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.

Peripheral blood mononuclear cell responses to heat shock proteins and their derived synthetic peptides in juvenile idiopathic arthritis patients.

BACKGROUND: Sequence homology and cross reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsps might be involved in the etiopathogenesis of autoimmune diseases. OBJECTIVE: In our study we stimulated peripheral blood mononuclear cells (PBMC) of patients with juvenile idiopathic arthritis (JIA) and healthy controls with various hsp-derived peptides together with the whole molecules of corresponding hsp. METHODS: PBMC were cultured with recombinant human hsp60 (rh-hsp60), rh-hsp70, Mycobacterium bovis hsp65 (M.bovis hsp65), P562-571 human hsp60, P180-188 M. bovis hsp65, P450-463 human hsp70 and P545-554 cytokeratin derived synthetic peptides. Cell responses were measured after incorporation of (3)H-thymidine and expressed as stimulation indices. RESULTS AND CONCLUSION: We found elevated proliferative response to rh-hsp60, M. bovis hsp65 and P562-571 human hsp60 derived peptide in patients with JIA polyarthritis. Significantly elevated proliferation to P180-188 M. bovis hsp65 was found in JIA lasting more than 2 years. None of the particular clinical characteristics (RF, ANA, HLA B27 and disease activity) seemed to be associated with hsp or hsp-derived synthetic peptide proliferative response in the JIA cohort.

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.

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). (51)Cr-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.

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

An in vitro skin 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.

Anti-keratin antibodies in patients with juvenile idiopathic arthritis.

OBJECTIVE: We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). METHODS: An indirect immunofluorescence test with rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients'sera. RESULTS: Overall 30/60 patients with JIA had sera positiveforAKA (50%, p=0.0005) ranging from 1:20 to 1:160 dilutions. Using the classification criteria for childhood idiopathic arthritis, AKA occurred in 2/7 patients with systemic disease (28.6%), in 13/30 patients with RF negative polyarthritis (43.3%, p=0.008) and in 12/18 RF positive polyarthritis (66.7%, p=0.002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence ofAKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 16/29 patients (55.2%) with severe JIA and in 11/26 patients (42.3%) with non-severe disease. We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 44.4% patients with active JIA and in 45.9% patients in the complete or near remission. CONCLUSION: Our data suggest that AKA are present in patients with JIA. However no correlation with severity or disease activity was observed.

Class II alleles in juvenile arthritis in Czech children.

OBJECTIVE: Patients with pauciarticular and polyarticular onset rheumatoid factor (RF) negative juvenile arthritis (JA) have been reported to have a variety of HLA associations. The reason for the differences found in several recent studies is not known. We compare a new series of patients investigated in Prague, Czechoslovakia with those we reported from Dallas. METHODS: Czech patients with JA (N = 153) were classified clinically using the same criteria as in our studies in Dallas. The RF negative group included 56 patients that had persistent pauciarticular disease, 42 pauciarticular with polyarticular course and 39 with polyarticular onset. RF was present in 13 additional patients. HLA class II alleles were determined by oligotyping as previously described from our laboratory. RESULTS: DRB1*0801 was increased and DRB1*0701 was decreased in all the RF negative groups. The persistent pauciarticular group was associated with DRB1*11 and DPB1*0201 and lacked the association with DRB1*1301 seen in Dallas. Also found in Prague and not in Dallas were an increase in the frequency of DR2 in pauciarticular patients with early conversion and of DRB1*1201 in patients with iritis. Certain HLA associations (DRB1*0801, DPB1*0201) appear to be present in patients with JA in most studies; others (DRB1*1301, DPB1*0301) are more variable. CONCLUSION: The reason for differences in the HLA risk factors observed in our 2 populations is not known. Clinical heterogeneity not detected by our method seems the most likely explanation. Genetic and environmental factors may also play a role.

Metabolic bone status in young women with JCA.

The study was based on clinical, densitometric and biochemical evaluations and on a life-style questionnaire, applied in a cohort of 41 individuals. The mean age of the young women was 24.2 (18-36) years. The main point was to compare subgroups with (CS) an without (NCS) corticosteroid treatment (19 and 22 patients, respectively). There was no significant difference in age, weight and duration of JCA. Of the densitometric examinations, spine DXA (DPX-L LUNAR apparatus) yielded values significantly lower in CS than in NCS individuals (p = 0.05). Much more apparent was the difference in stiffness measurements in the calcanei performed by Achilles-LUNAR ultrasound instrument, again with lower values in CS women (p = 0.001) (Fig. 1, 2, 3). No significant differences were found between the two subgroups as regards blood levels of bone alkaline phosphatase, osteocalcin and tartrate-resistant acidic phosphatase, and urine hydroxyproline output. Menarche occurred at a mean age of 14.37 years in the CS subgroup (p = 0.01, against healthy population) and of 13.32 years in the NCS subgroup (not significant). The prevalence of fractures was enconsiderable in both subgroups. These findings are to be understood from the viewpoint of combined influences of both disease activity and corticotherapy in the CS patients with JCA.

HLA and juvenile rheumatoid arthritis.

HLA class II analysis in a group of 153 Czech children with juvenile rheumatoid arthritis by PCR and oligonucleotide hybridization demonstrated associations with several alleles. DRB1*0801 (RR = 5.3, p < 0.005) and DRB1 * 11 (RR = 2.2, p < 0.01) including all subtypes were shown to be increased in the rheumatoid factor-negative group (N = 137). The same results were observed in Italy, England and Norway. In patients with the pauciarticular onset with conversion to polyarticular within 3 years, a statistically significant increase in DR2 (RR = 10.1, p < 0.00005), mostly due to DRB1*1501, was found. In the iridocyclitis and antinuclear factor groups, susceptibility to DRB1*1201 was observed. There was a striking decrease in DRB1*0701 (RR = 0.3, p < 0.00005) in all groups. There was neither an increase in DRB1*1301 or DPB1*0301 nor a decrease in DRB1*04, as reported from other studies in Texas and Norway. The rheumatoid factor-positive group with polyarticular onset (N = 13) was associated with DRB1*04 (RR = 7.1, p < 0.005), as observed in adults. DPB1*0201 was increased in the persistent pauciarticular group (RR = 3.7, p < 0.0005). DPB1*0402 was decreased in all pauciarticular groups with or without conversion (RR = 0.3, p < 0.005). Taken together, there are not only genetic differences and clinical heterogeneity in juvenile rheumatoid arthritis patients but, also, common predisposing factors.

Metabolic bone status in young women with juvenile chronic arthritis.

A group of 26 young women (18-36 years of age) with juvenile chronic arthritis (JCA, duration 8-33 years) was investigated for bone metabolism and mineral status. Six of the patients were receiving longterm corticosteroid therapy, and 5 had received corticosteroid treatment in the past. Serum osteocalcin and urinary hydroxyproline were significantly elevated in 17 and 14 of the 26 patients, respectively, compared with healthy controls. Compared to controls, bone mineral density (BMD) in the lumbar spine was significantly lowered in 6 of 26 patients, all of whom were in the corticosteroid treated subgroup. No correlation was evident between any of the variables measured, except for the association of corticosteroid therapy with low BMD.

HLA class II alleles in juvenile dermatomyositis.

HLA class II alleles were investigated in 27 Czech patients (11 females and 16 males) with juvenile dermatomyositis. The immunogenetic investigation comprised determination of DRB1, DRB3, DRB5, DQA1, DQB1, and DPB1 alleles. Their prevalence was compared with that found in healthy Czech controls. No allele was found significantly more frequently in patients than in controls.

Clinical practice in pediatric rheumatology: a review of the Czechoslovak experience.

The use of corticosteroid therapy in the treatment of juvenile chronic arthritis in Czechoslovakia is summarized. The indispensability of corticosteroids in pediatric rheumatology is confirmed and some practical aspects of their application are discussed.

Sequences of HLA alleles associated with arthritis in adults and children.

Adult rheumatoid factor (RF) positive rheumatoid arthritis (RA) and RF positive arthritis of childhood are associated with DRB1*0401 in Caucasians, and DRB1*0405 in Orientals, and in Ashkenazi and nonAshkenazi Jews. Certain other DRB1 alleles (DRB1*0101,1001) which have a similar sequence in the 3rd hypervariable region of the 1st domain are also associated with RA, but they appear to function as weaker risk factors. The difference in the relative strength of the associations is likely to be due to structural differences in the 1st and 2nd variable regions of the first domain of these alleles. Similarities and differences in the HLA associations between North American Caucasoid patients with juvenile arthritis in Dallas, TX, USA, and in Prague, Czechoslovakia, are discussed.

Syndrome of secondary polymyositis with leukemia.

A boy with neurofibromatosis suffered mumps at the age of 5. A full-blown juvenile polymyositis developed shortly afterwards. First hematological consultation was done for monocytosis in peripheral blood at the age of 7. He suffered varicella at the age of 8. Diagnosis of acute nonlymphocytic leukemia with monosomy 7 was done before the age of 9. The boy died at the age of 10.