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

Activated by interferon gamma (IFN gamma)(50 U/ml) and lipopolysaccharide (50 ng/ml), mouse peritoneal macrophages were cocultured with the L1210 parental cell line (L1210/PRT) and its Adriamycin-, cisplatin-resistant cell lines (L1210/ADM, L1210/CDDP) for 24 h at effector: target (E:T) ratios of 10:1, 5:1 and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT)-cleavage assay, a new improved indirect MTT assay, and the colony-formation assay were used to quantify macrophage-mediated suppression of these non-adherent tumour targets. The results showed that the macrophages can produce formazan at a high level, which can interfere with the final results of a direct MTT assay. The new indirect MTT assay can avoid such interference because the effectors are separated from the targets before the assay is performed, so the real viability of the targets is reflected. An indirect MTT assay, as developed in this study, could be better than the direct assay for examining the suppressive effect of activated macrophages on non-adherent tumour cells in vitro. This study also revealed that all the L1210 cell lines can be suppressed significantly by the macrophages at E:T ratios of 10:1 and 5:1 while the two drug-resistant cell lines have lower survival rates at an E:T ratio of 10:1, indicating that they are more susceptible than their parental cell line.
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PMID:A new 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for testing macrophage cytotoxicity to L1210 and its drug-resistant cell lines in vitro. 139 44

After activation by interferon-gamma (INF-gamma) and lipopolysaccharide(LPS), mouse peritoneal macrophages were cocultured with P388 parental cell line (P388/PRT) and its adriamycin (ADM)-, cisplatin(CDDP)-, cyclophosphamide(CPM)-, and mitomycin-C(MMC)-resistant cell lines for one day at effector:target ratios (E:T) of 10:1, 5:1, and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) cleavage assay and a new indirect MTT assay as well as clonogenic assay were used to quantitate activated macrophage-mediated cytotoxicity to these non-adherent leukemia targets. The results revealed that all the P388 cell lines can be suppressed efficiently by activated macrophages, but P388 CPM- and MMC-resistant cell lines (P388/CPM, P388/MMC) were more susceptible than P388/PRT while P388 ADM- and CDDP-resistant cell lines (P388/ADM, P388/CDDP) shared equal level of survival rates with P388/PRT. This study also showed that both non-activated and activated macrophages can produce formazan in a high level, which can interfere with the final results of direct MTT assay. The new indirect MTT assay can avoid such interference by separating the effectors from the targets before performing the MTT assay and reflects the real viability of the targets so the indirect MTT assay developed in this study could be a better way to examine cytostatic and cytotoxic effect of activated macrophages on non-adherent tumor cells in vitro.
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PMID:Differential macrophage-mediated cytotoxicity to P388 leukemia cells and its drug-resistant cells examined by a new MTT assay. 146 25

Reactive nitrogen intermediates are important in the anti-tumor and anti-microbial activities of rodent macrophages, but it is not known whether this is the case for human macrophages. In the present study, nitrite concentrations in vitro were used as an indicator of reactive nitrogen intermediate production by mouse, rat, and human macrophages. Human macrophages derived by culturing peripheral blood monocytes did not consistently produce detectable nitrite levels in response to any stimulus examined. Human macrophages were viable and metabolically active as indicated by the MTT assay, and their respiratory burst response to phorbol myristate acetate was increased following incubation with Interferon-gamma, as expected for typical macrophages. In contrast, rat or mouse peritoneal macrophages produced nitrite concentrations of approximately 20-100 microM in response to lipopolysaccharide, Interferon-gamma, or both. These results demonstrate substantial differences in the production of nitrites by rodent and human macrophages. Because of the heterogeneity among macrophage populations, these findings may not be applicable to all human macrophage populations, but they suggest a need for caution in extrapolating from rodent studies regarding the role of reactive nitrogen intermediates in anti-tumor or anti-microbial functions of human macrophages.
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PMID:Evaluation of nitrite production by human monocyte-derived macrophages. 149 65

Cultures derived from human corneo-scleral rims remaining after a central corneal button had been removed for transplantation, revealed two types of cells on light microscopy: One with typical epithelial morphology and the other resembling fibroblasts. Both cell types contained keratin filaments in early passage and were therefore considered epithelial in nature. The fibroblast-like cells were designated fibroblast-like epithelial cells (FLE) while the typical epithelial cells were referred to as E-type. Both E and FLE cells constitutively produced an IL-1-like factor as determined by thymocyte proliferation assay and IL-2 induction in EL-4 lymphoma cells. Moreover, the supernatants from these cells potentiated concanavalin A (Con A)-primed mitochondrial oxidative metabolism in thymocytes, as indicated by the tetrazolium salt reduction assay (MTT) and this effect was significantly neutralized with monoclonal anti-IL-1 beta. The release of biologically active IL-1 beta by the FLE cells is another characteristic (in addition to the presence of keratin) distinguishing these cells from fibroblasts which do not release biologically active IL-1 beta. Using an ELISA, specific for IL-1 beta, there was clear cut evidence for increased production of this cytokine by FLE cells in response to human recombinant gamma-interferon (IFN-gamma), Staphylococcus aureus, and lipopolysaccharide (LPS) in combination with silica (LPS/silica). Time studies with IFN-gamma and LPS/silica demonstrated that enhanced production was time dependent and that IL-1 beta was primarily cell associated. The results indicate that human corneal E- and FLE-type cells can produce and release IL-1 and that FLE cells can be induced by inflammatory mediators to increase production of IL-1 beta.
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PMID:Interferon-gamma, Staphylococcus aureus, and lipopolysaccharide/silica enhance interleukin-1 beta production by human corneal cells. 248 99

The B9 assay is known to be a specific and sensitive assay for the estimation of interleukin-6 activity. This assay was found to be compromised by lipopolysaccharide in concentrations > or = 40 ng lipopolysaccharide per ml. The lipopolysaccharide stimulates proliferation of the B9 cell line in a dose-dependent manner both when measuring the proliferation by thymidine incorporation and when using the MTT assay. However the LPS dose-response curve is different compared to the dose-response curve for IL-6. A sample containing 100 ng LPS/ml but no IL-6 would be estimated erroneously to contain 12 pg IL-6. The interference of lipopolysaccharide is totally abolished by the addition of polymyxin B to the samples but the addition has no effect on the IL-6 induced proliferation.
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PMID:Lipopolysaccharide in concentrations above 40 ng/ml stimulates proliferation of the IL-6-dependent B9 cell line. 771 31

A blastogenesis microassay employing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was adapted to measure blastogenic responses of lymphocytes from the chicken's head-associated lymphoid tissues (i.e., the harderian gland and conjunctiva-associated lymphoid tissue) to T- and B-cell mitogens. Lymphocytes isolated from peripheral blood, spleen, and the harderian gland had highly significant (P < 0.01) responses to T- and B-cell mitogens compared with control lymphocytes cultured without mitogens. Cultured lymphocytes obtained from the harderian gland had highly significant mitogenic responses to the T-cell mitogen concanavalin A (25 to 100 micrograms/ml) and to the B-cell mitogen Salmonella typhimurium lipopolysaccharide (1.25 to 5.0 micrograms/ml) compared with the control lymphocytes. Mitogenic responses of cultured lymphocytes obtained from the conjunctiva-associated lymphoid tissue could not be measured within the given parameters of the blastogenesis microassay. This was primarily due to the low yield of lymphocytes, which proved to be a limiting factor. The ability of the MTT blastogenesis microassay to detect blastogenic responses of the harderian gland to mitogens may be indicative of its usefulness for measuring cell-mediated immunity responses to other antigens.
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PMID:Mitogenic responses of the head-associated lymphoid tissues of the chicken. 779 67

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

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

We studied the plasma levels of tumor necrosis factor-alpha (TNF-alpha) interleukin-6 (IL-6) as well as the in vitro production of these cytokines by peripheral blood mononuclear cells, in 10 patients with amebic liver abscess (ALA) and seven healthy controls. TNF-alpha was measured using a bioassay with L929 fibroblasts; IL-6 was quantitated by the B9 cell line assay. In both assays, the number of viable cells was estimated by the conversion of MTT to formazan. TNF-alpha plasma levels were nondetectable (< 20 pg/mL) in ALA patients, as well as in the majority of healthy controls. The in vitro production of TNF induced by lipopolysaccharide was significantly decreased in ALA patients. Most ALA patients (8/10) had increased plasma levels of IL-6. Furthermore, the spontaneous production of IL-6 in vitro was significantly increased in ALA patients compared to controls. In the acute stage of the ALA, a negative relationship was found between the raised plasma levels of IL-6 and the in vitro diminished production of TNF-alpha. After recovery, ALA patients showed both normal plasma levels and in vitro production of TNF and IL-6. Our data corroborate previous reports regarding plasma levels of TNF in ALA, and suggest that E. histolytica induces the in vivo production of IL-6 through a TNF-independent pathway. The raised levels of IL-6 might in turn down-regulate the production of TNF in ALA patients.
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PMID:Plasma levels and in vitro production of tumor necrosis factor-alpha and interleukin-6 in patients with amebic liver abscess. 797 44

In an analysis of nitric oxide (.NO) production and toxicity, chicken macrophage-generated .NO inhibited mitochondrial activity in both .NO-producing macrophages themselves and lymphoid tumor targets. However, differences in targeting of mitochondrial toxicity were observed among these cells. Two chicken macrophage cell lines, HD11 and MQ-NCSU, produced .NO (measured as nitrite) dependent upon concentrations of L-arginine and bacterial endotoxin (lipopolysaccharide). Mitochondrial activity was negatively correlated with the amount of .NO produced. Using a modified MTT assay, .NO induced suppression in two mitochondrial complexes. Mitochondrial activity was significantly suppressed among HD11 cells receiving LPS alone (complex I, 63.0 +/- 5.5% suppression; complex II, 27.9 +/- 5.2%). In contrast, mitochondrial activities in samples receiving LPS plus inhibitor, NG-nitro-L-arginine methyl ester (NAME; 5 mM) or 2,4-diamino-6-hydroxypyrimidine (DAHP; 5 mM), were not significantly different from control values. When HD11 macrophages were cocultured with lymphoblastoid tumor targets, RECC-CU60 (T cell) or LSCC-RP9 (B cell), adding LPS (1 microgram/ml), tumor cell mitochondrial activity was significantly suppressed. In the generator macrophages, complex I was more suppressed than complex II, whereas in lymphoid targets no such difference was observed. These results indicate that .NO inhibits complex I and II mitochondrial activity but that differential targeting can occur among chicken leukocyte populations.
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PMID:Nitric oxide (.NO)-induced mitochondrial injury among chicken .NO-generating and target leukocytes. 802 70


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