Project description:We performed total RNA-Seq and compared expression levels of genes of whole blood cells isolated from patients after kidney transplantation with stable graft function, antibody mediated- and t cell mediated graft rejection. Overall design: Whole blood cells were isolated from 6 patients with stable graft function, 6 patients with histologically verified antibody mediated graft rejection episode and 4 patients with histologically verified T cell mediated graft rejection after kidney transplantation. Total RNA was extracted and cDNA libraries for total RNA sequencing were generated using “TruSeq® Stranded Total RNA Library” kit (Illumina, San Diego, CA, USA).
Project description:Investigation of whole genome gene expression level changes in Inflammatory bowel disease rats after MSC transplantation, compared to IBD control rats, and to explore the mechanism of MSC transplantation. A four chip study using total RNA recovered from two separate IBD rats after MSC transplantation and two separate IBD control rats. Each chip measures the expression level of 26,419 genes from normal rat and IBD rat treated with MSC transplantation.
Project description:We have studied the genes activated in human liver transplantation to identify potential target genes for the prevention or treatment of related injuries. In a first protocol, in order to evaluate the effect of Ischemia-Reperfusion Injury (IRI) on gene expression profile, we compared gene expression levels in transplanted-reperfused livers versus basal values in donor livers, identifying 795 genes significantly modified in human liver after transplantation. Some genes are likely to be completely activated by IRI, as they are not expressed at all in basal livers. In a second protocol, in order to identify gene dysregulations already present in donor livers, which might affect gene expression profile after transplantation, gene expression evaluated in the first study was compared to control livers (espression data retrieved from ArrayExpress database). About 900 genes in donor livers are dysregulated if compared to the control condition. At least 400 of them remain dysregulated or become more and more dysregulated after transplantation. Experiment Overall Design: For the first protocol, two biopsies were collected from each liver: 1 biopsy before explantation from the donor, immediately after opening, before ice was applied, and 1 biopsy about 2-3 hours after liver reperfusion in the recipient organism. Gene expression of 5 transplanted livers was compared to that of 5 donor livers. Experiment Overall Design: In the second protocol gene expression both of transplanted and donor livers was compared to gene expression data from 5 control livers retrieved from ArrayExpress repository SAMPLE ID: E-AFMX-11
Project description:Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferases (DNMT) inhibitor which induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in G0-G1 phase and decreasing the production of proinflammatory cytokines such as TNFα and IFNγ. This effect was not due to a pro-apoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes which induce cell growth arrest, such as DCUN1D2, U2AF2, GADD45B or p53. In spite of being also up-regulated, we did not find any effect of 5-azaC on the methylation pattern of FOXP3. Finally, the administration of 5-azaC at 60 and 84 hours post-transplant prevented the development of GVHD leading to a significant increase in survival in a fully mismatched BMT mouse model. In conclusion, the current study shows the effect of 5-azaC in T-lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways which must be explored in order to prevent graft-versus-host disease. Overall design: In order to elucidate the mechanisms involved in the effect of 5-azaC on T-cells we performed Affymetrix gene expression assays in T-cells untreated versus treated with 5-azaC at 1 μM after 2 and 4 days of culture, making a total of 12 samples (coming from time-series studies of 3 healthy individuals)
Project description:We have studied the genes activated in human liver transplantation to identify potential target genes for the prevention or treatment of related injuries. In a first protocol, in order to evaluate the effect of Ischemia-Reperfusion Injury (IRI) on gene expression profile, we compared gene expression levels in transplanted-reperfused livers versus basal values in donor livers, identifying 795 genes significantly modified in human liver after transplantation. Some genes are likely to be completely activated by IRI, as they are not expressed at all in basal livers. In a second protocol, in order to identify gene dysregulations already present in donor livers, which might affect gene expression profile after transplantation, gene expression evaluated in the first study was compared to control livers (espression data retrieved from ArrayExpress database). About 900 genes in donor livers are dysregulated if compared to the control condition. At least 400 of them remain dysregulated or become more and more dysregulated after transplantation. Overall design: For the first protocol, two biopsies were collected from each liver: 1 biopsy before explantation from the donor, immediately after opening, before ice was applied, and 1 biopsy about 2-3 hours after liver reperfusion in the recipient organism. Gene expression of 5 transplanted livers was compared to that of 5 donor livers. In the second protocol gene expression both of transplanted and donor livers was compared to gene expression data from 5 control livers retrieved from ArrayExpress repository SAMPLE ID: E-AFMX-11
Project description:Immune responses induced by ongoing and/or past infections prevent the establishment of transplantation tolerance in experimental animal models. How host-pathogen interactions influence allograft tolerance in humans has not been investigated before. The spontaneous development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection constitutes a unique setting to address this question. We conducted a clinical trial of immunosuppression withdrawal in stable HCV-infected liver recipients to elucidate: i) the mechanisms through which allograft tolerance is established in the presence of an ongoing inflammatory response; and ii) how is influenced by anti-HCV heterologous immune responses. Enrolled patients gradually discontinued their immunosuppressive drugs over 6-9 months, and those who maintained normal allograft status 12 months after drug withdrawal were considered operationally tolerant. Successful drug withdrawal was associated with intra-hepatic over-expression of type I interferon and immune-regulatory genes, and correlated with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8+ T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced inflammatory gene expression and the scope of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data indicate that in humans persistent viral infections do not necessarily preclude the development of transplantation tolerance. At least in HCV-infected liver allografts, mechanisms associated with the capacity of the virus to evade adaptive immunity could contribute to the restraining of alloimmune responses and the establishment of transplantation tolerance. Transcriptomic study from the following liver tissue samples: 12 tolerant before immunosuppression (IS), 13 non-tolerant before IS, 4 from non-tolerant at the time of rejection, 13 from tolerant patients 12 months after IS discontinuation. Additionally, 8 liver tissue samples obtained from healthy living liver donors undergoing partial hepatectomy were included as non-transplanted controls.
Project description:Gut microbiota dysbiosis characterizes systemic metabolic alteration, yet its causality is debated. To address this issue, we transplanted antibiotic-free conventional wild-type mice with either dysbiotic (“obese”) or eubiotic (“lean”) gut microbiota and fed them either a NC or a 72%HFD. We report that, on NC, obese gut microbiota transplantation reduces hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non-transplanted mice. Of note, this phenotype is blunted in conventional NOD2KO mice. By contrast, lean microbiota transplantation did not affect hepatic gluconeogenesis. In addition, obese microbiota transplantation changed both gut microbiota and microbiome of recipient mice. Interestingly, hepatic gluconeogenesis, PEPCK and G6Pase activity were reduced even once mice transplanted with the obese gut microbiota were fed a 72%HFD, together with reduced fed glycaemia and adiposity compared to non-transplanted mice. Notably, changes in gut microbiota and microbiome induced by the transplantation were still detectable on 72%HFD. Finally, we report that obese gut microbiota transplantation may impact on hepatic metabolism and even prevent HFD-increased hepatic gluconeogenesis. Our findings may provide a new vision of gut microbiota dysbiosis, useful for a better understanding of the aetiology of metabolic diseases. all livers are from NC-fed mice only. Overall design: 6-wk-old C57Bl/6 male mice were fed a normal chow (NC) for 4 weeks. Mice have been transplanted with the vehicle (reduced PBS), or lean or obese gut microbiota. At the sacrifice by cervical dislocation, livers were dissected and snap-frozen in liquid nitrogen per each mouse.
Project description:Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2-3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. Here we describe a novel site for islet transplantation, the inguinal subcutaneous white adipose tissue. In this site, transplanted islets are engrafted as clusters and function to reverse diabetes in mice. Importantly, transplanted islets can be visualized by CT and are easily retrievable, and allograft rejection is preventable by blockade of co-stimulatory signals. Of much interest, the efficiency of islet transplantation is superior to the liver, with increased mass of transplanted β cells. Furthermore, transplanted human islets function to reverse diabetes in immunodeficient mice. Thus, this adipose tissue site may be ideal for clinical islet transplantation. Overall design: Gene expression of was observed before and after islet translplantation
Project description:The aim of this study was to investigate the role of infiltrating macrophages in renal allograft fibrosis. Forty-six protocol renal allograft biopsies obtained one-year after transplantation were stained with Sirius Red to quantify fibrosis and double stained with CD68 and CD206 to identify the proportion of alternately activated (M2) macrophages. 23 protocol biopsies obtained 12 months post transplant were analyzed for gene expression by microarray, which was correlated with macrophage infiltration and the severity of fibrosis. Phenotypic analysis showed 92% of infiltrating macrophages exhibited an M2 phenotype with CD68+CD206+ dual staining. Gene microarrays demonstrated a distinct alloimmune response despite the lack of rejection and inflammatory infiltrate with upregulation of interferon-γ-response genes. This suggests that following initiation of Th1 driven macrophage proliferation or infiltration, M2 macrophages contribute to tubular injury and progression of fibrosis. 23 protocol renal biopsies were obtained from patients at 12 month post transplant. The study population was divided into two groups according to the number of infiltrating macrophages (CD68 positive cells) (Group I: Recipients with a low number of infiltrating macrophages, CD68 positive cells < 400/mm2; Group II: Recipients with a high number of infiltrating macrophages, CD68 positive cells ≥ 400/mm2). Additional analyses were undertaken by dividing the group into those with fibrosis (ci score >1) and those without. To correlate gene expression with kidney fibrosis, or intensity of CD68 infiltrate, Spearman correlations analysis of the gene expression data with 12 month IFTA was performed and the correlation co-efficiency and its p value calculated. Gene Ontology enrichment and IPA pathway and network analysis (Ingenuity System Inc.) were performed on the associated genes. All p-values were two-sided, and p < 0.05 was considered significant.
Project description:The biopsy samples obtained at implantation segregated in 2 distinct groups according to donor origin, with a cluster of 319 unique identified genes higher expressed in DD compared to LD kidneys, and 329 genes lower expressed (false discovery rate <5%). Using pathway analysis software a significant local renal overrepresentation of complement genes in DD implantation biopsies was identified. Complement gene expression in DD kidneys related both to donor death and cold ischemia duration, and was associated with a slower onset of renal allograft function. In post-transplantation protocol biopsies, there was a continued overexpression of complement genes, regardless of donor source. The local renal complement gene expression variability in post-transplantation biopsies correlated with renal graft function. This study demonstrates a significant and clinically relevant local overexpression of complement genes in DD kidneys at engraftment and continuous functionally important regulation of complement gene expression after transplantation, regardless of donor source. Targeted therapy interfering with complement activation is an attractive therapeutic target that deserves further investigation in solid organ transplantation. Experiment Overall Design: A total of 95 human renal allograft protocol biopsies were included in this study, 28 biopsies (14 DD, 14 LD) obtained at implantation prior to revascularization and 67 protocol biopsies obtained after transplantation. Whole genome expression profiles were assessed using microarrays.