc.

Angiogenesis plays a key role in the pathogenesis of psoriasis. New blood vessel formation occurs early during plaque lesion development in psoriatic skin, and sometimes precedes disease recurrence. TNF-α, a well established mediator of inflammation in psoriasis, up-regulates the transcription of several pro-angiogenic chemokines that are over-expressed in psoriatic skin and serum, promoting microvascular modifications in psoriatic plaque. Adalimumab is a fully human monoclonal antibody that blocks the interaction between TNF-α and its cell surface receptor, thus inhibiting the TNF-α dependent inflammatory cascade. The aims of this study were to investigate several angiogenic parameters involved in the pathogenesis of psoriasis, and to evaluate the ability of adalimumab to modulate them. Fifteen patients affected by psoriasis received Adalimumab 40 mg EOW for twelve weeks and were evaluated at baseline (T0) and after treatment (T12) for the following parameters: i) new blood vessels formation in lesional skin assessed by intra-vital video-capillaroscopy analysis (IVCP) and histology; ii) VEGF and Factor VIII expression in lesional skin detected by immunohistochemistry; iii) serological levels of several angiogenic chemokines detected by luminex assay. At baseline, newly formed capillaries in psoriatic plaque correlated with skin expression of VEGF and factor VIII and with serum levels of angiopoietin-2, IL-8, PDGF-BB, VEGF, but not with serum levels of follistatin, TNF-α, G-CSF, HGF, FGF, PECAM, IFN-γ, and TGF-α. All patients responded to adalimumab, reached PASI 50, and 70% achieved PASI 75 after twelve weeks of treatment. Although adalimumab administration for twelve weeks caused a dramatic decrease of new blood vessel formation, confirmed by IVCP (p<0.05), histology and immunohistochemical (p<0.05) analysis, we did not observe a parallel significant reduction of angiogenic chemokines in the serum. However, a positive correlation between the density of newly formed blood vessels in lesional skin and the serum levels of angiopoietin-2, IL-8, PDGF-BB, and VEGF, was still persisting after treatment.

No abstract

Biosensors are versatile tools for monitoring molecular interactions. Herein, we report a novel assay designed to detect anti-platelet-derived growth factor receptor α (PDGFRα) autoantibodies in the serum of patients affected by systemic sclerosis (SSc), an autoimmune disease of the connective tissue. The assay was based on resonant mirror sensing, and used recombinant PDGFRα as molecular "bait" for anti-PDGFRα autoantibodies (IgG class). The selection and optimization of the analytical procedure required a preliminary investigation on the preservation of the native-like conformation of the receptor following the immobilization procedure. The PDGFRα-based biosensor was used to test IgG purified from sera of SSc patients and healthy controls (HC). The specificity and the reversibility of the interaction permitted a rapid analysis, a single detection test requiring only a few minutes. Remarkably, this assay could discriminate between SSc patients and HC. This receptor-based biosensor, based on the reversible interaction between a blocked macromolecule and soluble ligands, and with major advantages such as the rapidity, the reusability of the capturing surface and the low cost per single test, could represent a promising approach for the diagnosis of SSc and other diseases characterized by anti-receptor autoantibodies or other bioactive ligands to cellular receptors.