Project description:Acute allograft rejection is a leading cause for the failure of organ allotransplantation. Identifying the genes involved in the rejection process provides clues to study the mechanisms, and to provide specific gene targets for monitoring, predicting and preventing acute allograft rejection. Using a mice model of skin acute allograft rejection and SAGE method, we analyzed gene expression in the CD4+ T cells of the mice, the cell type known to play critical roles in acute allograft rejection. Our study identifies 402 SAGE tags significantly different from these from the control. From these SAGE tags, we identified 91 increasingly and 85 decreasingly expressed genes, and many genes have not been linked with acute allograft rejection before. Functional classification of these genes shows that apoptosis, transcription regulation, cell growth and maintenance and signal transduction are among the most frequently changed functional groups. Our study provides a genome-wide view for the genes involving acute allograft rejection in the CD4+ T cells, and indicates that acute allograft rejection involves multiple genes in different functional categories. The genes identified from the study provide candidates for further studying the mechanisms and for monitoring, predicting and preventing acute allograft rejection. Female mice of 6-10 week age were used for the study. BALB/c mice (H-2d) and BALB/c severe combined immunodeficient (SCID) mice were from the animal facility of Shandong University (Jinan, China). C57BL/6 (H-2b, B6) were from Vitalriveri Co. ltd (Beijing, China). Mice were maintained in a pathogen-free animal environment during the experimental process. The use of mice for the study was approved by the Institutional Animal Experimental Committee, and animal care and surgical procedures were performed in compliance with the standard animal experimental protocols of Shandong University School of Medicine. The process of generating skin acute allograft rejection followed the established procedure (12). Briefly, dorsal skin of C57BL/6 was transplanted onto the dorsal thorax of BALB/c SCID mice under sterile condition (allotransplant). As a control, dorsal skin of BALB/c mice was transplanted to BALB/c SCID mice (autotransplant). After 28 days of transplantation, CD4+ T cells were harvested from the spleens of BALB/c mice by using the mouse CD4+ T cells enrichment columns (R&D) with the purity >90% as measured by flow cytometry and 8x106 purified CD4+ T cells were adoptively injected into each transplanted mice via the lateral tail vein. The skin graft in each transplanted mice was then monitored daily. The skin graft was considered to fully reject when more than 50% of the skin became necrosis. SAGE Analysis Upon 14 days post CD4+ T cell transferring, CD4+ T cells were collected from the spleens of five mice in each group with adaptive CD4 T cell transferring using the same process described above. Total RNA was extracted from the purified cells by using Trizol reagent (Invitrogen) and mRNA was purified from the total RNA using oligo dT beads (Invitrogen). cDNA was synthesized by using MMLV reverse transcriptase and oligo(dT) primers (MBI-add full name). SAGE libraries from 4 allotransplant and autotransplant groups were constructed following the standard procedure (13). For each SAGE library, 1,200 sequences were collected through an ABI 3730 DNA sequencer (Applied BioSystems). SAGE tags were extracted from the sequences by using the SAGE2000 software (Invitrogen).

Project description:CONTEXT Slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms are thought to contribute, but the most promising targets for timely intervention have not been identified. OBJECTIVE In the current study we seek to determine the driving force behind progressive histological damage of renal allografts, without the interference of donor pathology, delayed graft function and acute graft rejection. DESIGN We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. PATIENTS Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection. RESULTS In this highly cross-validated study, we demonstrate the significant association of established, ongoing and future chronic histological damage with regulation of adaptive immune gene expression (T-cell and B-cell transcript sets) and innate immune response gene expression (dendritic cell, NK-cell, mast cell and granulocyte transcripts). We demonstrate the ability of gene expression analysis to perform as a quantitative marker for ongoing inflammation with a wide dynamic range: from subtle subhistological inflammation prior to development of chronic damage, over moderate subclinical inflammation associated with chronic histological damage, to marked inflammation of Banff-grade acute T-cell mediated rejection. CONCLUSION Progressive chronic histological damage after kidney transplantation is associated with significant regulation of both innate and adaptive immune responses, months before the histological lesions appear. This study therefore corroborates the hypothesis that quantitative inflammation below the diagnostic threshold of classic T-cell or antibody-mediated rejection is associated with early subclinical stages of progressive renal allograft damage. We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection.

Project description:BACKGROUND: Assessment of gene expression in peripheral blood may provide a noninvasive screening test for allograft rejection. We hypothesized that changes in peripheral blood expression profiles would correlate with biopsy-proven rejection and would resolve after treatment of rejection episodes. METHODS AND RESULTS: We performed a case-control study nested within a cohort of 189 cardiac transplant patients who had blood samples obtained during endomyocardial biopsy (EMB). Using Affymetrix HU133A microarrays, we analyzed whole-blood expression profiles from 3 groups: (1) control samples with negative EMB (n=7); (2) samples obtained during rejection (at least International Society for Heart and Lung Transplantation grade 3A; n=7); and (3) samples obtained after rejection, after treatment and normalization of the EMB (n=7). We identified 91 transcripts differentially expressed in rejection compared with control (false discovery rate <0.10). In postrejection samples, 98% of transcripts returned toward control levels, displaying an intermediate expression profile for patients with treated rejection (P<0.0001). Cluster analysis of the 40 transcripts with >25% change in expression levels during rejection demonstrated good discrimination between control and rejection samples and verified the intermediate expression profile of postrejection samples. Quantitative real-time polymerase chain reaction confirmed significant differential expression for the predictive markers CFLAR and SOD2 (UniGene ID No. 355724 and No. 384944). CONCLUSIONS: These data demonstrate that peripheral blood expression profiles correlate with biopsy-proven allograft rejection. Intermediate expression profiles of treated rejection suggest persistent immune activation despite normalization of the EMB. If validated in larger studies, expression profiling may prove to be a more sensitive screening test for allograft rejection than EMB. Experiment Overall Design: Case- control study with three groups. Patients with rejection (r1-r7), follow-up samples after treatment of rejection (post1-7), and controls with no rejection (con1-7)

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