ABSTRACT
Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification.
Subject(s)
Polymerase Chain Reaction/methods , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/metabolism , Biomarkers , Biopsy , DNA Primers , Humans , Liver/metabolism , Liver/pathology , RNA-Directed DNA Polymerase/genetics , Reproducibility of Results , Sensitivity and Specificity , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The aim of this work was to investigate the mechanism of spontaneous rat liver allograft tolerance. Liver allografts from a LEW donor into DA recipient (LEW-->DA) or of PVG-->DA were spontaneously tolerated (TOL) across a complete MHC mismatch. In contrast, DA-->LEW or PVG-->LEW liver allografts were rejected in 10 to 15 days (REJ). We examined whether donor cell migration to recipient lymphoid tissues might be associated with TOL. Many donor cells were observed in draining (celiac) lymph nodes (LN) and spleen, reaching a peak on day 1 and then decreasing rapidly thereafter. Irradiation of liver donors, which we have previously shown to delete tolerance, significantly reduced the number of donor leukocytes in recipient lymphoid tissues. While this suggested an association between donor cell migration and tolerance, the number, distribution, and type of donor cells in recipient lymphoid tissues of REJ was similar to those of TOL. Expression of cytokine mRNA in LN and spleen showed an early increase in the expression of IL-2 and IFN-gamma mRNA on day 1 and then a rapid decrease to constitutive levels. Spleen and LN levels of IL-6, IL-10, TNF-alpha, or TGF-beta mRNA showed much less up-regulation than IL-2 or IFN-gamma. Paradoxically, there was greater expression of IL-2 and IFN-gamma mRNA in TOL lymphoid tissues than in REJ, and this superinduction was partially prevented by donor irradiation. Superinduction of IL-2 and IFN-gamma was, therefore, more closely associated with TOL than was donor cell migration. This was confirmed by treatment of TOL recipients with a short course of methylprednisolone, which reduced survival of subsequent donor strain skin grafts. This finding has implications for treatment of human liver transplants and is evidence for a novel pathway of transplant tolerance.
