Edoardo Melilli

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 (nu2009=u200939) or preemptive treatment (nu2009=u200998). 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 (pu2009<u20090.05). 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 (AUCu2009>u20090.70). 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.

Hepcidin is not useful as a biomarker for iron needs in haemodialysis patients on maintenance erythropoiesis-stimulating agents

BACKGROUNDnIt has been suggested that hepcidin may be useful as a tool for managing iron therapy in haemodialysis (HD) patients on erythropoiesis-stimulating agents (ESA).nnnMETHODSnWe used SELDI-TOF mass spectrometry assay to measure serum hepcidin-25 (Hep-25) and hepcidin-20 (Hep-20) in 56 adult HD patients on maintenance ESA to assess their ability to predict haemoglobin (Hb) response after 1 g intravenous iron (62.5 mg ferric gluconate at 16 consecutive dialysis sessions) and their relationship with markers of iron status, inflammation and erythropoietic activity.nnnRESULTSnAt multivariate analysis (in a model that also included Hb, reticulocyte, ESA dose, HFE genotype, soluble transferrin receptor [sTfR] and C-reactive protein), Hep-25 independently correlated with ferritin (β = 0.03, P = 0.01) and the percentage of hypochromic red blood cells [%Hypo] (β = 1.84, P = 0.01), suggesting that Hep-25 may be a useful biomarker for iron stores and bone marrow iron availability. Hep-20 correlated independently with Hep-25 (β = 0.159, P <u20090.001) and ferritin (β = 0.006, P = 0.05), suggesting that it may be a useful additional biomarker for iron stores. On receiver operating characteristics curve analysis, neither Hep-25 nor Hep-20 significantly predicted who will increase their Hb after iron loading (AUC = 0.52 ± 0.09 and 0.54 ± 0.08, P = 0.612), and the same applied to ferritin and transferrin saturation (AUC = 0.55 ± 0.08 and 0.59 ± 0.08, P = 0.250), whereas %Hypo and reticulocyte Hb content were significant predictors (AUC = 0.84 ± 0.05 and 0.70 ± 0.08, P <u20090.01). At multivariate logistic regression analysis, %Hypo was the only biomarker independently associated with iron responsiveness.nnnCONCLUSIONSnAlthough our study suggests an important role for hepcidin in regulating iron homeostasis in HD patients on ESA, our findings do not support its utility as a predictor of iron needs, offering no advantage over established markers of iron status.

Intragraft regulatory T cells in protocol biopsies retain foxp3 demethylation and are protective biomarkers for kidney graft outcome.

Presence of subclinical rejection (SCR) with IF/TA in protocol biopsies of renal allografts has been shown to be an independent predictor factor of graft loss. Also, intragraft Foxp3+ Treg cells in patients with SCR has been suggested to differentiate harmful from potentially protective infiltrates. Nonetheless, whether presence of Foxp3 Treg cells in patients with SCR and IF/TA may potentially protect from a deleterious graft outcome has not yet been evaluated. This is a case‐control study in which 37 patients with the diagnosis of SCR and 68 control patients with no cellular infiltrates at 6‐month protocol biopsies matched for age and time of transplantation were evaluated. We first confirmed that numbers of intragraft Foxp3‐expressing T cells in patients with SCR positively correlates with Foxp3 demethylation at the Treg‐specific demethylation region. Patients with SCR without Foxp3+ Treg cells within graft infiltrates showed significantly worse 5‐year graft function evolution than patients with SCR and Foxp3+ Treg cells and those without SCR. When presence of SCR and IF/TA were assessed together, presence of Foxp3+ Treg could discriminate a subgroup of patients showing the same graft outcome as patients with a normal biopsy. Thus, presence of Foxp3+ Treg cells in patients with SCR even with IF/TA is associated with a favorable long‐term allograft outcome.

Prospective assessment of antidonor cellular alloreactivity is a tool for guidance of immunosuppression in kidney transplantation.

Current characterization of the immune risk in renal transplant patients is only focused on the assessment of preformed circulating alloantibodies; however, alloreactive memory T cells are key players in mediating allograft rejection. Immune monitoring of antidonor alloreactive memory/effector T cells using an IFN-γ Elispot has been shown to distinguish patients at risk for immune-mediated graft dysfunction, suggesting a potential tool for immunosuppression individualization. In this nonrandomized study, we prospectively assessed donor and nondonor T-cell alloreactivity in 60 highly alloreactive patients receiving calcineurin inhibitor-based immunosuppression and in non-T-cell alloreactive transplant recipients treated with a calcineurin inhibitor-free regimen. The impact was evaluated using 1-year allograft outcome. We found a strong association between ongoing antidonor T-cell alloreactivity and histological lesions of acute T cell-mediated rejection in 6-month protocol biopsies, distinguishing those patients with better 1-year graft function, regardless of immunosuppression regimen. Interestingly, evidence for enhanced immune regulation, driven by circulating Foxp3-demethylated regulatory T cells, was only observed among patients achieving antidonor T-cell hyporesponsiveness. Thus, prospective evaluation of donor-specific T-cell sensitization may add crucial information on the alloimmune state of transplanted patients to be used in daily clinical practice.

In Search of an Optimal Bedside Screening Program for Arteriovenous Fistula Stenosis

BACKGROUND AND OBJECTIVESnGuidelines recommend systematically screening for stenosis using various methods, but no studies so far have compared all of the options. A prospective blinded study was performed to compare the performance of several bedside tests performed during dialysis in diagnosing angiographically proven >50% fistula stenosis.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnIn an unselected population of 119 hemodialysis patients with mature fistulas, physical examination (PE) was conducted; dynamic and derived static venous pressure (VAPR), blood pump flow/arterial pressure (Qb/AP) ratio, recirculation (R), and access blood flow (Qa) were measured; and angiography was performed.nnnRESULTSnAngiography identified 59 stenotic fistulas: 43 stenoses were located upstream from the venous needle (inflow stenosis), 12 were located downstream (outflow stenosis), and 4 were located at both sites. The optimal tests for identifying an inflow stenosis were Qa < 650 ml/min and the combination of a positive PE or Qa < 650 ml/min (accuracy 80% and 81%, respectively), the latter being preferable because it was more sensitive (85% versus 65%, respectively) for a comparable specificity (79% versus 89%, respectively). The best tests for identifying outflow stenosis were PE and VAPR, with no difference between the two (accuracy 91% and 85%, sensitivity 75% and 81%, specificity 93% and 86%, respectively), the former being preferable because it was more reproducible, easier to perform, and applicable to all fistulas.nnnCONCLUSIONSnThis study showed that fistula stenosis can be detected and located during dialysis with a moderate-to-excellent accuracy using PE and Qa measurement as screening procedures.

Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation

The accurate evaluation of donor-specific antibodies (DSAs) has allowed a precise identification of sensitized patients at risk of antibody-mediated rejection (ABMR). However, the scale of the humoral response is not always fully addressed, as it excludes the complete memory B-cell (mBC) pool such as that caused by antigen-specific mBC. Using a novel B-cell ELISpot assay approach, we assessed circulating mBC frequencies against class I and II HLA antigens in highly sensitized and nonsensitized patients in the waiting list for kidney transplantation. Also, kidney transplant patients undergoing ABMR were evaluated for the presence of donor-specific mBCs both at the time of rejection and before transplantation. For this purpose, 278 target HLA-sp antigens from 70 patients were studied and compared to circulating HLA-sp antibodies. Both class I and II HLA-sp mBC frequencies were identified in highly sensitized individuals but not in nonsensitized and healthy individuals, many years after first sensitization. Also, high donor-specific mBC responses were clearly found both during ABMR and before transplantation, regardless of circulating DSA. The higher the donor-specific mBC response, the more aggressive the allograft rejection. Thus, assessing donor-specific mBC frequencies may be relevant to better refine patient alloimmune-risk stratification, and provides new insight into the mechanisms of the adaptive humoral alloimmune response taking place in kidney transplantation.

Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy.

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.

Bedside Screening for Fistula Stenosis Should Be Tailored to the Site of the Arteriovenous Anastomosis

BACKGROUND AND OBJECTIVESnGiven different sites of stenosis and access blood flow rates (Qa), the criteria for diagnosing fistula stenosis might vary according to anastomotic site. To test this, we analyzed the database of a prospective blinded study seeking an optimal bedside screening program for fistula stenosis.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnSeveral methods used during dialysis (physical examination [PE], dynamic and derived static venous pressure [VAPR], dialysis blood pump flow/arterial pressure ratio, and Qa measurement) to diagnose angiographically-proven >50% stenosis were assessed in an unselected population of hemodialysis patients with mature fistulae (43 at the wrist [distal fistulae], 76 at mid-forearm or the elbow [proximal fistulae]).nnnRESULTSnPrevalence of inflow stenosis was uninfluenced by anastomotic site, whereas outflow stenoses were more prevalent in proximal fistulae. The best test for inflow stenosis was Qa <650 ml/min in distal fistulae and a combination of a positive PE and Qa <900 ml/m in proximal fistulae. In proximal fistulae, PE and VAPR >0.5 were both equally highly diagnostic of outflow stenosis. Tailoring choice of test to site of the anastomosis may also contain the screening-associated workload, by reducing the need to perform PE and measure VAPR, compared with a screening approach regardless of the access location.nnnCONCLUSIONSnOur study shows that an effective bedside screening program with ≥85% accuracy for fistula stenosis can be tailored to the site of the anastomosis, Qa being the tool of choice for the wrist, and PE alone or combined with Qa and VAPR measurements for more proximally-located accesses.

Posttransplant peripheral blood donor–specific interferon-γ enzyme-linked immune spot assay differentiates risk of subclinical rejection and de novo donor-specific alloantibodies in kidney transplant recipients

Noninvasive diagnosis of kidney allograft inflammation in transplant recipients with stable graft function (subclinical rejection) could permit more effective therapy and prevent later development of de novo anti-donor HLA antibodies and/or graft dysfunction. Here we tested whether quantifying posttransplant donor-specific alloreactive T-cells by IFN-γ ELISPOT assay noninvasively detects subclinical T-cell mediated rejection and/or predicts development of anti-donor HLA antibodies. Using an initial cross-sectional cohort of 60 kidney transplant patients with six-month surveillance biopsies, we found that negative donor-specific IFN-γ ELISPOT assays accurately ruled out the presence of subclinical T-cell mediated rejection. These results were validated using a distinct prospective cohort of 101 patients where donor-specific IFN-γ ELISPOT results at bothxa0three- and six-months posttransplant significantly differentiated patients with subclinical T-cell mediated rejection at six months, independent of other clinical variables (odds ratio 0.072, 95% confidence interval 0.008-0.653). The posttransplant donor-specific IFN-γ ELISPOT results independently associated with subsequent development of significant anti-donor HLA antibodies (0.085, 0.008-0.862) and with significantly worse two-year function (estimated glomerular filtration rate) compared to patients with a negative test. Thus, posttransplant immune monitoring by donor-specific IFN-γ ELISPOT can assess risk for developing subclinical T-cell mediated rejection and anti-donor HLA antibodies, potentially limiting the need for surveillance biopsies. Our study provides a guide for individualizing immunosuppression to improve posttransplant outcomes.

De novo use of a generic formulation of tacrolimus versus reference tacrolimus in kidney transplantation: evaluation of the clinical results, histology in protocol biopsies, and immunological monitoring.

The use of generic formulations of immunosuppressive drugs in renal transplantation has been and still is a controversial subject. The lack of clinical studies about safety and efficacy in transplant patients is one of the factors restricting the diffusion of generic drugs in the renal transplant field. Since March 2013, our transplant unit has incorporated generic tacrolimus (Adoport®; Sandoz), replacing the one we were currently using (Prograf®; Astellas). When carrying out our retrospective analysis comparing the two different formulations, we evaluated several clinical results: tacrolimus trough concentrations (C0) at 5–7 days; 1, 3, and 6 months post‐transplantation; concentration/dose ratio at 6 months; acute rejection incidence; delayed graft function (DGF); renal function (as CKD‐EPI); and proteinuria at 6 months in 120 patients (1:1 ratio of Prograf® versus Adoport®), noticing no important differences. We also evaluated the results of protocol biopsies at 6 months in a subgroup of patients, thus verifying the safety and efficacy of this particular generic drug versus the reference product on a histological basis as well. No difference in the development of dnDSA (de novo donor‐specific antibody) was found between the two groups.