Pavla Vavrincova

Anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis.

We analysed the presence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin (AKA) antibodies of the IgG class in sera of patients with defined juvenile idiopathic arthritis (JIA) of various subgroups with more than one year duration of the disease. Enzyme-linked immunosorbent assay (Immunoscan RA, Eurodiagnostica, The Netherlands) and an indirect immunofluorescence (IIF) test on rat oesophagus substrate (ImmuGloTM, Immco Diagnostics, Buffalo, USA) were used for the detection and quantification of anti-CCP and AKA antibodies in 140 patients with JIA (64 male and 76 female) aged 2-47 years (median 16.5 years). Overall, anti-CCP were found in 7/140 (5.0%) patients including 3/52 RF negative polyarthritis, 2/18 RF positive polyarthritis, 1/15 enthesitis related arthritis and 1/5 unclassifiable arthritis. AKA were detected in 40/140 patients (28.6%, p =0.04) including 2/11 systemic arthritis, 2/32 oligoarthritis, 18/52 patients with RF negative polyarthritis (34.6%, p =0.01), 14/18 RF positive polyarthritis (77.8%, p =0.000002), 2/15 enthesitis related arthritis and 2/3 psoriatic arthritis. While simultaneous negativity for AKA and anti-CCP occurred in most (97/140; 69.3%) studied cases, simultaneous antibody positivity was found only in few (4/140; 2.9%) studied samples. We conclude that while AKA measured using IIF on rat esophagus can be detected approximately in one third of patients with definite JIA with more than 1 year duration of the disease, only rare occurrence of anti-CCP was observed. We conclude that AKA seem to be partly useful to confirm JIA diagnosis, however, useless to follow-up severity or activity in JIA patients. Anti-CCP do not have any additional value in JIA cohort in comparison to RA where their diagnostic and prognostic importance was reported.

Heat shock protein 70 membrane expression on fibroblast‐like synovial cells derived from synovial tissue of patients with rheumatoid and juvenile idiopathic arthritis

Objective: To screen fibroblast‐like synovial cells derived from synovial tissue of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) patients for the membrane expression of the heat shock protein Hsp70. Methods: We performed flow cytometric (fluorescence‐activated cell sorting, or FACS) analysis on fibroblast‐like synovial cells of 15 RA patients and three JIA patients to investigate Hsp70 membrane expression. Skin fibroblasts derived from the operation wound (n = 4) and peripheral blood mononuclear cells (PBMC) of seven RA and three JIA patients were also tested. Peripheral blood lymphocytes (PBL) and skin fibroblasts of 10 healthy individuals were used as negative controls. Results: A significantly higher percentage of Hsp70 membrane expression was found on fibroblast‐like synovial cells derived from arthritis‐affected joints in RA patients (mean 47.7%) when compared with autologous skin fibroblasts (mean 9.5%, p<0.001) and control skin fibroblasts (mean 5.6%, p<0.001) or autologous PBL (mean CD45/Hsp70‐positive 10.4%, p<0.001) and control PBL (mean CD45/Hsp70‐positive 7.7%, p<0.001). A high percentage of Hsp70 membrane expression was also observed on fibroblast‐like synovial cells derived from three patients with JIA (mean 35.2%) when compared with autologous PBL (mean CD45/Hsp70‐positive 10.4%). Synovial cells derived from non‐affected joints in a patient with RA who underwent synovectomy for trauma showed low expression of Hsp70 (10.9%). Conclusion: Fibroblast‐like synovial cells derived from patients with severe course of RA and JIA are strongly positive for membrane‐expressed Hsp70.

Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls

Objective: Heat shock proteins (Hsps) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this work was to study Hsp mRNA and protein levels to determine whether they can be used to differentiate between RA, osteoarthritis (OA), and healthy controls. Methods: Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 mRNA expression was analysed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 24 RA, 11 OA, and 21 healthy controls. Hsp70 and HspBP1 protein levels were measured in serum using an enzyme-linked immunosorbent assay (ELISA). Results: Hsp gene expression profiles differ significantly between inflammatory (RA) and non-inflammatory (OA) joint diseases, showing significantly increased Hsp27 and Hsp90α mRNA levels in RA synovial tissues. Up-regulated Hsp60 and Hsp90α together with down-regulated Hsp70 and elevated HspBP1/Hsp70 mRNA ratios can be used to differentiate between RA patients and healthy individuals through analysis of peripheral blood samples. Despite increased HspBP1 levels in RA sera, Hsp70 levels and the HspBP1/Hsp70 protein ratio remained identical in the RA patients and healthy individuals, which may contribute to the inhibition of Hsp70 anti-apoptotic activity. Conclusion: Hsp gene expression analysis can be implemented as a new diagnostic approach to facilitate differentiation between RA, OA, and healthy controls.

Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA.

Abstract Background: We examined antibodies against 60-, 65- and 70-kDa heat shock proteins (HSPs) in paediatric healthy individuals, patients with juvenile idiopathic arthritis (JIA) and those undergoing allogeneic stem-cell transplantation for various malignant and non-malignant diseases. Methods: Western blotting and ELISA were used to examine HSP-directed humoral immune responses. Results: Using ELISA we detected anti-Hsp60, -Hsp65 and -Hsp70 IgG antibodies in patient sera before, during and after conditioning and at all post-transplant times, as well as in JIA patients and controls. Western blotting showed positivity for anti-Hsp60 and anti-Hsp65 antibodies in all samples with a HSP concentration of 0.5μg/lane. However, anti-Hsp70 antibodies were not detected at all when both sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were used, except for one JIA patient, for whom a positive signal was only achieved in native PAGE when Hsp70 was increased to 2μg/lane and serum dilution decreased to 1:10. Conclusion: Western blotting is convenient for the detection of anti-Hsp60 and anti-Hsp65 antibodies, but it is not sensitive enough for the detection of anti-Hsp70 antibodies. ELISA, which is more sensitive, might be preferentially used to screen anti-Hsp60, -Hsp65 and -Hsp70 antibodies in sera of children with various disorders.

In vitro Autoreactivity against Skin and Synovial Cells in Patients with Juvenile Idiopathic and Rheumatoid Arthritis

In the present study we compared specific lysis of various autologous target cells in patients with juvenile idiopathic arthritis JIA; n = 8) or rheumatoid arthritis RA; n = 17) with those of healthy controls (n = 15). 51Cr-release cytotoxic assay with autologous peripheral blood mononuclear cells as effector cells was used. When compared with controls, effector cells of patients with JIA or RA were found to lyse significantly autologous synovial cells (p < 0.0005) and epidermal keratinocytes (p < 0.0005), however, no difference was found for autologous dermal fibroblasts.

Humoral Response Against Mycobacterium bovis Hsp65 Derived Fragments in Children and Young People with Various Disorders

Abstract Using Western blotting, we investigated IgG antibodies against Mycobacterium bovis heat shock protein 65 (MB-Hsp65) fragments produced by cleavage with cyanogen bromide (CNBr) in 10 healthy controls, 11 patients with juvenile idiopathic arthritis (JIA), and 10 children with various diseases before haematopoietic stem cell transplantation (HSCT). CNBr cleaved MB-Hsp65 to three larger fragments: P1-163, P191-285, and P290-534. Sera of JIA patients and those before HSCT reacted with individual MB-Hsp65 fragments P1-163 and P290-534 significantly more frequently when compared with healthy controls. These results suggested that the key B-cell epitopes of MB-Hsp65 might be located on the aforementioned sequences.

In vitro autoreactivity against skin in rheumatoid arthritis: are peripheral blood mononuclear cells of patients with rheumatoid arthritis able to lyze autologous keratinocytes?

Abstract An in vitro explant model was originally developed to predict the occurrence and severity of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplants. In previous studies we reported that peripheral blood mononuclear cells of patients with rheumatoid arthritis were able to induce graft-versus-host-like histopathological changes when co-cultured in vitro with autologous skin explants. The aim of the present study was to verify if observed skin damage was really of autoimmune origin. Using a 51chromium release cytotoxic assay we found that peripheral blood mononuclear cells of patients lyzed autologous keratinocytes (n=5 patients with rheumatoid arthritis) but not autologous lymphoblasts (n=4 with rheumatoid arthritis, n=8 patients with juvenile idiopathic arthritis). No specific lysis of keratinocytes or lymphoblasts was observed in healthy controls (n=15). We hypothesize that autologous peripheral blood mononuclear cells might recognize similar autoantigen(s) expressed on epidermal cells, which gives rise to an autoimmune response in the synovium.