Characterization of the effects of asparaginase from escherichia coli and a glutaminase-free asparaginase from vibrio succinogenes on specific cell-mediated cytotoxicity

Asparaginases isolated from Escherichia coli and Erwinia carotovora are effective in the treatment of acute lymphoblastic leukemia in man. During treatment with either of these enzymes, patients frequently experience pronounced toxicity, including liver and pancreatic dysfunction and immunosuppression. The capabilities of these enzymes to hydrolyze L-glutamine, an important amino acid in mammalian intermediary metabolism, have led investigators to suggest that the glutaminase activity may be responsible for the observed toxicities. Unlike other asparaginases, an enzyme isolated in our laboratory from Vibrio succinogenes, has been shown to be a potent antilymphoma agent and to be highly specific for L-asparagine. Our previous work has established that the glutaminase-free asparaginase from V. succinogenes does not suppress the in vivo humoral or cell-mediated immune responses of mice to sheep red blood cells, whereas the E. coli enzyme abolishes these responses. In this study, we describe the mechanisms by which E. coli asparaginase suppresses specific antibody-dependent cell-mediated cytotoxicity. An analysis of the kinetics of appearance of the specific cell-mediated cytotoxic response of spleen cells after immunization with sheep red blood cells revealed that it paralleled the IgG plaque-forming cell response. In addition, 18-h culture supernatants from immune spleen cells conferred upon non-immune cells the ability to specifically lyse sheep red blood cells. Pretreatment of immune supernatants with protein A-Sepharose 4B absorbed out the soluble arming factor. An analysis of E. coli asparaginase-induced suppression of specific antibody-dependent cell-mediated cytotoxicity revealed that a decreased synthesis of specific IgG was responsible for the observed reduction in cytotoxic reactivity. When culture supernatants from spleen cells of immunized animals not treated with asparaginase were added to spleen cells from E. coli asparaginase-treated mice, however, there was an increased cytotoxic response. This suggested that there was an increase in the number or ratio of effector cells for the specific antibody-dependent cell-mediated cytotoxic response in spleens of enzyme-treated animals. Through the characterization of the immunosuppressive effects of two asparaginases—one having the catalytic capability to hydrolyze L-glutamine and the other lacking this catalytic activity—we now have evidence that the immunosuppressive effects of E. coli asparaginase on specific antibody-dependent cell-mediated cytotoxicity cannot be the result of asparagine depletion alone. Our data demonstrate that the glutaminase-free asparaginase from V. succinogenes is not immunosuppressive and suggest that an asparaginase with high substrate specificity for L-asparagine may be a safer treatment for leukemia patients.     

Long-term effect of motor cortical repetitive transcranial magnetic stimulation [correction

Repetitive transcranial magnetic stimulation (rTMS) recently has been assessed as a noninvasive treatment modality for movement and psychiatric disorders, whereas the mechanism underlying the therapeutic effects is not fully understood. Studies in rodents showed lasting functional changes in some selected regions, such as limbic-associated structures, but unfocused brain stimulation did not clarify the regional effects. To address the topographical and temporal profiles of the effects on glucose metabolism in primate brain, we performed rTMS and repeated (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) before, during, and up to 16 days after rTMS in anesthetized cynomologous monkeys. We delivered a total of 2,000 pulses of 5 Hz-rTMS over the right precentral gyrus using a small-sized eight-figured coil that induced a localized electrical field. Voxel-based analysis in a standard space of the macaque brain showed statistically robust changes in FDG uptake: a decrease in the motor/premotor cortices and an increase in the limbic-associated areas involving the anterior/posterior cingulate, and orbitofrontal cortices. Interestingly, these uptake changes continued for at least 8 days and the magnitude of the lasting effects in the limbic-related areas was negatively correlated across subjects with those in the motor/premotor cortices. The results demonstrate that motor rTMS has a long-term lasting effect on motor-related regions and distant limbic-related areas via functional connections.     

A Phase II Trial of Imatinib Mesylate as Maintenance Therapy for Patients With Newly Diagnosed C-kit–positive Acute Myeloid Leukemia

IntroductionAdults with acute myeloid leukemia (AML) have a high rate of remission; however, more than 50% relapse. C-kit is expressed in approximately 60% of patients with de novo AML and represents a potential therapeutic target.Materials and MethodsPatients with newly diagnosed AML received 12 months of imatinib mesylate as maintenance therapy after the completion of post-remission therapy. The primary objective was to determine whether this approach improved progression-free survival (defined as no relapse and no death) compared with historical controls.ResultsThe median progression-free survival of patients < 60 years of age was 52.1 months (historical control, 13 months) and for patients ≥ 60 years of age was 10.7 months (historical control, 8 months). The median level of AF1q expression was high (9.59), and 84% of patients had moderate or high levels of drug-resistance factors.ConclusionsImatinib maintenance therapy may improve the outcome of newly diagnosed patients with AML who are < 60 years of age.     

Innate-like Cytotoxic Function of Bystander-Activated CD8+ T Cells Is Associated with Liver Injury in Acute Hepatitis A

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MIR29B regulates expression of MLLT11 (AF1Q), an MLL fusion partner, and low MIR29B expression associates with adverse cytogenetics and poor overall survival in AML

MLLT11, an MLL fusion partner, is a poor prognostic biomarker for paediatric acute myeloid leukaemia (AML), adult normal cytogenetics AML, and adult myelodysplastic syndrome. MLLT11 is highly regulated during haematopoietic progenitor differentiation and development but its regulatory mechanisms have not been defined. In this study, we demonstrate by transfection experiments that MIR29B directly regulates MLLT11 expression in vitro. MIR29B expression level was also inversely related to MLLT11 expression in a cohort of 56 AML patients (P<0·05). AML patients with low MIR29B/elevated MLLT11 expression had poor overall survival (P=0·038). Therefore, MIR29B may be a potential prognostic biomarker for AML patients.     

Glutaminase-free asparaginase from vibrio succinogenes: An antilymphoma enzyme lacking hepatotoxicity

Successful treatment of neoplastic disease has been impeded by the lack of therapeutic agents which specifically destroy tumor cells. Enzymes which selectively deplete substrates required by tumor cells, but not by normal tissue, could improve therapeutic indices dramatically. Presently, microbial asparaginases are used clinically for treating acute lymphocytic leukemia. While these enzymes should destroy neoplastic cells and spare normal tissues, their use is accompanied by many toxic effects and immunosuppression. The administration of Escherichia coli or Erwinia carotovora asparaginase depletes circulating glutamine as well as asparagine. It has been suggested that this glutaminase activity may be responsible for the observed toxicities. We have isolated a glutaminase-free asparaginase from Vibrio succinogenes with potent antilymphoma activity. Previously, we demonstrated that administration of Vibrio asparaginase does not cause the immunosuppression of humoral or cell-mediated responses associated with treatment by other microbial enzymes. In the present communication we have evaluated the hepatotoxic effects of different asparaginases since liver damage is the major toxicity associated with treatment by these microbial enzymes. BALB/c mice treated with 50 IU of E. coli asparaginase daily for 4 days exhibited diffuse microfatty infiltration within hepatocytes throughout the liver. Cross-sections of liver from V. succinogenes asparaginase-treated mice appeared normal as compared to specimens from control animals. Quantitation of the total amount of extractable lipid from the livers of E. coli asparaginase-treated animals indicated a 45% and 127% increase in lipid concentration as compared to controls after 4 and 5 days of treatment, respectively. The Vibrio enzyme did not cause a change in extractable lipid concentration as compared to control animals. Plasma antithrombin III activity and albumin, triglyceride and cholesterol concentrations all decreased in E. coli asparaginase-treated mice, confirming hepatotoxicity. No modifications in plasma proteins were observed in mice treated with asparaginase from V. succinogenes. The plasma lipids did decrease minimally but only the levels of cholesterol were shown to be statistically significant from those of controls. The data strongly support the concept that specific asparagine depletion is not significantly hepatotoxic. More importantly, the asparaginase from V. succinogenes may serve as a potent antileukemic agent without causing damage to normal tissues.