Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-γ or TNF-α. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IκB kinase/NF-κB pathway, evidenced by no degradation of IκBα and no translocation of NF-κB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.

Aims/hypothesis Expression of T helper (Th)1 cytokine mRNA in pregnant women is known to be inversely correlated with serum human chorionic gonadotropin (hCG). Type 1 diabetes is a Th1-mediated autoimmune disease, in which intervention at an early stage of the autoimmune process can prevent disease progression. We hypothesised that immune modulation by treating young NOD mice with hCG may prevent diabetes. Methods Female NOD mice were treated with hCG or recombinant hCG from 3 to 15 weeks of age and the incidence of diabetes and development of insulitis was determined. CD4 + and CD8 + T cell populations, T cell proliferation, cytokine production and CD4 + CD25+ regulatory T cells were examined and adoptive transfer experiments were performed.Results Both purified and recombinant hCG prevented development of diabetes in NOD mice. hCG decreased the proportion and number of CD4 + and CD8 + T cells and inhibited T cell proliferative responses against beta cell antigens. hCG treatment suppressed IFN-γ production, but increased IL-10 and TGF-β production in splenocytes stimulated with anti-CD3 antibody. hCG treatment also suppressed TNF-α production in splenocytes stimulated with lipopolysaccharide. + /CD4 + T cell ratio, thus preventing autoimmune diabetes in NOD mice.

Glutamic acid decarboxylase (GAD) is considered to be one of the strongest candidate autoantigens involved in triggering beta-cell-specific autoimmunity. The majority of recent onset type 1 diabetes patients and pre-diabetic subjects have anti-GAD antibodies in their sera, as do nonobese diabetic (NOD) mice, one of the best animal models for human type I diabetes. Immunization of young NOD mice with GAD results in the prevention or delay of the disease as a result of tolerizing autoreactive T cells. Autoimmune diabetes can also be prevented by the suppression of GAD expression in antisense GAD transgenic mice backcrossed with NOD mice for seven generations. These results support the hypothesis that GAD plays an important role in the development of T-cell-mediated autoimmune diabetes. However, there is some controversy regarding the role of GAD in the pathogenesis of diabetes. Whether GAD truly plays a key role in the initiation of this disease remains to be determined. The examination of the development of insulitis and diabetes in beta-cell-specific GAD knockout NOD mice will answer this remaining question.

Aims/hypothesis Type 2 diabetes mellitus is a common agedependent disease. We discovered that male offspring of non-diabetic C57BL/6 and DBA/2 mice, called JYD mice, develop type 2 diabetes when they grow old. JYD mice show characteristics of insulin resistance, hyperglycaemia and hyperinsulinaemia in old age without obesity. We postulated that the mechanism of age-dependent type 2 diabetes in this model relates to caveolin-1 status in skeletal muscle, which appears to regulate insulin sensitivity in the mice. Methods We compared insulin sensitivity in aged C57BL/6 and JYD mice using glucose and insulin tolerance tests and 18 Ffluorodeoxyglucose positron emission tomography. We also determined insulin signalling molecules and caveolin proteins using western blotting, and altered caveolin-1 levels in skeletal muscle of C57BL/6 and JYD mice using viral vector systems, to examine the effect of this on insulin sensitivity. Results In 30-week-old C57BL/6 and JYD mice, the basal levels of IRS-1, Akt and peroxisome proliferator-activated receptor-γ decreased, as did insulin-stimulated phosphorylation of Akt and insulin receptor β. However, caveolin-1 was only increased about twofold in 30-week-old JYD mice as compared with 3-week-old mice, whereas an eightfold increase was seen in C57BL/6 mice. Downregulation of caveolin-1 production in C57BL/6 mice caused severe impairment of glucose and insulin tolerance. Upregulation of caveolin-1 in aged diabetic JYD mice significantly improved insulin sensitivity with a concomitant increase of glucose uptake in the skeletal muscle. Conclusions/interpretation The level of skeletal muscle caveolin-1 is correlated with the progression of age-dependent type 2 diabetes in JYD mice.

AIM:To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO). METHODS:Cytotoxic effects of VCA on COLO cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro and tumor-killing effects in vivo. To study the mechanisms involved, the expression of various pro-caspases, antiapoptotic proteins, and death receptors was determined by western blot. To determine which death receptor is involved in VCA-induced apoptosis of COLO cells, cytotoxicity was examined by MTT assay after treatment with agonists or antagonists of death receptors. RESULTS:VCA killed COLO cells in a time-and dosedependent manner and induced complete regression of tumors in nude mice transplanted with COLO cells. Treatment of COLO cells with VCA activated caspase-2, -3, -8, and -9 and decreased expression of anti-apoptotic molecules including receptor interacting protein, nuclear factor-κB, X-linked inhibitor of apoptosis protein, and Akt/protein kinase B. We then examined the involvement of death receptors in VCA-induced apoptosis. Only tumor necrosis factor receptor 1, among the death receptors examined, was involved in apoptosis of COLO cells, evidenced by inhibition of VCA-induced apoptosis and decreased activation of caspases, particularly caspase-8, by tumor necrosis factor receptor 1 antagonizing antibody. CONCLUSION:VCA-induced apoptotic COLO cell death is due to the activation of caspases and inhibition of antiapoptotic proteins, in part through the tumor necrosis factor receptor 1 signaling pathway.