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BACKGROUND AND AIMS TOMOGRAM, a European multicenter study, recently identified a new cohort of kidney transplant recipients (KTR) with operational tolerance (OT) with the aim to identify new transcriptomic signatures of OT. METHOD RNA sequencing of peripheral blood was evaluated in 15 OT KTR identified by TOMOGRAM, 23 stable KTR (≥15 years, STA), 14 KTR with transplant glomerulopathy (≥1 year, CR), 14 CyA-treated primary GN patients and 14 healthy controls (HC). The ability to discriminate OT from others was analyzed using three classifiers (GLMNET, voomNSC and SVM-RFE) with 10-fold cross-validation and visualized by principal component analysis (PCA) of molecular archetypes. RESULTS Peripheral blood transcriptome of OT patients is similar to the STA group (AUC 0.8). Also, in PCA of molecular archetypes, direction of correlation suggested similarity between STA and OT and their anti-correlation to CR (Figure 1). Top 10 transcripts differentiated OT from CR were assessed in prospective independent cohort (n = 396) to predict premature graft loss. Multivariable Cox regression model based on the expression of 5 of those transcripts at 3 time-points was associated with graft loss at 3 years with an AUC = 0.815. CONCLUSION Identification of the OT peripheral molecular signature among stable KTR is not feasible. Instead, novel peripheral molecular markers associated with premature graft loss were identified.

The ELISpot assay is a sensitive technique applied to assess cytokine-producing memory/effector T cells and human leukocyte antigens (HLA)-specific IgG-producing B cells. Besides the fact that the method is laborious and is difficult to standardise between laboratories, it may provide valuable information on the immune response of recipients before and after organ transplantation. In this article, we briefly review the recent literature and discuss the clinical significance of the ELISpot assay in predicting the risk and incidence of allograft rejection and survival.

The aim of the study was to characterize by molecular profiling two glomerular diseases: IgA nephropathy (IgAN) and focal segmental glomerulosclerosis (FSGS) and to identify potential molecular markers of IgAN and FSGS progression. The expressions of 90 immune-related genes were compared in biopsies of patients with IgAN (n=33), FSGS (n=17) and in controls (n=11) using RT-qPCR. To identify markers of disease progression, gene expression was compared between progressors and non-progressors in 1 year follow-up. The results were verified on validation cohort of patients with IgAN (n=8) and in controls (n=6) using laser-capture microdissection, that enables to analyze gene expression separately for glomeruli and interstitium. In comparison to controls, patients with both IgAN and FSGS, had lower expression of BAX (apoptotic molecule BCL2-associated protein) and HMOX-1 (heme oxygenase 1) and higher expression of SELP (selectin P). Furthermore, in IgAN higher expression of PTPRC (protein-tyrosine phosphatase, receptor-type C) and in FSGS higher expression of BCL2L1 (regulator of apoptosis BCL2-like 1) and IL18 compared to control was observed. Validation of differentially expressed genes between IgAN and controls on another cohort using laser-capture microdissection confirmed higher expression of PTPRC in glomeruli of patients with IgAN. The risk of progression in IgAN was associated with higher expression EDN1 (endothelin 1) (AUC=0.77) and FASLG (Fas ligand) (AUC=0.82) and lower expression of VEGF (vascular endothelial growth factor) (AUC=0.8) and in FSGS with lower expression of CCL19 (chemokine (C-C motif) ligand 19) (AUC=0.86). Higher expression of EDN1 and FASLG along with lower expression of VEGF in IgAN and lower expression of CCL19 in FSGS at the time of biopsy can help to identify patients at risk of future disease progression.

Background Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. Methods In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12 months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3 months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. Results Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B ( GZMB ) transcript at POD 14 and perforin 1 ( PRF1 ) transcript at POD 7. The higher interleukin 2 ( IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14 days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3-month biopsies among groups. Conclusions The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.

BK polyomavirus infection is the important cause of virus-related nephropathy following kidney transplantation. BK virus reactivates in 30%–80% of kidney transplant recipients resulting in BK virus-related nephropathy in 1%–10% of cases. Currently, the molecular processes associated with asymptomatic infections in transplant patients infected with BK virus remain unclear. In this study we evaluate intrarenal molecular processes during different stages of BKV infection. The gene expression profiles of 90 target genes known to be associated with immune response were evaluated in kidney graft biopsy material using TaqMan low density array. Three patient groups were examined: control patients with no evidence of BK virus reactivation (n=11), infected asymptomatic patients (n=9), and patients with BK virus nephropathy (n=10). Analysis of biopsies from asymptomatic viruria patients resulted in the identification of 5 differentially expressed genes (CD3E,CD68,CCR2,ICAM-1, andSKI) (P<0.05), and functional analysis showed a significantly heightened presence of costimulatory signals (e.g.,CD40/CD40L;P<0.05). Gene ontology analysis revealed several biological networks associated with BKV immune control in comparison to the control group. This study demonstrated that asymptomatic BK viruria is associated with a different intrarenal regulation of several genes implicating in antiviral immune response.

Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping, and gene annotation enrichment analysis. Results were further validated using quantitative RT-PCR on the independent larger cohort. Microarray screening proved homogeneity of gene transcripts in hemodialysis patients compared with the transplant cohort and primary hyperparathyroidism; therefore, further experiments were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes database in ribosome structure ( P = 3.70 × 10−18). Next, quantitative RT-PCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF; P < 0.001), calcyclin-binding protein ( CACYBP; P < 0.05), and exocyst complex component 8 ( EXOC8; P < 0.05) and lower expression of peptidylprolyl cis/ trans-isomerase and NIMA-interacting 1 (PIN1; P < 0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. In conclusion, our study suggests the RANGRF transcript, which controls RNA metabolism, to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript, along with PIN1 transcript, which influences parathyroid hormone secretion, may represent new therapeutical targets to cure secondary hyperparathyroidism.

BACKGROUND Regulatory T cells have been suggested to down-regulate the alloimmune response. The aim of this prospective open study was to evaluate the effects of different inductive agents on peripheral blood regulatory T cells in kidney transplant patients and to analyse their association with short-term graft outcome. METHODS Regulatory and effector T cell numbers in peripheral blood were determined by flow cytometry in 71 prospectively followed kidney transplant recipients at postoperative day 0, 7, 14, 21, 28, 60 and 90. Patients were treated with a calcineurin inhibitor-based triple immunosuppression with polyclonal rabbit anti-thymocyte globulin (rATG, n = 28), basiliximab, the anti-CD25 monoclonal antibody (n = 18) or without induction (controls, n = 25). Flow cytometry data were correlated to rejection incidence. RESULTS Compared to controls, CD4(+)CD25(+)FoxP3(+) regulatory T-cell expansion among CD4(+) T cells was noticed in the rATG group at all post-transplant time-points by Day 14 (P < 0.001). A significant decrease in Treg frequency (P < 0.001) and concurrently a transient increase of CD4(+)CD25(low/-)FoxP3(+) population were observed in basiliximab-treated patients 7-60 days post-transplantation. Biopsy-proven acute rejection occurred in 16.7% of controls, 10.7% of the rATG group and in 11.1% of the basiliximab group. Higher CD4(+)FoxP3(+)/CD8(+)CD45RA(+)CD62L(-) ratios were observed repeatedly in those patients after basiliximab induction who were rejection free (P < 0.01). CONCLUSIONS In this study, the rATG induction therapy was associated with an expansion of regulatory cells. Sustained high CD4(+)FoxP3(+)/Teff ratios were associated with the absence of rejection after basiliximab induction.

Polyomavirus BK (BKV) is a common human polyomavirus that rarely causes clinical symptoms in immunocompetent individuals. However, BK virus reactivation occurs in 20-40% of kidney transplant patients and 1-10% of cases present with BK virus-associated nephropathy (BKVN) and reduced kidney allograft survival. In this study, 120 consecutive renal allograft recipients were monitored for BK virus replication by real-time PCR (qPCR) in the blood and urine during the first year post-transplantation and risk factors for BK viremia, viruria, and polyoma BKV-associated nephropathy were evaluated. Receiver operating characteristic curve analysis was used to determine the cutoff points for assessing the risk of developing BKVN. In total, 1,243 samples were tested. BK-DNAuria >10(7) copies/ml and BK-DNAemia >10(4) copies/ml were found in 25.8% and 5% of the samples screened, respectively, during the 12 month follow-up period. BKVN was confirmed histologically in 3/120 patients and viremic patients were treated with dialysis for longer time periods and had higher levels of panel [corrected] reactive antibodies. Patients with viruria were also treated longer with dialysis and had impaired graft function 12 months post-transplantation. Patients with sustained viruria exhibited more acute rejection episodes than patients with transient viruria. Using receiver operating characteristic curve analysis, the cutoff point for viremia and viruria was redefined to 10(3) copies/ml serum for BK viremia and a cutoff point of 6.7 × 10(7) copies/ml in urine. In conclusion, polyoma BK viremia and viruria are frequent findings in kidney transplant recipients that warrant intensive monitoring as a means of preventing graft failure [corrected].

On the basis of reviewed literature here we describe models of tolerance and summarize the evidence of circulating biomarkers suitable for the assessment of immunological risk in organ transplantation. We focused on results of evaluation of specific peripheral immune cell populations and transcripts in peripheral blood of operationally tolerant liver and kidney transplant recipients. Validation of described markers to define potentially tolerant patients before their use in clinical trials is critical.

Background. Induction therapy is associated with excellent short-term kidney graft outcome. The aim of this study was to evaluate differences in the intragraft transcriptome after successful induction therapy using two rabbit antithymocyte globulins. Methods. The expression of 376 target genes involved in tolerance, inflammation, T- and B-cell immune response, and apoptosis was evaluated using the quantitative real-time reverse-transcriptase polymerase chain reaction (2 ― ΔΔCt ) method in kidney graft biopsies with normal histological findings and stable renal function, 3 months posttransplantation after induction therapy with Thymoglobulin, ATG-Fresenius S (ATG-F), and a control group without induction therapy. Results. The transcriptional pattern induced by Thymoglobulin differed from ATG-F in 18 differentially expressed genes. Down-regulation of genes involved in the nuclear factor- K B pathway (TLR4, MYD88, and CD209), costimulation (CD80 and CTLA4), apoptosis (NLRP1), chemoattraction (CCR10), and dendritic cell function (CLEC4C) was observed in the biopsies from patients treated with Thymoglobulin. A hierarchical clustering analysis clearly separated the Thymoglobulin group from the ATG-F group, while the control group had a similar profile as the Thymoglobulin group. Conclusions. Despite normal morphology in graft biopsy taken 3 months posttransplantation, the intrarenal transcriptome differed in patients treated with induction therapy using different rATGs. In the Thymoglobulin high-risk group, the transcriptome profile was identical to the low-risk group. Therefore, the down-regulation of the nuclear factor- K B pathway after Thymoglobulin induction in vivo is likely to explain the clinical success of this biologic.

Cytomegalovirus (CMV) infection influences both short and long term outcomes in immunosuppressed organ transplant recipients. The aim of this study was to evaluate the effect of different induction immunosuppression regimens on CMV specific T cell response in patients with already established CMV immunity. In 24 seropositive living donor kidney recipients, the frequency of CMV specific T cells was determined by ELISPOT (Enzyme-Linked ImmunoSpot) assay prior and 6 months after transplantation. Recipients’ peripheral blood mononuclear cells were stimulated with immediate-early (IE1) and phosphoprotein 65 (pp65) CMV-derived peptide pools and the number of cells producing interferon gamma (IFN-γ) was assessed. Patients received quadruple immunosuppression based either on depletive rabbit antithymocyte globulin (rATG) or non-depletive basiliximab induction and tacrolimus/mycophenolate mofetil/steroids. Patients with rATG induction received valgancyclovir prophylaxis. No effects of different induction agents on CMV specific T cell immunity were found at sixth month after kidney transplantation. There were no associations among dialysis vintage, pretransplant CMV specific T cell immunity, and later CMV DNAemia. Similarly, no effect of CMV prophylaxis on CMV specific T cell immunity was revealed. This study shows no effect of posttransplant immunosuppression on CMV specific T cell immunity in living donor kidney transplant recipients with CMV immunity already established, regardless of lymphocyte depletion and CMV prophylaxis.