Polymorphisms of the GSTT1 and GSTM1 genes in women of central Serbia: Absence of association with uterine myoma

Since glutathione S-transferase (GST) enzymes are involved in cellular protection, we aimed to determine the distribution of GSTT1 and GSTM1 null genotypes in women in central Serbia in order to assess the risk of development of uterine myoma. The study consisted of 34 clinically diagnosed uterine myoma patients and 35 healthy control women. Analyses of GST polymorphism were carried out by multiplex PCR. Our results showed no significant differences in the GSTT1 and GSTM1 null genotypes between the patients and controls. Using the GSTT1 positive/GSTM1 positive combination as reference, there was no statistically significant risk of uterine myoma with the combination of GSTT1 null and GSTM1 null genotypes. We conclude that polymorphism of both GSTT1 and GSTM1 genes, alone or in combination, did not present the main risk for uterine myoma in women from central Serbia.[Projekat Ministarstva nauke Republike Srbije, br. III41010 i br. ON 175103

Effect of interleukin-17 on nitric oxide production in murine fibroblast-like cell line L929

The ability of interleukin-17 (IL-17) to induce nitric oxide (NO) synthesis in murine L929 fibroblasts was investigated. L929 cells were incubated with IL-17 and/or LPS, interferon-g (IFN-gama), interleukin-1 (IL-1), or dibutyryl-cAMP. NO production was assessed indirectly, by measuring nitrite accumulation in 24 h culture supernatants. L929 fibroblasts did not produce NO constitutively, nor IL-17 alone induced NO production. Of all other stimuli tested, only IFN-gama significantly up regulated nitrite level in L929 cell cultures. However, when IL-17 was applied simultaneously with each of the tested stimuli, NO synthesis was markedly elevated, thus indicating involvement of distinct signaling pathways of NO induction by IL-17 and other tested agents. Production of NO by IL-17+LPS-treated L929 cells was dependent on synthesis and activity of inducible NO synthase (iNOS), as indicated by abrogation of nitrite accumulation with protein synthesis inhibitor cycloheximide or selective inhibitor of iNOS, aminoguanidine. A role for protein tyrosine kinase (PTK) and transcription factor NF-kB in iNOS activation is suggested by reduction of NO synthesis with PTK inhibitor genistein and an inhibitor of NF-kB activation, pyrrolidine dithiocarbamate (PDTC). In contrast, protein kinase C inhibitor staurosporine was ineffective in blocking IL-17+LPS-induced NO production in L929 cells. Taken together, our results for the first time showed an active participation of IL-17 in co-induction of fibroblast NO synthesis with LPS, cytokines (IL-1, IFN-gama), or cAMP analogue, and suggested that IL-17 up-regulated NO synthesis in fibroblasts through mechanisms involving PTK and NF-kB activation.Ispitan je uticaj interleukina-17 (IL-17) na produkciju azot monoksida (NO) od strane L929 fibroblasta. L929 ćelije su inkubirane sa IL-17 i/ili LPS-om, interferonom-gama (IFN-gama), interleukinom-1 (IL-1), ili dibutiril-cAMP-om. Produkcija NO ispitana je indirektno, merenjem koncentracije nitrita akumuliranih nakon 24 h u supernatantu ćelijskih kultura. Kultivisani L929 fibroblasti nisu produkovali NO konstitutivno, niti je sam IL-17 indukovao NO produkciju. Od ostalih ispitanih agenasa samo je IFN-gama značajno povećao nivo nitrita u kulturama L929 ćelija. Međutim, kada je IL-17 primenjen sa bilo kojim od testiranih agenasa, došlo je do snažnog povećanja NO sinteze, što je ukazalo da su signalni putevi indukcije NO sa IL-17 i ostalim navedenim agensima različiti. Sprečavanje akumulacije nitrita inhibitorom proteinske sinteze cikloheksimidom ili selektivnim inhibitorom iNOS aminoguanidinom u ćelijama stimulisanim sa IL-17+LPS ukazalo je da NO produkcija zavisi od sinteze i aktivnosti enzima inducibilne NO sinteze (iNOS). Inhibitor protein tirozin kinaze (PTK), genistein i inhibitor aktivacije transkripcionog faktora NF-kB, pirolidin ditiokarbamat (PDTC), takođe su redukovali sintezu NO. Nasuprot tome, inhibitor protein kinaze C, staurosporin, nije blokirao NO produkciju L929 ćelija stimulisanih sa IL-17+LPS. U celini, naši rezultati su po prvi put pokazali aktivno učešće IL-17 u koindukciji NO sinteze sa LPS-om, citokinima (IL-1, IFN-gama), ili cAMP analogom u fibroblastima i ukazali da stimulacija NO sinteze sa IL-17 uključuje mehanizme zavisne od PTK i NF-kB.nul

Interactions between infections and immune-inflammatory cells in type 1 diabetes mellitus and inflammatory bowel diseases: evidences from animal models

Type 1 diabetes mellitus (T1D) and inflammmtory bowel diseases (IBD) are multifactorial disorders of autoimmune origin. Several microbial agents have been reported to be associated with the development of type 1 diabetes and inflammatory bowel diseases in animal models by different mechanisms. These models which resemble the phenotype of the human disease they mimic, can be very useful to identify important pathogenetic mechanisms, as well as therapeutical targets to treat the disease. This review is focused on the immune inflammatory pathways which are considered to be associated with the pathogenesis T1D and IBD in transgenic mice.nul

Tumor cell-specific inhibition of inducible nitric oxide synthase activation by tiazofurin

The effects of tiazofurin (TR) on proliferation and cytokine-induced nitric oxide (NO) production in the L929 fibrosarcoma cell line and murine embryonic fibroblasts were investigated. Treatment with TR inhibited the growth of nonconfluent L929 cells in a dose-dependent manner. TR, at concentrations not affecting cell viability or proliferation, markedly decreased IFN-gamma + LPS-induced expression of inducible NO synthase (iNOS) mRNA and, subsequently. NO production in confluent L929 cultures. However, TR did not interfere with the IFN-gamma -triggered expression of mRNA for IRF-1, an important iNOS transcription factor, implying that TR interferes with some other intracellular pathway involved in iNOS induction triggered by IFN-gamma + LPS. In contrast to the results obtained in L929 cells, iNOS mRNA expression induced by IFN-gamma + LPS in murine embryonic fibroblasts was resistant to TR, indicating a tumor-selective action of this agent. (C) 2001 Elsevier Science B.V. All rights reserved.nul

Deletion of macrophage migration inhibitory factor promotes obesity-associated insulin resistance while attenuating inflammation in mice fed a high-fat diet

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STAT1 is required for iNOS activation, but not IL-6 production in murine fibroblasts

The role of transcription factor STAT1 in production of pro-inflammatory mediators nitric oxide (NO) and IL-6 was examined in murine embryonic fibroblasts. While cells from wild-type animals released large amounts of NO after stimulation with IFN-gamma in combination with LPS, TNF-alpha or IL-1, their STAT1-deficient counterparts failed to synthesise detectable levels of this free radical gas. Inability of STAT1(-/-) fibroblasts to produce NO was accompanied by complete absence of mRNA for iNOS and its transcription factor IRF-1, both readily upregulated in wild-type cells. However, treatment with cytokines (IFN-gamma, TNF-alpha, IL-1, IL-17) significantly increased IL-6 generation in STAT1-deficient fibroblasts. These results indicate that STAT1 activation and subsequent IRF-1 transcription are required for induction of iNOS, but not IL-6 in murine fibroblasts. (C) 2001 Academic Press.nul

Mycophenolic acid inhibits activation of inducible nitric oxide synthase in rodent fibroblasts

Mycophenolate mofetil (MMF) is an immunosuppressive drug that acts as a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH). MMF has recently been shown to inhibit the enzymatic activity of inducible NO synthase (iNOS) and subsequent production of the cytotoxic free radical nitric oxide (NO) in endothelial cells. We here investigated the effect of bioactive MMF compound mycophenolic acid (MPA) on iNOS-mediated NO synthesis in fibroblasts, which are important source of NO in rheumatoid arthritis and during rejection of solid organ transplants. MPA exerted dose-dependent inhibition of NO synthesis, measured as nitrite accumulation, in IFN-gamma + LPS-stimulated L929 mouse fibroblast cell line and rat primary fibroblasts. The effect of MPA was not mediated through interference with IMPDH-dependent synthesis of iNOS co-factor BH4 and subsequent suppression of iNOS enzymatic activity, as direct BH4 precursor sepiapterin failed to block the action of the drug. MPA suppressed the IFN-gamma + LPS-induced expression of fibroblast iNOS protein, as well as mRNA for iNOS and its transcription factor IRF-1, as assessed by cell-based ELISA and semiquantitative RT-PCR, respectively. MPA suppression of fibroblast NO release, iNOS, and IRF-1 activation, was efficiently prevented by exogenous guanosine, indicating that the drug acted through reduction of IMPDH-dependent synthesis of guanosine nucleotides. These results suggest that MPA inhibits NO production in fibroblasts by blocking guanosine nucleotide-dependent expression of iNOS gene, through mechanisms that might involve the interference with the induction of iNOS transcription factor IRF-1.nul

The role of macrophage migration inhibitory factor in obesity-associated type 2 diabetes

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Deletion of macrophage migration inhibitory factor promotes obesity-associated insulin resistance while attenuating inflammation in mice fed a high-fat diet

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STAT1 is required for iNOS activation, but not IL-6 production in murine fibroblasts

The role of transcription factor STAT1 in production of pro-inflammatory mediators nitric oxide (NO) and IL-6 was examined in murine embryonic fibroblasts. While cells from wild-type animals released large amounts of NO after stimulation with IFN-gamma in combination with LPS, TNF-alpha or IL-1, their STAT1-deficient counterparts failed to synthesise detectable levels of this free radical gas. Inability of STAT1(-/-) fibroblasts to produce NO was accompanied by complete absence of mRNA for iNOS and its transcription factor IRF-1, both readily upregulated in wild-type cells. However, treatment with cytokines (IFN-gamma, TNF-alpha, IL-1, IL-17) significantly increased IL-6 generation in STAT1-deficient fibroblasts. These results indicate that STAT1 activation and subsequent IRF-1 transcription are required for induction of iNOS, but not IL-6 in murine fibroblasts. (C) 2001 Academic Press.nul