Denisa Zlacka

Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells

Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Methods: Herein, Hsp70 cell surface and mRNA expression were studied in human fibroblast-like synovial cells, dermal fibroblasts and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow-cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of 17 healthy controls were tested. Results: Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p = 0.002; healthy controls: average 6.0%, median 4.5%, p = 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p < 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p = 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p = 0.005). Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p < 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p = 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p < 0.001). Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p < 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p < 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p < 0.001). Conclusion: Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors. As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.

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.

Fetal cells of mesenchymal origin in cultures derived from synovial tissue and skin of patients with rheumatoid arthritis

The transplacental cell transfer naturally takes place during pregnancy and occurs bi-directionally between the mother and fetus. Using real-time polymerase chain reaction (PCR) assay and sex determining region Y (SRY) gene as a marker, we examined the presence of male fetal cells in cell cultures derived from synovial tissues and skin dermis in women with prior pregnancy history suffering from rheumatoid arthritis (RA) who underwent synovectomy. Male DNA was detected in synovial cell samples derived from carpal, hip, metacarpophalangeal and metatarsophalangeal joints in five out of 13 (38.5%) patients with RA in a frequency range of 0.02-62.55 (mean 12.17) male cells per 10,000,000 total cells. SRY gene positivity was found as well in skin fibroblast cultures in four out of 10 (40.0%) RA patients in a frequency range of 3.26-43.47 (mean 15.42) male cells per 10,000,000 total cells, respectively. The difference in a frequency of fetal-derived male cells between both the cohorts did not achieve the statistical difference (p=0.77). We conclude that persisting male fetal cells are able to grow from non-inflamed tissues as well as from those which have many features characteristic of a stressed tissue. We conclude that persisting male fetal cells are also able to proliferate in cell culture since their presence was detected even in consecutive passages.

Antibodies to 60, 65 and 70 kDa heat shock proteins in pediatric allogeneic stem cell transplant recipients.

Abstract:  Allogeneic SCT remains the only means of cure for many patients with various malignant disorders as well as non‐malignant diseases. Infection together with severe aGvHD may result in a significant incidence of transplant‐related morbidity and mortality. Current evidence suggests that hSPS represent major immunodominant antigens in many pathogens and therefore might play an important role in the pathogenesis of GvHD. We investigated the levels of total Ig, IgG and IgM isotype antibodies to rh‐hsp60, recombinant Mycobacterium bovis hsp65 and stress‐inducible rh‐hsp70 in sera of pediatric patients undergoing SCT by using ELISA. We studied whether humoral immune responses to hSPS follow transplant‐related complications, bacterial and fungal infection. Anti‐hsp antibodies were detected in patients’ sera before conditioning, over the course of conditioning and all the time post‐transplant. We found no correlation between anti‐hsp antibodies and the occurrence and severity of GvHD and/or other transplant‐related complications like graft failure, hemorrhagic cystitis and capillary leakage syndrome. However, elevated anti‐hsp antibodies involving IgM and IgG isotypes were found to be associated with bacterial and fungal infection depending on etiological agents. We demonstrated de novo humoral response to hSPS in a cohort of patients with actual infection caused by Klebsiella pneumoniae (anti‐hsp60, anti‐hsp65 and anti‐hsp70), Pseudomonas aeruginosa (anti‐hsp60, anti‐hsp70) and Aspergillus fumigatus (anti‐hsp65). We conclude that anti‐hsp antibodies might be produced after SCT in relation to infection depending on etiological agents; however, transplant‐related complications by themselves had a little impact.

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.