Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.
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PMID:Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines. 896 Aug 25

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.
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PMID:Induction of guanosine triphosphate-cyclohydrolase by follicle-stimulating hormone enhances interleukin-1 beta-stimulated nitric oxide synthase activity in granulosa cells. 897

Recent evidence indicates that membrane-bound costimulatory molecules of the B7 family are important for T-cell activation and are upregulated in IFN gamma-stimulated human microglia and in multiple sclerosis active lesions. In this study we have performed a detailed analysis of B7-1 and B7-2 expression and regulation in cultured mouse glial cells using immunocytochemical and semi-quantitative reverse transcriptase-polymerase chain reaction techniques. In an immortalized mouse microglial cell line (BV-2), expression of B7-1 and B7-2 was enhanced by interferon-gamma (IFN gamma). IFN gamma was a weak inducer of B7-2 mRNA and immunoreactivity in microglia primary cultures obtained from the neonatal mouse brain, whereas lipopolysaccharide, tumour necrosis factor-alpha, colony-stimulating factors and interleukin-1 beta did not affect microglial B7-2 expression. Combined IFN gamma and lipopolysaccharide treatment very effectively upregulated the B7-2 gene expression and immunoreactivity in microglia, but not in astrocytes. In both glial cell types, expression of B7-1 was not induced by any of the above agents. Among known microglia/macrophage deactivators, interleukin-10, prostaglandin E2 and cAMP-elevating agents, but not transforming growth factor-beta 1 and interleukin-4, inhibited B7-2 transcripts and immunoreactivity in IFN gamma/LPS-stimulated microglia, thus suggesting possible paracrine and autocrine mechanisms for regulating the expression of this important T-cell costimulatory signal in the brain.
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PMID:Analysis of B7-1 and B7-2 costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin-10, prostaglandin E2 and cyclic AMP-elevating agents. 900 48

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.
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PMID:Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide. 902 28

Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.
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PMID:Identification and characterization of the dTDP-rhamnose biosynthesis and transfer genes of the lipopolysaccharide-related rfb locus in Leptospira interrogans serovar Copenhageni. 902 10

Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.
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PMID:Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs. 905 43

Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-gamma, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-gamma (MIX) strongly inhibits the cNOS activity, as determined by measuring [3H]citrulline production. In addition, the effect of MIX is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MIX does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction.
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PMID:Bacterial lipopolysaccharide plus interferon-gamma elicit a very fast inhibition of a Ca2+-dependent nitric-oxide synthase activity in human astrocytoma cells. 906 11

IL-1beta, IL-6, IL-8 and TNF-alpha production by PMNL from 21 HIV-infected (HIV+), including 11 full-blown AIDS, and 20 HIV-uninfected (HIV-) subjects (matched for age and sex to HIV+ ones) was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. PMNL from both categories of subjects were strongly stimulated in their actual cytokine production by a mannoprotein fraction (MP-F2) of Candida albicans, as well as by the bacterial lipopolysaccharide (LPS). These stimulatory effects were apparently due to increased cytokine gene expression and were substantially reversed by the physiological inhibitor IL-10. However, PMNL from HIV+ subjects showed increased IL-6 and TNF-alpha gene expression and produced more IL-6 and TNF-alpha than PMNL from HIV- controls, under similar stimulation conditions. This difference could not be attributed to a given stage of HIV infection, any associated medication, or to a generalized increase of gene expression, as quantitatively similar beta-actin and IL-1beta transcripts were detected. Moreover, no significant difference in IL-8 production by the PMNL from HIV+ and HIV- subjects was observed. Our studies suggest that PMNL from HIV+ subjects might add to other cellular sources of IL-6 and TNF-alpha (e.g. monocytes-macrophages) in contributing to the cytokine-dysregulated pattern typical of the HIV+ patient.
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PMID:Responsiveness of human polymorphonuclear cells (PMNL) to stimulation by a mannoprotein fraction (MP-F2) of Candida albicans; enhanced production of IL-6 and tumour necrosis factor-alpha (TNF-alpha) by MP-F2-stimulated PMNL from HIV-infected subjects. 906 16

Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.
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PMID:Species differences in NO formation by rat and hamster alveolar macrophages in vitro. 911 52

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection. TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of IFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal Mphi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG.1.6) monoclonal antibodies. TDM-primed Mphi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of Mphi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited Mphi in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal Mphi through IFN-gamma induction.
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PMID:Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages. 911 75


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