Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization
| Autor: | Madhavan Nair, J. S. Lwebuga-Mukasa, Stanley A. Schwartz, J. F. Greden, Ziad Kronfol, Niranjan M. Kumar |
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| Rok vydání: | 1996 |
| Předmět: |
Microbiology (medical)
Lipopolysaccharides Clinical Biochemistry Immunology Gene Expression In situ hybridization Biology Peripheral blood mononuclear cell Polymerase Chain Reaction chemistry.chemical_compound Gene expression Immunology and Allergy Humans In Situ Hybridization Whole blood Ethanol Tumor Necrosis Factor-alpha RNA-Directed DNA Polymerase Molecular biology Reverse transcription polymerase chain reaction chemistry Leukocytes Mononuclear Tumor necrosis factor alpha RNA extraction Research Article |
| Zdroj: | Clinical and diagnostic laboratory immunology. 3(4) |
| ISSN: | 1071-412X |
| Popis: | We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction. |
| Databáze: | OpenAIRE |
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