Anlun Ma

Antioxidant Therapy for Prevention of Inflammation, Ischemic Reperfusion Injuries and Allograft Rejection

Oxidative stress results from an imbalance, an excess of oxidants, depletion of antioxidants or failure to repair oxidative damage induced by reactive oxygen species. A vast amount of evidence implicates oxygen-derived free radicals and high-energy oxidants as mediators in many pathological conditions of inflammation, shock, and organ responses to ischemia/reperfusion, which arise during a number of clinical surgical interventions, including transplant graft rejection and coronary bypass surgery, and in such diseases as, diabetes, atherosclerosis, hypertension, organ ischemia/reperfusion, cardiovascular inflammation, cardiac/brain infarction, cancer, pulmonary emphysema and autoimmune diseases. To eliminate or attenuate oxidative stress, antioxidant therapies have been developed and may be of great help to these patients. This review describes recent developments in the field of oxidative stress research and antioxidant function, summarizes new pharmacological strategies that are ongoing in antioxidant therapy with small molecules, free radical-scavenging enzymes, superoxide dismutases, catalase mimetics, flavonoids, vitamins and poly polymerase inhibitors, and presents experimental and clinical evidence of the role of antioxidants in diseases.

Dysfunction of IL-10-producing type 1 regulatory T cells and CD4+CD25+ regulatory T cells in a mimic model of human multiple sclerosis in Cynomolgus monkeys

CD4(+)CD25(+) Treg and IL-10(+) Tr1 cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Dysfunction of Tregs appears to be a critical factor in the pathogenesis of autoimmune diseases. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of CNS, where CD4(+) T cells result in nervous tissue damage. The aim of this study was to investigate the protective role of Treg and Tr1 cells in a mimic model of human MS in Cynomolgus monkeys. This study indicated the suppressive capacity of Tregs from MS monkeys was impaired compared with naive controls. The population of CD4(+)CD25(+) Tregs was decreased in acute stage of MS. However, they showed a restored function and percentage in remitting monkeys. In stable phase, CD4(+)CD25(+) Tregs differentially expressed elevated level of CD62P cell adhesion molecule which contributes to the mechanism by which Treg cells inhibit CD4(+) T cell responses. On the other hand, the percentage of CD4(+)IL-10(+) Tr1 and suppressive function of Tr1 cells were found reduced in MS monkeys. IL-10 secretion was diminished almost 9-fold in active MS, and recovered in active MS. This deficit in IL-10 secretion was specific to CD3/CD46, but not to CD3/CD28 stimulation. The concentrations of IFN-gamma secreted by CD3/CD46-activated T cells were also not affected. These results demonstrate that Tregs are dysfunctional in Cynomolgus monkey with MS. Loss of regulatory function appears to be an important factor in the pathogenesis of MS. Hence, to develop new approaches for induction of Tregs in vivo may be beneficial for the clinical treatment in autoimmune diseases.

Significant prolongation of renal allograft survival by delayed combination therapy of FK778 with tacrolimus in nonhuman primates

Background. Malononitrilamide 715 (FK778) is a new class of low-molecular-weight immunosuppressant that is a derivative of the active metabolite of leflunomide, A77 1726. In this study, the authors evaluated the combined effect of FK778 with tacrolimus in prevention of renal allograft rejection in Vervet monkeys. Methods. Male Vervet monkeys were obtained from Caribbean Primates Ltd. Donor and recipient monkeys were from different breeding colonies. Eleven groups (n≥4 per group) were involved in this study. FK778 and tacrolimus were administered orally for 60 days according to protocol. Results. Naive controls rejected renal grafts, with a median survival time (MST) of 8.0 days in group 1. When recipient monkeys were treated with tacrolimus 1.0 mg/kg/day in group 2 or FK778 2.5 mg/kg/day in group 3, the MST was 16.0 days (P =0.001) and 11.0 days (P =0.266), respectively. Combination therapy of these two agents at the same doses immediately after transplantation resulted in an MST of 25.0 days (P =0.016) in group 4. When tacrolimus was initiated immediately after transplantation and FK778 treatment was delayed until day 7 after surgery in group 5, recipient survivals were significantly prolonged to 38.0 days (P =0.02). These results were repeatable when FK778 5.0 mg/kg/day (9.0 days, P =0.544 in group 6) was combined with tacrolimus 1.0 mg/kg/day immediately after transplantation (8.0 days, P =0.339) in group 7, or when FK778 was delayed 7 days (60.0 days, P =0.002) in group 8. Furthermore, it was also repeatable when FK778 10 mg/kg/day was combined with tacrolimus 1.0 mg/kg/day with a 7-day delay. Conclusions. A significant prolongation of renal allograft survival was produced when FK778 administration was delayed by 7 days combined with tacrolimus in Vervet monkeys.

Adoptive transfer of CD4+CD25+ regulatory cells combined with low-dose sirolimus and anti-thymocyte globulin delays acute rejection of renal allografts in Cynomolgus monkeys

Although donor alloantigen specific Treg cells play an important role in transplant tolerance, therapeutic applications are limited by their low frequency. In this study, isolated Tregs from Cynomolgus monkeys were efficiently expanded by a co-culture system, and maintained suppressive function on the proliferation of CD4(+) effector cells in vitro. Adoptive transfer of expanded donor alloantigen specific Tregs without any immunosuppressants could prolong survival of MHC-mismatched allografts in Cynomolgus monkeys. To reach the feasibility of clinical transplantation, our objectives focused on whether exposure of monkey Tregs to immunosuppressants could preserve suppressive function in vitro and in vivo. The results showed that low-dose sirolimus selectively expanded Tregs, increased the expression of CD25(bright) and Foxp3 markers, and suppressed TCR- or allo-antigens induced CD4(+) T cell proliferation in vitro. In vivo, after pre-treated with anti-thymocyte globulin (ATG) for consecutive 3days, a 14-day therapy of adoptive infusion of donor alloantigen-specific Tregs combined with low-dose sirolimus delayed acute rejection of renal allografts in Cynomolgus monkeys, showing an MST of 48.5days as compared with those of untreated and sirolimus-treated monkeys (7days and 22days). The frequencies of CD4(+)CD25(bright) T cells were significantly elevated in mesenteric lymph nodes vs. those in inguino lymph nodes and peripheral blood. In summary, expanded donor alloantigen specific Tregs exposed to sirolimus can preserve inhibition in vitro and in vivo. Tregs are more resistant to sirolimus than other T cells. Expanded donor alloantigen specific Tregs combined with sirolimus and ATG prolongs renal allograft survival in monkeys, suggesting that sirolimus might be one of the best synergists for Tregs therapy.

Combined therapy of CD4(+)CD25(+) regulatory T cells with low-dose sirolimus, but not calcineurin inhibitors, preserves suppressive function of regulatory T cells and prolongs allograft survival in mice.

Our previous study proved that sirolimus is a potent immunosuppressant which induces long-term allograft survival depends on persistence of alloantigens. CD4(+)CD25(+) regulatory T (Treg) cells are potent suppressors in transplantation. Our objectives focus on whether combined-therapy of Tregs with immunosuppressants could prolong allograft survival in mice. The study showed that inhibition of Tregs was maintained by co-cultured with sirolimus (1 nM) in vitro, but not tacrolimus (1 nM) or CsA (1 nM). When the concentration was increased >100 nM, suppression was fallen. Based on the ability of sirolimus to target effector T cells, but retaining the inhibition of Tregs, an adoptive infusion of donor alloantigen specific Tregs combined with 30-day sirolimus (1 mg/kg) and 3-day ATG (20 mg/kg) was found to prolong heart allograft survival in mice. Even though the cell numbers of CD4(+) T cells were found to decrease in sirolimus-treated mice, sirolimus selectively enhanced the numbers of CD4(+)CD25(+) cells and increased the expression of Foxp3 in spleens and lymph nodes, respectively, in recipients. However, combined therapy with low-dose CsA (5 mg/kg) or tacrolimus (1 mg/kg) reduced significantly the expression of Foxp3 and failed to prolong the allograft survival. In summary, expanded Tregs exposed to sirolimus can survive, proliferate, and preserve inhibition in vitro. Tregs are more resistant to sirolimus than other T cells. Combined with Tregs, sirolimus rather than calcineurin inhibitors, prolongs the allograft survival. Sirolimus may be the best copartner for Tregs therapy. It also suggests calcineurin-dependent signals may be required in the development of Tregs.

Effects of ASKP1240 combined with tacrolimus or mycophenolate mofetil on renal allograft survival in Cynomolgus monkeys.

Background Blocking the CD40-CD154 signal pathway has previously shown promise as a strategy to prevent allograft rejection. In this study, the efficacy of a novel fully human anti-CD40 monoclonal antibody—ASKP1240, administered as a monotherapy or combination therapy (subtherapeutic dose of tacrolimus or mycophenolate mofetil), on the prevention of renal allograft rejection was evaluated in Cynomolgus monkeys. Methods Heterotopic kidney transplants were performed in ABO-compatible, stimulation index 2.5 or higher in the two-way mixed lymphocyte reaction monkey pairs. Animals were divided into 12 groups and observed for a maximum of 180 days. Histopathologic, hematology, and biochemistry analyses were conducted in all groups. Cytokine release (interleukin [IL]-2, IL-4, IL-5, IL-6, tumor necrosis factor, and interferon-&ggr;) was investigated in several groups. Results ASKP1240 prolonged renal allograft survival in a dose-dependent manner in monotherapy. Low-dose (2 mg/kg) or high-dose (5 mg/kg) ASKP1240, in combination with mycophenolate mofetil (15 mg/kg) or tacrolimus (1 mg/kg), showed a significantly longer allograft survival time compared with monotherapy groups. No obvious side effects including drug-related thromboembolic complications were found. Cytokine release was not induced by ASKP1240 administration. Conclusion The present study indicates that ASKP1240, alone or in combination with other immunosuppressive drugs, could be a promising antirejection agent in organ transplantation.

Pharmacokinetics and Pharmacodynamics of ASKP1240, a Fully Human Anti-CD40 Antibody, in Normal and Renal Transplanted Cynomolgus Monkeys

Background The purpose of this study was to evaluate the serum concentration of ASKP1240 (pharmacokinetics [PK]) and the CD40 occupancy of ASKP1240 (pharmacodynamics [PD]) in normal and renal transplanted Cynomolgus monkeys to clarify the PK/PD relationship. Methods In a 70-day study, two ASKP1240 doses (2 and 5 mg/kg) were evaluated in normal and transplanted monkeys. Full doses were administered during the induction phase, and half doses were administered during the maintenance phase. The PK and PD were assessed using ELISA and FACS assays. Results The serum concentration and receptor occupancy of ASKP1240 reached their maximum levels rapidly after the first dose and remained at an almost saturated rate during the induction phase. They then decreased gradually during the maintenance phase in all of the groups. The serum concentration and duration of full receptor occupancy were dose dependent in the normal and transplanted monkeys. On day 70 after therapy with 5 mg/kg ASKP1240, the transplanted monkeys presented a significantly lower occupancy of the CD40 receptors compared with the normal animals (5.5%±14.1% vs. 72.8%±3.4%). The serum concentration of ASKP1240 was also strongly correlated with the occupancy of the ASKP1240 receptors. Conclusion This study showed strong positive PK/PD relationships in renal transplanted and normal monkeys. The results may thus serve as a guide for optimal dosage and timing of ASKP1240 therapy in clinical trials and will propel the translation of ASKP1240 therapeutics from the bench to preclinical and clinical trials.

Cryopreservation of vascularized ovary: An evaluation of histology and function in rats

Cryopreservation of organs has been investigated to sustain the reproductive function of patients undergoing sterilizing chemotherapy and radiotherapy or reproductive surgery. A modified protocol for whole organ cryopreservation was described and the outcome of cryopreservative ovaries was evaluated, and apoptosis of cryopreservative cells stored for different time period and the viability of cryopreserved cells stored at different temperature was examined in rats. Lewis rat ovarian grafts were perfused for 30 min at 0.35 ml/min with M2 medium containing 0.1M fructose and increasing concentrations of 0–1.5M dimethylsulfoxide, cooled to −140°C controlled by a computerized program, and stored in liquid nitrogen (−196°C) for 24 hours. After being thawed, ovaries were transplanted to syngeneic recipients after bilateral oophorectomy. Graft functions were monitored postoperatively. The major findings were that: 1) A 100% survival rate of rat ovaries was achieved in this study. Ovarian hormone secretion recovered in 80% rats which had received cryopreservative ovarian grafts. Postoperative serum estradiol levels in the cryopreservative graft group were lower than in the sham surgery control, but much higher than in the bilateral oophorectomy group. 2) Histological examination of cryopreservative ovarian grafts showed preantral and antral follicles. Two gestations were obtained. 3) Estradiol levels remained low in ovariectomized rats while in the oophorectomized rats given cryopreservative ovarian grafts levels started to rise after 14 ± 3 days. 4) The average viability in the cells from cryopreservative ovary organ (−196°C) was about 71 ± 18% compared to 90 ± 9% of fresh cells. This success should encourage further improvement of cryopreservative techniques for large organs.

Combination therapy of malononitrilamide FK778 with tacrolimus on cell proliferation assays and in rats receiving renal allografts.

Background. Malononitrilamide FK778, an analogue of leflunomide’s active metabolite, is a promising novel small molecule with immunosuppressive and immunomodulatory properties. In this study, we evaluated the ability of combination therapy of FK778 with tacrolimus to inhibit lymphocyte proliferation and to prevent acute allograft rejection. Methods. Proliferation assay was used to evaluate the effect of FK778 plus tacrolimus on murine splenocytes, monkey lymphocytes, and human peripheral blood mononuclear cells, after activation with T or B cell–specific mitogens. A rat kidney transplantation model was used to evaluate the ability of FK778 combined with tacrolimus to prolong allograft survival. Median-effect principle and combination index (CI) were used to determine synergism, summation, or antagonism. Results. A total of 58 combinations of FK778 plus tacrolimus were evaluated. Of the combinations tested, 82.8% (24/29) produced additive to synergistic effects in B cells, whereas 79.3% (23/29) produced moderate antagonistic effects in T cells. A concomitant 14-day therapy of FK778 (10 mg/kg/day) and tacrolimus (1 mg/kg/day) synergistically prolonged renal allograft survival to 25.5±5.9 days (CI=0.458). However, when addition of FK778 to tacrolimus therapy was delayed to day 7 after transplantation, a strong synergism was obtained (mean survival time=74.9±14.8 days, CI<0.001). Conclusions. This study demonstrates that the combination of FK778 with tacrolimus in vitro produces synergistic inhibition on B-cell proliferation but not on T cell proliferation in mice, nonhuman primates, and humans. When the addition of FK778 treatment was delayed to day 7 after transplantation, a strong synergism was produced in prolongation of renal allograft survival in the rat.

Effect of a novel inducible nitric oxide synthase inhibitor in prevention of rat chronic aortic rejections.

Background. Cytotoxic nitric oxide (NO) is produced during ischemia-reperfusion injury and acute and chronic rejection in allografts by expression of inducible (i) NO synthase (NOS). Therefore, continuous inhibition of iNOS may prevent early graft dysfunction and immune injury (rejection) and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity that works by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. Methods. Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n≥6) were involved in this study. FR260330, tacrolimus, or both were administered orally for 14 or 90 days, according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. Results. Both low and high doses of FR260330- or tacrolimus-treated grafts showed significantly decreased intima/(intima+media) ratios at day 90 compared with placebo controls. Combination therapy of low-dose FR260330 with low-dose tacrolimus produced a significant decrease of intima/(intima+media) ratios with intact endothelium compared with placebo controls. Anti–α-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. Conclusions. A selective inhibitor of NOS, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose FR260330 with tacrolimus produces significant protection of immune injury and may serve to improve long-term graft survival and function.