dLLME-µSPE extraction coupled to HPLC-ESI-MS/MS for the determination of F2α-IsoPs in human urine.

Oxidative stress is a pathological condition characterized by an imbalance between body's antioxidant defenses and oxidizing agents, resulting in damage of endogenous molecules. These products can be used as markers of oxidative conditions; in particular, isoprostanes (IsoPs) come from the reaction of arachidonic acid with reactive oxygen species (ROS) and are currently defined as gold markers of oxidative stress in urine. Our main goal was the development of a reliable analytical method for the determination and quantification of the IsoPs in human urine by dispersive Liquid-Liquid Micro Extraction (dLLME) coupled with micro Solid Phase Extraction (µSPE) clean-up and HPLC-MS/MS analysis. The selected compounds are present in very small concentration in urine, furthermore, due to relevant matrix effect, they are challenging for ESI-MS/MS analysis. This approach provided selectivity and sensitivity for 8-isoprotaglandine F2α (8-iso-PGF2α), the "gold" OS marker, together with the main isomers. dLLME extraction allowed a significant enrichment factor and µSPE clean-up provided the removal of ion-suppressing compounds from the sample resulting in low matrix effect. The chromatographic separation was also challenging as the target compounds possess very similar chemical characteristics, so experimental conditions were carefully tuned. The reported method represents a useful tool for the detection of IsoPs in urine taking advantage of the combination of dLLME extraction and µSPE clean-up; overall recoveries were above 50 % and matrix effects were ≤15 %, with LOQs ranging between 0.020 and 0.060 ng mL-1. The procedure is easy to use and rapid allowing the removal of interfering compounds and matrix effect maintaining a highly sensitive determination.

Mycophenolate Mofetil Withdrawal With Conversion to Everolimus to Treat BK Virus Infection in Kidney Transplant Recipients.

BACKGROUND: BK virus (BKV) is a significant post-transplant infection. Mammalian target of rapamycin inhibitors (mTORis) reduce BKV large T antigen expression in vitro and are associated with lower rates of BKV infection when used as de novo immunosuppression in clinical studies. METHODS: We performed a prospective, single-center, randomized, open label pilot trial to evaluate the impact of mycophenolate mofetil (MMF) withdrawal with conversion to the mTORi everolimus versus a 50% reduction of the MMF dose for the treatment of BKV infection after kidney transplantation. Patients maintained on tacrolimus, MMF, and corticosteroids that developed BK viremia or BK viruria ≥1 × 106 copies/mL were eligible. The primary endpoint was a >50% reduction of BK viruria or clearance of viremia at 3 months postrandomization. RESULTS: Forty patients were enrolled and randomized in a 1:1 manner; 11 (55%) and 8 patients (40%) reached the primary endpoint in the everolimus group and the MMF group, respectively (P = .53). Of those with BK viremia at the time of enrollment, 8 of 16 (50%) and 5 of 15 (33.3%) cleared the viremia by month 3 in the everolimus conversion and MMF dose reduction groups, respectively (P = .47). CONCLUSION: Conversion from MMF to everolimus in BKV infection demonstrated a trend toward improved viral clearance but did not reach statistical significance.

Ten-year outcomes in a randomized phase II study of kidney transplant recipients administered belatacept 4-weekly or 8-weekly.

In the phase II IM103-100 study, kidney transplant recipients were first randomized to belatacept more-intensive-based (n = 74), belatacept less-intensive-based (n = 71), or cyclosporine-based (n = 73) immunosuppression. At 3-6 months posttransplant, belatacept-treated patients were re-randomized to receive belatacept every 4 weeks (4-weekly, n = 62) or every 8 weeks (8-weekly, n = 60). Patients initially randomized to cyclosporine continued to receive cyclosporine-based immunosuppression. Cumulative rates of biopsy-proven acute rejection (BPAR) from first randomization to year 10 were 22.8%, 37.0%, and 25.8% for belatacept more-intensive, belatacept less-intensive, and cyclosporine, respectively (belatacept more-intensive vs cyclosporine: hazard ratio [HR] = 0.95; 95% confidence interval [CI] 0.47-1.92; P = .89; belatacept less-intensive vs cyclosporine: HR = 1.61; 95% CI 0.85-3.05; P = .15). Cumulative BPAR rates from second randomization to year 10 for belatacept 4-weekly, belatacept 8-weekly, and cyclosporine were 11.1%, 21.9%, and 13.9%, respectively (belatacept 4-weekly vs cyclosporine: HR = 1.06, 95% CI 0.35-3.17, P = .92; belatacept 8-weekly vs cyclosporine: HR = 2.00, 95% CI 0.75-5.35, P = .17). Renal function trends were estimated using a repeated-measures model. Estimated mean GFR values at year 10 for belatacept 4-weekly, belatacept 8-weekly, and cyclosporine were 67.0, 68.7, and 42.7 mL/min per 1.73 m2 , respectively (P<.001 for overall treatment effect). Although not statistically significant, rates of BPAR were 2-fold higher in patients administered belatacept every 8 weeks vs every 4 weeks.

Polyclonal Regulatory T Cell Therapy for Control of Inflammation in Kidney Transplants.

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6-month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 106 (319, 321, and 363.8 × 106 ) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow-up biopsies and urinary biomarkers. Nearly 1 × 109 (0.932, 0.956, 1.565 × 109 ) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy-related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non-immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen-reactive Tregs for the treatment of inflammation in kidney transplants.

B Cell Receptor Genes Associated With Tolerance Identify a Cohort of Immunosuppressed Patients With Improved Renal Allograft Graft Function.

We previously reported that two B cell receptor genes, IGKV1D-13 and IGKV4-1, were associated with tolerance following kidney transplantation. To assess the potential utility of this "signature," we conducted a prospective, multicenter study to determine the frequency of patients predicted tolerant within a cohort of patients deemed to be candidates for immunosuppressive minimization. At any single time point, 25-30% of patients were predicted to be tolerant, while 13.7% consistently displayed the tolerance "signature" over the 2-year study. We also examined the relationship of the presence of the tolerance "signature" on drug use and graft function. Contrary to expectations, the frequency of predicted tolerance was increased in patients receiving tacrolimus and reduced in those receiving corticosteroids, mycophenolate mofetil, or Thymoglobulin as induction. Surprisingly, patients consistently predicted to be tolerant displayed a statistically and clinically significant improvement in estimated glomerular filtration rate that increased over time following transplantation. These findings indicate that the frequency of patients consistently predicted to be tolerant is sufficiently high to be clinically relevant and confirm recent findings by others that immunosuppressive agents impact putative biomarkers of tolerance. The association of a B cell-based "signature" with graft function suggests that B cells may contribute to the function/survival of transplanted kidneys.

Evaluation of the effect of tofacitinib exposure on outcomes in kidney transplant patients.

Tofacitinib fixed-dose regimens attained better kidney function and comparable efficacy to cyclosporine (CsA) in kidney transplant patients, albeit with increased risks of certain adverse events. This post-hoc analysis evaluated whether a patient subgroup with an acceptable risk-benefit profile could be identified. Tofacitinib exposure was a statistically significant predictor of serious infection rate. One-hundred and eighty six kidney transplant patients were re-categorized to above-median (AME) or below-median (BME) exposure groups. The 6-month biopsy-proven acute rejection rates in AME, BME and CsA groups were 7.8%, 15.7% and 17.7%, respectively. Measured glomerular filtration rate was higher in AME and BME groups versus CsA (61.2 and 67.9 vs. 53.9 mL/min) at Month 12. Fewer patients developed interstitial fibrosis and tubular atrophy (IF/TA) at Month 12 in AME (20.5%) and BME (27.8%) groups versus CsA (48.3%). Serious infections occurred more frequently in the AME group (53.0%) than in BME (28.4%) or CsA (25.5%) groups. Posttransplant lymphoproliferative disorder (PTLD) only occurred in the AME group. In kidney transplant patients, the BME group preserved the clinical advantage of comparable acute rejection rates, improved renal function and a lower incidence of IF/TA versus CsA, and with similar rates of serious infection and no PTLD.

Long-term belatacept exposure maintains efficacy and safety at 5 years: results from the long-term extension of the BENEFIT study.

The Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial randomized patients receiving a living or standard criteria deceased donor kidney transplant to a more (MI) or less intensive (LI) regimen of belatacept or cyclosporine A (CsA). The 5-year results of the long-term extension (LTE) cohort are reported. A total of 456 (68.5% of intent-to-treat) patients entered the LTE at 36 months; 406 patients (89%) completed 60 months. Between Months 36 and 60, death occurred in 2%, 1% and 5% of belatacept MI, belatacept LI and CsA patients, respectively; graft loss occurred in 0% belatacept and 2% of CsA patients. Acute rejection between Months 36 and 60 was rare: zero belatacept MI, one belatacept LI and one CsA. Rates for infections and malignancies for Months 36-60 were generally similar across belatacept groups and CsA, respectively: fungal infections (14%, 15%, 12%), viral infections (21%, 18%, 16%) and malignancies (6%, 6%, 9%). No new posttransplant lymphoproliferative disorder cases occurred after 36 months. Mean calculated GFR (MDRD, mL/min/1.73 m(2) ) at Month 60 was 74 for belatacept MI, 76 for belatacept LI and 53 for CsA. These results show that the renal function benefit and safety profile observed in belatacept-treated patients in the early posttransplant period was sustained through 5 years.

Polyomavirus BK replication in de novo kidney transplant patients receiving tacrolimus or cyclosporine: a prospective, randomized, multicenter study.

Polyomavirus BK (BKV)-associated nephropathy causes premature kidney transplant (KT) failure. BKV viruria and viremia are biomarkers of disease progression, but associated risk factors are controversial. A total of 682 KT patients receiving basiliximab, mycophenolic acid (MPA), corticosteroids were randomized 1:1 to cyclosporine (CsA) or tacrolimus (Tac). Risk factors were analyzed in 629 (92.2%) patients having at least 2 BKV measurements until month 12 posttransplant. Univariate analysis associated CsA-MPA with lower rates of viremia than Tac-MPA at month 6 (10.6% vs. 16.3%, p = 0.048) and 12 (4.8% vs. 12.1%, p = 0.004) and lower plasma BKV loads at month 12 (3.9 vs. 5.1 log(10) copies/mL; p = 0.028). In multivariate models, CsA-MPA remained associated with less viremia than Tac-MPA at month 6 (OR 0.60; 95% CI 0.36-0.99) and month 12 (OR 0.33; 95% CI 0.16-0.68). Viremia at month 6 was also independently associated with higher steroid exposure until month 3 (OR 1.19 per 1 g), and with male gender (OR 2.49) and recipient age (OR 1.14 per 10 years) at month 12. The data suggest a dynamic risk factor evolution of BKV viremia consisting of higher corticosteroids until month 3, Tac-MPA compared to CsA-MPA at month 6 and Tac-MPA, older age, male gender at month 12 posttransplant.

Randomized phase 2b trial of tofacitinib (CP-690,550) in de novo kidney transplant patients: efficacy, renal function and safety at 1 year.

In this Phase 2b study, 331 low-to-moderate risk de novo kidney transplant patients (approximately 60% deceased donors) were randomized to a more intensive (MI) or less intensive (LI) regimen of tofacitinib (CP-690, 550), an oral Janus kinase inhibitor or cyclosporine (CsA). All patients received basiliximab induction, mycophenolic acid and corticosteroids. Primary endpoints were: incidence of biopsy-proven acute rejection (BPAR) with a serum creatinine increase of ≥0.3 mg/dL and ≥20% (clinical BPAR) at Month 6 and measured GFR at Month 12. Similar 6-month incidences of clinical BPAR (11%, 7% and 9%) were observed for MI, LI and CsA. Measured GFRs were higher (p < 0.01) at Month 12 for MI and LI versus CsA (65 mL/min, 65 mL/min vs. 54 mL/min). Fewer (p < 0.05) patients in MI or LI developed chronic allograft nephropathy at Month 12 compared with CsA (25%, 24% vs. 48%). Serious infections developed in 45%, 37% and 25% of patients in MI, LI and CsA, respectively. Anemia, neutropenia and posttransplant lymphoproliferative disorder occurred more frequently in MI and LI compared with CsA. Tofacitinib was equivalent to CsA in preventing acute rejection, was associated with improved renal function and less chronic allograft histological injury, but had side-effects at the doses evaluated.

Three-year outcomes from BENEFIT, a randomized, active-controlled, parallel-group study in adult kidney transplant recipients.

The clinical profile of belatacept in kidney transplant recipients was evaluated to determine if earlier results in the BENEFIT study were sustained at 3 years. BENEFIT is a randomized 3 year, phase III study in adults receiving a kidney transplant from a living or standard criteria deceased donor. Patients were randomized to a more (MI) or less intensive (LI) regimen of belatacept, or cyclosporine. 471/666 patients completed ≥3 years of therapy. A total of 92% (MI), 92% (LI), and 89% (cyclosporine) of patients survived with a functioning graft. The mean calculated GFR (cGFR) was ∼21 mL/min/1.73 m(2) higher in the belatacept groups versus cyclosporine at year 3. From month 3 to month 36, the mean cGFR increased in the belatacept groups by +1.0 mL/min/1.73 m(2) /year (MI) and +1.2 mL/min/1.73 m(2) /year (LI) versus a decline of -2.0 mL/min/1.73 m(2) /year (cyclosporine). One cyclosporine-treated patient experienced acute rejection between year 2 and year 3. There were no new safety signals and no new posttransplant lymphoproliferative disorder (PTLD) cases after month 18. Belatacept-treated patients maintained a high rate of patient and graft survival that was comparable to cyclosporine-treated patients, despite an early increased occurrence of acute rejection and PTLD.

Sotrastaurin, a novel small molecule inhibiting protein-kinase C: randomized phase II study in renal transplant recipients.

Sotrastaurin, a selective protein-kinase-C inhibitor, blocks early T-cell activation through a calcineurin-independent mechanism. In this study, de novo renal transplant recipients with immediate graft function were randomized 1:2 to tacrolimus (control, n = 44) or sotrastaurin (300 mg b.i.d.; n = 81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary endpoint was the composite of treated biopsy-proven acute rejection (BPAR), graft loss, death or lost to follow-up at month 3. The main safety assessment was estimated glomerular filtration rate (eGFR); modification of diet in renal disease (MDRD) at month 3. Composite efficacy failure at month 3 was higher for the sotrastaurin versus control regimen (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p = 0.003), which led to early study termination. Median (± standard deviation [SD]) eGFR was higher for sotrastaurin versus control at all timepoints from day 7 (month 3: 59.0 ± 22.3 vs. 49.5 ± 17.7 mL/min/1.73 m(2) , p = 0.006). The most common adverse events were gastrointestinal disorders (control: 63.6%; sotrastaurin: 88.9%) which led to study-medication discontinuation in two sotrastaurin patients. This study demonstrated a lower degree of efficacy but better renal function with the calcineurin-inhibitor-free regimen of sotrastaurin+MPA versus the tacrolimus-based control. Ongoing studies are evaluating alternative sotrastaurin regimens.

Immunosuppression with belatacept-based, corticosteroid-avoiding regimens in de novo kidney transplant recipients.

Current immunosuppressive regimens in renal transplantation typically include calcineurin inhibitors (CNIs) and corticosteroids, both of which have toxicities that can impair recipient and allograft health. This 1-year, randomized, controlled, open-label, exploratory study assessed two belatacept-based regimens compared to a tacrolimus (TAC)-based, steroid-avoiding regimen. Recipients of living and deceased donor renal allografts were randomized 1:1:1 to receive belatacept-mycophenolate mofetil (MMF), belatacept-sirolimus (SRL), or TAC-MMF. All patients received induction with 4 doses of Thymoglobulin (6 mg/kg maximum) and an associated short course of corticosteroids. Eighty-nine patients were randomized and transplanted. Acute rejection occurred in 4, 1 and 1 patient in the belatacept-MMF, belatacept-SRL and TAC-MMF groups, respectively, by Month 6; most acute rejection occurred in the first 3 months. More than two-thirds of patients in the belatacept groups remained on CNI- and steroid-free regimens at 12 months and the calculated glomerular filtration rate was 8-10 mL/min higher with either belatacept regimen than with TAC-MMF. Overall safety was comparable between groups. In conclusion, primary immunosuppression with belatacept may enable the simultaneous avoidance of both CNIs and corticosteroids in recipients of living and deceased standard criteria donor kidneys, with acceptable rates of acute rejection and improved renal function relative to a TAC-based regimen.

Islet transplantation in type 1 diabetics using an immunosuppressive protocol based on the anti-LFA-1 antibody efalizumab.

The applicability of islet transplantation as treatment for type 1 diabetes is limited by renal and islet toxicities of currently available immunosuppressants. We describe a novel immunosuppressive regimen using the antileukocyte functional antigen-1 antibody efalizumab which permits long-term islet allograft survival while reducing the need for corticosteroids and calcineurin inhibitors (CNI). Eight patients with type 1 diabetes and hypoglycemic unawareness received intraportal allogeneic islet transplants. Immunosuppression consisted of antithymocyte globulin induction followed by maintenance with efalizumab and sirolimus or mycophenolate. When efalizumab was withdrawn from the market in mid 2009, all patients were transitioned to regimens consisting of mycophenolate and sirolimus or mycophenolate and tacrolimus. All patients achieved insulin independence and four out of eight patients became independent after single-islet transplants. Insulin independent patients had no further hypoglycemic events, hemoglobin A1c levels decreased and renal function remained stable. Efalizumab was well tolerated and no serious adverse events were encountered. Although long-term follow-up is limited by discontinuation of efalizumab and transition to conventional imunnosuppression (including CNI in four cases), these results demonstrate that insulin independence after islet transplantation can be achieved with a CNI and steroid-free regimen. Such an approach may minimize renal and islet toxicity and thus further improve long-term islet allograft survival.

A phase III study of belatacept-based immunosuppression regimens versus cyclosporine in renal transplant recipients (BENEFIT study).

Belatacept, a costimulation blocker, may preserve renal function and improve long-term outcomes versus calcineurin inhibitors in kidney transplantation. This Phase III study (Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial) assessed a more intensive (MI) or less intensive (LI) regimen of belatacept versus cyclosporine in adults receiving a kidney transplant from living or standard criteria deceased donors. The co-primary endpoints at 12 months were patient/graft survival, a composite renal impairment endpoint (percent with a measured glomerular filtration rate (mGFR) <60 mL/min/1.73 m(2) at Month 12 or a decrease in mGFR > or =10 mL/min/1.73 m(2) Month 3-Month 12) and the incidence of acute rejection. At Month 12, both belatacept regimens had similar patient/graft survival versus cyclosporine (MI: 95%, LI: 97% and cyclosporine: 93%), and were associated with superior renal function as measured by the composite renal impairment endpoint (MI: 55%; LI: 54% and cyclosporine: 78%; p < or = 0.001 MI or LI versus cyclosporine) and by the mGFR (65, 63 and 50 mL/min for MI, LI and cyclosporine; p < or = 0.001 MI or LI versus cyclosporine). Belatacept patients experienced a higher incidence (MI: 22%, LI: 17% and cyclosporine: 7%) and grade of acute rejection episodes. Safety was generally similar between groups, but posttransplant lymphoproliferative disorder was more common in the belatacept groups. Belatacept was associated with superior renal function and similar patient/graft survival versus cyclosporine at 1 year posttransplant, despite a higher rate of early acute rejection.

A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT study).

Recipients of extended criteria donor (ECD) kidneys are at increased risk for graft dysfunction/loss, and may benefit from immunosuppression that avoids calcineurin inhibitor (CNI) nephrotoxicity. Belatacept, a selective costimulation blocker, may preserve renal function and improve long-term outcomes versus CNIs. BENEFIT-EXT (Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial-EXTended criteria donors) is a 3-year, Phase III study that assessed a more (MI) or less intensive (LI) regimen of belatacept versus cyclosporine in adult ECD kidney transplant recipients. The co-primary endpoints at 12 months were composite patient/graft survival and a composite renal impairment endpoint. Patient/graft survival with belatacept was similar to cyclosporine (86% MI, 89% LI, 85% cyclosporine) at 12 months. Fewer belatacept patients reached the composite renal impairment endpoint versus cyclosporine (71% MI, 77% LI, 85% cyclosporine; p = 0.002 MI vs. cyclosporine; p = 0.06 LI vs. cyclosporine). The mean measured glomerular filtration rate was 4-7 mL/min higher on belatacept versus cyclosporine (p = 0.008 MI vs. cyclosporine; p = 0.1039 LI vs. cyclosporine), and the overall cardiovascular/metabolic profile was better on belatacept versus cyclosporine. The incidence of acute rejection was similar across groups (18% MI; 18% LI; 14% cyclosporine). Overall rates of infection and malignancy were similar between groups; however, more cases of posttransplant lymphoproliferative disorder (PTLD) occurred in the CNS on belatacept. ECD kidney transplant recipients treated with belatacept-based immunosuppression achieved similar patient/graft survival, better renal function, had an increased incidence of PTLD, and exhibited improvement in the cardiovascular/metabolic risk profile versus cyclosporine-treated patients.

The efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients.

Late-onset cytomegalovirus (CMV) disease is a significant problem with a standard 3-month prophylaxis regimen. This multicentre, double-blind, randomized controlled trial compared the efficacy and safety of 200 days' versus 100 days' valganciclovir prophylaxis (900 mg once daily) in 326 high-risk (D+/R-) kidney allograft recipients. Significantly fewer patients in the 200-day group versus the 100-day group developed confirmed CMV disease up to month 12 posttransplant (16.1% vs. 36.8%; p < 0.0001). Confirmed CMV viremia was also significantly lower in the 200-day group (37.4% vs. 50.9%; p = 0.015 at month 12). There was no significant difference in the rate of biopsy-proven acute rejection between the groups (11% vs. 17%, respectively, p = 0.114). Adverse events occurred at similar rates between the groups and the majority were rated mild-to-moderate in intensity and not related to study medication. In conclusion, this study demonstrates that extending valganciclovir prophylaxis (900 mg once daily) to 200 days significantly reduces the incidence of CMV disease and viremia through to 12 months compared with 100 days' prophylaxis, without significant additional safety concerns associated with longer treatment. The number needed to treat to avoid one additional patient with CMV disease up to 12 months posttransplant is approximately 5.

KDIGO clinical practice guideline for the care of kidney transplant recipients: a summary

The 2009 Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guideline on the monitoring, management, and treatment of kidney transplant recipients is intended to assist the practitioner caring for adults and children after kidney transplantation. The guideline development process followed an evidence-based approach, and management recommendations are based on systematic reviews of relevant treatment trials. Critical appraisal of the quality of the evidence and the strength of recommendations followed the Grades of Recommendation Assessment, Development, and Evaluation (GRADE) approach. The guideline makes recommendations for immunosuppression and graft monitoring, as well as prevention and treatment of infection, cardiovascular disease, malignancy, and other complications that are common in kidney transplant recipients, including hematological and bone disorders. Limitations of the evidence, especially the lack of definitive clinical outcome trials, are discussed and suggestions are provided for future research. This summary includes a brief description of methodology and the complete guideline recommendations but does not include the rationale and references for each recommendation, which are published elsewhere.