Data on genotypic distribution and linkage disequilibrium of several ANRIL polymorphisms in hemodialysis patients.

A long non-coding RNA called ANRIL located on chromosome 9p21.3 has been identified as a novel genetic factor associated with cardiovascular disease. Investigation of several single nucleotide polymorphisms (SNPs) of Noncoding Antisense RNA in the INK4 Locus (ANRIL) gene are of particular interest. This article reports data related to the research article entitled: "Association of ANRIL gene polymorphisms with major adverse cardiovascular events in hemodialysis patients" (Arbiol-Roca et al. [1]). Data presented show the genotypic distribution of four selected ANRIL SNPs: rs10757278, rs4977574, rs10757274 and rs6475606 in a cohort constituted by 284 hemodialysis patients. This article analyzes the Hardy-Weinberg disequilibrium of each studied SNP, and the linkage disequilibrium between them.

Association of ANRIL gene polymorphisms with major adverse cardiovascular events in hemodialysis patients.

BACKGROUND: Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD). Single nucleotide polymorphisms (SNPs) in ANRIL gene have been associated with higher cardiovascular morbidity and mortality in general population. The main objective was to ascertain whether ANRIL polymorphisms could identify risk of major adverse cardiovascular event (MACE) in patients starting on hemodialysis (HD). METHODS: This was a prospective observational cohort study. 284 CKD patients starting on HD were included in the study and followed until achievement of the primary end-point (MACE) or end of the study. All patients were genotyped for four ANRIL SNPs (rs10757278, rs4977574, rs10757274 and rs6475606). Kaplan-Meier curves and multivariate Cox survival analyses, together with multiple logistic regression were used to analyze the association between ANRIL SNPs and MACE. RESULTS: We found that ANRIL SNP rs10757278 was a representative SNP of a strong linkage disequilibrium block and showed significant genotypic associations with MACE in hemodialysis patients. Homozygous patients for the risk allele (GG) showed 2.17 (1.05-4.49) fold increased risk of MACE during hemodialysis than carriers of the protective allele (AA or AG). Diabetes mellitus was a strong enhancer of this effect. CONCLUSIONS: Our results indicate that ANRIL polymorphisms may confer risk to development of MACE in incident patients on hemodialysis.

Complete Regression of Psoriatic Arthritis After Belatacept Conversion in a Highly HLA-Sensitized Kidney Transplant Patient.

Costimulatory inhibitors (i.e. abatacept and belatacept) effectively abrogate T lymphocyte activation and proliferation and have been shown to be effective for disease control in certain autoimmune disorders as well as in preventing allograft rejection in kidney transplantation. Whether such immunomodulatory agents may be useful for the control of autoimmune flares and allograft acceptance, while avoiding the need of additional strong immunosuppressants, has not been shown. Here, we report the first case of a 47-year-old man affected by a serious debilitating form of psoriatic arthritis that presented during the course of a third, high immunological-risk kidney transplantation. Three years after transplantation, the patient benefited by switching from tacrolimus- to belatacept-based therapy, without additional immunosuppression, by showing complete regression of the arthritic symptoms as well as no progression of severe radiological lesions, which leaded to the recovery of disability and functional impairment. Remarkably, the treatment with belatacept in association with mycophenolate mofetil and steroids also provided a stable normal allograft function over time and abrogated the development of de novo circulating donor-specific alloantibodies after 4 years of follow-up.

Long-Term Outcomes in Belatacept- Versus Cyclosporine-Treated Recipients of Extended Criteria Donor Kidneys: Final Results From BENEFIT-EXT, a Phase III Randomized Study.

In the Belatacept Evaluation of Nephroprotection and Efficacy as First-Line Immunosuppression Trial-Extended Criteria Donors (BENEFIT-EXT), extended criteria donor kidney recipients were randomized to receive belatacept-based (more intense [MI] or less intense [LI]) or cyclosporine-based immunosuppression. In prior analyses, belatacept was associated with significantly better renal function compared with cyclosporine. In this prospective analysis of the intent-to-treat population, efficacy and safety were compared across regimens at 7 years after transplant. Overall, 128 of 184 belatacept MI-treated, 138 of 175 belatacept LI-treated and 108 of 184 cyclosporine-treated patients contributed data to these analyses. Hazard ratios (HRs) comparing time to death or graft loss were 0.915 (95% confidence interval [CI] 0.625-1.339; p = 0.65) for belatacept MI versus cyclosporine and 0.927 (95% CI 0.634-1.356; p = 0.70) for belatacept LI versus cyclosporine. Mean estimated GFR (eGFR) plus or minus standard error at 7 years was 53.9 ± 1.9, 54.2 ± 1.9, and 35.3 ± 2.0 mL/min per 1.73 m2 for belatacept MI, belatacept LI and cyclosporine, respectively (p < 0.001 for overall treatment effect). HRs comparing freedom from death, graft loss or eGFR <20 mL/min per 1.73 m2 were 0.754 (95% CI 0.536-1.061; p = 0.10) for belatacept MI versus cyclosporine and 0.706 (95% CI 0.499-0.998; p = 0.05) for belatacept LI versus cyclosporine. Acute rejection rates and safety profiles of belatacept- and cyclosporine-based treatment were similar. De novo donor-specific antibody incidence was lower for belatacept (p ≤ 0.0001). Relative to cyclosporine, belatacept was associated with similar death and graft loss and improved renal function at 7 years after transplant and had a safety profile consistent with previous reports.

Contribution of Population Pharmacokinetics to Dose Optimization of Ganciclovir-Valganciclovir in Solid-Organ Transplant Patients.

Treatment of solid-organ transplant (SOT) patients with ganciclovir (GCV)-valganciclovir (VGCV) according to the manufacturer's recommendations may result in over- or underexposure. Bayesian prediction based on a population pharmacokinetics model may optimize GCV-VGCV dosing, achieving the area under the curve (AUC) therapeutic target. We conducted a two-arm, randomized, open-label, 40% superiority trial in adult SOT patients receiving GCV-VGCV as prophylaxis or treatment of cytomegalovirus infection. Group A was treated according to the manufacturer's recommendations. For group B, the dosing was adjusted based on target exposures using a Bayesian prediction model (NONMEM). Fifty-three patients were recruited (27 in group A and 26 in group B). About 88.6% of patients in group B and 22.2% in group A reached target AUC, achieving the 40% superiority margin (P< 0.001; 95% confidence interval [CI] difference, 47 to 86%). The time to reach target AUC was significantly longer in group A than in group B (55.9 ± 8.2 versus 15.8 ± 2.3 days,P< 0.001). A shorter time to viral clearance was observed in group B than in group A (12.5 versus 17.6 days;P= 0.125). The incidences of relapse (group A, 66.67%, and group B, 9.01%) and late-onset infection (group A, 36.7%, and group B, 7.7%) were higher in group A. Neutropenia and anemia were related to GCV overexposure. GCV-VCGV dose adjustment based on a population pharmacokinetics Bayesian prediction model optimizes GCV-VGCV exposure. (This study has been registered at ClinicalTrials.gov under registration no. NCT01446445.).

Use of mycophenolate in ANCA-associated renal vasculitis: 13 years of experience at a university hospital.

INTRODUCTION: Standard therapy with corticosteroids (CS) and cyclophosphamide (CYC) followed by azathioprine has been shown to improve renal and patient survival in ANCA-associated renal vasculitis (rAAV). Mycophenolate mofetil (MF) has been progressively introduced for the treatment of rAAV in the last years because of its immunosuppressive efficacy combined with a lower toxicity profile. In this study, we retrospectively analyse the results of the introduction of MF for maintenance and induction therapy in rAAV in our institution from 2001 to 2013. RESULTS: We reported 67 patients treated with MF as a maintenance treatment, divided by baseline serum creatinine (>500 µmol/L: Group 1 and <500 µmol/L: Group 2) and treatment schedule. Twenty-nine of the 67 patients were also treated with MF as induction treatment, mostly in Group 2. During the follow-up (2 years after the diagnosis) creatinine levels for serum glomerular filtration rate, ANCA titres, C-reactive protein and percentage of haematuria decreased in all groups. In Group 2, parameters and also relapse rates were similar at 24 months in patients treated with CYC or MF as an induction treatment (Subgroups 2a and 2b, respectively). Median dose of MF in maintenance treatment was 1000 mg daily and prednisone dose was tapered to 10 mg daily from Month 3. After 24 months, 82% of patients remained on MF therapy, 18% had discontinued the treatment, seven of them due to medical indication and two because of gastrointestinal intolerance. The percentage of patients that started renal replacement therapy was irregular in Group 1 depending on the subgroup (25-100%), and 10% in Group 2. Adverse effects, such as neutropenia, infections and neoplasia, were more prevalent in groups treated with CYC. CONCLUSION: In conclusion, in our patients with rAAV, MF demonstrated to be an effective and well-tolerated option for maintenance treatment. As an induction treatment, MF seems to be similar to CYC for patients with moderate renal failure in the diagnosis.

Human adipose tissue-derived mesenchymal stem cells abrogate plasmablast formation and induce regulatory B cells independently of T helper cells.

Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B-cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue-derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact-dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19(+) CD27(high) CD38(high) antibody-producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B-cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL-10-producing CD19(+) CD24(high) CD38(high) B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B-cell response in immune disease.

Novel once-daily extended-release tacrolimus (LCPT) versus twice-daily tacrolimus in de novo kidney transplants: one-year results of Phase III, double-blind, randomized trial.

This Phase III randomized trial examined efficacy and safety of a novel once-daily extended-release tacrolimus formulation (LCP-Tacro [LCPT]) versus twice-daily tacrolimus in de novo kidney transplantation. Primary efficacy end point was proportion of patients with treatment failure (death, graft failure, biopsy-proven acute rejection or lost to follow-up) within 12 months. Starting doses were, LCPT: 0.17 mg/kg/day and tacrolimus twice-daily: 0.1 mg/kg/day; 543 patients were randomized, LCPT: n = 268; tacrolimus twice-daily: n = 275. At 12 months treatment failure was LCPT: 18.3% and tacrolimus twice-daily: 19.6%; the upper 95% CI of the treatment difference was +5.27%, below the predefined +10% noninferiority criteria. There were no significant differences in the incidence of individual efficacy events or adverse events. Target tacrolimus trough levels were more rapidly achieved in the LCPT group. Following initial dose, 36.6% of patients in the LCPT group had rapidly attained trough levels within 6-11 ng/mL versus 18.5% of tacrolimus twice-daily patients; majority of tacrolimus twice-daily patients (74.7%) had troughs <6 ng/mL compared with 33.5% in the LCPT group. Overall, cumulative study dose was 14% lower for LCPT. Results suggest that use of once-daily LCPT in de novo kidney transplantation is efficacious and safe. Lower LCPT dose reflects the improved absorption provided by the novel formulation.

Low serum mannose-binding lectin levels are associated with inflammation and apoptosis in early surveillance allograft biopsies.

INTRODUCTION: Mannose-binding lectin (MBL) is a protein of the innate immune system that participates in host defense and the tissue injury/repair process, enhancing the clearance of apoptotic cells by macrophages. The aim is to characterize the relationship between pre-transplant MBL levels, histological lesions and number of apoptotic cells in early surveillance renal allograft biopsies. PATIENTS AND METHODS: Consecutive renal transplant recipients were recruited and MBL levels were classified into tertiles. The first tertile was considered the low MBL group. Surveillance biopsies were done during the first 6 months and were evaluated according to Banff criteria. Renal inflammatory infiltrates were studied by immunohistochemical techniques. Apoptosis was studied using morphological methods in renal tubular cells and was expressed as the number of apoptotic cells/mm(2). RESULTS: MBL was determined in 126 patients and a surveillance biopsy with sufficient tissue was obtained in 41 of them. Patients with low pre-transplant MBL levels showed a higher acute Banff index (3.14 ± 1.96 vs. 1.88 ± 1.56, p = 0.044) and an increased proportion of biopsies with tubular cell apoptosis The proportion of biopsies with tubular cell apoptosis was higher in patients with low pre-transplant MBL levels in comparison with patients with high MBL levels (4.3 ± 3.6 versus 0.2 ± 0.9 p = 0.012) and increased interstitial number of inflammatory cells and significantly the macrophages/mm(2) (109 ± 118 vs. 32 ± 46; p = 0.04). CONCLUSION: Low pre-transplant serum MBL levels are associated with more severe inflammation and increased apoptosis in early surveillance renal allograft biopsies suggesting that MBL modulates renal inflammation after transplantation.

Sterile leukocyturia is associated with interstitial fibrosis and tubular atrophy in kidney allograft protocol biopsies.

Kidney allograft interstitial fibrosis and tubular atrophy (IF/TA) is associated with a poorer renal function and outcome. In the current clinical practice, an early diagnosis can only be provided by invasive tests. We aimed to investigate the association of sterile leukocyturia with Banff criteria histological findings in kidney allograft protocol biopsies. We studied 348 allograft biopsies from two different European countries performed at 8.5 + 3.5 months after transplantation. In these cases, the presence of sterile leukocyturia (Leuc+, n = 70) or no leukocyturia (Leuc-, n = 278) was analyzed and related to Banff elementary lesions. Only IF/TA was significantly different between Leuc+ and Leuc- groups. IF/TA was present in 85.7% of Leuc+ and 27.7% of Leuc- patients (p < 0.001). IF/TA patients had higher serum creatinine and presence of proteinuria (p < 0.05). Independent predictors of IF/TA were donor age, donor male sex, serum creatinine and Leuc+ (hazard ratio 18.2; 95% confidence interval, 8.1-40.7). The positive predictive value of leukocyturia for predicting IF/TA was 85.7% whereas the negative predictive value was 72.3%. These studies suggest that leukocyturia is a noninvasive and low-cost test to identify IF/TA. An early diagnosis may allow timely interventional measures directed to minimize its impact and improve graft outcome.

Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients.

Assessment of donor-specific alloreactive memory/effector T cell responses using an IFN-γ Elispot assay has been suggested to be a novel immune-monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross-validation data of the IFN-γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN-γ plates assessing intra- and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count-settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN-γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen-specific primed/memory T cell alloresponses. Interestingly, significant high donor-specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF-γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.

Pretransplant immediately early-1-specific T cell responses provide protection for CMV infection after kidney transplantation.

Cytomegalovirus (CMV) infection is still a major complication after kidney transplantation. Although cytotoxic CMV-specific T cells play a crucial role controlling CMV survival and replication, current pretransplant risk assessment for CMV infection is only based on donor/recipient (IgG)-serostatus. Here, we evaluated the usefulness of monitoring pre- and 6-month CMV-specific T cell responses against two dominant CMV antigens (IE-1 and pp65) and a CMV lysate, using an IFN-γ Elispot, for predicting the advent of CMV infection in two cohorts of 137 kidney transplant recipients either receiving routine prophylaxis (n = 39) or preemptive treatment (n = 98). Incidence of CMV antigenemia/disease within the prophylaxis and preemptive group was 28%/20% and 22%/12%, respectively. Patients developing CMV infection showed significantly lower anti-IE-1-specific T cell responses than those that did not in both groups (p < 0.05). In a ROC curve analysis, low pretransplant anti-IE-1-specific T cell responses predicted the risk of both primary and late-onset CMV infection with high sensitivity and specificity (AUC > 0.70). Furthermore, when using most sensitive and specific Elispot cut-off values, a higher than 80% and 90% sensitivity and negative predictive value was obtained, respectively. Monitoring IE-1-specific T cell responses before transplantation may be useful for predicting posttransplant risk of CMV infection, thus potentially guiding decision-making regarding CMV preventive treatment.

Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5'-triphosphate-binding cassette transporters far beyond an efflux pump.

This study examines adenosine 5'-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50 ): P-glycoprotein inhibition using valspodar (PSC833) 5 µM, CAS 115104-28-4 (MK571) 50 µM and probenecid 2·5 µM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC-transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia.

Immunomodulatory effect of mesenchymal stem cells on B cells.

The research on T cell immunosuppression therapies has attracted most of the attention in clinical transplantation. However, B cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. Indeed, humoral immune responses can lead to renal allograft rejection even in patients whose cell-mediated immune responses are well controlled. On the other hand, newly studied B cell subsets with regulatory effects have been linked to tolerance achievement in transplantation. Better understanding of the regulatory and effector B cell responses may therefore lead to new therapeutic approaches. Mesenchymal stem cells (MSC) are arising as a potent therapeutic tool in transplantation due to their regenerative and immunomodulatory properties. The research on MSCs has mainly focused on their effects on T cells and although data regarding the modulatory effects of MSCs on alloantigen-specific humoral response in humans is scarce, it has been demonstrated that MSCs significantly affect B cell functioning. In the present review we will analyze and discuss the results in this field.

Long-term results of biopsy-guided selection and allocation of kidneys from older donors in older recipients.

In our old-for-old program, we discard or allocate older extended criteria donor kidneys to single (SKT) or dual kidney transplantation (DKT) depending on histological Remuzzi's score in recipients older than 60 years. Here, we analyze the long-term results of this program and try to identify independent predictors of patient and graft survival. Between December 1996 and January 2008, we performed 115 SKT and 88 DKT. Discard rate was 15%. Acute rejection incidence was higher in SKT than in DKT (22.6% vs. 11.4%, p = 0.04). Renal function was better in DKT than in SKT up to 5 years after transplantation. Surgical complications were frequent in DKT. Ten-year cumulative graft survival was significantly lower in the SKT group (31% vs. 53%, p = 0.03). In SKT, histological score 4 provided similar graft survival than 3 or less, whereas in DKT score 4, 5 or 6 displayed similar outcome. Finally, independent predictors of graft survival were history of major adverse cardiac event and 1-year serum creatinine, rather than SKT or DKT. In conclusion, this biopsy-guided old-for-old strategy resulted in acceptable long-term graft survival. Our results suggest that DKT should be considered for scores of 5 or 6 only.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes.

AIMS/HYPOTHESIS: We previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marrow-derived cells are also involved in this HGF-induced reparative process. METHODS: We have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals. RESULTS: We find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman's capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation. CONCLUSIONS/INTERPRETATION: Altogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Three-year outcomes from BENEFIT-EXT: a phase III study of belatacept versus cyclosporine in recipients of extended criteria donor kidneys.

Recipients of extended-criteria donor (ECD) kidneys have poorer long-term outcomes compared to standard-criteria donor kidney recipients. We report 3-year outcomes from a randomized, phase III study in recipients of de novo ECD kidneys (n = 543) assigned (1:1:1) to either a more intensive (MI) or less intensive (LI) belatacept regimen, or cyclosporine. Three hundred twenty-three patients completed treatment by year 3. Patient survival with a functioning graft was comparable between groups (80% in MI, 82% in LI, 80% in cyclosporine). Mean calculated GFR (cGFR) was 11 mL/min higher in belatacept-treated versus cyclosporine-treated patients (42.7 in MI, 42.2 in LI, 31.5 mL/min in cyclosporine). More cyclosporine-treated patients (44%) progressed to GFR <30 mL/min (chronic kidney disease [CKD] stage 4/5) than belatacept-treated patients (27-30%). Acute rejection rates were similar between groups. Posttransplant lymphoproliferative disorder (PTLD) occurrence was higher in belatacept-treated patients (two in MI, three in LI), most of which occurred during the first 18 months; four additional cases (3 in LI, 1 in cyclosporine) occurred after 3 years. Tuberculosis was reported in two MI, four LI and no cyclosporine patients. In conclusion, at 3 years after transplantation, immunosuppression with belatacept resulted in similar patient survival, graft survival and acute rejection, with better renal function compared with cyclosporine. As previously reported, PTLD and tuberculosis were the principal safety findings associated with belatacept in this study population.