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Impact of Alpha Interferon and Ribavirin on the Function of Maturing Dendritic Cells. Eleanor Barnes, 2004.Alpha interferon and ribavirin are required in combination to achieve a sustained virological response in the treatment of hepatitis C virus (HCV) infection . Alpha interferon has direct antiviral activity and also enhances HCV-specific T-cell responses . Ribavirin has little direct activity against HCV but reduces hepatic inflammation . It is therefore likely that these drugs in combination have hitherto unidentified immunological effects . In the present study we investigated the effects of alpha interferon and ribavirin on dendritic cell (DC) maturation and cytokine production induced by double-stranded RNA in vitro . Alpha interferon alone enhanced the expression of HLA class I, HLA class II, and CD86 on immature DCs but did not stimulate full DC maturation, which requires the expression of CD83 . Alpha interferon enhanced the production of interleukin 12 p70 [IL-12(p70)] and tumor necrosis factor alpha (TNF-  ) but had no effect on IL-10 production . In contrast, ribavirin at physiological doses had no effect on DC maturation but markedly suppressed the production of TNF-, IL-10, and IL-12(p70) . The suppression of cytokines by ribavirin cannot be explained by the induction of DC apoptosis or cell death . Quantitative PCR confirmed that cytokine suppression occurs at the level of mRNA . The suppression of IL-12(p70) and TNF- in maturing DCs may explain the reduction in hepatic inflammation observed during ribavirin monotherapy . Combination alpha interferon-ribavirin therapy may alter the cytokine profile of maturing DCs overall by suppressing IL-10 production but maintaining IL-12(p70) and TNF- production, a pattern that would favor viral elimination through downstream effects on T cells .
Gene Islands Integrated into tRNAGly Genes Confer Genome Diversity on a Pseudomonas aeruginosa Clone. Karen D. Larbig, 2002.Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P . aeruginosa chromosome . The ca . 110-kb large hypervariable region located near the lipH gene in two members of the predominant P . aeruginosa clone C, strain C and strain SG17M, was sequenced . In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs . The gene islands are integrated into conserved tRNAGly genes and have a bipartite structure . The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance . The second part is made up mostly of ORFs of yet-unknown function . Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands . We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat . Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts. Tillmann Lueders, 2003.Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes . It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP . In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis . PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens . Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments . SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool . SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers . Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used . Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias . In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool .
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