OBJECTIVES: To dissect the role of toll-like receptor (TLR) signalling and B cell survival/proliferating factors in the crosstalk between rheumatoid arthritis synovial fibroblasts (RASF) and B cells. METHODS: RASF, rheumatoid arthritis dermal fibroblasts (RADF) and osteoarthritis synovial fibroblasts (OASF) were analysed for the expression of B cell survival/proliferating factors BAFF and APRIL in resting conditions and upon stimulation with TLR2/TLR3/TLR4 ligands. Unswitched IgD+ B cells were co-cultured with RASF/OASF/RADF in the presence/absence of TLR ligands and with/without BAFF/APRIL blocking antibodies. Activation-induced cytidine deaminase (AID) mRNA expression, I-C and I-C circular transcripts (CTs; markers of ongoing class-switching to IgG and IgA) and IgM/A/G production were measured to assess functional activation of B cells. RESULTS: TLR3 and to a lesser extent TLR4, but not TLR2 stimulation, induced up to 1000-fold BAFF mRNA and increased soluble BAFF release. APRIL was less significantly regulated by TLR3. Resting and TLR3-stimulated RASF released higher levels of BAFF/APRIL compared with RADF. TLR3 stimulation of RASF but not RADF in co-culture with B cells strongly enhanced AID expression, I-C and I-C CTs and class-switching to IgG/IgA. Blockade of BAFF/APRIL signalling completely inhibited TLR3-induced, RASF-dependent expression of AID, CTs and the secretion of IgG/IgA. CONCLUSIONS: RASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, class-switch recombination and IgG/IgA production in IgD+ B cells. Overall, this work highlights a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the rheumatoid arthritis synovium. Conclusions. RASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, CSR and IgG/IgA production in IgD+ B cells. Overall, we highlight a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the RA synovium.
Metaphase comparative genomic hybridisation (CGH) studies indicate that chromosomes 4, 5, 6, 13, 14, 15, and 18 are frequently deleted in primary ovarian cancers (OC). Therefore we used microcell-mediated chromosome transfer (MMCT) to establish the functional effects of transferring normal copies of these chromosomes into two epithelial OC cell lines (TOV112D and TOV21G). The in vitro neoplastic phenotype (measured as anchorage dependent and independent growth and invasion) was compared between recipient OC cell lines and multiple MMCT hybrids. Chromosomes 6 and 18 showed strong evidence of functional, neoplastic suppression for multiple hybrids in both cell lines. We also found evidence in one cancer cell line suggesting that chromosomes 4, 13 and 14 may also cause functional suppression. Array CGH and microsatellite analyses were used to characterise the extent of genomic transfer in chromosome 6 and 18 hybrids. A 36Mb deletion on chromosome 6 in two hybrids from one cell line mapped the candidate region proximal to 6q15 and distal to 6q22.2; and an approximately 10Mb candidate region spanning the centromere on chromosome 18, was identified in two hybrids from the other cell line. These data confirm reported functional effects of chromosome 6 in OC cell lines; but to our knowledge, this is the first time that functional suppression for chromosome 18 has been reported. This suggests that these chromosomes may harbour tumour suppressor like genes. The future identification of these genes may have a significant impact on the understanding and treatment of the disease, the identification of novel therapeutic targets
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