Humoral response against Mycobacterium bovis Hsp65 derived fragments in children and young people with various disorders.

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.

Expression of heat shock protein receptors on fibroblast-like synovial cells derived from rheumatoid arthritis-affected joints.

We examined the membrane expression of inducible Hsp70 and HSP receptors like TLR2, TLR4, CD14, CD36, CD40 and CD91 on fibroblast-like synovial cells (SC) derived from synovial tissue in 23 patients with rheumatoid arthritis (RA), who underwent synovectomy by using flow cytometric analysis. For comparison, autologous skin fibroblasts (SF) derived from the operation wound were tested. Significantly higher Hsp70 expression was found on synovial cells than on skin fibroblasts (median SC 21.4% x SF 5.0%, P < 0.001). Both synovial cells and skin fibroblasts expressed high levels of cell surface CD91 (median SC 80.2% x SF 79.2%), however, no or low levels of CD14, CD40, TLR2, TLR4 and CD36. Further, we observed high co-expression of CD91 and Hsp70 on RA synovial cells (median 18.6%), while skin fibroblasts showed only background Hsp70 expression (median 3.9%, P < 0.001). Since we demonstrated the high prevalence of inducible Hsp70 in RA synovial fluids, we speculate that Hsp70 might be captured onto the membrane of synovial cells from the extracellular space via the CD91 receptor. The significance of the Hsp70 interaction with synovial cells via CD91 remains undefined, but may mediate other non-immune purposes.

Antibodies against M. Bovis 65 KDa Heat Shock Protein and Its P180-188 Epitope in Sera of Patients with Juvenile Idiopathic Arthritis.

We screened the levels of antibodies to M. bovis hsp65 and the 180-188 epitope by using ELISA in a cohort of 72 juvenile idiopathic arthritis (JIA) patients and 38 healthy controls. We analysed an association between antibody levels and rheumatoid factor, antinuclear antibodies, human leukocyte antigen B27 and the severity and the duration of the disease. The majority of anti-hsp65 antibodies in a cohort of JIA patients were of the IgG isotype (54.2%) with IgM (13.9%) antibodies increased to a lesser degree. IgG antibodies to M. bovis hsp65 (P<0.001) and the 180-188 epitope (P<0.001) were significantly increased in all of three disease onset types when compared with healthy controls. The highest levels of IgG antibodies to M. bovis hsp65 and its P180-188 epitope were observed in oligoarthritis and in patients with no X-ray changes and functional limitation, while the lowest antibody levels were detected in patients with the most severe stage of articular damage. When antibody levels to M. bovis hsp65 and the 180-188 epitope were examined within patient and control populations, significantly higher levels of IgG and IgM antibodies to M. bovis hsp65 were observed in both JIA (P<0.001) and healthy control (P<0.001) cohorts. These findings may suggest that the high levels of IgG antibodies to M. bovis hsp65 and its P180-188 epitope would reflect the least serious cases of JIA. Since IgM antibodies to M. bovis hsp65 and P180-188 M. bovis hsp65 epitope exceeded the control level in a few patients with JIA we believe they are not of concern.

Frequency of anti-hsp60, -65 and -70 antibodies in sera of patients with juvenile idiopathic arthritis.

Cross-reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsp might be involved in the etiopathogenesis of autoimmune diseases. We investigated antibodies to recombinant human hsp60, recombinant Mycobacterium bovis hsp65 and to stress-inducible recombinant human hsp70 using enzyme-linked immunosorbent assay (ELISA) in sera of 209 juvenile idiopathic arthritis (JIA) patients and 50 healthy controls. Anti-hsp60 antibodies did not exceed the control level in any JIA patient. The numbers of JIA patients (16/209, 7.6%) who raised anti-hsp65 antibodies was equal to healthy controls (4/50, 8%). Elevated levels of antibodies against hsp70 were found in a cohort of patients with JIA (36.8%) when compared with age-matched healthy individuals (2%). These antibodies were predominantly of IgG isotype in systemic disease and IgM isotype in oligoarthritis. In polyarthritis both IgG and IgM antibodies frequently occurred. Significantly higher anti-hsp70 antibody levels were found in RF-positive JIA patients. The levels of anti-hsp70 antibodies correlated with the severity of disease evaluated on the basis of Steinbrocker's functional classification and rtg staging system. No association between anti-hsp70 antibody levels and ANA, HLA B27 and disease duration (less than 2 years x more than 2 years) was observed except IgM anti-hsp70 antibody where significantly higher levels were also detected in HLA B27-positive patients. The prevalence of anti-hsp70 antibodies is much higher in JIA patients when compared with healthy controls, suggesting their possible role in pathological mechanism of the disease.

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

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 0.5 microg/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 2 microg/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.

Association of single nucleotide polymorphisms within cytokine genes with juvenile idiopathic arthritis in the Czech population.

OBJECTIVE: To examine the possible association of juvenile idiopathic arthritis (JIA) with polymorphisms within cytokine genes in the Czech population. METHODS: In a case-control study, genotypes of 130 patients with JIA (63 male, 67 female; age at onset 7.6 +/- 4.4 yrs; 43 oligoarticular, 72 polyarticular, 15 systemic form) were compared to 102 healthy unrelated blood donors. Using the polymerase chain reaction technique with sequence-specific primers from the 13th IHWG workshop, we analyzed 19 single nucleotide polymorphisms within 12 different cytokine genes [interleukin (IL)-1a, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, transforming growth factor (TGF)-beta, interferon (IFN)-g], and related molecules (IL-1R, IL-1RA, IL-4Ra). Genotype frequencies were compared using chi-square analysis, and the significance level was corrected for the number of independent tests. RESULTS: Significant positive association was found for the G allele of the IL-4 -1098 T/G polymorphism, which was carried by 10% of cases and 25% of controls [odds ratio (OR) 0.32, 95% confidence interval (CI) 0.16-0.67, corrected p = 0.038). Also, a nonsignificant increase in the frequency of the IL-1beta +3962 C allele was detected in cases (96%) versus controls (84%) (OR 4.65, 95% CI 1.64-13.2, corrected p = 0.091). We did not replicate previously found associations with the IL-1a, IL-6, IL-10, and IL-1RA polymorphisms. CONCLUSION: Our study showed association with JIA for the IL-4 -1098 T/G polymorphism. It also underlines the genetic contribution of IL-1 polymorphisms to the pathogenesis of JIA, as another polymorphism within the IL-1beta may influence the risk of the disease.