Project description:The ability to identify a robust means for both pediatric and adult liver transplant recipients for minimization and weaning of immunosuppression is greatly needed. We analyzed 298 samples from pediatric (n=83) and published adult (n=57) liver transplant recipients, and the public available cell or tissue specific samples (n=158). Biomarkers were first identified from the integration of the cross-platform analysis of Stanford pediatric and adult studies and validated in independent samples from UCLA on microarray. Furthermore, Q-PCR was performed for targets validation and delivers a robust diagnostic assay of predicting potential liver tolerance after liver transplantation. Thirteen unique genes were identified (FDR<5%) using nearest shrunken centroid classification methods as a minimum gene set to cross-validate and predict Stanford pediatric tolerance samples with 1misclassification, 5 misclassification for adult samples, and 100% sensitivity and 83% specificity for an independent UCLA pediatric samples in microarray platform. These subset of tolerant specific genes are highly expressed CD56+ natural killer cells (p=0.03). Furthermore, the subset can be narrowed down to 3 key genes with combined ROC=0.988 for the liver tolerance prediction from Q-PCR verification and 64% MIS and STA were predicted as tolerance with the >90% prediction probability. Specific peripheral transcriptional programs can be identified in operational tolerance in pediatric recipients of liver allografts, distinct from those previously identified in adult operationally tolerant liver recipients, and may provide a means to non-invasively monitor patients in a serial manner for immunosuppression minimization. These genes are highly expressed in specific peripheral blood lymphocyte subsets, and their coordinated may support the maintenance of operational tolerance in children, following liver transplantation.
Project description:The ability to identify a robust means for both pediatric and adult liver transplant recipients for minimization and weaning of immunosuppression is greatly needed. We analyzed 298 samples from pediatric (n=83) and published adult (n=57) liver transplant recipients, and the public available cell or tissue specific samples (n=158). Biomarkers were first identified from the integration of the cross-platform analysis of Stanford pediatric and adult studies and validated in independent samples from UCLA on microarray. Furthermore, Q-PCR was performed for targets validation and delivers a robust diagnostic assay of predicting potential liver tolerance after liver transplantation. Thirteen unique genes were identified (FDR<5%) using nearest shrunken centroid classification methods as a minimum gene set to cross-validate and predict Stanford pediatric tolerance samples with 1misclassification, 5 misclassification for adult samples, and 100% sensitivity and 83% specificity for an independent UCLA pediatric samples in microarray platform. These subset of tolerant specific genes are highly expressed CD56+ natural killer cells (p=0.03). Furthermore, the subset can be narrowed down to 3 key genes with combined ROC=0.988 for the liver tolerance prediction from Q-PCR verification and 64% MIS and STA were predicted as tolerance with the >90% prediction probability. Specific peripheral transcriptional programs can be identified in operational tolerance in pediatric recipients of liver allografts, distinct from those previously identified in adult operationally tolerant liver recipients, and may provide a means to non-invasively monitor patients in a serial manner for immunosuppression minimization. These genes are highly expressed in specific peripheral blood lymphocyte subsets, and their coordinated may support the maintenance of operational tolerance in children, following liver transplantation. Two centers data was included, which are 41 samples from Stanford as the training set and 44 samples from UCLA as the test set. 26 samples from Stanford and 15 samples from UCLA were used in microarray Agilent whole human genome microarray
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:The goal is to identify new molecules implicated in tolerance, to determine the implication of these molecules in immune responses to transplantation by gene expression comparison of 27,088 individual rat genes between tolerated kidney allotransplant and syngeneic kidney transplant.