Lucile E. Wrenshall

A Randomized 2×2 Factorial Trial, Part 1: Single-Dose Rabbit Antithymocyte Globulin Induction May Improve Renal Transplantation Outcomes

Background We conducted a randomized and unblinded 2×2 sequential-factorial trial, composed of an induction arm (part 1) comparing single-dose (SD) versus divided-dose rabbit antithymocyte globulin (rATG), and a maintenance arm (part 2) comparing tacrolimus minimization versus withdrawal. We report the long-term safety and efficacy of SD-rATG induction in the context of early steroid withdrawal and tacrolimus minimization or withdrawal. Methods Patients (n=180) received 6 mg/kg rATG, SD or four alternate-day doses (1.5 mg/kg/dose), with early steroid withdrawal and tacrolimus or sirolimus maintenance. After 6 months targeted maintenance levels were tacrolimus, 2 to 4 ng/mL and sirolimus, 4 to 6 ng/mL or, if calcineurin inhibitor–withdrawn, sirolimus 8 to 12 ng/mL with mycophenolate mofetil 2 g two times per day. Primary endpoints were renal function (abbreviated modification of diet in renal disease) and chronic graft histopathology (Banff). Secondary endpoints included patient survival, graft survival, biopsy-proven rejection, and infectious or noninfectious complications. Results Follow-up averaged longer than 4 years. Tacrolimus or sirolimus and mycophenolate mofetil exposure was identical between groups. The SD-rATG associated with improved renal function (2-36 months; P<0.001) in deceased donor recipients. The SD-rATG associated with quicker lymphocyte, CD4 T cell, and CD4-CD8 recovery and fewer infections. Cox multivariate hazard modeling showed divided-dose–rATG (P=0.019), deceased donor (P=0.003), serious infection (P=0.0.018), and lower lymphocyte count (P=0.001) associated with increased mortality. Patients with all four covariates showed a 27-fold increased likelihood of death (P=0.00002). Chronic graft histopathology, rejection rates, and death-censored graft survival were not significantly different between groups. Conclusion The SD-rATG induction improves the 3-year renal function in recipients of deceased donor kidneys. This benefit, along with possibly improved patient survival and fewer infections suggest that how rATG is administered may impact its efficacy and safety.

Heparan Sulfate Proteoglycan Metabolism and the Fate of Grafted Tissues.

Tissue and organ transplants between genetically distinct individuals are always or nearly always rejected. The universality and speed of transplant rejection distinguishes this immune response from all others. Although this distinction is incompletely understood, some efforts to shed light on transplant rejection have revealed broader insights, including a relationship between activation of complement in grafted tissues, the metabolism of heparan sulfate proteoglycan and the nature of immune and inflammatory responses that ensue. Complement activation on cell surfaces, especially on endothelial cell surfaces, causes the shedding heparan sulfate, an acidic saccharide, from the cell surface and neighboring extracellular matrix. Solubilized in this way, heparan sulfate can activate leukocytes via toll like receptor-4, triggering inflammatory responses and activating dendritic cells, which migrate to regional lymphoid organs where they spark and to some extent govern cellular immune responses. In this way local ischemia, tissue injury and infection, exert systemic impact on immunity. Whether or in what circumstances this series of events explains the distinct characteristics of the immune response to transplants is still unclear but the events offer insight into the inception of immunity under the sub-optimal conditions accompanying infection and mechanisms by which infection and tissue injury engender systemic inflammation.

A Double-Blind, Double-Dummy, Flexible-Design Randomized Multicenter Trial: Early Safety of Single- Versus Divided-Dose Rabbit Anti-Thymocyte Globulin Induction in Renal Transplantation

A previous nonblinded, randomized, single‐center renal transplantation trial of single‐dose rabbit anti‐thymocyte globulin induction (SD‐rATG) showed improved efficacy compared with conventional divided‐dose (DD‐rATG) administration. The present multicenter, double‐blind/double‐dummy STAT trial (Single dose vs. Traditional Administration of Thymoglobulin) evaluated SD‐rATG versus DD‐rATG induction for noninferiority in early (7‐day) safety and tolerability. Ninety‐five patients (randomized 1:1) received 6 mg/kg SD‐rATG or 1.5 mg/kg/dose DD‐rATG, with tacrolimus‐mycophenolate maintenance immunosuppression. The primary end point was a composite of fever, hypoxia, hypotension, cardiac complications, and delayed graft function. Secondary end points included 12‐month patient survival, graft survival, and rejection. Target enrollment was 165 patients with an interim analysis scheduled after 80 patients. Interim analysis showed primary end point noninferiority of SD‐rATG induction (p = 0.6), and a conditional probability of <1.73% of continued enrollment producing a significant difference (futility analysis), leading to early trial termination. Final analysis (95 patients) showed no differences in occurrence of primary end point events (p = 0.58) or patients with no, one, or more than one event (p = 0.81), or rejection, graft, or patient survival (p = 0.78, 0.47, and 0.35, respectively). In this rigorously blinded trial in adult renal transplantation, we have shown SD‐rATG induction to be noninferior to DD‐rATG induction in early tolerability and equivalent in 12‐month safety. (Clinical Trials.gov #NCT00906204.)

Loss of Renal Allografts Secondary to Candida Vascular Complications in Two Recipients from the Same Donor

Infections remain a major cause of morbidity and mortality in transplant patients. Organ recipients are also susceptible to donor-derived pathogens and the majority of donor infections are easily treatable. Rarely, some pathogens have produced life-threatening complications by compromising the vascular anastomosis. In this case series we report loss of two kidney allografts secondary to vascular complications due to Candida albicans. Both recipients received grafts from a common donor, in whom Candida bacteremia in the donor was not apparent at the time of organ acceptance but became apparent on delayed cultures.

Oxidized LDL phagocytosis during foam cell formation in atherosclerotic plaques relies on a PLD2–CD36 functional interdependence

The uptake of cholesterol carried by low‐density lipoprotein (LDL) is tightly controlled in the body. Macrophages are not well suited to counteract the cellular consequences of excess cholesterol leading to their transformation into “foam cells,” an early step in vascular plaque formation. We have uncovered and characterized a novel mechanism involving phospholipase D (PLD) in foam cell formation. Utilizing bone marrow‐derived macrophages from genetically PLD deficient mice, we demonstrate that PLD2 (but not PLD1)‐null macrophages cannot fully phagocytose aggregated oxidized LDL (Agg‐Ox‐LDL), which was phenocopied with a PLD2‐selective inhibitor. We also report a role for PLD2 in coupling Agg‐oxLDL phagocytosis with WASP, Grb2, and Actin. Further, the clearance of LDL particles is mediated by both CD36 and PLD2, via mutual dependence on each other. In the absence of PLD2, CD36 does not engage in Agg‐Ox‐LDL removal and when CD36 is blocked, PLD2 cannot form protein–protein heterocomplexes with WASP or Actin. These results translated into humans using a GEO database of microarray expression data from atheroma plaques versus normal adjacent carotid tissue and observed higher values for NFkB, PLD2 (but not PLD1), WASP, and Grb2 in the atheroma plaques. Human atherectomy specimens confirmed high presence of PLD2 (mRNA and protein) as well as phospho‐WASP in diseased arteries. Thus, PLD2 interacts in macrophages with Actin, Grb2, and WASP during phagocytosis of Agg‐Ox‐LDL in the presence of CD36 during their transformation into “foam cells.” Thus, this study provides new molecular targets to counteract vascular plaque formation and atherogenesis.

A Randomized 2x2 Factorial Clinical Trial of Renal Transplantation: Steroid-Free Maintenance Immunosuppression with Calcineurin Inhibitor Withdrawal after Six Months Associates with Improved Renal Function and Reduced Chronic Histopathology

Introduction The two most significant impediments to renal allograft survival are rejection and the direct nephrotoxicity of the immunosuppressant drugs required to prevent it. Calcineurin inhibitors (CNI), a mainstay of most immunosuppression regimens, are particularly nephrotoxic. Until less toxic antirejection agents become available, the only option is to optimize our use of those at hand. Aim To determine whether intensive rabbit anti-thymocyte globulin (rATG) induction followed by CNI withdrawal would individually or combined improve graft function and reduce graft chronic histopathology–surrogates for graft and, therefore, patient survival. As previously reported, a single large rATG dose over 24 hours was well-tolerated and associated with better renal function, fewer infections, and improved patient survival. Here we report testing whether complete CNI discontinuation would improve renal function and decrease graft pathology. Methods Between April 20, 2004 and 4-14-2009 we conducted a prospective, randomized, non-blinded renal transplantation trial of two rATG dosing protocols (single dose, 6 mg/kg vs. divided doses, 1.5 mg/kg every other day x 4; target enrollment = 180). Subsequent maintenance immunosuppression consisted of tacrolimus, a CNI, and sirolimus, a mammalian target of rapamycin inhibitor. We report here the outcome of converting patients after six months either to minimized tacrolimus/sirolimus or mycophenolate mofetil/sirolimus. Primary endpoints were graft function and chronic histopathology from protocol kidney biopsies at 12 and 24 months Results CNI withdrawal (on-treatment analysis) associated with better graft function (p <0.001) and lower chronic histopathology composite scores in protocol biopsies at 12 (p = 0.003) and 24 (p = 0.013) months, without affecting patient (p = 0.81) or graft (p = 0.93) survival, or rejection rate (p = 0.17). Conclusion CNI (tacrolimus) withdrawal at six months may provide a strategy for decreased nephrotoxicity and improved long-term function in steroid-free low immunological risk renal transplant patients. Trial Registration ClinicalTrials.gov NCT00556933

A Randomized 2x2 Factorial Trial: Part 1, Single-Dose rATG Induction Improves Long-Term Renal Transplant Outcomes.: Abstract# 1369

1368 Outcomes Associated With Steroid Avoidance and Alemtuzumab Among Kidney Recipients in the United States. O. Serrano,1 P. Friedmann,1 S. Ahsanuddin,2 N. Swerdlow,1 A. Ben Yaacov,3 L. Kayler.1 1Department of Surgery, Montefiore Medical Center, Bronx, NY; 2Department of Medicine, Case Western Reserve University, Cleveland, OH; 3Department of Surgery, Rabin Medical Center, Petah Tikva, Israel. Alemtuzumab (AZ) is a humanized anti-CD52 monoclonal antibody used as an induction agent in kidney transplantation. Few studies have reported long-term outcomes of AZ compared to other agents in the context of steroid avoidance. Scientifi c Registry of Transplant Recipients (SRTR) data was accessed to identify all de novo adult kidney transplant recipients from 2003 to 2010 who received maintenance immunosuppression with tacrolimus and mycophenolate mofetil and induction with AZ (n=3035, 28.8%), IL-2 receptor antibody blockers (IL-2RAb, n=1165, 11.0%), or thymoglobulin (THYMO, n=6354, 60.4%). Exclusions included donor age <6 years, other types of maintenance immunosuppression, research study participation within 6 months of transplantation, HCV, or HIV. To avoid bias due to changes in therapy in response to worsening clinical course, we used an intentionto-treat analysis. On univariate analysis, death-censored graft survival (DCGS) was lower for patients receiving AZ (Figure). On multivariate analysis both AZ (aHR 1.35, 95% CI 1.16-1.57) and IL2-RAb (aHR 1.28, 95% CI 1.02-1.60) were associated with signifi cantly lower DCGS compared to THYMO. Differences were not observed in 1-year acute rejection in AZ (8.2%; aHR 0.99, 95% CI 0.84-1.17) or IL2-RAb (7.4%; aHR 1.03, 95% CI 0.80-1.31) treated groups compared to THYMO (8.0%). There were also no differences in overall patient survival in AZ (aHR 1.10, 95% CI 0.94-1.27) or IL2-RAb (aHR 1.17, 95% CI 0.96-1.41) treated groups. The differences in DCGS among AZ-treated patients were not apparent when the analysis was restricted to a more recent cohort (2007 to 2010) (aHR 1.11, 95% CI 0.89-1.38). In the context of early steroid elimination, induction with AZ results in worse graft survival, but not patient survival or 1-year acute rejection. Graft survival with AZ is similar to other induction strategies when assessing more recent cohorts suggesting that the expertise at centers utilizing AZ has improved over time. Abstract# 1369 A Randomized 2x2 Factorial Trial: Part 1, Single-Dose rATG Induction Improves Long-Term Renal Transplant Outcomes. R. Stevens,1 K. Foster,2 C. Miles,2 T. Rigley,1 A. Kalil,2 L. Wrenshall.1 1Wright State University, Dayton; 2University of Nebraska Medical Center, Omaha. Background We conducted a randomized/unblinded 2x2 sequential-factorial trial of intensive induction with single-dose (SD) vs. divided-dose (DD) rabbit anti-thymocyte globulin (rATG) and early steroid withdrawal (ESW) (Part 1), and Part 2, tacrolimus minimization vs. withdrawal. Here we report the long-term Part 1 safety and effi cacy of SD-rATG induction combined with ESW and tacrolimus/sirolimus (tac/ srl) minimization. Methods 180 patients received 6 mg/kg rATG, either single-dose or four alternate-day doses (1.5 mg/kg/dose), with ESW and tac/srl maintenance. Maintenance immunosuppression after 6 months was minimized in patients without evidence of rejection. Targeted maintenance levels were tac, 2-4 ng/ml and srl, 4-6 ng/ml. Primary endpoints were renal function and graft histopathology. Results Follow-up averaged 51.8 ± 15.1 months; tac/srl levels reached 4.6 ± 2.3/6.6 ± 2.7 ng/ml. SD-rATGassociated signifi cantly with fewer patient deaths and infections, quicker lymphocyte, CD4 T-cell, and CD4/CD8 recovery, and better renal function (all donors, days 1-4 and weeks 3-26; deceased donors, months 6-36). The risk of multiple infections decreased in association with rising absolute CD4 counts and CD4/CD8 ratio. In a multivariate regression model fewer occurrences of severe viral infections associated with SD-rATG induction and higher 12-month absolute lymphocyte counts. Chronic graft histopathology in protocol biopsies progressed from 12 to 24 months but was not signifi cantly different between groups. Induction groups had no differences in rejection rates or death-censored graft survival. Conclusions Intensive SD-rATG induction improves patient survival and infection rates, with better graft function among deceased-donor kidney recipients. 1369 A Randomized 2x2 Factorial Trial: Part 1, Single-Dose rATG Induction Improves Long-Term Renal Transplant Outcomes. R. Stevens,1 K. Foster,2 C. Miles,2 T. Rigley,1 A. Kalil,2 L. Wrenshall.1 1Wright State University, Dayton; 2University of Nebraska Medical Center, Omaha. Background We conducted a randomized/unblinded 2x2 sequential-factorial trial of intensive induction with single-dose (SD) vs. divided-dose (DD) rabbit anti-thymocyte globulin (rATG) and early steroid withdrawal (ESW) (Part 1), and Part 2, tacrolimus minimization vs. withdrawal. Here we report the long-term Part 1 safety and effi cacy of SD-rATG induction combined with ESW and tacrolimus/sirolimus (tac/ srl) minimization. Methods 180 patients received 6 mg/kg rATG, either single-dose or four alternate-day doses (1.5 mg/kg/dose), with ESW and tac/srl maintenance. Maintenance immunosuppression after 6 months was minimized in patients without evidence of rejection. Targeted maintenance levels were tac, 2-4 ng/ml and srl, 4-6 ng/ml. Primary endpoints were renal function and graft histopathology. Results Follow-up averaged 51.8 ± 15.1 months; tac/srl levels reached 4.6 ± 2.3/6.6 ± 2.7 ng/ml. SD-rATGassociated signifi cantly with fewer patient deaths and infections, quicker lymphocyte, CD4 T-cell, and CD4/CD8 recovery, and better renal function (all donors, days 1-4 and weeks 3-26; deceased donors, months 6-36). The risk of multiple infections decreased in association with rising absolute CD4 counts and CD4/CD8 ratio. In a multivariate regression model fewer occurrences of severe viral infections associated with SD-rATG induction and higher 12-month absolute lymphocyte counts. Chronic graft histopathology in protocol biopsies progressed from 12 to 24 months but was not signifi cantly different between groups. Induction groups had no differences in rejection rates or death-censored graft survival. Conclusions Intensive SD-rATG induction improves patient survival and infection rates, with better graft function among deceased-donor kidney recipients.

Identification of a cytotoxic form of dimeric interleukin-2 in murine tissues.

Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include “traditional” IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems.