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GENDER SPECIFIC INFLUENCES ON EFFECTOR T-CELL FUNCTION AND PROLIFERATION: 2-METHOXYESTRADIOL MODULATES CELLULAR REJECTION
Abstracts
normothermic beating state and transitioned into working mode for assessment of myocardial function by measuring the cardiac index (mL/minute/gram heart tissue) achieved at a left atrial pressure of 8 mmHg and an aortic diastolic pressure of 40 mmHg. RESULTS: Hearts sustained an equivalent period of warm ischemia (5 C¼281, 25 C¼291, 35 C¼271 minutes, p¼0.50) prior to IR. During IR coronary blood flow (5 C¼48353, 25 C¼72260, 35 C¼90636 mL/min, p<0.01) and coronary sinus lactate concentration (5 C¼ 0.730.06, 25 C¼1.330.03, 35 C¼1.750.15 umol/L, p<0.01) differed among treatment groups. Greater preservation of endothelial cell integrity (electron microscopy endothelial injury score: 5 C¼3.20.5, 25 C¼1.80.2, 35 C¼1.70.3, p¼0.01) and less myocardial injury (troponin I: 5 C¼916, 25 C¼6416, 35 C¼577 pg/ mL/gram, p¼0.04) were evident in hearts reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of indexed cardiac output (5 C¼3.90.8, 25 C¼6.20.4, 35 C¼6.50.0.6 mL/min/ gram, p<0.01). CONCLUSION: Avoidance of profound hypothermia during IR with a normokalemic adenosine-lidocaine cardioplegia minimizes injury and improves the functional recovery of DCD hearts.
384 GENDER SPECIFIC INFLUENCES ON EFFECTOR T-CELL FUNCTION AND PROLIFERATION: 2-METHOXYESTRADIOL MODULATES CELLULAR REJECTION JG Luc, JY Zhao, ED Michelakis, DH Freed, J Nagendran Edmonton, Alberta BACKGROUND:
Gender differences are seen in many disease processes. There is a growing awareness for gender-specific hormonal regulation of immunomodulatory roles however, the mechanism remains unexplained. 2-Methoxyestradiol (2ME2) is an endogenous metabolite of estrogen that is nonestrogenic and is found in lower levels in men than women. 2ME2 has been studied in cancer as an antimitotic-agent that is beneficial by its selectivity in targeting only actively proliferating cancer cells without toxicity to normal cells. As its effect in a transplant rejection setting remains unknown, we hypothesized that 2ME2 can inhibit activated T-cell function. METHODS: Human peripheral blood mononuclear cells (Cedarlane) were cultured and pre-treated with 2ME2 overnight (18H) before activation with Cell Stimulation Cocktail (4H) (eBioscience). The cultured medium was collected for ELISA assays and whole-cell-lysates were collected for western immunoblotting. Five day cultured cells were stained with CellTrace Violet proliferation dye for flow cytometry (FCM). Annexin V/PI assays were also analyzed by FCM. RESULTS: Markers of T-cell activation, TNF-a (p¼0.0025) and IFN-g (p¼0.0216) levels in 2ME2 treated activated T-
S251
cells were not significantly reduced relative to controls. When compared to controls, activated T-cell proliferation was significantly blunted upon treatment with 2ME2, with an observed 10% decrease in apoptosis, no change in necrotic events, and a 30% decrease in caspase-9 activity. The ageinduced senescent marker p21 was preserved whereas the stress-induced senescent marker p16 was completely ablated showing that the T cells were not being harmfully stressed through a senescent pathway. CONCLUSION: Our study is the first to show that 2ME2 is able to effect immunosuppression of activated T-cells in a rejection setting causing them to not proliferate, not apoptose and not undergo stress-induced senescence. Continued and near baseline production of cytokines show that the T-cells remain healthy where they are needed in the innate response. As 2ME2 has a low side effect profile, it may be a possible oral immunomodulatory adjunctive therapy for individuals undergoing cardiac transplantation. CIHR, CNTRP
385 IMPACT OF INITIAL ACIDIC REPERFUSION ON THE FUNCTIONAL RECOVERY OF DCD HEARTS DURING EX VIVO HEART PERFUSION CW White, Y Li, E Ambrose, A Müller, H Le, J Thliveris, TW Lee, RC Arora, G Tian, J Nagendran, L Hryskho, DH Freed Winnipeg, Manitoba BACKGROUND:
Donor hearts following circulatory death (DCD) have been proposed as an alternative source of organs for transplant. DCD hearts experience significant ischemia during the hypoxemic cardiac arrest and warm-ischemic standoff period that ethically define death. Subsequent initial reperfusion (IR) causes intracellular sodium and calcium overload, which play an important role in the pathogenesis of ischemia-reperfusion injury. However, initial acidic reperfusion may minimize sodium and calcium overload, limit ischemia-reperfusion injury, and optimize functional recovery. We investigated the impact of IR pH on the recovery of myocardial function during ex vivo heart perfusion (EVHP). METHODS: Twenty-two pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed and then hearts were procured and reperfused with an oxygenated, normokalemic, adenosine-lidocaine, cardioplegia with a pH of 6.4 (N¼6), 6.9 (N¼8), or 7.4 (N¼8) for 3 minutes at 35 C. Hearts were then perfused ex vivo in a normothermic beating state and transitioned into working mode for assessment of myocardial function by measuring the cardiac index (mL/minute/gram heart tissue) achieved at a left atrial pressure of 8 mmHg and an aortic diastolic pressure of 40 mmHg. RESULTS: Hearts sustained an equivalent period of warm ischemia (6.4¼27.00.7, 6.9¼27.50.3, 7.4¼27.90.4
normothermic beating state and transitioned into working mode for assessment of myocardial function by measuring the cardiac index (mL/minute/gram heart tissue) achieved at a left atrial pressure of 8 mmHg and an aortic diastolic pressure of 40 mmHg. RESULTS: Hearts sustained an equivalent period of warm ischemia (5 C¼281, 25 C¼291, 35 C¼271 minutes, p¼0.50) prior to IR. During IR coronary blood flow (5 C¼48353, 25 C¼72260, 35 C¼90636 mL/min, p<0.01) and coronary sinus lactate concentration (5 C¼ 0.730.06, 25 C¼1.330.03, 35 C¼1.750.15 umol/L, p<0.01) differed among treatment groups. Greater preservation of endothelial cell integrity (electron microscopy endothelial injury score: 5 C¼3.20.5, 25 C¼1.80.2, 35 C¼1.70.3, p¼0.01) and less myocardial injury (troponin I: 5 C¼916, 25 C¼6416, 35 C¼577 pg/ mL/gram, p¼0.04) were evident in hearts reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of indexed cardiac output (5 C¼3.90.8, 25 C¼6.20.4, 35 C¼6.50.0.6 mL/min/ gram, p<0.01). CONCLUSION: Avoidance of profound hypothermia during IR with a normokalemic adenosine-lidocaine cardioplegia minimizes injury and improves the functional recovery of DCD hearts.
384 GENDER SPECIFIC INFLUENCES ON EFFECTOR T-CELL FUNCTION AND PROLIFERATION: 2-METHOXYESTRADIOL MODULATES CELLULAR REJECTION JG Luc, JY Zhao, ED Michelakis, DH Freed, J Nagendran Edmonton, Alberta BACKGROUND:
Gender differences are seen in many disease processes. There is a growing awareness for gender-specific hormonal regulation of immunomodulatory roles however, the mechanism remains unexplained. 2-Methoxyestradiol (2ME2) is an endogenous metabolite of estrogen that is nonestrogenic and is found in lower levels in men than women. 2ME2 has been studied in cancer as an antimitotic-agent that is beneficial by its selectivity in targeting only actively proliferating cancer cells without toxicity to normal cells. As its effect in a transplant rejection setting remains unknown, we hypothesized that 2ME2 can inhibit activated T-cell function. METHODS: Human peripheral blood mononuclear cells (Cedarlane) were cultured and pre-treated with 2ME2 overnight (18H) before activation with Cell Stimulation Cocktail (4H) (eBioscience). The cultured medium was collected for ELISA assays and whole-cell-lysates were collected for western immunoblotting. Five day cultured cells were stained with CellTrace Violet proliferation dye for flow cytometry (FCM). Annexin V/PI assays were also analyzed by FCM. RESULTS: Markers of T-cell activation, TNF-a (p¼0.0025) and IFN-g (p¼0.0216) levels in 2ME2 treated activated T-
S251
cells were not significantly reduced relative to controls. When compared to controls, activated T-cell proliferation was significantly blunted upon treatment with 2ME2, with an observed 10% decrease in apoptosis, no change in necrotic events, and a 30% decrease in caspase-9 activity. The ageinduced senescent marker p21 was preserved whereas the stress-induced senescent marker p16 was completely ablated showing that the T cells were not being harmfully stressed through a senescent pathway. CONCLUSION: Our study is the first to show that 2ME2 is able to effect immunosuppression of activated T-cells in a rejection setting causing them to not proliferate, not apoptose and not undergo stress-induced senescence. Continued and near baseline production of cytokines show that the T-cells remain healthy where they are needed in the innate response. As 2ME2 has a low side effect profile, it may be a possible oral immunomodulatory adjunctive therapy for individuals undergoing cardiac transplantation. CIHR, CNTRP
385 IMPACT OF INITIAL ACIDIC REPERFUSION ON THE FUNCTIONAL RECOVERY OF DCD HEARTS DURING EX VIVO HEART PERFUSION CW White, Y Li, E Ambrose, A Müller, H Le, J Thliveris, TW Lee, RC Arora, G Tian, J Nagendran, L Hryskho, DH Freed Winnipeg, Manitoba BACKGROUND:
Donor hearts following circulatory death (DCD) have been proposed as an alternative source of organs for transplant. DCD hearts experience significant ischemia during the hypoxemic cardiac arrest and warm-ischemic standoff period that ethically define death. Subsequent initial reperfusion (IR) causes intracellular sodium and calcium overload, which play an important role in the pathogenesis of ischemia-reperfusion injury. However, initial acidic reperfusion may minimize sodium and calcium overload, limit ischemia-reperfusion injury, and optimize functional recovery. We investigated the impact of IR pH on the recovery of myocardial function during ex vivo heart perfusion (EVHP). METHODS: Twenty-two pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed and then hearts were procured and reperfused with an oxygenated, normokalemic, adenosine-lidocaine, cardioplegia with a pH of 6.4 (N¼6), 6.9 (N¼8), or 7.4 (N¼8) for 3 minutes at 35 C. Hearts were then perfused ex vivo in a normothermic beating state and transitioned into working mode for assessment of myocardial function by measuring the cardiac index (mL/minute/gram heart tissue) achieved at a left atrial pressure of 8 mmHg and an aortic diastolic pressure of 40 mmHg. RESULTS: Hearts sustained an equivalent period of warm ischemia (6.4¼27.00.7, 6.9¼27.50.3, 7.4¼27.90.4