Quantitative Detection Of Immune Effector Molecules And Its Application To Acute Rejection Of Human Renal Allografts

To defne the relationship between gene expression of immune effector molecules and acute rejection of human renal allografts, the magnitude of Fas,FasL,GB,P and TIA-1 mRNA were quantfied by competitive PCR in samples obtained for surveillance or to dignose the etiology of graft dysfunction.For this purpore, we developed a multispecific control fragment that consisted of the complementary 5' and 3' primer sequences of the 5 genes and f3 -actin gene, the fragment was cloned in PKF3.First, intragraft gene transcript levels were determined, results were compared with the histological diagnosis based upon Banff criteria. A constant volume of sample-derived RT-cDNA was co-amplified with series dilutions of known amount of each competitive internal control, the PCR products were seperated by agarose gel electrophoresis, and the ratio of densities reflected the initial amount of cDNA, all results were expressed as the ratio of picograms of target gene expression to picograms of 13 -actin. The study demonstrated that: (l)Intragraft expression of FasL, GB,P and TIA-1 but not Fas was significantly heightened during acute rejection compared with chronic and no evidence of rejection, expression of Fas was similar among different categories. (2)A direct correlation existed between the histological severity of acute rejection and the expression of mRNA encording genes with the exception of Fas. (3)A simutaneous PCR evaluation of FasL,GB and hA-I identified acute rejection with extrodinary Sensitivity and specificity, especially when any two of the three genes were simultaneously up-regulated.Using this PCR-based molecular approach,we have also analgzed FasL,GB,P and TIA-1 genes expression from peripheral blood leukocytes, a pattern similar to that of tissues was shown. In so doing, we have established a noninvasive means of monitoring for acute rejection.Early posttransplant surveilance specimens of peripheral blood leukocytes revealed that FasL,GB,P and TIA- 1 mRNA levels were slightly higher and showed a tendency to decline within the first week, the expression was probably predictive for later development of rejection episodes. The magnitude of genes associated with acute rejection apparently went down after initiation of effective antirejection therapy.