Mary Jane Elliott

Chimerism and Tolerance Without GVHD or Engraftment Syndrome in HLA-Mismatched Combined Kidney and Hematopoietic Stem Cell Transplantation

Durable chimerism and donor-specific tolerance can be safely achieved without GVHD in HLA-mismatched donor/recipient pairs. Teaching Tolerance According to Greek mythology, the Chimera was a fire-breathing creature made of parts from different animals: the body of a lioness, a snake’s head at the end of the tail, and the head of the goat. Sightings of this fearsome beast portended any of a number of terrible disasters. In the context of organ transplantation, a “chimera” can indicate both desirable and disastrous outcomes. For example, hematopoietic chimerism, in which the immune cells in the graft recipient come from both the host and the donor, may promote graft tolerance, but may also cause graft-versus-host disease (GVHD), in which the donor immune cells attack the healthy tissue of the host. Leventhal et al. now report mixed chimerism and tolerance without the negative side effects of GVHD or engraftment syndrome in a phase 2 clinical trial of combined kidney and hematopoietic transplantation. Leventhal et al. used a combination of mobilized cells enriched for hematopoietic stem cells and graft-facilitating cells—which are composed largely of plasmacytoid precursor dendritic cells—with nonmyeloablative conditioning in conjunction with kidney transplant from major histocompatibility complex–mismatched, nonrelated donors and recipients. Five of eight kidney transplant recipients exhibited durable chimerism and were weaned off immunosuppressive therapies by 1 year after transplantation, with no signs of GVHD or engraftment syndrome. If confirmed in larger patient cohorts, this approach to transplantation could free some patients from the difficulties associated with lifelong immunosuppression and add transplantation as a viable option for patients for whom no matched donors exist. As with the Chimera of legend, mixed chimerism may be a harbinger of things to come—albeit hopefully a brighter future for transplant patients. The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSCs) and tolerogenic graft facilitating cells (FCs) combined with nonmyeloablative conditioning; this approach resulted in engraftment, durable chimerism, and tolerance induction in recipients with highly mismatched related and unrelated donors. Eight recipients of human leukocyte antigen (HLA)–mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200-centigray total body irradiation, and cyclophosphamide followed by posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from five of six loci with related donors to one of six loci with unrelated donors. The absolute neutrophil counts reached a nadir about 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism at 1 month ranged from 6 to 100%. The conditioning was well tolerated, with outpatient management after postoperative day 2. Two subjects exhibited transient chimerism and were maintained on low-dose tacrolimus monotherapy. One subject developed viral sepsis 2 months after transplant and experienced renal artery thrombosis. Five subjects experienced durable chimerism, demonstrated immunocompetence and donor-specific tolerance by in vitro proliferative assays, and were successfully weaned off all immunosuppression 1 year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.

Efficacy and Tolerability of Evolocumab vs Ezetimibe in Patients With Muscle-Related Statin Intolerance: The GAUSS-3 Randomized Clinical Trial

IMPORTANCE Muscle-related statin intolerance is reported by 5% to 20% of patients. OBJECTIVE To identify patients with muscle symptoms confirmed by statin rechallenge and compare lipid-lowering efficacy for 2 nonstatin therapies, ezetimibe and evolocumab. DESIGN, SETTING, AND PARTICIPANTS Two-stage randomized clinical trial including 511 adult patients with uncontrolled low-density lipoprotein cholesterol (LDL-C) levels and history of intolerance to 2 or more statins enrolled in 2013 and 2014 globally. Phase A used a 24-week crossover procedure with atorvastatin or placebo to identify patients having symptoms only with atorvastatin but not placebo. In phase B, after a 2-week washout, patients were randomized to ezetimibe or evolocumab for 24 weeks. INTERVENTIONS Phase A: atorvastatin (20 mg) vs placebo. Phase B: randomization 2:1 to subcutaneous evolocumab (420 mg monthly) or oral ezetimibe (10 mg daily). MAIN OUTCOME AND MEASURES Coprimary end points were the mean percent change in LDL-C level from baseline to the mean of weeks 22 and 24 levels and from baseline to week 24 levels. RESULTS Of the 491 patients who entered phase A (mean age, 60.7 [SD, 10.2] years; 246 women [50.1%]; 170 with coronary heart disease [34.6%]; entry mean LDL-C level, 212.3 [SD, 67.9] mg/dL), muscle symptoms occurred in 209 of 491 (42.6%) while taking atorvastatin but not while taking placebo. Of these, 199 entered phase B, along with 19 who proceeded directly to phase B for elevated creatine kinase (N = 218, with 73 randomized to ezetimibe and 145 to evolocumab; entry mean LDL-C level, 219.9 [SD, 72] mg/dL). For the mean of weeks 22 and 24, LDL-C level with ezetimibe was 183.0 mg/dL; mean percent LDL-C change, -16.7% (95% CI, -20.5% to -12.9%), absolute change, -31.0 mg/dL and with evolocumab was 103.6 mg/dL; mean percent change, -54.5% (95% CI, -57.2% to -51.8%); absolute change, -106.8 mg/dL (P < .001). LDL-C level at week 24 with ezetimibe was 181.5 mg/dL; mean percent change, -16.7% (95% CI, -20.8% to -12.5%); absolute change, -31.2 mg/dL and with evolocumab was 104.1 mg/dL; mean percent change, -52.8% (95% CI, -55.8% to -49.8%); absolute change, -102.9 mg/dL (P < .001). For the mean of weeks 22 and 24, between-group difference in LDL-C was -37.8%; absolute difference, -75.8 mg/dL. For week 24, between-group difference in LDL-C was -36.1%; absolute difference, -71.7 mg/dL. Muscle symptoms were reported in 28.8% of ezetimibe-treated patients and 20.7% of evolocumab-treated patients (log-rank P = .17). Active study drug was stopped for muscle symptoms in 5 of 73 ezetimibe-treated patients (6.8%) and 1 of 145 evolocumab-treated patients (0.7%). CONCLUSIONS AND RELEVANCE Among patients with statin intolerance related to muscle-related adverse effects, the use of evolocumab compared with ezetimibe resulted in a significantly greater reduction in LDL-C levels after 24 weeks. Further studies are needed to assess long-term efficacy and safety. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01984424.

Tolerance Induction in HLA Disparate Living Donor Kidney Transplantation by Donor Stem Cell Infusion: durable chimerism predicts outcome

Background We recently reported that durable chimerism can be safely established in mismatched kidney recipients through nonmyeloablative conditioning followed by infusion of a facilitating cell (FC)-based hematopoietic stem cell transplantation termed FCRx. Here we provide intermediate-term follow-up on this phase II trial. Methods Fifteen human leukocyte antigen–mismatched living donor renal transplant recipients underwent low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression, consisting of tacrolimus and mycophenolate, was weaned over 1 year. Results All but one patient demonstrated peripheral blood macrochimerism after transplantation. Engraftment failure occurred in a highly sensitized (panel reactive antibody [PRA] of 52%) recipient. Chimerism was lost in three patients at 2, 3, and 6 months after transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other two had PRA greater than 20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full-dose immunosuppression. Complete immunosuppression withdrawal at 1 year after transplantation was successful in all patients with durable chimerism. There has been no graft-versus-host disease or engraftment syndrome. Renal transplantation loss occurred in one patient who developed sepsis following an atypical viral infection. Two subjects with only transient chimerism demonstrated subclinical rejection on protocol biopsy despite donor-specific hyporesponsiveness. Conclusions Low-intensity conditioning plus FCRx safely achieved durable chimerism in mismatched allograft recipients. Sensitization represents an obstacle to successful induction of chimerism. Sustained T-cell chimerism is a more robust biomarker of tolerance than donor-specific hyporeactivity.

Lipid-lowering efficacy of the PCSK9 inhibitor evolocumab (AMG 145) in patients with type 2 diabetes: a meta-analysis of individual patient data

BACKGROUND Patients with type 2 diabetes have increased cardiovascular risk. PCSK9 monoclonal antibodies have been shown to reduce LDL cholesterol and other lipids, but specific efficacy for patients with diabetes is unknown. We compared the effect of the PCSK9 inhibitor evolocumab on lipid parameters in patients with and without type 2 diabetes. METHODS We did a random-effects meta-analysis of randomised clinical trials comparing the efficacy of evolocumab, placebo, and ezetimibe to improve lipid parameters in adult patients (age 18-80 years) with or without type 2 diabetes. We searched MEDLINE and Embase to identify eligible 12-week, phase 3 trials published between Jan 1, 2012, and Feb 28, 2015. We excluded trials that included patients who had homozygous familial hypercholesterolaemia. All analyses were based on individual participant data. We used DerSimonian and Laird random-effects meta-analyses to compare the mean changes from baseline in concentrations of LDL cholesterol, non-HDL cholesterol, total cholesterol, triglycerides, lipoprotein(a), and HDL cholesterol at 12 weeks for evolocumab, placebo, and ezetimibe. We also assessed the effect of evolocumab therapy compared with placebo across subgroups of patients based on glycaemia, insulin use, renal function, and cardiovascular disease status at baseline. RESULTS Three trials met our inclusion criteria, and included 413 patients with type 2 diabetes and 2119 patients without type 2 diabetes. In patients with type 2 diabetes evolocumab caused mean reductions in LDL cholesterol concentration that were 60% (95% CI 51-69) versus placebo and 39% (32-47) versus ezetimibe. In patients without type 2 diabetes, evolocumab caused mean reductions in LDL cholesterol that were 66% (62-70) versus placebo and 40% (36-45) versus ezetimibe. In patients with type 2 diabetes, evolocumab was associated with reductions in non-HDL cholesterol (55% [47-63] vs placebo and 34% [26-41] vs ezetimibe), total cholesterol (38% [32-44] vs placebo and 24% [16-31] vs ezetimibe), and lipoprotein(a) (31% [25-37] vs placebo and 26% [16-35] vs ezetimibe), and an increase in HDL cholesterol (7% [4-11] vs placebo and 8% [4-13] vs ezetimibe). Findings were similar across diabetes subgroups based on glycaemia, insulin use, renal function, and cardiovascular disease status. INTERPRETATION Evolocumab markedly reduces atherogenic lipoproteins in patients with type 2 diabetes, an effect that is consistent across subgroups and similar to that seen in patients without type 2 diabetes. Results from ongoing cardiovascular outcome trials of PCSK9 inhibitors will provide additional data to inform the use of these drugs in patients with type 2 diabetes. FUNDING Amgen.

Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants.

Nineteen subjects have more than 18 months’ follow-up in a phase IIb tolerance protocol in HLA–mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4+ and CD8+ T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.

Adenovirus-mediated E2F-1 gene transfer efficiently induces apoptosis in melanoma cells.

E2F‐1 is a transcription factor that stimulates cellular proliferation and cell cycle progression from G1 to S‐phase. Somewhat paradoxically, E2F‐1 also has the properties of a tumor suppressor. Overexpression of E2F‐1 has been shown to induce apoptosis in some cancer cells. In the current study, the effect of adenovirus‐mediated E2F‐1 gene transfer on human melanoma cell growth was investigated.

E2F-1 Gene Therapy Induces Apoptosis and Increases Chemosensitivity in Human Pancreatic Carcinoma Cells

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing β-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl XL, Bax or Bak following gene or ‘chemogene’ therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.

Bcl-2 inhibits early apoptotic events and reveals post-mitotic multinucleation without affecting cell cycle arrest in human epithelial tumor cells exposed to etoposide

Abstract Defective apoptotic mechanisms are considered to play a role in both the development of malignancy and resistance to chemotherapeutic drugs. The Bcl-2 family of proteins regulate the cellular commitment to survive or die when challenged with various apoptotic stimuli. Purpose: The purpose of this study was to identify the point at which Bcl-2 interrupts the apoptotic cascade initiated following exposure of human tumor cells to etoposide. Methods: A stable Bcl-2-expressing HeLa-transfected clonal cell line, along with its control-vector-transfected counterpart, were utilized in this study. Following etoposide exposure, cells were examined for cell cycle arrest, formation of hyperdiploid cells, apoptotic DNA degradation, loss of plasma membrane integrity, levels of expression of members of the Bcl-2 protein family, caspase activation, degradation of poly(ADP-ribose) polymerase and movement of Bax from cytosol to cellular membrane fractions. Results: Caspase activation, poly(ADP-ribose) polymerase degradation and Bax membrane insertion were initiated rapidly following etoposide removal, concomitantly with cell cycle arrest. Whereas Bcl-2 had no effect on etoposide-induced cell arrest, it interrupted all aspects of apoptosis, including activation of caspases, poly(ADP-ribose) polymerase degradation, DNA fragmentation and loss of plasma membrane integrity. Surprisingly, Bcl-2 also blocked Bax membrane insertion. In addition, Bcl-2 also prevented the increase in cellular levels of Bak, Bax and Bcl-xL, along with degradation of actin and Bax. However, inhibition of etoposide-induced apoptosis by Bcl-2 resulted in the accumulation of giant, multinucleated cells that eventually lost the ability to exclude trypan blue without apoptotic morphology or DNA degradation. Conclusions: These results indicate that biochemical apoptotic processes are initiated concomitant with etoposide-induced cell cycle arrest and are interrupted by Bcl-2 overexpression. However, the aberrant mitotic events induced by etoposide are sufficient to kill these cells even in the absence of apoptosis.

Evaluation of the efficacy, safety and glycaemic effects of evolocumab (AMG 145) in hypercholesterolaemic patients stratified by glycaemic status and metabolic syndrome

To examine the lipid and glycaemic effects of 52 weeks of treatment with evolocumab.

Additive effect of adenovirus-mediated E2F-1 gene transfer and topoisomerase II inhibitors on apoptosis in human osteosarcoma cells.

Recently, it has been demonstrated that Etoposide, a topoisomerase II inhibitor, can induce apoptosis in MDM2-overexpressing tumor cells by inhibition of MDM2 synthesis. We have previously shown that E2F-1 overexpression induces apoptosis of MDM2-overexpressing sarcoma cells, which is related to the inhibition of MDM2 expression. Therefore, the present study was designed to investigate the in vitro and in vivo effect of combined treatment of adenovirus-mediated E2F-1 and topoisomerase II inhibitors on the growth inhibition and apoptosis in human sarcoma cells. Two human sarcoma cell lines, OsACL and U2OS, were treated with topoisomerase II inhibitors (Etoposide and Adriamycin), alone or in combination with adenoviral vectors expressing β-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1). E2F-1 expression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at a low dose (multiplicity of infection, 2) markedly increased the sensitivity of human sarcoma cells to topoisomerase II inhibitor treatment. This cooperative effect of E2F-1 and topoisomerase II inhibitors was less marked in SAOS-2 cells (p53 and pRb null). Topoisomerase II inhibitors also cooperated with E2F-1 overexpression to enhance tumor cell killing in an in vivo model using xenografts in nude mice. When combined with Adriamycin or Etoposide, E2F-1 adenovirus therapy resulted in approximately 95% and 85% decrease in tumor size, respectively, compared to controls (P<.05). These results suggest a new chemosensitization strategy that is effective in MDM2-overexpressing tumors and may have clinical utility. Cancer Gene Therapy (2001) 8, 241–251