Gene Expression in Highly Sensitised Patients Waiting for a Kidney Transplant: A Real Time - PCR Analysis Approach

The presence of alloantibodies against human HLA class I (A, B, C) and class II (DR, DQ) antigens in transplant recipients waiting for a first or subsequent kidney transplant has a significant negative impact on graft outcome, with increased acute and chronic rejection rates. Higly sensitised patients, displaying a wide antibody reactivity with PRA (panel reactive antibodies) values >85%, are hardly transplantable because of lacking of any window of permissive HLA antigens. In addition to major immunological allostimuli such pregnancies, blood transfusions and previous failed transplants also factors including alterations of apoptotic and anti-inflammatory mechanisms could play some role in determining the hyperimmunization status. In this study we have utilized a real -time PCR array to analyze in hyperimmunized patients, waiting for a first or a subsequent transplant, the expression of a number of cytokine and lymphokine genes as well as of genes responsible for the synthesis of membrane glycoprotein receptors, soluble ligands/ cytoplasmic proteins involved in transduction pathways and activation or apoptotic signals. A custom made real-time PCR array comprising 27 relevant genes and 5 five control genes was purchased from Qiagen. cDNAs were obtained from peripheral blood mononuclear cells of six hyperimmunized dialysis patients and from four non immunized dialysis controls and amplified with Chromo 4 real-time PCR detector (Biorad) by means of SYBR® green dye technology. Data were analysed with a PCR Array data analysis software. Hyperimmunized patients were found to have a significant increase, as compared to controls, in the expression of the following genes: IL-2, IL-9, IL-21, IFN-γ, JAK2, STAT-1,CD28, CD256(APRIL); a significant decrease of the following genes were also detected:TNSF-8 (TNF-related apoptosis inducing ligand),IL10, IL-6, IL-1A. Taken together the above results indicate that coexistence of an hyperexpression of genes involved in T and B cell activation/antibody production and of a downregulation of other genes that control apoptotic and anti-inflammatory pathways can contribute to the establishment of hyperimmunization.