Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages by Jeju plant extracts: Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be responsible for the vasodilation and hypotension observed during septic shock and inflammation. Thus, inhibitors of iNOS may be useful candidates for the treatment of inflammatory diseases accompanied by the overproduction of NO. In this study, we prepared alcoholic extracts of Jeju plants and screened them for their inhibitory activity against NO production in lipopolysaccharide (LPS)-activated macrophages. Among the 260 kinds of plant extract tested, 122 extracts showed potent inhibitory activity towards NO production by more than 25% at a concentration of 100 μg/mL. Plants such asMalus sieboldii, Vaccinium oldhamii, Corylus hallaisanensis, Carpinus laxiflora, Styrax obassia, andSecurinega suffruticosashowed the most potent inhibition (above 70%) at a concentration of 100 μg/mL. The cytotoxic effects of the plant extracts were determined by colorimetric MTT assays and most plant extracts exhibited only moderate cytotoxicity at 100 μg/mL. Therefore, these plants should be considered promising candidates for the further purification of bioactive compounds and would be useful for the treatment of inflammatory diseases accompanying overproduction of NO.

Objective: Rheumatoid arthritis is a disease that, pathologically, is characterized by the progressive growth and invasion of the synovial pannus into the surrounding cartilage and bone. Many cytokines, including transforming growth factor β1 (TGFβ1), have been implicated in this process, but their mode of action is incompletely understood. The goal of the present study was to better understand the downstream signaling pathways of TGFβ in fibroblasts.Methods: The role of phosphatidylinositol 3‐kinase (PI 3‐kinase) was determined by chemical inhibition with LY294002 or wortmannin. Activation of protein kinase B (Akt), c‐Jun N‐terminal kinases (JNKs), and extracellular signal–regulated kinases (ERKs) was evaluated by Western blot analysis using phospho‐specific antibodies.Results: Exposure of fibroblasts to TGFβ rapidly induced activation of a kinase, Akt, that is known to inhibit apoptosis by a variety of pathways. Activation of Akt was blocked by the specific PI 3‐kinase inhibitor, LY294002, indicating that TGFβ‐mediated phosphorylation of Akt was dependent on PI 3‐kinase activation. This activation pathway was relatively selective for Akt, since inhibition of PI 3‐kinase failed to substantially modify activation of ERKs or JNKs in synovial fibroblasts. Inhibition of the PI 3‐kinase/Akt pathway resulted in impaired proliferation of synovial fibroblasts and partial attenuation of the protective effect of TGFβ on Fas‐mediated apoptosis.Conclusion: TGFβ exerts its growth and antiapoptotic effects on fibroblasts, at least in part, by activation of the PI 3‐kinase/Akt pathway.

To characterize the nature of autoimmune disease-inducing T cells in the target organ, oligoclonal expansion of spinal cord T cells of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined by complementarity-determining region 3 (CDR3) size spectratyping. It is known that TCR of in vitro-established myelin basic protein-specific T cell clones and lines have a short CDR3 and that the amino acid sequence in this region is highly preserved. On the basis of these findings, we analyzed 22 spectratypes of the TCR β-chain (Vβ1–20). Among them, only Vβ8.2 and Vβ17 showed oligoclonal expansion of TCR with a short CDR3 at the early stage of EAE. More interestingly, the spectratype profile of Vβ8.2 seen at the early stage was preserved throughout the course of EAE, whereas that of Vβ17 became more diverse at the peak stage of the disease. Analysis of nucleotide and predicted amino acid sequences of Vβ8.2 CDR3 derived from the spectratypes revealed that the clones with CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population not only at the early stage but also at the peak and recovery stages (71, 71, and 60%, respectively). These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus, CDR3 size spectratyping is a powerful tool for the screening of disease-inducing T cells in an autoimmune disease of unknown pathomechanism.