Integrating APOL1 Gene Variants Into Renal Transplantation: Considerations Arising From the American Society of Transplantation Expert Conference.

Thirteen percent of individuals of African ancestry express two variant copies of the gene encoding apolipoprotein 1 (APOL1) that has been associated with an increased risk of end-stage renal disease (ESRD) in the general population. Limited studies suggest that the survival of transplanted kidneys from donors expressing two APOL1 risk alleles is inferior to that of kidneys from donors with zero or one risk allele. In living kidney donation, two case reports describe donors expressing two APOL1 risk alleles who developed ESRD. Given the potential impact of APOL1 variants on the utility and safety of kidney transplantation and living kidney donation, the American Society of Transplantation convened a meeting with the goals of summarizing the current state of knowledge with respect to transplantation and APOL1, identifying knowledge gaps and studies to address these gaps, and considering approaches to integrating APOL1 into clinical practice. The authors recognize that current data are not sufficient to support traditional evidence-based guidelines but also recognize that it may require several years to generate the necessary data. Thus, approaches as to how APOL1 might currently be integrated into the clinical decision-making process were considered. This report summarizes the group's deliberations.

APOL1 Polymorphisms in a Deceased Donor and Early Presentation of Collapsing Glomerulopathy and Focal Segmental Glomerulosclerosis in Two Recipients.

Two common polymorphisms in APOL1 (G1 and G2) are conserved in persons of African ancestry, and the presence of two polymorphisms (commonly referred to as risk variants) has been identified as a risk factor for chronic kidney disease and focal seg-mental glomerulosclerosis. In kidney transplantation, deceased donors with two APOL1 risk variants carry an increased risk of renal allograft failure in the recipient. An emerging question is whether these data should influence deceased donor assessment or be used to refine prediction of allograft survival. We present the first detailed report of two cases of recipient glomerular disease in the first year following transplant from a deceased donor later defined as carrying two APOL1 risk variants. A possible "second hit" predisposing to renal disease in these recipients is discussed, one with active cytomegalovirus infection concurrent with collapsing glomerulopathy and renal failure and the other with chronic, slowly healing wound infection and focal segmental glomeru-losclerosis but stable renal function. In retrospect, awareness of the donor APOL1 risk alleles would not have influenced donor selection and ultimately did not influence posttransplant management. These case reports inform further discussion of the value of APOL1 testing for deceased donors.

Clinical Significance of Pretransplant Donor-Specific Antibodies in the Setting of Negative Cell-Based Flow Cytometry Crossmatching in Kidney Transplant Recipients.

Antibodies to donor-specific HLA antigens (donor-specific antibodies [DSA]) detected by single-antigen bead (SAB) analysis prior to kidney transplant have been associated with inferior graft outcomes. However, studies of pretransplant DSA, specifically in the setting of a negative flow cytometry crossmatch (FCXM) without desensitization therapy, are limited. Six hundred and sixty kidney and kidney-pancreas recipients with a negative pretransplant FCXM from September 2007 to August 2012 without desensitization therapy were analyzed with a median follow-up of 4.2 years. All patients underwent cell-based FCXM and SAB analysis on current and historic sera prior to transplantation. One hundred and sixty-two patients (24.5%) had DSA detected prior to transplant. One-year acute rejection rates were similar in DSA-positive versus DSA-negative patients (15.4% vs. 11.4%, respectively; p = 0.18) and were higher in those with DSA mean fluorescence intensity (MFI) greater than or equal to 3000 in multivariable analysis (p = 0.046). The estimated glomerular filtration rate (eGFR) at 3 and 4 years was lower in the DSA(+) versus the DSA(-) group (p = 0.050 at 3 years) without an impact on 5-year death-censored graft survival (89.0% vs. 90.6%, respectively; p = 0.53). Timing (current or historic) of DSA detection did not alter these findings. In conclusion, pretransplant DSA in the setting of a negative FCXM confers minimal immunologic risk in the intermediate term, does not necessitate desensitization therapy and should not represent a barrier to renal transplant.

First-in-human study of the safety and efficacy of TOL101 induction to prevent kidney transplant rejection.

TOL101 is a murine IgM mAb targeting the αß TCR. Unlike other T cell targets, the αß TCR has no known intracellular signaling domains and may provide a nonmitogenic target for T cell inactivation. We report the 6-month Phase 2 trial data testing TOL101 in kidney transplantation. The study was designed to identify a dose that resulted in significant CD3 T cell modulation (<25 T cell/mm(3) ), to examine the safety and tolerability of TOL101 and to obtain preliminary efficacy information. Thirty-six patients were enrolled and given 5-10 daily doses of TOL101; 33 patients completed dosing, while three discontinued after two doses due to a self-limiting urticarial rash. Infusion adjustments, antihistamines, steroids and dose escalation of TOL101 reduced the incidence of the rash. Doses of TOL101 above 28 mg resulted in prolonged CD3 modulation, with rapid recovery observed 7 days after therapy cessation. There were no cases of patient or graft loss. Few significant adverse events were reported, with one nosocomial pneumonia. There were five biopsy-confirmed acute cellular rejections (13.9%); however, no donor-specific antibodies were detected. Overall TOL101 was well-tolerated, supporting continued clinical development using the dose escalating 21-28-42-42-42 mg regimen.

Molecular classifiers for acute kidney transplant rejection in peripheral blood by whole genome gene expression profiling.

There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with excellent function and normal histology (TX; n = 46), AR (n = 63) and ADNR (n = 39), from two independent cohorts were analyzed with DNA microarrays. We applied a new normalization tool, frozen robust multi-array analysis, particularly suitable for clinical diagnostics, multiple prediction tools to discover, refine and validate robust molecular classifiers and we tested a novel one-by-one analysis strategy to model the real clinical application of this test. Multiple three-way classifier tools identified 200 highest value probesets with sensitivity, specificity, positive predictive value, negative predictive value and area under the curve for the validation cohort ranging from 82% to 100%, 76% to 95%, 76% to 95%, 79% to 100%, 84% to 100% and 0.817 to 0.968, respectively. We conclude that peripheral blood gene expression profiling can be used as a minimally invasive tool to accurately reveal TX, AR and ADNR in the setting of acute kidney transplant dysfunction.

The effect of everolimus versus mycophenolate upon proteinuria following kidney transplant and relationship to graft outcomes.

Although mTOR inhibitor use has been associated with proteinuria in kidney transplant recipients, dose dependency and impact on allograft function are unknown. In a post hoc analysis, we compared rates of proteinuria 3 months posttransplant among everolimus (EVR) and mycophenolate (MPA) treatment arms and used a time-dependent model to correlate the risk of proteinuria to EVR trough levels up to 24 months posttransplant. eGFR and graft loss was compared by proteinuria status at 3 months. Of 833 randomized patients, 24%, 36% and 19% of lower exposure EVR (1.5 mg/day), higher exposure EVR (3.0 mg/day) and MPA-treated patients had proteinuria ≥ 300 mg/g Cr at 3 months, respectively. EVR 1.5 was not associated with an increase in risk of proteinuria (HR 1.20; p = 0.19) unlike EVR 3.0 (HR 1.84; p < 0.001) versus MPA. EVR trough levels >8 ng/mL were significantly associated with proteinuria compared to 3-8 ng/mL (HR 1.86; p < 0.001). Those patients with proteinuria at 3 months and those who developed proteinuria thereafter had lower eGFR and higher graft loss at 24 months, regardless of treatment arm. We identify a dose-dependent effect of EVR with the risk of proteinuria; however, its independent impact upon eGFR and graft survival at 2 years was not evident.

Wound events in kidney transplant patients receiving de novo everolimus: a pooled analysis of three randomized controlled trials.

Data were pooled from three prospective, multicenter trials in which 1996 de novo kidney transplant recipients were randomized to everolimus 1.5 or 3.0 mg or mycophenolic acid (MPA), with cyclosporine and steroids. Wound healing complications reported as adverse events were retrospectively reviewed in a blinded manner. The incidence of wound healing adverse events was 17.6% (351 of 1996) by day 90 and was similar for everolimus 1.5 mg (16.6% [110 of 661]) vs. MPA (14.3% [95 of 665]) (p = 0.255), but higher with everolimus 3.0 mg (21.8% [146 of 670]) (p < 0.001 vs. MPA). Similar results were observed for wound healing complications reported as serious adverse events. The 12-month incidence of lymphocele was 11.2% with everolimus 1.5 mg and 8.9% with MPA (p = 0.171), but lymphocele reported as a serious adverse event were more frequent with everolimus 1.5 mg (6.5% vs. 3.5%; p = 0.012). The hazard ratio (HR) for any wound healing complication vs. MPA was not significantly higher for everolimus <3 ng/mL (HR 1.33; 95% CI 0.94-1.88; p = 0.104), but increased to 1.46 (95% CI 1.12-1.90; p = 0.005) for 3-8 ng/mL and 1.69 (95% CI 1.20-2.38; p = 0.002) for >8 ng/mL. These results suggest that de novo kidney transplant patients receiving an initial everolimus dose of 1.5 mg do not appear to have a pronounced increased risk of wound healing complications vs. patients receiving MPA.

Novel immunosuppressive agents in kidney transplantation.

The last several decades have seen a substantial decrease in the prevalence of acute allograft rejection in kidney transplant recipients, while equivalent improvements in long-term graft function have not been realized. As a result, the primary focus of new immunosuppressive drug development has expanded to include ease of use, improved side effect profiles, and reduced nephrotoxicity in addition to the more traditional goal of improved short-term outcomes. A number of novel drugs are currently under investigation in Phase I, II, or III clinical trials primarily to replace the nephrotoxic but highly effective calcineurin inhibitors. ISA247 (voclosporine) is a cyclosporine (CsA) analog with reduced nephrotoxicity in Phase III study. AEB071 (sotrastaurin), a protein kinase C inhibitor, and CP-690550, a JAK3 inhibitor, are small molecules in Phase II studies. Everolimus is derived from the mTOR inhibitor sirolimus and is in Phase III study. Belatacept is a humanized antibody that inhibits T-cell costimulation and has shown encouraging results in multiple Phase II and III trials. Alefacept and Efaluzimab are humanized antibodies that inhibit T-cell adhesion and are in Phase I and II clinical trials. This article reviews the mechanisms of action as well as published and preliminary results of the Phase I-III clinical trials involving these novel immunosuppressive agents.

Projected long-term demographic trends and aggregate personal saving in the United States.

The potential effect of the changing age distribution of the U.S. population on the level of aggregate saving is examined. "The Modigliani-Brumberg life-cycle model predicts a relationship between age distribution and the aggregate saving ratio via the population growth rate, which affects the ratio of earner/savers to retiree-dissavers. Aging resulting from slower population growth will result in a decline in the aggregate saving rate. This paper utilizes empirical age-saving relations, together with projected long-run population age distributions, to estimate the age distribution effect on aggregate personal saving. Results predict a much smaller decline in the saving ratio than is generated by the basic life-cycle model."

Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML.

Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (allo-HCT) can cure patients with AML in CR. However, relapse after RIC allo-HCT may indicate heterogeneity in the stringency of CR. Strict definition of CR requires no evidence of leukemia by both morphologic and flow cytometric criteria. We re-evaluated 85 AML patients receiving RIC allo-HCT in CR to test if a strict definition of CR had direct implications for the outcome. These patients had leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. Eight (9.4%) had persistent leukemia by flow cytometric criteria at allo-HCT. The patients with immunophenotypic persistent leukemia had a significantly increased relapse (hazard ratio (HR): 3.7; 95% confidence interval (CI): 1.3-10.3, P=0.01) and decreased survival (HR: 2.9; 95% CI: 1.3-6.4, P<0.01) versus 77 patients in CR by both morphology and flow cytometry. However, the pre-allo-HCT bone marrow (BM) blast count (that is, 0-4%) was not significantly associated with risks of relapse or survival. These data indicate the presence of leukemic cells, but not the BM blast count affects survival. A strict morphologic and clinical lab flow cytometric definition of CR predicts outcomes after RIC allo-HCT, and therefore is critical to achieve at transplantation.

Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.

BACKGROUND: Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. METHODS: Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). RESULTS: Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE). CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.

Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection.

Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.