Joshua J. Augustine

Use of Sirolimus in Solid Organ Transplantation

Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that inhibits cell cycle progression and has proven to be a potent immunosuppressive agent for use in solid organ transplant recipients. The drug was initially studied as an adjunct to ciclosporin (cyclosporine) to prevent acute rejection in kidney transplant recipients. Subsequent studies have shown efficacy when combined with a variety of other immunosuppressive agents. The most common adverse effects of sirolimus are hyperlipidaemia and myelosuppression. The drug has unique antiatherogenic and antineoplastic properties, and may promote immunological tolerance and reduce the incidence of chronic allograft nephropathy. Although sirolimus is relatively non-nephrotoxic when administered as monotherapy, it pharmacodynamically enhances the toxicity of calcineurin inhibitors. Ironically, the drug has been used to facilitate calcineurin inhibitor-free protocols designed to preserve renal function after solid organ transplantation. Whether sirolimus can be used safely over the long term with low doses of calcineurin inhibitors requires further study. The use of sirolimus as a corticosteroid-sparing agent also remains to be proven in controlled trials. Postmarketing studies have revealed a number of unforeseen adverse effects including impaired wound healing and possibly proteinuria, oedema, pneumonitis and thrombotic microangiopathy. Overall, sirolimus is a powerful agent when used judiciously with other available immunosuppressants. As is true for all immunosuppressive drugs available for treatment of solid organ transplant recipients, the efficacy of the drug must be balanced against its considerable adverse effects.

Pre-Transplant IFN-γ ELISPOTs Are Associated with Post-Transplant Renal Function in African American Renal Transplant Recipients

Final crossmatch testing is routinely used to assess the risk of antibody‐mediated graft injury/rejection post‐transplant. Analogously, we postulated that quantitative measurements of anti‐donor effector/memory T cells pre‐transplant would independently assess post‐transplant risk. To address this hypothesis, we determined the frequencies of pre‐transplant, donor‐specific interferon‐γ (IFN‐γ) enzyme‐linked immunosorbent spots (ELISPOTs) and correlated the results with post‐transplant outcomes in 37 African American recipients of deceased donor kidney transplants treated with tacrolimus‐ and sirolimus‐based immunosuppression.

Comparative Effects of Sirolimus and Mycophenolate Mofetil on Erythropoiesis in Kidney Transplant Patients

Anemia and erythrocytosis (PTE) are common after kidney transplantation. We sought to determine the influence of sirolimus compared to mycophenolate mofetil (MMF) on post‐transplant erythropoiesis.

Pretransplant cellular alloimmunity as assessed by a panel of reactive T cells assay correlates with acute renal graft rejection

Background. The panel reactive antibody test (PRA) is an established method for assessing posttransplant risk of immune-mediated graft injury. The panel of reactive T cell assay (PRT) in which transplant candidates’ peripheral blood mononuclear cells are tested for reactivity to a panel of allogenic stimulator cells by the IFN-&ggr; enzyme-linked immunosorbent spot assay analogously assesses the strength of the pretransplant effector–memory alloreactive T cell repertoire. Methods. PRT assays were performed in 30 kidney transplant candidates and results were correlated with acute rejection (AR). A positive PRT assay was defined as a response to at least 75% of the stimulators tested. Results. A positive pretransplant PRT test was observed in 11 of 30 (37%) patients, and AR within 1 year posttransplantation was seen in 7 of 30 (23%) subjects. Six of the seven (86%) patients with AR were PRT-positive (P=0.01) whereas only one of seven (14%) patients with a PRA greater than 15% had AR. The mean pretransplant PRT percentage was 40% for patients with no AR versus 81% for patients with AR (P=0.01). Estimated glomerular filtration rate (mL/min/1.73 m2) showed a trend towards a lower value in PRT-positive (48±15) versus PRT-negative (55±13) individuals. Conclusions. The data suggest that pretransplant PRT screening can identify patients at risk for posttransplant cellular immune mediated graft injury despite the absence of humoral allosensitization. Once confirmed by larger prospective trials, PRT screening could be used to guide clinical decision-making with regard to choosing donor organs and individualizing immunosuppression regimens.

Steroid Sparing in Kidney Transplantation: Changing Paradigms, Improving Outcomes, and Remaining Questions

The widely known adverse effects of long-term therapy with corticosteroids have motivated increasing interest in steroid-free immunosuppression for kidney transplant recipients. Results from recent trials that used newer immunosuppressants to facilitate elimination of steroids suggest better short-term results than were achieved in an earlier era. However, the best results have been reported in uncontrolled trials of low-risk patients or in randomized trials with relatively short periods of follow-up. Increasingly, the therapeutic paradigm has shifted from late withdrawal of steroids to very early withdrawal after transplantation or even complete avoidance. Induction antibody therapy has been used routinely in the most successful trials that involved early steroid withdrawal or avoidance. Although the outcomes of kidney transplant recipients who are treated with steroid-free immunosuppression are improving steadily, there still is room for concern in recommending this strategy as a standard of practice.

Differences in proteinuria and graft function in de novo sirolimus-based vs. calcineurin inhibitor-based immunosuppression in live donor kidney transplantation.

Background. Calcineurin inhibitor(CNI)-free protocols using sirolimus (SRL) in kidney transplantation have proven effective, although reports have linked SRL to proteinuria. We sought to investigate this link and its impact on graft function. Methods. We retrospectively analyzed 184 live donor kidney transplant recipients who exclusively received de novo CNI-based (n=106) or SRL-based (n=78) regimens. Estimated glomerular filtration rate (GFR) and semi-quantitative dipstick proteinuria measurements were obtained at one, six, 12, and 24 months and six and 12 months, respectively. Results. SRL-treated patients had higher frequencies of proteinuria (≥1+) at 6 months (40.8% vs. 21.4%, P=0.006) and 12 months (37.8% vs. 18.4%, P=0.004) than those treated with CNI. Independent predictors of proteinuria at 12 months were GFR at one month (OR 0.62 per 10 ml/min/1.73m2, P<0.001), delayed graft function (OR 11.5, P=0.02), and a SRL-based regimen (OR 4.18, P=0.002). By univariable analysis, SRL vs. CNI patients had higher GFR at each point. SRL-treated patients without proteinuria had higher GFR at 12 months compared to CNI-treated patients with and without proteinuria (66 vs. 50 or 56 ml/min/1.73m2, P<0.05). No difference in GFR was seen between SRL-treated patients with proteinuria vs. CNI-treated patients without proteinuria (57 vs. 56 ml/min/1.73m2, P>0.05). Absence of proteinuria and a SRL-based regimen remained independently associated FSwith higher GFR at 12 months by multivariable analyses. Conclusions. De novo SRL-based immunosuppression is associated with a higher frequency of semi-quantitative proteinuria, however, estimated graft function at 1 year posttransplant remains superior to that of CNI-treated patients. Nevertheless, the long-term implications of these findings need to be determined.

Effects of influenza immunization on humoral and cellular alloreactivity in humans

Background. Alloreactive T cells and anti-human leukocyte antigen antibodies mediate transplant injury. Environmental exposures, including vaccinations, may activate the alloimmune repertoire leading to accelerated allograft injury. To test whether vaccination impacts human alloimmunity, we analyzed humoral and cellular immune reactivity in subjects undergoing influenza vaccination. Methods. We serially obtained blood samples from 30 healthy subjects and 8 kidney and 9 lung transplant recipients who received influenza vaccination, and from 20 healthy unvaccinated controls. We measured cellular and humoral anti-influenza responses, anti-human leukocyte antigen antibodies, and alloreactive T-cell immunity (interferon-&ggr; ELISPOT) at 0, 2, 4, and 12 weeks after vaccination. Results. Vaccination induced influenza-reactive humoral and cellular responses in control subjects and in transplant recipients. Only two of 30 vaccinated volunteers developed new alloantibodies, but none of the transplant patients. Vaccination also specifically and significantly augmented cellular alloimmunity based on reactivity to a panel of stimulators in both healthy subjects and in transplant recipients within 4 weeks of vaccination. The enhanced cellular alloresponse waned toward prevaccine levels by week 12. Conclusion. Our findings newly demonstrate that influenza vaccination can have a significant impact on the potency of the alloimmune repertoire. Because the strength of the alloresponse influences long-term graft function, our results suggest that further investigation of alloimmune monitoring after vaccination is needed.

Hemodialysis Vintage, Black Ethnicity, and Pretransplantation Antidonor Cellular Immunity in Kidney Transplant Recipients

Prolonged exposure to dialysis before transplantation and black ethnicity are known risk factors for acute rejection and graft loss in kidney transplant recipients. Because the strength of the primed antidonor T cell repertoire before transplantation also is associated with rejection and graft dysfunction, this study sought to determine whether hemodialysis (HD) vintage and/or black ethnicity affected donor-directed T cell immunity. An enzyme-linked immunosorbent spot (ELISPOT) assay was used to measure the frequency of peripheral T cells that expressed IFN-gamma in response to donor stimulator cells before transplantation in 100 kidney recipients. Acute rejection occurred in 38% of ELISPOT (+) patients versus 14% of ELISPOT (-) patients (P = 0.008). The median (HD) vintage was 46 mo (0 to 125 mo) in ELISPOT (+) patients versus 24 mo (0 to 276 mo) in ELISPOT (-) patients (P = 0.009). Black recipients had a greater median HD vintage (55 versus 14 mo in nonblack recipients; P < 0.001). Black recipients with less HD exposure had a low incidence of an ELISPOT (+) test, similar to nonblack recipients. Among variables examined, only HD vintage remained a significant positive correlate with an ELISPOT (+) result (odds ratio per year of HD 1.3; P = 0.003). These data suggest that the risk for developing cross-reactive antidonor T cell immunity increases with longer HD vintage, providing an explanation for the previously observed relationship between increased dialysis exposure and worse posttransplantation outcome. Longer HD vintage may also explain the increased T cell alloreactivity that previously was observed in black kidney recipients.

Preferential Benefit of Antibody Induction Therapy in Kidney Recipients With High Pretransplant Frequencies of Donor-Reactive Interferon-γ Enzyme-Linked Immunosorbent Spots

Background. The decision to use induction antibody therapy in kidney transplantation is often based on perceived patient risk, as no objective biomarker has been shown to predict the effectiveness of such therapy. Because pretransplant T-cell alloreactivity has been shown to increase the risk of poor posttransplant outcome, and because induction therapy is directed at alloreactive T cells, we hypothesized that antibody induction would preferentially benefit patients with high pretransplant antidonor T-cell immunity. Methods. In a retrospective analysis of 130 patients who had enrolled in an immune monitoring study, we correlated acute rejection rates, renal allograft function, and use of antibody induction therapy with donor-reactive interferon-γ enzyme-linked immunosorbent spot (ELISPOT) frequencies assessed pre and postkidney transplantation. Results. Of the 32 ELISPOT (+) patients, eight received induction therapy and had no rejection. Of the remaining 24 ELISPOT (+) patients with no induction therapy, acute rejection occurred in 11 (46%), (P=0.02). Twelve month glomerular filtration rate was significantly higher in the eight patients who received induction therapy (P=0.0001). Posttransplant conversion to a negative ELISPOT assay occurred in 86% of patients who received induction therapy vs. 35% of patients who did not (P=0.02). In the ELISPOT (−) cohort, acute rejection rates (∼15%) and glomerular filtration rates were similar in the 98 patients regardless of induction therapy. Conclusions. Our results suggest that antibody induction therapy preferentially benefits kidney transplant candidates with strong pretransplant donor-reactive cellular immunity. If confirmed prospectively, pretransplant ELISPOT assessments could be used to guide decision making regarding induction therapy.

Independent of nephrectomy, weaning immunosuppression leads to late sensitization after kidney transplant failure

Background Patients returning to dialysis therapy after renal transplant failure have a high rate of human leukocyte antigen antibody sensitization, and sensitization has been linked to allograft nephrectomy. We hypothesized that nephrectomy for cause is a consequence of weaning immunosuppression and that weaning leads to sensitization even in the absence of nephrectomy. Methods We examined outcomes in 300 consecutive patients with kidney allograft failure and survival of more than 30 days after failure. We analyzed a subset of 119 patients with a low panel reactive antibody (PRA) before transplantation and follow-up PRA testing at 6 to 24 months after failure (late PRA). Results By late PRA testing, 56% of patients were highly sensitized (class I or II PRA ≥80%). On multivariate analysis controlling for human leukocyte antigen matching, allograft nephrectomy, and other variables, weaning of immunosuppression predicted high sensitization (odds ratio, 14.34; P=0.004). In a subset of patients, the percentage of those who were highly sensitized increased from 21% at the time of failure on immunosuppressive therapy to 68% by late PRA after weaning (P<0.001). Conversely, patients who maintained immunosuppression showed minimal sensitization after failure. Transplant nephrectomy was required in 41% of patients who weaned immunosuppression versus 0% of the 24 patients who maintained immunosuppression with calcineurin inhibitor therapy after failure (P<0.001). Conclusions Weaning immunosuppression was a triggering event leading to late rejection and allograft nephrectomy and was an independent predictor of alloantibody sensitization after kidney allograft failure.