| Background:Solid organ transplantation has become increasingly important as a treatment for end-stage organ function failure. Despite optimal HLA matching and advances in immunosuppressive therapy, acute rejection is one of the key factors which determine long-term graft function and survival in renal transplant patients. Timely detection and treatment of rejection is therefore, an important goal in the post-transplant surveillance. The standard care with serum creatinine measurements and biopsy upon allograft dysfunction implies that acute rejection is detected in an advanced stage. Therefore, rapid noninvasive biomarkers of rejection are needed to improve the management of these patients. Tumor necrosis factor receptor (TNFR)/tumor necrosis factor (TNF) superfamily members can control diverse aspects of immune function. Research over the past10years has shown that one of the most important and prominent interactions in this family are that between OX40(CD134) and its partner OX40L (CD252). Recent work has demonstrated that signaling through the OX40co-stimulatory receptor can augment CD4and CD8T-cell expansion, differentiation, and the generation of memory cells. Objective:In this study, we will focus specifically on OX40and OX40L, to investigate whether testing for OX40and OX40L mRNA may provide a simple, safer, noninvasive, clinically important molecule marker for predicting acute rejection risk after renal transplantation. Also we want to provide new theoretical basement for the induction and maintenance of transplantation immune tolerance through this study. Methods:The first part:We performed a prospective study on40end-stage renal disease patients who received kidney allograft at orient organ transplant center of Tianjin First Central Hospital. Of these,20patients had biopsy-proven acute rejection (acute rejection group) and20patients had the normal renal function (non rejection group). Test of the all patients yielded no evidence of viral infection, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. The control group consisted of20 healthy individuals. The expression levels of OX40, OX40L mRNA in fresh peripheral blood mononuclear cells (PBMCs) were measured by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with three groups, and to compared the diagnostic value of OX40and OX40L for diagnosing acute rejection. The second part:T helper (Th)1(CD4+IFN-y+) cells, Th2(CD4+IL-4+) cells and Th3(CD4+TGF-β+) cells were determined using flow cytometry analysis. To compared the correlations between Th cells and the levels of OX40, OX40L mRNA. The third part:CD95+CD3+cells and CD38+CD8+cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period. The fourth part:The fresh PBMCs were isolated from acute rejection group by Ficoll density gradient centrifugation, and were divided into two subgroups (all cultured in medium containing RPMI1640,10% Fetal bovine serum):①Anti-CD3MoAb stimulated group (anti-CD3MoAb2μg/mL),②Recombinant human OX40-IgGl fusion protein treated group (anti-CD3MoAb2μg/mL+ recombinant human OX40-IgGl fusion protein10μg/mL). We studied Thl, Th2cytokine concentration upon anti-CD3MoAb stimulated group and recombinant human OX40-IgG1fusion protein treated group in acute rejection recepients in vitro. PBMCs were incubated at37℃,5%CO2for48h. The Thl, Th2cytokine concentrations of culture supernatant were detected by Enzyme Linked-Immuno-Sorbent Assay (ELISA). Results:The first part:The levels of OX40mRNA on PBMCs showed significantly elevated in acute rejection group compared with that in non rejection group and normal control group (P<0.001), there was no significant difference of OX40mRNA levels between non acute rejection group and normal control group (P=0.585). The levels of OX40L mRNA on PBMCs showed significantly elevated in acute rejection group compared with that in normal control group (^=0.016), there was no significant difference of OX40L mRNA levels between acute rejection group and non rejection group (P=0.305), and between non rejection group and normal control group (P=0.153). The area under the receiver operating characteristic (ROC) curve in the diagnosis of acute rejection was0.929(95%CI0.860-0.997, P<0.001) for OX40 (best cutoff,7.5×106copy/μL; sensitivity80%; specificity92.5%; positive predictive value (PPV)84.2%; negative predictive value (NPV)90.2%), and0.638(95%CI0.485-0.791, P=0.083) for OX40L (best cutoff,4.2×103copy/μL; sensitivity40%; specificity77.5%; PPV47%; NPV72.1%), respectively. OX40mRNA was considered good predictive marker. The mRNA levels of OX40, OX40L did not significantly correlate with serum creatinine (Cr) levels in acute rejection group. The second part:The percentages of Thl cells (CD4+IFN-y+), Th2cells (CD4+IL-4+), Th3cells (CD4+TGF-β+) were no any significant difference in non rejection group compared with that in normal control group (P=1.000). The percentage of Thl cells were significantly upregulated in acute rejection group compared with that in non rejection group or normal control group (P<0.001). There was no significant difference between Th2cells, Th3cells in peripheral blood in acute rejection group and non rejection group (P=0.307, P=0.990, respectively). There was a positive correlation between the mRNA levels of OX40, OX40L and Thl cells in acute rejection group. The third part:CD95+CD3+cells were drastically increased in the acute rejection group compared with that in non rejection group or in the normal control group (P<0.01), there was no significant difference between non rejection group and normal control group (P>0.05). CD38+CD8+cells were drastically increased in the renal acute rejection group compared with that in the normal control group (P<0.05), there was no significant difference between renal acute rejection group and non rejection group, and between non rejection group and normal control group (P>0.05). There was no significant difference among liver acute rejection group, non rejection group, and normal control group (P>0.05). The fourth part: Determination of Thl, Th2cytokine secretion of the48h of recombinant human OX40-IgG1fusion protein treated group, which showed that the recombinant human OX40-IgGl fusion protein treated group reduced significantly the secretion and production of IFN-y and IL-4in culture supernatant of PBMCs when compared with that with anti-CD3MoAb stimulated group (P<0.05, P<0.01, resprectively). Conclusion:1. This study presents the first evidence that the OX40-OX40L mRNA is associated with acute allograft rejection after renal transplantation. OX40mRNA maybe a safer, noninvasive molecule marker for predicting acute rejection in PBMCs.2. Upregulations of OX40, OX40L mRNA, which increase Thl cells, will cause the acute rejection after renal transplant.3. CD38, CD95molecules may involve the immunological procedures of the transplantation recipients.4. It is indicated that, in vitro, blockage of the interaction of OX40and OX40L is a new strategy to be considered for inducing and keeping transplantation immune tolerance and requires further investigation. |
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