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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results indicate that CAEV infection changes caprine monocyte cytokine responsivity.
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PMID:Caprine arthritis encephalitis virus infection changes caprine blood monocyte responsiveness to lipopolysaccharide stimulation in vitro. 785 74

The purpose of this study was to compare the ability of fresh and cryopreserved mononuclear cells to generate thrombin, induce fibrin formation and finally resolve the fibrin formed, when exposed to plasma. Peripheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diacetate and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were seeded, stimulated with lipopolysaccharide(LPS), and exposed to a standard heparinized overlay plasma. Plasma was harvested at intervals (0-7 days). Thrombin generation and fibrin formation were measured by quantification of prothrombin fragment (F1 + 2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin (ogen) degradation products (FbDP and FgDP). Recovery of cells after thawing was about 80%, and the viability of fresh and cryopreserved PBM was > 95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothombinase activity (F1 + 2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there were significant variations between the donors in all the parameters measured. We conclude that cryopreservation of human blood mononuclear cells is possible with maintainance of the potential of the cells to mediate coagulation in plasma upon LPS stimulation, whereas the fibrin resolving capacity apparently is reduced by the preservation procedure.
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PMID:Procoagulant and profibrinolytic activities of cryopreserved human monocytes. 787 96

Influenza B virus has been aetiologically linked to Reye Syndrome (RS), but the mechanism(s) by which this pathogen could disrupt liver metabolism and produce the hepatic mitochondrial injury characteristic of the syndrome are unknown. In this study, two mechanisms by which infection of hepatocytes with influenza B virus could disrupt cellular metabolism were investigated. (1) virus-induced increase in pro-oxidant iron with subsequent iron-induced lipid peroxidation (LP) and (2) increased membrane permeability. Hep G2 cells, a well-differentiated continuous human liver cell line derived from a hepatoblastoma, were infected with allantoic-fluid derived influenza B Lee/40 virus (AFDV) at a multiplicity of infection of 10 for 24 h; productive infection was confirmed by both haemagglutination of chick erythrocytes and by plaque assay. Infection of Hep G2 cells preloaded with 59Fe-transferrin resulted in increased release of 59Fe (153 +/- 17% of controls, P < 0.03). However, the iron released did not result in increased LP (assessed by thiobarituric acid reactive substances; TBARS). To confirm that this lack of of increase in TBARS was not due to insensitivity of the cell line to pro-oxidant iron, cells were exposed to 15 microM iron ascorbate for 60 min. Production of TBARS was increased (122 +/- 4% of controls, P < 0.0003). Release of 51Cr from infected cells was also increased (128 +/- 12% of controls, P < 0.05); thus the infected cells exhibited a generalized increase in membrane permeability. However, infection did not depress mitochondrial respiration (as assessed by the formation of MTT-f3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan. To determine if the combination of viral infection and soluble products of activated macrophages would affect mitochondrial respiration, infected hepatocytes were exposed to the supernatant fluid from THP-1 cells which had previously been incubated with lipopolysaccharide at 100 ng ml-1 for 18 h. This supernate did depress the formation of MTT-f (81 +/- 5% of controls, P < 0.03). We conclude that influenza B virus does productively infect Hep G2 cells, and does increase hepatocyte membrane permeability. This effect does not impair mitochondrial respiration directly. However, infection does act in concert with soluble products of activated macrophages to depress hepatic mitochondrial respiration. Whether this interaction can be explained by virus-induced permeability changes and/or other effects of infection deserves further investigation.
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PMID:Activated THP-1 cells depress mitochondrial respiration in Hep G2 cells infected with influenza B virus. 787 29

Medullary thick ascending limb of Henle's loop (mTALH) tubules, isolated from kidneys of male Sprague-Dawley rats, expressed the gene for tumor necrosis factor (TNF) and released this cytokine when challenged with lipopolysaccharide (LPS). The TNF produced was biologically active, as determined by cytotoxic activity present in supernatants from LPS-stimulated mTALH, using the TNF-sensitive murine fibrosarcoma cell line, WEHI 164. The amount of TNF produced, approximately 75 nM, has previously been shown to affect ion transport in the mTALH. The TNF-mediated cytotoxicity (and ion transport effects) were completely neutralized with a polyclonal anti-TNF antisera. Further, immunoprecipitation experiments demonstrated that the 17 kDa TNF monomer was formed by de novo protein synthesis. In contrast, the mTALH did not produce the related cytokine, lymphotoxin (LT). Production of TNF was confirmed by demonstrating the accumulation of a 1.6 kb TNF mRNA by Northern blot analysis; mRNA for LT was not detected. Expression of the TNF gene in the mTALH was confirmed by the polymerase chain reaction (PCR). Southern blot analysis and ethidium bromide staining of the resultant PCR products revealed the expected 276 bp sequence of TNF DNA for the mTALH. We have demonstrated that mTALH tubules stimulated with LPS express the gene for TNF, but not LT, and release biologically active TNF. TNF is an important mediator of septic shock and may contribute to changes in renal function associated with endotoxemia. Production of TNF by the mTALH may be an important autocrine regulatory mechanism for this nephron segment.
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PMID:TNF production by the medullary thick ascending limb of Henle's loop. 793 28

Injury results in altered hepatocyte protein synthesis including the production of acute-phase reactants. Evidence suggests that these hepatocyte products regulate macrophage function; however, their role in liver macrophage-mediated hepatocyte dysfunction following a second insult is poorly characterized. We hypothesize that IL-6-stimulated hepatocyte products alter liver macrophage responses to lipopolysaccharide, contributing to enhanced hepatocyte dysfunction. To test this hypothesis, hepatocytes, obtained by liver collagenase digestion, were treated with rIL-6 (murine, 300 units/ml) for 24 hr, and then liver macrophages, obtained by perfusion and pronase digestion, were added to establish cocultures. Cocultures were then stimulated with endotoxin (LPS, Escherichia coli O111:B4, 10 micrograms/ml) and hepatocyte dysfunction was assessed by determining secretory protein synthesis ([35S]methionine labeling, trichloracetic acid precipitation, and SDS-PAGE) and energy metabolism [mitochondrial respiration using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye]. Cultures of hepatocytes alone stimulated with IL-6, LPS, or sequential IL-6 followed by LPS demonstrate no difference in total secretory protein synthesis or mitochondrial respiration. In contrast, hepatocyte-liver macrophage cocultures demonstrate significantly reduced total secretory protein synthesis following sequential IL-6 followed by LPS ([35S]methionine cpm x 10(3): control, 33.8 +/- 8.5; LPS, 25.8 +/- 6.3; IL-6/LPS, 15.7 +/- 6.4; P < 0.05 vs control). This effect is specific to IL-6 since sequential TNF-alpha followed by LPS did not result in significant suppression of secretory protein synthesis. One-dimensional SDS-PAGE of labeled coculture secretory proteins demonstrates qualitative changes following sequential insult in vitro compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequential insult enhances liver macrophage-signaled hepatocyte dysfunction. 804 Nov 36

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor alpha (TNF alpha) or recombinant interleukin 1 beta (IL-1 beta) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1-50 ng/ml of TNF alpha, growth being up to 400% more than in the control culture. The effect of TNF alpha depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF alpha, IL-1 beta inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF alpha and IL-1 beta and between TNF alpha and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.
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PMID:Effects of tumour necrosis factor alpha and interleukin 1 beta on the proliferation of cultured glomerular epithelial cells. 805 51

A number of recent studies suggest that cells of the immune system, e.g. peripheral blood mononuclear cells (PBMC), can synthesize and process POMC and secrete POMC-derived peptides, such as ACTH and endorphins, upon immune and hormonal challenges. From this, it has been proposed that POMC-derived peptides originating from lymphoid cells can function as hormones, for instance in a lymphoid-adrenal axis. In view of the important physiological implications of this proposal, the present study was designed to investigate the expression of the POMC gene in human PBMC and the production by these cells of alpha-, beta-, and gamma-endorphins (alpha E, beta E, and gamma E) peptides that are established end products of the posttranslational processing of POMC. PBMC of individual donors were used uncultured (fresh cells) or cultured for 24 and 48 h in the presence and absence of Concanavalin-A (Con-A), bacterial lipopolysaccharide, phytohemagglutinin, or CRH, and vasopressin, conditions that reportedly stimulate POMC activity in those cells, to investigate the presence of POMC transcripts by analysis of total RNA with Northern blotting and the reverse transcriptase polymerase chain reaction (RT-PCR). Large scale preparations containing over 10(9) cells (fresh, cultured with and without Con-A) originating from several donors were examined for the presence of POMC transcripts by analysis of poly(A)+ RNA on Northern blots and for the presence of alpha E, beta E, and gamma E by gel filtration over Sephadex G-75 and reverse phase HPLC, followed by assay of the fractions in four endorphin RIA systems with different specificities. On the Northern blots of total RNA, no POMC transcripts were detectable. In poly(A)+ RNA preparations, no full-length POMC mRNA was found, and it was estimated that the concentration of POMC mRNA, if present, was below approximately 0.005 transcript/cell in Con-A-stimulated cells and still lower in unstimulated cells. In accord with literature data, an 800- to 900-nucleotide POMC transcript was detected in cultured PBMC, and the levels of this transcript were stimulated by Con-A. In all samples analyzed with RT-PCR, a transcript spanning most of exons 2 and 3 was detectable only on Southern blots of the RT-PCR product, but not on agarose gels stained with ethidium bromide. Chromatographic analysis of endorphin immunoreactivities in cell extracts revealed no qualitative differences between the immunoreactive profiles of fresh PBMC or PBMC cultured with or without Con-A.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Analysis of proopiomelanocortin (POMC) messenger ribonucleic acid and POMC-derived peptides in human peripheral blood mononuclear cells: no evidence for a lymphocyte-derived POMC system. 840 37

The effects of bovine leukemia virus (BLV) infection on cytokine activity of bovine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared to supernatants of LPS-stimulated monocytes from BLV-negative cows, supernatants from BLV-positive cows contained about four times more interleukin-1 beta (IL-1 beta) (as measured by an enzyme-linked immunosorbent assay (ELISA) for bovine IL-1 beta). Despite their higher IL-1 beta concentration, supernatants from BLV-positive cows stimulated proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide) assay similar to supernatants from BLV-negative cows, but showed about 30% less IL-1 activity than supernatants from BLV-negative cows on the IL-1-dependent cell line LBRM-33 1 A-5, and about five times more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results demonstrate that BLV infection changes the cytokine response of bovine monocytes to LPS stimulation in vitro. The results are consistent with the assumption that BLV infection leads to the production and secretion of a soluble IL-1 inhibitor by LPS-stimulated peripheral blood monocytes.
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PMID:Effect of bovine leukemia virus infection on bovine peripheral blood monocyte responsiveness to lipopolysaccharide stimulation in vitro. 853 18

A reverse transcription PCR assay with porcine cytokine-specific primers was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1 beta. The specificities of the cDNA PCR products were confirmed by sequence analysis on the basis of known porcine cytokine gene sequences. The reverse transcription PCR assay was also used to study cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemiluminescence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control unstimulated cells. Thus, TNF-alpha mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control cell cultures. No differences in TNF-alpha transcription levels between LPS-stimulated and control cells were observed after incubation for 24 or 55 h. Enhancements of IL-6 and IL-1 beta mRNA levels were also observed in the cultures stimulated with LPS for 6 and 24 h compared with the cytokine mRNA levels in control cell cultures. The presence of cytokine mRNA transcripts in the LPS-stimulated macrophage cultures correlated with the detection of these soluble cytokines by the bioassays. In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.
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PMID:Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization. 857 26

Syntheses of the pentasaccharide 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6- tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2- ulopyranosidonic acid and of the tetrasaccharide 3,4,6-tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside, both as its methyl and 2-(4-trifluoro-acetamidophenyl)ethyl glycoside, are described. These oligosaccharides correspond to structures found in the lipopolysaccharide of Moraxella catarrhalis and were needed for biological experiments aimed at producing antibodies against the bacteria. The best way to introduce the glucopyranosyl groups into the 3-, 4-, and 6-positions of the branched target compounds was found to be a one-step reaction using a 3,4,6-triol as acceptor and 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl bromide as donor in a silver trifluoromethanesulfonate-promoted coupling. The spacer arm, necessary for the formation of immunoactive glycoconjugates, was introduced into the glucose moiety via a dimethyl(methylthio)sulfonium trifluoromethanesulfonate-promoted reaction using the ethyl thioglucoside as donor, whereas for Kdo, the acetylated glycal derivative, methyl 4,5,7,8-tetra-O-acetyl-2,6-anhydro-3-deoxy-D-manno-oct-2-enonate, was used as donor and phenylselenyl trifluoromethanesulfonate as a stereocontrolling promoter.
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PMID:Synthesis of 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6-tri-O-beta-D- glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2-ulopyrano sid onic acid, a highly branched pentasaccharide corresponding to structures found in lipopolysaccharides from Moraxella catarrhalis. 859 Apr 46


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