Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization

M. P N Nair, N. M. Kumar, Ziad Kronfol, J. F. Greden, J. S. Lwebuga-Mukasa, S. A. Schwartz

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6 Citations (Scopus)

Abstract

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) 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-α 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 μg/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-α 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-α. These studies confirm our earlier finding that alcohol suppressed the production of TNF-α 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.

Original languageEnglish
Pages (from-to)392-398
Number of pages7
JournalClinical and Diagnostic Laboratory Immunology
Volume3
Issue number4
Publication statusPublished - 1996
Externally publishedYes

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ASJC Scopus subject areas

  • Microbiology (medical)
  • Immunology and Allergy
  • Clinical Biochemistry
  • Immunology

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