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Post-IL2 receptor blockade increases fas ligand expression and apoptosis in allospecifically-activated human helper T cells
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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
P100. Soluble Plasma Factor in Trauma Patients Following Injury Alters Platelet Activation. J. M. Kehoe, M.D., R. C. Gosselin, T. Paglieroni, R. Jacoby, J. T. Owings, M.D., UC Davis Health System. We have previously observed striking differences in the percentage of activated platelets identified following injury in two similar trauma populations. When a monoclonal antibody (MoAb) to CD61 (glycoprotein Ib-IX) was used to identify platelets, the percentage of activated platelets was higher than when MoAb to CD42a (glycoprotein IIb-IIIa) was used to identify platelets. This discordance is not seen in other non-trauma patient populations. We hypothesized that a plasma component generated by severe injury was responsible for this finding. Fifteen trauma patients, defined as ISS ⬎ 9, had blood drawn within 24 hours of injury. Flow cytometry was used to identify platelets via MoAbs to both CD42a and CD61. Activated platelets were identified with the MoAb PAC-1 (binds the active conformation of GPIIb-IIIa). This experiment was then repeated while adding platelet-free plasma from each trauma patient to ABO-matched blood from healthy controls. Lastly, experiments were repeated with the addition of a MoAb that blocks the platelet Fc-gamma receptor, to preclude in-vitro platelet activation (known to occur via this receptor). Platelet activation as expressed by PAC-1 binding was significantly higher when CD61 was used to identify platelets rather than CD42a (p ⫽ 0.02, Wilcoxon Signed Rank test). Adding trauma plasma to platelets from healthy controls resulted in the same discordance (p ⫽ 0.01). Fc-gamma blockade did not significantly change this discordance. In trauma patients, flow cytometric evaluation of platelet activation is influenced by the MoAb used to identify platelets. Discordance in platelet activation occurred in healthy control with the addition of trauma plasma, and was not fc-gamma dependent. These data suggest the existence of a unique soluble component in the plasma of trauma patients that affects the platelet membrane in the GPIIb-IIIa region. P101. Mitochondrial Calcium Uptake During Ischemia is Regulated by Cytosolic ATP:ADP Ratio. A. Wakata, B.S., A. E. Belous, M.D., Ph.D., C. D. Knox, B.A., J. M. Pierce, B.S., I. B. Nicoud, B.S., C. D. Anderson, M.D., C. W. Pinson, M.D., M.B.A., R. S. Chari, M.D. Vanderbilt University Medical Center. Prolonged ischemia causes functional impairment of mitochondria by irreversible Ca 2⫹ overload, which leads to induction of permeability transition and initiation of apoptosis. It has been reported that cytosolic adenine nucleotides regulate mitochondrial Ca 2⫹ uptake in a concentration dependent manner, through a mechanism which does not require hydrolysis. In ischemia, cytosolic [ATP] is decreased and [ADP] is increased. We hypothesized that during ischemia, the fluctuations in the ATP:ADP ratio, rather than absolute changes in either, govern mitochondrial Ca 2⫹ uptake. Methods: Mitochondria were isolated from rat livers by differential centrifugation and incubated at 37°C in Mg 2⫹-free medium containing 0.2 M 45Ca 2⫹, 1 mM pyruvate, and 1 mM malate. Mitochondrial Ca 2⫹ uptake was deter-
mined by scintillation counting in the presence of varying ATP:ADP (3:1, 1:1, 1:3) and each alone (0, 0.1, 1, 3 mM). All measurements were performed in triplicate on mitochondria prepared from 4 separate rats. Statistical analysis was performed using Student t-test, with p ⬍ 0.05 taken as significant. Results: 1) ATP and ADP alone at low concentration each had a strong regulatory effect on mitochondrial Ca 2⫹ accumulation: both ATP and ADP increased Ca 2⫹ uptake at concentrations ⬍ 0.1 mM and ⬍ 1 mM, respectively. 2) At [ATP] ⬎ 0.1 mM and [ADP] ⬎ 1 mM, however, there was a decrease in Ca 2⫹ uptake. 3) The individual effects of ATP and ADP on mitochondrial Ca 2⫹ uptake were not additive when combined: Ca 2⫹ uptake was increased by about 12-fold at ATP:ADP ratio of 1:3, while it was only increased by 3-fold at 3:1. Conclusion: These data indicate that mitochondrial Ca 2⫹ uptake is significantly enhanced when the extramitochondrial ATP:ADP is low; thus, ischemic conditions facilitate mitochondrial Ca 2⫹ uptake. Maintenance of a cytosolic ATP:ADP ⬎ 3 during ischemia, however, will minimize mitochondrial Ca 2⫹ overload and may minimize subsequent cellular damage.
P102. Post-IL2 Receptor Blockade Increases fas Ligand Expression and Apoptosis in Allospecifically-Activated Human Helper T Cells. K. J. Woodside, M.D., Y. Liu, G. C. Hunter, M.D., J. A. Daller, M.D., Ph.D. University of Texas Medical Branch & University of Rochester. Introduction: Pre-interleukin-2 receptor (IL-2R) blockade with cyclosporine (CsA) abrogates apoptosis of activated allospecific lymphocytes. The mammalian target of rapamycin (mTOR) is a postIL-2R signaling molecule with anti-apoptotic effects. We examined the effects of rapamycin (RPM) and CsA on apoptosis and surface expression of the apoptotic mediators fas and fasL in human mixed lymphocyte cultures (MLC). Methods: Peripheral blood lymphocytes were isolated by Ficoll-Paque gradient centrifugation. Responder and irradiated stimulator cells were co-cultured with logfold concentrations of RPM or CsA. Cells were stained with PE-labeled anti-CD4 antibody and annexin-V and 7-AAD, FITC-labeled anti-fas antibody, or FITC-labeled anti-fasL antibody and analyzed by flow cytometry. Cells staining for annexin-V, but excluding 7-AAD, were interpreted as apoptotic. Results: While CsA treatment decreased CD4⫹ T cell surface expression of fasL by up to 60% by day 7, RPM increased fasL express by up to 40%. Interestingly, fas surface expression was decreased by both RPM (up to 83%) and CsA (up to 89%). RPM increased CD4⫹ T cell apoptosis by up to twofold, while CsA decreased apoptosis by up to 50%. Conclusions: Post-IL-2R blockade with RPM increases apoptosis of allospecifically-activated helper T cells. This effect is consistent with the antiapoptotic function of mTOR, and may explain the dissimilar apoptotic effects when compared to pre-IL-2R blockade with CsA. The increase in fasL expression, an apoptotic effector molecule, on the cell surface in response to post-IL2R blockade suggests a potential mechanism for this effect. P103. Post-Transplant Total Lymphoid Irradiation Prolongs the Survival of Rat Liver Allografts. H. Obara, M.D.,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
P100. Soluble Plasma Factor in Trauma Patients Following Injury Alters Platelet Activation. J. M. Kehoe, M.D., R. C. Gosselin, T. Paglieroni, R. Jacoby, J. T. Owings, M.D., UC Davis Health System. We have previously observed striking differences in the percentage of activated platelets identified following injury in two similar trauma populations. When a monoclonal antibody (MoAb) to CD61 (glycoprotein Ib-IX) was used to identify platelets, the percentage of activated platelets was higher than when MoAb to CD42a (glycoprotein IIb-IIIa) was used to identify platelets. This discordance is not seen in other non-trauma patient populations. We hypothesized that a plasma component generated by severe injury was responsible for this finding. Fifteen trauma patients, defined as ISS ⬎ 9, had blood drawn within 24 hours of injury. Flow cytometry was used to identify platelets via MoAbs to both CD42a and CD61. Activated platelets were identified with the MoAb PAC-1 (binds the active conformation of GPIIb-IIIa). This experiment was then repeated while adding platelet-free plasma from each trauma patient to ABO-matched blood from healthy controls. Lastly, experiments were repeated with the addition of a MoAb that blocks the platelet Fc-gamma receptor, to preclude in-vitro platelet activation (known to occur via this receptor). Platelet activation as expressed by PAC-1 binding was significantly higher when CD61 was used to identify platelets rather than CD42a (p ⫽ 0.02, Wilcoxon Signed Rank test). Adding trauma plasma to platelets from healthy controls resulted in the same discordance (p ⫽ 0.01). Fc-gamma blockade did not significantly change this discordance. In trauma patients, flow cytometric evaluation of platelet activation is influenced by the MoAb used to identify platelets. Discordance in platelet activation occurred in healthy control with the addition of trauma plasma, and was not fc-gamma dependent. These data suggest the existence of a unique soluble component in the plasma of trauma patients that affects the platelet membrane in the GPIIb-IIIa region. P101. Mitochondrial Calcium Uptake During Ischemia is Regulated by Cytosolic ATP:ADP Ratio. A. Wakata, B.S., A. E. Belous, M.D., Ph.D., C. D. Knox, B.A., J. M. Pierce, B.S., I. B. Nicoud, B.S., C. D. Anderson, M.D., C. W. Pinson, M.D., M.B.A., R. S. Chari, M.D. Vanderbilt University Medical Center. Prolonged ischemia causes functional impairment of mitochondria by irreversible Ca 2⫹ overload, which leads to induction of permeability transition and initiation of apoptosis. It has been reported that cytosolic adenine nucleotides regulate mitochondrial Ca 2⫹ uptake in a concentration dependent manner, through a mechanism which does not require hydrolysis. In ischemia, cytosolic [ATP] is decreased and [ADP] is increased. We hypothesized that during ischemia, the fluctuations in the ATP:ADP ratio, rather than absolute changes in either, govern mitochondrial Ca 2⫹ uptake. Methods: Mitochondria were isolated from rat livers by differential centrifugation and incubated at 37°C in Mg 2⫹-free medium containing 0.2 M 45Ca 2⫹, 1 mM pyruvate, and 1 mM malate. Mitochondrial Ca 2⫹ uptake was deter-
mined by scintillation counting in the presence of varying ATP:ADP (3:1, 1:1, 1:3) and each alone (0, 0.1, 1, 3 mM). All measurements were performed in triplicate on mitochondria prepared from 4 separate rats. Statistical analysis was performed using Student t-test, with p ⬍ 0.05 taken as significant. Results: 1) ATP and ADP alone at low concentration each had a strong regulatory effect on mitochondrial Ca 2⫹ accumulation: both ATP and ADP increased Ca 2⫹ uptake at concentrations ⬍ 0.1 mM and ⬍ 1 mM, respectively. 2) At [ATP] ⬎ 0.1 mM and [ADP] ⬎ 1 mM, however, there was a decrease in Ca 2⫹ uptake. 3) The individual effects of ATP and ADP on mitochondrial Ca 2⫹ uptake were not additive when combined: Ca 2⫹ uptake was increased by about 12-fold at ATP:ADP ratio of 1:3, while it was only increased by 3-fold at 3:1. Conclusion: These data indicate that mitochondrial Ca 2⫹ uptake is significantly enhanced when the extramitochondrial ATP:ADP is low; thus, ischemic conditions facilitate mitochondrial Ca 2⫹ uptake. Maintenance of a cytosolic ATP:ADP ⬎ 3 during ischemia, however, will minimize mitochondrial Ca 2⫹ overload and may minimize subsequent cellular damage.
P102. Post-IL2 Receptor Blockade Increases fas Ligand Expression and Apoptosis in Allospecifically-Activated Human Helper T Cells. K. J. Woodside, M.D., Y. Liu, G. C. Hunter, M.D., J. A. Daller, M.D., Ph.D. University of Texas Medical Branch & University of Rochester. Introduction: Pre-interleukin-2 receptor (IL-2R) blockade with cyclosporine (CsA) abrogates apoptosis of activated allospecific lymphocytes. The mammalian target of rapamycin (mTOR) is a postIL-2R signaling molecule with anti-apoptotic effects. We examined the effects of rapamycin (RPM) and CsA on apoptosis and surface expression of the apoptotic mediators fas and fasL in human mixed lymphocyte cultures (MLC). Methods: Peripheral blood lymphocytes were isolated by Ficoll-Paque gradient centrifugation. Responder and irradiated stimulator cells were co-cultured with logfold concentrations of RPM or CsA. Cells were stained with PE-labeled anti-CD4 antibody and annexin-V and 7-AAD, FITC-labeled anti-fas antibody, or FITC-labeled anti-fasL antibody and analyzed by flow cytometry. Cells staining for annexin-V, but excluding 7-AAD, were interpreted as apoptotic. Results: While CsA treatment decreased CD4⫹ T cell surface expression of fasL by up to 60% by day 7, RPM increased fasL express by up to 40%. Interestingly, fas surface expression was decreased by both RPM (up to 83%) and CsA (up to 89%). RPM increased CD4⫹ T cell apoptosis by up to twofold, while CsA decreased apoptosis by up to 50%. Conclusions: Post-IL-2R blockade with RPM increases apoptosis of allospecifically-activated helper T cells. This effect is consistent with the antiapoptotic function of mTOR, and may explain the dissimilar apoptotic effects when compared to pre-IL-2R blockade with CsA. The increase in fasL expression, an apoptotic effector molecule, on the cell surface in response to post-IL2R blockade suggests a potential mechanism for this effect. P103. Post-Transplant Total Lymphoid Irradiation Prolongs the Survival of Rat Liver Allografts. H. Obara, M.D.,