ABSTRACT
Signal transducer and activator of transcription 4 (STAT4), a STAT family member, mediates interleukin 12 (IL12) signal transduction. IL12 is known to be related to calorie-restricted status. In the central nervous system, IL12 also enhances the production of nitric oxide (NO), which regulates food intake. In this study, the expression of neuronal NO synthase (Nos1), which is also related to food intake, was investigated in the hypothalamic areas of Stat4 knockout (KO) mice using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, a marker for neurons expressing Nos1 enzyme. Western blots were also performed to evaluate Nos1 and Fos expression. Wild-type Balb/c (WT group, n=10 male) and Stat4 KO mice (Stat4 KO group, n=8 male) were used. The body weight and daily food intake in the WT group were 22.4+/-0.3 and 4.4 g per day, while those in the Stat4 KO group were 18.7+/-0.4 and 1.8 g per day, respectively. Stat4 mice had lower body weight and food intake than Balb/c mice. Optical intensities of NADPH-d-positive neurons in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of the Stat4 KO group were significantly higher than those of the WT group. Western blotting analysis revealed that the hypothalamic Nos1 and Fos expression of the Stat4 KO group was up-regulated, compared to that in the WT group. These results suggest that Stat4 may be related to the regulation of food intake and expression of Nos1 in the hypothalamus.
Subject(s)
Animals , Humans , Mice , Blotting, Western , Body Weight , Central Nervous System , Eating , Hypothalamic Area, Lateral , Hypothalamus , Interleukin-12 , Mice, Knockout , NAD , Neurons , Nitric Oxide , Nitric Oxide Synthase , Paraventricular Hypothalamic Nucleus , Signal Transduction , STAT4 Transcription FactorABSTRACT
BACKGROUND: IL-18 was originally cloned as a IFN-gamma inducing factor in primed T cells. In synergy with IL-12, IL-18 has been shown to induce strikingly high levels of IFN-gamma production by T cells and to enhance Th1 development. Also this cytokine exerts induction of Th2 development through IL-4 induction. METHODS: Resting CD4+ T cells were sorted by negative selection and activated by anti-CD3 plus anti-CD28 Ab. Expression of IL-12 binding sites, IL-18 binding sites, IL-18Ralpha, and GATA-3 mRNA were analysed by FACS and RT-PCR, respectively. RESULTS: Resting CD4+ T cells expressed IL-18Ralpha chain but not IL-18 binding sites, suggesting a lack of IL-18Rbeta expression. IL-18Ralpha was maintained on the Th1 and Th2 committed cells. IL-18 binding sites were induced on the Th1 but not Th2 cells. Exposure of these cells to IL-18 led to up-regulation of GATA-3 mRNA expression only in Th2 committed cells. To elucidate the relationship between IL-18Ralpha expression and GATA-3 induction by IL-18, Th1 and Th2 committed cells were further cultured in medium with or without IL-12 for 2 days. IL-12 binding sites were maintained on the Th1 and Th2 cells regardless of IL-12 treatment, but IL-18Ralpha expression was rapidly down-regulated on the IL- 12-untreated Th2 cells which did not induce GATA-3 mRNA expression followed by IL-18 stimulation. CONCLUSION: IL-12 supports expression of IL-18Ralpha and GATA-3 mRNA expression was induced by IL-18 through IL-18Ralpha without expression of IL-18 binding site in Th2 cells.
Subject(s)
Binding Sites , Clone Cells , Interleukin-12 , Interleukin-18 , Interleukin-4 , RNA, Messenger , T-Lymphocytes , Th2 Cells , Up-RegulationABSTRACT
BACKGROUND: Fibroblast functions both as a structural element and as a vital immunoregulatory cell. Fibroblasts regulate inflammation through governing of chemokine expression. In order to elucidate the mechanisms by which the expressions of chemokines were regulated, the co-stimulatory effects of Th1 and proinflammatory cytokines were compared using nasal mucosal fibroblasts. METHODS: Human nasal mucosa was obtained from surgery for septal deviation and the growth of fibroblasts was established. Fibroblasts from 4th to 6th passage were stimulated with various combinations of cytokines. To inhibit selected signaling pathways, fibroblasts were pretreated with cyclosporin A, wortmannin, staurosporine, and dexamethasone prior to the stimulation with cytokines. The supernatants were collected and chemokines were detected with a sandwich enzyme-linked immunosorbent assay. RESULTS: TNF-alpha/IFN-gamma-induced production of RANTES was inhibited by all inhibitors used. MCP-1 was produced constitutively and TNF-alpha-induced or TNF-alpha/IFN-gamma-induced production of MCP-1 was not inhibited by cyclosporin A or wortmannin, but by stauroporine or dexamethasone. All inhibitors used in this experiment inhibited TNF-alpha/IFN-gamma-induced or IL-1beta/IFN-gamma-induced production of MCP-2 in nasal mucosal fibroblasts. Although staurosporine or dexamethasone showed strong inhibitory effects, cyclosporin A or wortmannin did not inhibit the production of MCP-3 by IL-1beta/IFN-gamma treatment. CONCLUSION: Chemokines were strongly induced by stimulation of cytokines in combination and showed different pattern of inhibition by the inhibitors. Therefore, it was assumed that cytokines acted on multiple pathways or on unknown pathways which converged to gene-specific transcription factors
Subject(s)
Humans , Chemokine CCL5 , Chemokines , Cyclosporine , Cytokines , Dexamethasone , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Inflammation , Nasal Mucosa , Staurosporine , Transcription FactorsABSTRACT
BACKGROUND AND OBJECTIVES: Fibroblasts interact with eosinophils and play a key role in the pathogenesis of airway diseases. The aims of this study were to investigate whether Th1 or Th2 type cytokines can induce mRNA and protein expression of eotaxin and RANTES in human nasal fibroblast (HNF) and to verify the correlation between the stimulation of different cytokines and chemokines in HNF. Materials and Methods: Cultured HNF were stimulated by IL-13, TNF-alpha, IFN-gamma, IL-13 with TNF-alpha, IL-13 with IFN-gamma, TNF-alpha with IFN-gamma for 6, 24 and 48 hours. In addition, HNF were stimulated by different concentration of IL-13 (0.2, 2, 20, 200 ng/ml). MRNA expression of eotaxin and RANTES were revealed by RT-PCR and protein of eotaxin and RANTES were revealed by ELISA. RESULTS: TNF-alpha and IFN-gammar induced mRNA and protein expression of RANTES in HNF and they synergistically induced protein expression of RANTES. RANTES expression increased in a time dependent manner. IL-13 induced mRNA and protein expression of eotaxin in HNF and it synergistically reacted with TNF-alpha or IFN-gamma. The effects of IL-13 on mRNA and protein expression of eotaxin increased in a concentration dependent manner. CONCLUSION: Th1 or Th2 type cytokines induce mRNA and protein expression of eotaxin and RANTES in human nasal fibroblasts. TNF-alpha or IFN-gamma induce more RANTES than eotaxin but IL-13 induces more eotaxin than RANTES. There may be some synergic effects of cytokines for mRNA and protein expression of chemokines.
Subject(s)
Humans , Chemokine CCL5 , Chemokines , Cytokines , Enzyme-Linked Immunosorbent Assay , Eosinophils , Fibroblasts , Interleukin-13 , RNA, Messenger , Tumor Necrosis Factor-alphaABSTRACT
We have cloned the mouse neurotensin/neuromedin N (NT/N) gene from the murine mast cell line C1.MC/C57.1 for the first time. The murine NT/N cDNA clone consisted of 765 nucleotides and coded for 169 peptide residues with an N-terminal signal peptide, and the C-terminal region contained of one copy of neurotensin (NT) and one copy of neuromedin N (NN). Total of four Lys-Arg dibasic motifs were present; one each at the middle of the open reading frame, at the N-terminal of NN, at the C-terminal of NT, and between NN and NT. Amino acid sequence analysis of the mouse NT/N revealed 90% homology to that of the rat NT/N gene. NT/N is expressed in routine mast cell lines (C1.MC/C57.1 and P815), but not in murine bone marrow-derived mast cells (BMMCs), murine macrophage cell line (RAW 264.7), nor in murine T cell line (EL-4). NT/N mRNA in C1.MC/C57.1 is highly inducible by IgE cross-linking, phorbol myristate acetate, neurotensin, and substance P. Following the treatment of demethylating agent, 5-azacytidine (5-azaC), the NT/N gene was induced in BMMCs in response to IgE cross-linking. 5-azaC-treated BMMCs did not express the NT/N gene without additional stimuli. These findings suggested that the regulation of NT/N gene expression was dependent on the effects of not only gene methylation but also enhancer and/or repressor proteins acting on the NT/N promoter.
Subject(s)
Animals , Mice , Rats , Azacitidine , Cell Line , Clone Cells , DNA, Complementary , Gene Expression , Immunoglobulin E , Macrophages , Mast Cells , Methylation , Neurotensin , Nucleotides , Open Reading Frames , Protein Sorting Signals , Repressor Proteins , RNA, Messenger , Sequence Analysis, Protein , Substance P , Tetradecanoylphorbol AcetateABSTRACT
BACKGROUND: Disseminated intravascular coagulation (DIC) is a clinicopathologic syndrome for widespread intravascular coagulation. DIC occurs when the processes that regulate coagulation break down. Staphylococcal infection is one of the causes of DIC. Staphylococcus aureus produces coagulase that can clot plasma. There are two different tests to detect the coagulase; a tube test for free coagulase and a slide test for bound coagulase or clumping factor. The goal of the present study is to evaluate the characteristics of coagulase in Staphylococcus aureus for establishment of an in-vitro model for DIC. METHODS: Coagulase tests were performed by mixing plasma with two-fold diluted culture broths, culture supernatant and culture filtrates. Coagulase activity was expressed as the reciprocal of the highest dilution titer. Cell pellets treated with normal saline, ethyl alcohol, and aceton were used for the clumping tests. Platelet aggregation tests were conducted using a culture broth and a concentrated culture supernatant. A fibrinogen binding protein was isolated from sonificated bacteria and thus, determined the molecular weight. RESULTS: Coagulase activity was higher in the broth culture than in the culture supernatant and culture filtrate. Coagulase activity decreased after incubation at 37degrees C for 24 hours but culture filtrates were clumped after boiling at 100degrees C for 10 minutes. Alcohol and aceton did not inhibit the clumping test. S. aureus induced platelet aggregation but a concentrated culture filtrate did not induce platelet aggregation. Molecular weight of fibrinogen binding protein was 57 kDa. CONCLUSIONS: It is suggested that the plasma clot was due to free coagulase or a clumping factor. Free coagulase is different from a clumping factor. We concluded that the pathogenesis of DIC in the staphylococcal infection was due to platelet aggregation triggered by a clumping factor or coagulase with other substances.
Subject(s)
Bacteria , Carrier Proteins , Coagulase , Dacarbazine , Disseminated Intravascular Coagulation , Ethanol , Fibrinogen , Molecular Weight , Plasma , Platelet Aggregation , Staphylococcal Infections , Staphylococcus aureus , StaphylococcusABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Clone Cells , Cloning, Organism , Mast CellsABSTRACT
In order to explore the potential of ascorbic acid supplementation for the prevention and treatment of herpes simplex viral diseases, plaque reduction assays were performed. Ascorbic acid as well as copper chloride/ferric chloride were added to wells containing Vero cells infected with herpes simplex virus type 1 (HSV-1), and the infectivity of HSV-1 was determined. Since copper and iron are major transition metals in human plasma, near the normal human plasma concentrations of them were used for experiments. When Cu(II) and Fe(III) were applied, there were no significant differences between virus control and Cu(II)/Fe(III)-treated groups. But, when appropriate concentrations of ascorbic acid were added to wells, meaningful differences between control and ascorbate-treated groups were found. In the presence of Cu(II)/Fe(III) at 5.8/3.7 muM, 72-h treatment with ascorbate at 50 muM reduced HSV-1 infections to 10.77%+/-4.25% (P 0.05). Current recommended dietary allowance (RDA) of ascorbic acid is 60 mg/day, and the oral intake of 60 mg/day of ascorbic acid yields plasma ascorbic acid at 45 to 58 muM in a healthy adult man. Therefore, the results of this study suggest that the maintenance of appropriate level (more than 50 muM) of ascorbic acid in human plasma by appropriate amount (more than the RDA) of ascorbic acid supplementation may be helpful for the prevention and treatment of diseases caused by HSV-1 in an adult man. In addition, this study also suggests that ascorbic acid may be useful for the prophylaxis of fatal HSV-1 infections in neonates and the prevention of HSV-1 reactivation in immunocompromised hosts.
Subject(s)
Adult , Humans , Infant, Newborn , Ascorbic Acid , Copper , Herpes Simplex , Herpesvirus 1, Human , Immunocompromised Host , Iron , Metals , Plasma , Recommended Dietary Allowances , Simplexvirus , Vero Cells , Virus DiseasesABSTRACT
No abstract available.