Evaluation of SV40-transformed synovial fibroblasts in the study of rheumatoid arthritis pathogenesis.

The SV40 T antigen has been used to generate immortalized cells from rheumatoid arthritis (RA) synovial fibroblasts (RASFs) that are commonly used in lieu of primary RASFs. In the current study, we investigated the effect of stimulation by tissue necrosis factor alpha (TNF-α) and interleukin 17 (IL-17) on primary and immortalized RASFs in order to gauge the appropriateness of the use of immortalized RASFs, the MH7A cell line, in the study of RA pathogenesis. Changes in the levels of secretion and expression of 8 proteins associated with RA upon stimulation were assessed by multiplex immunoassay. IL-17 stimulation had a minimal impact on protein secretion and expression for primary and immortalized cells. Basic fibroblast growth factor (FGF-2) was not detectable for the primary cells but was detectable for the immortalized cells. In contrast, monocyte chemoattractant protein 1 (MCP-1) was detectable for primary cells but was undetectable for immortalized cells. In general, protein expression and secretion by cells stimulated with TNF-α were significantly increased. For primary cells, several proteins were below the limit of detection for unstimulated cells and cells stimulated with IL-17, while levels for TNF-α-stimulated cells were within the detectable range. For the same proteins, expression was observed for immortalized cells, regardless of stimulation, suggestive of constitutive activation of the NF-κB signaling pathway. The current study therefore provides strong evidence that immortalized and primary RASFs differ in regard to protein expression and secretion and therefore may not be appropriate for use in the study of RA pathogenesis.

Modulation of the response of rheumatoid arthritis synovial fibroblasts to proinflammatory stimulants with cyclic tensile strain.

Although physical therapy has been shown to be an effective method for treatment of rheumatoid arthritis, a thorough investigation on the impact of mechanical signals upon the complex cytokine network associated with pathogenesis has not yet been conducted. In the current study, our research group investigated the effect of mechanical stimulation on primary and immortalized rheumatoid arthritis synovial fibroblasts (RASFs) through analysis of secreted proteins using multiplex immunoassay. Equibiaxial tensile strain was applied to 2D cultures grown on collagen-coated, flexible silicone membranes at a magnitude of 10% and a frequency of 0.5Hz using the Flexcell System. After 24h, supernatant was removed and assayed for the following cytokines: IL-1beta, IL-6, IL-8, VEGF, FGF-2, GM-CSF, MCP-1, RANTES, TNF-alpha. The results were compared to unstimulated control groups. Mechanical stimulation alone only impacted secretion of IL-8 by primary RASFs. However, in the presence of proinflammatory mediators (TNF-alpha or IL-17), application of cyclic tensile strain increased secretion of a number of proteins by both primary and immortalized RASFs, although the responses were not analogous. In contrast, MCP-1 secretion was decreased when mechanical stimulation was applied in combination with IL-17 to primary cultures. In general, the study suggests that cyclic tensile strain can be used to modulate the effects of proinflammatory stimulants on RASFs; however, given the highly variable results, more research will be necessary to identify the pathways that are implicated in mechanotransduction.

Dendritic cell-based immunotherapy of prostate cancer: immune monitoring of a phase II clinical trial.

We assessed both non- and peptide-specific immune responses in prostate cancer patients before and after immunotherapy with dendritic cells exogenously pulsed with the prostate-specific membrane antigen-derived peptides, PSM-P1 and PSM-P2. For all subjects, we observed that clinical responses were strongly associated with two indicators of immunocompetence: skin test responses to recall antigens and cytokine secretion by T cells after nonspecific stimulation. In a subset of responders, we observed cytokine secretion or cytotoxicity against the immunizing peptides or an immunodominant epitope from an influenza recall antigen. The clinical results support the use of monitoring for overall immunocompetence to help determine why a patient has or has not responded to therapy. Moreover, it could be useful as an inclusion criterion to select those more likely to benefit from treatment.