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Negative regulation of erythropoiesis by caspasemediated cleavage of GATA-I
CURRENT LITERATURE not attributable to mPEG-indnced toxicity. In addition, cellular response to exogenous interleukin-2 (IL-2) had no proliferative effect in cells that were mPEG treated. Flow cytometric analysis of mPEG-modified cells showed a dramatic decrease in antibody recognition of T ceil molecules, CD2, 3, 4, 8, 28, 1 la, and 62L, as well as in antigen-presentation molecules CD80, 58, and 62L. The authors suggested, based on these and their previous findings, that the use of mPEG-modified blood or blood components could effectively decrease allosensitization rates in transfused patients and diminish the risk of graft-versus-host disease in immunosuppressed patients. Although these data are early in the development of "stealth" cells, they are of interest to both the transfusion and transplantation communities and are worthy of complete review by the readership. (C.H.)
Perioperative blood transfusion as a prognostic indicator in patients with hepatocellular carcinoma. Z Aeahara, K. Katayama, T. Itamoto, et al. World J Surg 23:676-680, 1999. The authors note that pretransplantation blood transfusions improve renal allograft survival, and thus transfusion-related irnrnunomodulation might have a beneficial effect. Conversely, they note that experimental and clinical studies show that transfusion-related immunomodulation may have a negative effect on postoperative outcome after primary surgery for a number of malignant neoplasms. Thus, they attempted to study the relationship of perioperative blood transfusion and clinical outcome in 175 patients with hepatocelhilar carcinoma who underwent resection during the period 1986 to 1994. Partial hepatectomy was performed in 23 (13.1%) patients with, and 152 (86.9%) patients without, blood transfusion. Their statistical analysis was controlled for age, gender, type of hepatitis, histological findings in the liver parenchyma, hepatic ftmctional reserve, numbers of tumors, tumor size, portal vein invasivehess, grade of histological differentiation, extent of hepatic resection, and distance from the surgical margin to the tumor. A Cox multivariate analysis was used to assess the relationship between perioperative blood transfusion recurrence of hepatocellular carcinoma after hepatectomy. This analysis indicated that blood transfusion was the most significant prognostic factor (P = .006) for recurrence in stage I and II patients. In contrast, portal vein invasion was the most significant factor (P = .006) in stage III and IV patients, but in this group blood transfusion was not significant. The extent of resection was nonsignificant in stage I and II patients comparing transfused and nontransfused groups. The cumulative cancer-free survival rates for patients who had received blood transfusion was significantly lower than that for patients who had not received blood transfusions (P = .003). These authors conclude that the data suggest that a perioperative blood transfusion is a significant prognostic indicator for patients with hepatocellular carcinoma who undergo hepatectomy and have stage I or II disease. The authors do note that recently improved techniques for hepatic resection with excellent intraoperative and postoperative management have decreased the operative mortality rate to 1% to 2%, and thus transfusion's effect on prognosis may be more clinically significant than before. The authors hypothesize that the nature of this effect is attributable to transfusion-related immunomodulation. This article is of some interest because it provides some (perhaps limited) additional data to support a role for perioperative blood transfusion and cancer recurrence. However, this
201 article suffers from the shortcoming found in many previous reports in which transfusion may simply be a marker for more advanced disease. The results of the study cannot be used to make any commentary on the possible success or failure of leukoreduction as a means to decrease immune suppression by transfusion. ( C.H. )
Prestorage and bedside leucofiltration of whole blood modulates storage-time-dependent suppression of in vitro TNFoL release. T. Mynster, J.H. Hammer, and H.J. Nielsen. Br J Haematol 106:248-251, 1999. This study was designed to help address the question of the pathophysiology of transfusion-related imnmnomodulation (TRIM). The investigators studied the effects of prestorage and bedside leukoreduction through filtration versus whole blood in the suppression of production of tumor necrosis factor-c~ (TNF-a). Nine units of whole blood were prestorage leukoreduced and stored for 35 days. An additional 27 units were studied that were filtered at the bedside; 9 at day 7, 9 at day 21, and 9 at day 35. Supernatants were collected from the units during storage and used in an Escherichia coli-lipopolysaccharide (LPS)-stimulated TNF release whole blood assay. This methodology allowed storage to be assessed as well as comparison of prestorage leukoreduction with that of the bedside. The authors' hypothesis was that prestorage leukoreduction would prevent storage-time-dependent extracellular accumulation of bioreactive substances, whereas bedside lenkoreduction would be less effective. It should be made clear that this study did not evaluate TNF-e~ release per se but rather the suppression of its release through bioactive substances in the supernatant of stored units. The results confirmed the hypothesis, that is, that supematants from freshly donated blood did not suppress TNF-e~ release. However, over time there was significant suppression of TNF-e~ release in units stored without lenkoreduction and in those leukorednced at the bedside. Units that were stored after prestorage leukoreduction maintained their ability to suppress TNF-e~ release. The authors conclude that prestorage leukoreduction of blood may be advantageous to reduce the in vitro suppressive effect that they studied by assaying TNF-e~ release. The importance of this study is difficult to address. It is hard to understand what substances might be responsible for suppressing TNF-e~ release. The authors include as potential candidates complement but have no description of other possible bioreactive substances. However, a number of factors are released by stored lenkocytes that could change inflammatory cytokines and lead to either immunosuppression or immune enhancement through cytokine networks. The pathogenic mechanisms of transfusion-related immunomodulation remain unclear, although it appears that wltite cells are critical to the development of this effect, and it may be mediated by cytokines. Therefore, this article is brought to the attention of the readership. (C.H.)
Negative regulation of erythropoiesis by caspasemediated cleavage of GATA-I. R. DeMaria, A. Zeuner, A. Eramo, et a/. N atu re 401:489-493, 1999. Under normal steady-state conditions, the average adult makes approximately 3 million erythrocytes every second. This enormous cellular production must be balanced by an equal destruction to prevent life-threatening anemia or polycythemia. Given the very high turnover rate, even small fractional errors in
202
CURRENT LITERATURE
the rate of red cell production would be disasterous. Although the mysteries of upregulation of erythrocyte production were largely shown by the discovery of erythropoietin, less has been known about the mechanism of negative regulation of erythropoiesis. This exciting study from Nature provides insight into red cell downregulation mechanisms. The investigators studied the GATA-1 transcription factor. GATA-1 is known to be the essential transcription factor to turn on many genes essential to erythroid production. Thus, downregulation of the level of GATA-1 will result in a corresponding downregulation of erythroid genes and red cell production. In this article, the investigators show that GATA-1 can be degraded by caspases. Caspases are a family of enzymes that are the major effectors of cellular apoptosis. These enzymes induce proteolytic cleavage adjacent to the aspartic acid of proteins carrying caspase recognition motifs. The investigators show that immature erythroid cells possess "death receptors." Stimulation of these receptors results in production of caspase and degradation of GATA-1. They further show that mature erythroblasts express ligands that bind to the death receptors on immature erythroid progenitors. Thus, excess production of mature erythroblasts results in negative feedback on immature erythroid progenitors. Evidence for the physiological significance of their proposed mechanism was provided by the development of a GATA-1 mutant mouse that was resistant to degradation by caspase. This mutant was insensitive to death receptor ligands and was insensitive to deprivation of erythropoietin. The death receptors expressed by immature erythroid progenitors are common to other apoptos]s systems. They include CD95 and tumor necrosis factor receptor 1, as well as TRAIL (TNF-related apoptosis inducing ligand) receptors. Antibodies capable of blocking CD95 ligand resulted in a dramatic increase in the level of GATA-1 in erythroblasts in culture. Immature erythroid cells cultured in the presence of mature erythroblasts displayed GATA-1 downregulation. The effect was reversed by transducing the immature cells with genes that block death receptor signaling. Using radiolabeled GATA-1 prepared by in vitro transcription, the investigators showed that GATA-1 was sensitive to proteolytic cleavage by caspase. This cleavage resulted in defined fragments that could be detected on Western blotting. To demonstrate the effect of death receptor binding on GATA-1 dowuregulation in cells, erythroblasts were treated for 12 hours with CD95 activation by an antibody. GATA-1 fragments of the size expected to result from caspase proteolysis were observed by immunoblot, Stimulation by tumor necrosis factor resulted in a similar result. The findings of this study are of interest to many aspects of erythroid regulation, including not only the anemia that accmnpanies acute and chronic inflammation but also erythroid overproduction in polycythemias and erythropoietin stimulation. Readers interested in the many physiological roles of the apoptosis process will be particularly interested in this paper. (S.D.)
administered platelet transfusions. Desmopressin acetate (DDAVP) was recognized years ago to improve platelet function among patients undergoing cardiac surgery. However, controlled trials evaluating the routine use of DDAVP to all cardiac patients failed to show benefit. The results of these studies suggested that DDAVP would be beneficial to some patients-the problem was identifying which ones. This study seeks to answer that question. The authors enrolled 203 patients scheduled for elective cardiac surgery into a prospective, doubleblind, placebo-controlled trial evaluating the effect of DDAVP versus placebo on the subset of patients who demonstrated an abnormal hemoSTATUS clot test. Patients were excluded (n = 30) if they required urgent surgery, had preexisting coagulation disorders, had been treated with fibrinolytic or antiplatelet drugs within 2 days of surgery, had microvascular bleeding intraoperatively resulting in transfusion, or had a normal postoperative hemoSTATUS assay. The hemoSTATUS assay is a bedside whole clot assay that measures the shortening of the kaolin-activated clotting time induced by platelet activating factor using four different concentrations of this agent. Results are expressed as a percentage of maximum clotting by a normal population. The lower the percentage (lower clot ratio), the worse the clotting. Among 173 patients, 101 had abnormally low hemoSTATUS results at the end of surgery after neutralization of heparin with protamine. These patients were then randomly treated with either DDAVP or placebo in a blinded fashion. The patients were then transferred to the postoperative care unit, where they received care blinded to the DDAVP-versusplacebo treatment given. Compared with the placebo group, the patients who received DDAVP had less blood loss in 24 hours (624 • 209 treatment v 1,028 -+ 682 placebo); received less transfusion of red blood cells, platelets, and fresh frozen plasma (FFP), and had fewer total blood donor exposures (1.56 + 0.31 treatment v 5.2 + 0.8 control). All differences, including total blood component use and use of each major blood component, were highly statistically significant (P < .001). Indeed, the study was prematurely terminated because the results between the 2 groups were so dramatic. Compared with the placebo group, the DDAVP-treated patients received 50% fewer RBCs, 95% fewer platelets, and 87% fewer units of FFP. The frequency of perioperative myocardial infarctions, pulmonary emboli, deep venous thrombosis, and postoperative mortality was not increased in the DDAVP group compared with placebo. This study from Washington University Medical Center in St Louis provides strong support for the selective use of DDAVP among those cardiac surgery patients who show an abnormal coagulation profile with the bedside assay used in this study. The observed effect of DDAVP treatment cornpared with placebo on blood loss and transfusion use was both statistically significant and clinically meaningful. Readers who participate in the postoperative care of adult elective cardiac surgery patients will want to take a closer look at this article. (S.D.)
Use of point-of-care test in identification of patients who can benefit from desmopressin during cardiac surgery: A randomized controlled trial. G.J. Despotis, V. Levine, R. Saleem, et aL La n cet 354:106-110, 1999.
Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. K. Enjyoji, J. Sevigny, Y. Lin, et al. Nature M e d
Each year in the United States an enormous number of platelet concentrates are transfused to patients who have undergone cardiac surgery. Although prolonged cardiopulmonary bypass is a well-recognized cause of platelet dysfunction, not all patients who are transfused require or receive any benefit from
Blood possesses the extraordinary property of existing as both a solid and a liquid at a single temperature. Even more extraordinary is the fact that under most circumstances the conversion from liquid to solid clot (and eventually back to liquid state) is directed to specific sites of vascular injury. Thus,
5:1010-1017, 1999.
Perioperative blood transfusion as a prognostic indicator in patients with hepatocellular carcinoma. Z Aeahara, K. Katayama, T. Itamoto, et al. World J Surg 23:676-680, 1999. The authors note that pretransplantation blood transfusions improve renal allograft survival, and thus transfusion-related irnrnunomodulation might have a beneficial effect. Conversely, they note that experimental and clinical studies show that transfusion-related immunomodulation may have a negative effect on postoperative outcome after primary surgery for a number of malignant neoplasms. Thus, they attempted to study the relationship of perioperative blood transfusion and clinical outcome in 175 patients with hepatocelhilar carcinoma who underwent resection during the period 1986 to 1994. Partial hepatectomy was performed in 23 (13.1%) patients with, and 152 (86.9%) patients without, blood transfusion. Their statistical analysis was controlled for age, gender, type of hepatitis, histological findings in the liver parenchyma, hepatic ftmctional reserve, numbers of tumors, tumor size, portal vein invasivehess, grade of histological differentiation, extent of hepatic resection, and distance from the surgical margin to the tumor. A Cox multivariate analysis was used to assess the relationship between perioperative blood transfusion recurrence of hepatocellular carcinoma after hepatectomy. This analysis indicated that blood transfusion was the most significant prognostic factor (P = .006) for recurrence in stage I and II patients. In contrast, portal vein invasion was the most significant factor (P = .006) in stage III and IV patients, but in this group blood transfusion was not significant. The extent of resection was nonsignificant in stage I and II patients comparing transfused and nontransfused groups. The cumulative cancer-free survival rates for patients who had received blood transfusion was significantly lower than that for patients who had not received blood transfusions (P = .003). These authors conclude that the data suggest that a perioperative blood transfusion is a significant prognostic indicator for patients with hepatocellular carcinoma who undergo hepatectomy and have stage I or II disease. The authors do note that recently improved techniques for hepatic resection with excellent intraoperative and postoperative management have decreased the operative mortality rate to 1% to 2%, and thus transfusion's effect on prognosis may be more clinically significant than before. The authors hypothesize that the nature of this effect is attributable to transfusion-related immunomodulation. This article is of some interest because it provides some (perhaps limited) additional data to support a role for perioperative blood transfusion and cancer recurrence. However, this
201 article suffers from the shortcoming found in many previous reports in which transfusion may simply be a marker for more advanced disease. The results of the study cannot be used to make any commentary on the possible success or failure of leukoreduction as a means to decrease immune suppression by transfusion. ( C.H. )
Prestorage and bedside leucofiltration of whole blood modulates storage-time-dependent suppression of in vitro TNFoL release. T. Mynster, J.H. Hammer, and H.J. Nielsen. Br J Haematol 106:248-251, 1999. This study was designed to help address the question of the pathophysiology of transfusion-related imnmnomodulation (TRIM). The investigators studied the effects of prestorage and bedside leukoreduction through filtration versus whole blood in the suppression of production of tumor necrosis factor-c~ (TNF-a). Nine units of whole blood were prestorage leukoreduced and stored for 35 days. An additional 27 units were studied that were filtered at the bedside; 9 at day 7, 9 at day 21, and 9 at day 35. Supernatants were collected from the units during storage and used in an Escherichia coli-lipopolysaccharide (LPS)-stimulated TNF release whole blood assay. This methodology allowed storage to be assessed as well as comparison of prestorage leukoreduction with that of the bedside. The authors' hypothesis was that prestorage leukoreduction would prevent storage-time-dependent extracellular accumulation of bioreactive substances, whereas bedside lenkoreduction would be less effective. It should be made clear that this study did not evaluate TNF-e~ release per se but rather the suppression of its release through bioactive substances in the supernatant of stored units. The results confirmed the hypothesis, that is, that supematants from freshly donated blood did not suppress TNF-e~ release. However, over time there was significant suppression of TNF-e~ release in units stored without lenkoreduction and in those leukorednced at the bedside. Units that were stored after prestorage leukoreduction maintained their ability to suppress TNF-e~ release. The authors conclude that prestorage leukoreduction of blood may be advantageous to reduce the in vitro suppressive effect that they studied by assaying TNF-e~ release. The importance of this study is difficult to address. It is hard to understand what substances might be responsible for suppressing TNF-e~ release. The authors include as potential candidates complement but have no description of other possible bioreactive substances. However, a number of factors are released by stored lenkocytes that could change inflammatory cytokines and lead to either immunosuppression or immune enhancement through cytokine networks. The pathogenic mechanisms of transfusion-related immunomodulation remain unclear, although it appears that wltite cells are critical to the development of this effect, and it may be mediated by cytokines. Therefore, this article is brought to the attention of the readership. (C.H.)
Negative regulation of erythropoiesis by caspasemediated cleavage of GATA-I. R. DeMaria, A. Zeuner, A. Eramo, et a/. N atu re 401:489-493, 1999. Under normal steady-state conditions, the average adult makes approximately 3 million erythrocytes every second. This enormous cellular production must be balanced by an equal destruction to prevent life-threatening anemia or polycythemia. Given the very high turnover rate, even small fractional errors in
202
CURRENT LITERATURE
the rate of red cell production would be disasterous. Although the mysteries of upregulation of erythrocyte production were largely shown by the discovery of erythropoietin, less has been known about the mechanism of negative regulation of erythropoiesis. This exciting study from Nature provides insight into red cell downregulation mechanisms. The investigators studied the GATA-1 transcription factor. GATA-1 is known to be the essential transcription factor to turn on many genes essential to erythroid production. Thus, downregulation of the level of GATA-1 will result in a corresponding downregulation of erythroid genes and red cell production. In this article, the investigators show that GATA-1 can be degraded by caspases. Caspases are a family of enzymes that are the major effectors of cellular apoptosis. These enzymes induce proteolytic cleavage adjacent to the aspartic acid of proteins carrying caspase recognition motifs. The investigators show that immature erythroid cells possess "death receptors." Stimulation of these receptors results in production of caspase and degradation of GATA-1. They further show that mature erythroblasts express ligands that bind to the death receptors on immature erythroid progenitors. Thus, excess production of mature erythroblasts results in negative feedback on immature erythroid progenitors. Evidence for the physiological significance of their proposed mechanism was provided by the development of a GATA-1 mutant mouse that was resistant to degradation by caspase. This mutant was insensitive to death receptor ligands and was insensitive to deprivation of erythropoietin. The death receptors expressed by immature erythroid progenitors are common to other apoptos]s systems. They include CD95 and tumor necrosis factor receptor 1, as well as TRAIL (TNF-related apoptosis inducing ligand) receptors. Antibodies capable of blocking CD95 ligand resulted in a dramatic increase in the level of GATA-1 in erythroblasts in culture. Immature erythroid cells cultured in the presence of mature erythroblasts displayed GATA-1 downregulation. The effect was reversed by transducing the immature cells with genes that block death receptor signaling. Using radiolabeled GATA-1 prepared by in vitro transcription, the investigators showed that GATA-1 was sensitive to proteolytic cleavage by caspase. This cleavage resulted in defined fragments that could be detected on Western blotting. To demonstrate the effect of death receptor binding on GATA-1 dowuregulation in cells, erythroblasts were treated for 12 hours with CD95 activation by an antibody. GATA-1 fragments of the size expected to result from caspase proteolysis were observed by immunoblot, Stimulation by tumor necrosis factor resulted in a similar result. The findings of this study are of interest to many aspects of erythroid regulation, including not only the anemia that accmnpanies acute and chronic inflammation but also erythroid overproduction in polycythemias and erythropoietin stimulation. Readers interested in the many physiological roles of the apoptosis process will be particularly interested in this paper. (S.D.)
administered platelet transfusions. Desmopressin acetate (DDAVP) was recognized years ago to improve platelet function among patients undergoing cardiac surgery. However, controlled trials evaluating the routine use of DDAVP to all cardiac patients failed to show benefit. The results of these studies suggested that DDAVP would be beneficial to some patients-the problem was identifying which ones. This study seeks to answer that question. The authors enrolled 203 patients scheduled for elective cardiac surgery into a prospective, doubleblind, placebo-controlled trial evaluating the effect of DDAVP versus placebo on the subset of patients who demonstrated an abnormal hemoSTATUS clot test. Patients were excluded (n = 30) if they required urgent surgery, had preexisting coagulation disorders, had been treated with fibrinolytic or antiplatelet drugs within 2 days of surgery, had microvascular bleeding intraoperatively resulting in transfusion, or had a normal postoperative hemoSTATUS assay. The hemoSTATUS assay is a bedside whole clot assay that measures the shortening of the kaolin-activated clotting time induced by platelet activating factor using four different concentrations of this agent. Results are expressed as a percentage of maximum clotting by a normal population. The lower the percentage (lower clot ratio), the worse the clotting. Among 173 patients, 101 had abnormally low hemoSTATUS results at the end of surgery after neutralization of heparin with protamine. These patients were then randomly treated with either DDAVP or placebo in a blinded fashion. The patients were then transferred to the postoperative care unit, where they received care blinded to the DDAVP-versusplacebo treatment given. Compared with the placebo group, the patients who received DDAVP had less blood loss in 24 hours (624 • 209 treatment v 1,028 -+ 682 placebo); received less transfusion of red blood cells, platelets, and fresh frozen plasma (FFP), and had fewer total blood donor exposures (1.56 + 0.31 treatment v 5.2 + 0.8 control). All differences, including total blood component use and use of each major blood component, were highly statistically significant (P < .001). Indeed, the study was prematurely terminated because the results between the 2 groups were so dramatic. Compared with the placebo group, the DDAVP-treated patients received 50% fewer RBCs, 95% fewer platelets, and 87% fewer units of FFP. The frequency of perioperative myocardial infarctions, pulmonary emboli, deep venous thrombosis, and postoperative mortality was not increased in the DDAVP group compared with placebo. This study from Washington University Medical Center in St Louis provides strong support for the selective use of DDAVP among those cardiac surgery patients who show an abnormal coagulation profile with the bedside assay used in this study. The observed effect of DDAVP treatment cornpared with placebo on blood loss and transfusion use was both statistically significant and clinically meaningful. Readers who participate in the postoperative care of adult elective cardiac surgery patients will want to take a closer look at this article. (S.D.)
Use of point-of-care test in identification of patients who can benefit from desmopressin during cardiac surgery: A randomized controlled trial. G.J. Despotis, V. Levine, R. Saleem, et aL La n cet 354:106-110, 1999.
Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. K. Enjyoji, J. Sevigny, Y. Lin, et al. Nature M e d
Each year in the United States an enormous number of platelet concentrates are transfused to patients who have undergone cardiac surgery. Although prolonged cardiopulmonary bypass is a well-recognized cause of platelet dysfunction, not all patients who are transfused require or receive any benefit from
Blood possesses the extraordinary property of existing as both a solid and a liquid at a single temperature. Even more extraordinary is the fact that under most circumstances the conversion from liquid to solid clot (and eventually back to liquid state) is directed to specific sites of vascular injury. Thus,
5:1010-1017, 1999.