A Salmonella Typhi ghost induced by the E gene of phage φX174 stimulates dendritic cells and efficiently activates the adaptive immune response

Previously, we genetically engineered a Salmonella Typhi bacterial ghost (STG) as a novel inactivated vaccine candidate against typhoid fever. The underlying mechanism employed by the ghost in stimulating the adaptive immune response remains to be investigated. In this study, we aimed to evaluate the immunostimulatory effect of STG on mouse bone marrow-derived dendritic cells (BMDCs) and its activation of the adaptive immune response in vitro. Immature BMDCs were stimulated with STG, which efficiently stimulated maturation events in BMDCs, as indicated by upregulated expressions of CD40, CD80, and major histocompatibility complex class II molecules on CD11⁺ BMDCs. Immature BMDCs responded to STG stimulation by significantly increasing the expression of interleukin (IL)-6, which might indicate the induction of dendritic cell maturation in vivo (p < 0.05). In addition, ghost-stimulated murine BMDCs showed significant expressions of interferon gamma and IL-4, which can drive the development of Th1 and Th2 cells, respectively, in co-cultured CD4⁺ T cells in vitro. These results suggest that STG can effectively stimulate maturation of BMDCs and facilitate subsequent immune responses via potent immunomodulatory cytokine responses.

Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates

Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10-4 to 10-6 M. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.

Antiobesity activity of a sphingosine 1-phosphate analogue FTY720 observed in adipocytes and obese mouse model

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.

Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cells via death receptor 5 upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, is one of the most promising candidates for cancer therapeutics. However, many osteosarcomas are resistant to TRAIL. Bisphosphonates are very effective in the treatment of bone problems associated with malignancies; the antitumor effects are due to the inhibition of protein prenylation that is essential for cell function and survival. The purpose of this study was to determine the effects of bisphosphonates on TRAIL-resistant MG 63 human osteosarcoma cells. The cells showed no response to TRAIL alone; however, pre-treatment with bisphosphonates significantly increased TRAIL-mediated apoptosis and cellular activation of caspase-3. Bisphosphonates significantly induced mRNA and protein expression of the TRAIL receptor, DR5. Bisphosphonates induced protein unprenylation in MG 63 cells; in addition, co-treatment with TRAIL also significantly increased protein unprenylation. Blocking of protein unprenylation using geranylgeraniol attenuated the cellular responses, including cell apoptosis and protein unprenylation induced by bisphosphonates and TRAIL. This is the first study to demonstrate that bisphosphonates markedly enhanced TRAIL-induced apoptosis in human osteosarcoma cells. These findings suggest that bisphosphonates may be a new and effective anticancer treatment with TRAIL proteins for TRAIL-resistant cancer cells.

Morphometric Studies in the Leydig Cell of Hemicastrated Rats / 체질인류학회지

The present study was conducted to investigate the influence of hemicastration and age at hemicastraion on the subsequent Leydig cell morphology and function of male rats. Sprague Dawley rats were left intact or hemicastrated at 20, 30, 40, 50, or 60 days of age (n=18 rats per group). At 100 days of age, all rats were sacrificed. Testes were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in Epon-araldite. Using 1 micrometer sections stained with methylene blue, qualitative and quantitative morphological studies were performed. Testis incubations were used to determine lutenizing hormone (LH; 100 ng/mL) stimulated testosterone secretory capacity per testis in vitro. Testosterone levels in the incubation medium, and testosterone and LH levels in serum of these six groups of rats were determined by radioimmunoassay. Body and testis weights were not changed by hemicastration between experimental and control groups. Volume density of seminiferous tubules, interstitium, and Leydig cells was not significantly affected by hemicastration. Absolute volume of seminiferous and interstitium was significantly increased in unilaterally castrated rats at 20, 30 and 40 days of age compared to control. Significant increases in the total number of Leydig cells per testis occurred in rats hemicastrated at 20, 30, 40 and 50 days of age compared to control. A significant increase in average volume of a Leydig cell was noted in the hemicastrated rats at 30 and 40 days compared to intact rats of the same age but was significantly decreased at 60 days of age. Serum testosterone levels and LH-stimulated testosterone production per testis were significantly (P<0.05) increased in the hemicastrated rats at 30 and 40 days. In summary, when rats were unilaterally castrated at 20, 30, 40, 50, and 60 days of age, those rats hemicastrated at 30 and 40 days showed compensatory hypertrophy/hypersecretion of Leydig cells when killed at 100 days of age. Especially, these data suggested that compensatory hypertrophy/hypersecretion of Leydig cells in rats hemicastrated around the time of puberty occurs in the remaining testis.

IL-2 Induced Nitric Oxide Synthesis by Tumor Cells in Corultures of Lymphocytes and Tumor Cells / 대한암학회지

PURPOSE: Nitric oxide (NO) synthesis has been known to be induced during interleukin-2 (IL-2) therapy. The present study was designed to elucidate mechanisms and roles of IL-2-induced NO synthesis in tumor cells. MATERIALS AND METHODS: Mechanisms of IL-2-induced NO synthesis were evaluated using in vitro culture systems of BALB/c mouse splenic lymphocytes and Meth-A tumor cells. Effects of IL-2-induced NO synthesis by Meth-A tumor cells on the tumor cell proliferation were also evaluated using an NO synthase inhibitor, N -monomethyl- L-arginine (MLA). RESULTS: Cultures of both lymphocytes alone and Meth-A tumor cells alone did not produce any significant amounts of nitrite, a stable metabolite of NO during IL-2 stimulation. In contrast, cocultures of lymphocytes and Meth-A tumor cells produced a large amount of nitrite during IL-2 stimulation. Addition of culture supernatants of lymphocytes incubated with IL-2 induced nitrite production in Meth-A tumor cell cultures. However, addition of culture supernatants of Meth-A tumor cells incubated with IL-2 did not induce nitrite production in lymphocyte cultures. Nitrite accumulation was markedly inhibited by addition of anti-interferon-y antibody, confirming the role of the cytokine in mediating tumor cell NO synthesis. MLA inhibited nitrite production by Meth-A tumor cells in a dose-dependent manner in the presence of culture supernatants of lymphocytes incubated with IL-2. Meth-A tumor cell nitrite production in the presence of increasing concentrations of MLA correlated inversely with tumor cell proliferation. CONCLUSION: NO synthesis can be induced by tumor cells by the secondarily released cytokines from lymphocytes during IL-2 stimulation. Autologous NO synthesized by tumor cells during IL-2 stimulation inhibits proliferation of tumor cells themselves.

Nitric Oxide Synthesis in Murine Peritoneal Macrophages by Antithrombin III / 대한혈액학회지

BACKGROUND: During the development of inflammatory responses, various cytokines including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) which activate macrophages to synthesize nitric oxide (NO) and hemostatic cascades are secreted. However, effects of various factors involved in hemostasis on NO synthesis have been little known. In this report, we studied the effects of antithrombin III (AT-III) on NO synthesis by thioglycollate-elicited peritoneal macrophages of BALB/c mouse. METHODS: Macrophages were obtained from BALB/c mice injected with 3% thioglycollate and cultured with or without various reagents. Nitrite concentration was measured using a modification of Griess reaction. Immunoblot analysis for inducible nitric oxide synthase (iNOS), AT-III receptor binding study and assay of TNF secretion were performed. RESULTS: AT-III alone did not induce nitrite synthesis by macrophages. However, AT-III induced nitrite synthesis in the presence of IFN-gamma in a dose dependant manner. iNOS was also expressed in macrophages by the treatment with IFN-gamma/AT-III. Addition of 0.5mM NG-monomethyl-L-arginine markedly inhibited IFN-gamma/AT-III-induced nirtite synthesis. Treatment with 5microgram/mL polymyxin B, which inactivates LPS, did not inhibit IFN-gamma/AT- III-induced nitrite synthesis, excluding inadvertent endotoxin contamination. Addition of rabbit anti-human AT-III polyclonal IgG antibodies, but not control rabbit IgG, inhibited IFN-gamma/ AT-III-induced nitrite production. Treatment of macrophages with heparin did not augment, but inhibited both IFN-gamma/AT-III- and IFN-gamma/LPS-induced nitrite synthesis. CONCLUSION: Our results indicated that AT-III acted as a modulator of macrophage activation for L-arginine dependent NO synthesis and that AT-III might be an important molecule in the regulation of immune responses by macrophage activation to induce NO synthesis as well as in the regulation of hemostasis. Heparin was not a cofactor in the AT-III-induced NO synthesis.