A comparison of the metabolic response to phagocytosis in human granulocytes and monocytes.

Recent studies indicate that oxygen radicals such as superoxide or singlet oxygen may be important in the functional activity of human granulocytes. We have examined the possible importance of these radicals in the functional capacity of human blood monocytes. Monocytes, like granulocytes, generate chemiluminescence during phagocytosis. Chemiluminescence is impaired 50-90% by superoxide dismutase, an enzyme which enhances the dismutation of superoxide to hydrogen peroxide. These results indicate that superoxide is related to the chemiluminescence generated by monocytes. Superoxide dismutase in a concentration which impaired chemiluminescence also impaired the staphylococcal killing by monocytes. Hexose monophosphate shunt activity and hydrogen peroxide production by granulocytes and monocytes were also evaluated. The oxidation of [1-14C]glucose was used as a measure of hexose monophosphate shunt activity and the oxidation of [14C]formate as an estimation of hydrogen peroxide production. The oxidation of both substrates by monocytes was increased during phagocytosis but, in contrast to results in granulocytes, was not further increased by the addition of superoxide dismutase. These data indicate that superoxide may be important in bactericidal activity of human monocytes. Our results also suggest that the metabolism of oxygen radicals in monocytes and granulocytes may be different.

Mechanisms of methylene blue stimulation of the hexose monophosphate shunt in erythrocytes.

The response of the hexose monophosphate shunt in erythrocytes was studied with the ionization chamber-electrometer apparatus to measure continuously 14CO2 derived from 14C-labeled substrates. The effect of methylene blue at high (0.1 mM) and low (1 muM) concentrations was evaluated under different gas mixtures; air, carbon monoxide, and 6% carbon monoxide in air. The latter gas mixture results in nearly 100% carboxyhemoglobin but provides a physiologic partial pressure of oxygen. The extent to which pentose is recycled through the shunt in response to methylene blue stimulation was examined with radioactive glucose substrates labeled on the first, second, and third carbon positions. Generation of hydrogen peroxide after stimulation of erythrocytes with methylene blue was evaluated by the catalase-aminotriazole trapping technique, [14C]formate oxidation, and oxidation of reduced glutatione. Stimulation of the shunt with 1 muM methylene blue was markedly impaired in the absence of oxyhemoglobin, but stimulation with 0.1 mM methylene blue was only slightly impaired under the carbon monoxide-air mixture. The higher concentration of methylene blue produced evidence of hydrogen peroxide generation of all three techniques. Despite the evidence for the involvement of oxygen, oxyhemoglobin, and hydrogen peroxide in the response to methylene blue, cells containing methemoglobin induced by sodium nitrite or from a patient with congenital methemoglobinemia responded normally to methylene blue in the absence of oxygen. These experiments indicate that the reactions induced by methylene blue in erythrocytes are more complex than generally thought and that high concentrations are associated with production of peroxide.

Phase II trial of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate in non-Hodgkin's lymphoma: prospective comparison of response with deoxycytidine kinase activity.

A phase II study of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate was done in non-Hodgkin's lymphoma with a loading dose/continuous intravenous infusion schedule, consisting of a 20 mg/m2 loading dose followed by a continuous i.v. infusion of 30 mg/m2/24 h for 48 h. The loading dose was held constant while the continuous i.v. dose was escalated or decreased as appropriate for toxicity. Twenty-six patients were entered on the study; 25 are evaluable for response. The patients' median age was 61 years (range 25 to 73); their mean performance status was 1.1. They had received a mean of 2.6 prior chemotherapeutic regimens, and six also had prior radiation therapy. There was one complete response lasting 9+ months, and there were seven partial responses lasting 20, 13, 11, 11, 10, 5, and 2 months (response rate 32%). Toxicity was acceptable and consisted mainly of myelosuppression. 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate is dephosphorylated in vivo and then is thought to be activated intracellularly to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. The rate-limiting enzyme is deoxycytidine kinase. Deoxycytidine kinase activity was determined on pretreatment tumor samples for correlation with response. There was no difference between the values for responders and nonresponders. There was a trend for higher values in more malignant histological subtypes.

Phase I trial of homoharringtonine administered by prolonged continuous infusion.

Cephalotaxine alkaloids have been extensively used in the Peoples Republic of China for treatment of acute leukemias and solid tumors (Yu-hua, L., Shu-fen, G., Fu-ying, Z., Shu-zhi, X., and Hui-lin, Z. Chin. Med. J., 96: 303-305, 1983). Several Phase I trials of homoharringtonine have been completed in the United States using either bolus administration or continuous infusion over a 5-day period. The major toxicities have been hypotension following rapid administration and myelosuppression when lower doses are infused over 5 to 7 days. None of these studies, however, reproduce the schedule used in China which is i.v. infusion of approximately 1 mg/day over 4-8 h for a period of 14-28 days or more, followed by a rest period of approximately 7-14 days. This study more closely reproduces that schedule as a Phase I trial by decreasing the daily dose of homoharringtonine and using a continuous infusion schedule to allow escalation of total days of treatment. Forty-eight patients entered the study. The final recommended dose of homoharringtonine is 1 mg/m2/day for 30 days followed by a 2-week rest period. The dose limiting toxicity of myelosuppression was severe and prolonged in some patients. Nonhematological toxicities were minimal and generally well tolerated. Patients should be followed with at least weekly blood counts and treatment interrupted pending full marrow recovery if the granulocyte count falls below 1,000/mm3 or the platelet count falls below 100,000/mm3.

A phase I trial of combination fludarabine monophosphate and chlorambucil in chronic lymphocytic leukemia: a Southwest Oncology Group study.

Fludarabine monophosphate is a new antimetabolite with demonstrated activity in chronic lymphocytic leukemia (CLL). We have investigated the practicality of utilizing fludarabine in combination with chlorambucil in a disease-specific phase I trial. Twenty-one patients with advanced and previously treated, relapsed or refractory CLL were treated with chlorambucil plus fludarabine. Chlorambucil was given day 1 at 15 or 20 mg/m2 per os and fludarabine days 1-5 at 10, 15, or 20 mg/m2 intravenously, every 28 days. We concluded that with chlorambucil 15 mg/m2, the maximum tolerated dose for fludarabine was 20 mg/m2 in this patient population with this scheduling. Dose-limiting toxicity was thrombocytopenia. A low incidence of peripheral neuropathy, rash, pulmonary fungal infection, and acute tumor lysis syndrome was also encountered. Although responses were observed, it was impossible from this study to determine whether the combination was better than fludarabine alone in this heavily pretreated population. This study does, however, demonstrate the feasibility of exploring the utility of such a combination in previously untreated patients. An intergroup phase III trial utilizing this combination has been initiated.

Acute thrombotic thrombocytopenic purpura. Another cause for hemolytic anemia and thrombocytopenia in cirrhosis.

Severe hemolytic anemia and thrombocytopenia developed in a 56-year-old man with Laennec's cirrhosis. Rather than having one or more of the expected causes for these problems associated with cirrhosis, he proved to have acute thrombotic thrombocytopenic purpura. Treatment with platelet antiaggregant drugs and exchange plasmapheresis was ineffective in controlling the disease. The presence of advanced liver disease may precipitate or complicate this unusual syndrome.

Howell-Jolly bodies. A clue to splenic infarction.

A 74-year-old woman with secondary erythrocytosis was found to have Howell-Jolly bodies in peripheral blood erythrocytes following acute splenic infarction. The Howell-Jolly bodies were a transitory finding and disappeared six days after the infarction, although the spleen remained abnormal by isotope scanning for several weeks. Careful inspection of the peripheral blood film may be an aid in the diagnosis of acute splenic infarction.

Leukemic reticuloendotheliosis. A study of the origin of the malignant cell.

A highly pure preparation of neoplastic cells from the spleen of a patient with leukemic reticuloendotheliosis was studied for function, membrane characteristics and glucose metabolism. Glass adherence and phagocytosis of small particles (latex and carbon black) were demonstrated with phase contrast microscopy. Staphylocidal activity was similar to that of normal monocytes. Immunofluorescent assays revealed nonspecific uptake of antiserums to immunoglobulins G (IgG), M (IgM), A (IgA) and kappa and kappa and lambda light chains. Rosette assays indicated the presence of receptors for IgG on the surface of all cells but no receptors for complement (C3) or sheep red blood cells. Glucose metabolic studies revealed a pattern that differed from that of normal monocytes or lymphocytes with intermediate values for glycolysis, low hexose monophosphate shunt activity and high Krebs cycle activity. Increments in tritiated (3H)-thymidine uptake and glucose metabolism in response to phytohemagglutinin stimulation were minimal (5 per cent of normal lymphocyte values) and no response was noted with pokeweed mitogen stimulation. These findings suggest that the leukemic reticuloendotheliosis cell most closely resembles cells of the monocyte-histiocyte series.

Acquired factor X deficiency with associated defects in platelet aggregation. A response to corticosteroid therapy.

A 79 year old white women presented with a severe bleeding disorder. Evaluation revealed a prothrombin time of 27.6 seconds (control, 11 seconds) and an activated partial thromboplastin time of 61 seconds. Specific clotting factor assays showed an isolated deficiency of factor X ranging from 7 to 12 per cent on three determinations. Platelet aggregation and bleeding time were also abnormal in response to epinephrine and collagen. Factor X levels and platelet aggregation returned to normal and bleeding stopped after institution of corticosteroid therapy.

Evaluating selected internship candidates and their subsequent performances.

The authors compared selection criteria with subsequent performances of housestaff on a medical service over a 13-year period. The selection criteria studied included class rank, grades in the four major clinical clerkships, scores on the National Board of Medical Examiners Part I examination, Alpha Omega Alpha membership, and numerical ranking by the intern selection committee. Only the last criterion correlated significantly (p = .0015) with subsequent housestaff performance. The synthesis of all available information by an intern selection committee is valuable in choosing candidates with future promise.

The effect of sodium azide on the chemiluminescence of granulocytes--evidence for the generation of multiple oxygen radicals.

It has been established that granulocytes generate superoxide (O-2) as well as hydrogen peroxide (H2O2) during phagocytosis. The chemiluminescence (CL) generated by phagocytes appears dependent on these oxygen radicals (or). however, recent studies suggest that oxygen molecules, including singlet oxygen (1O2) or hydroxyl radicals (OH-), may also be generated during phagocytosis and contribute to CL. We have tested this possibility by studying human granulocyte CL in the presence of 0.1 mM sodium azide, a known inhibitor of myeloperoxidase and catalase and a scavenger of 1O2. The effects of azide on CL were correlated with the effects of this compound on hexose monophosphate shunt (hmps) activity, nitroblue tetrazolium (NBT) dye reduction, formate oxidation, and cytochrome c reduction. CL generated by granulocytes during the phagocytosis of zymosan particles was markedly impaired by azide (24% to 47% of control values). On the other hand, phenomena dependent in part on the presence of O2 radicals, i.e., reduction of NBT dye and cytochrome c, were not impaired by the presence of azide. As would be expected, inhibition of catalase by azide virtually abolished the oxidation of formate, but the burst in HMPS activity associated with phagocytosis was augmented further. The latter observation indicated that azide did not impair generation of H2O2 but increased the relative amount detoxified via the HMPS. The experiment provides evidence that radicals other than O-2 and H2O2 are generated during phagocytosis and that these radicals are major contributors to the CL phenomenon.

The response of red cell hexose monophosphate shunt after sulfhydryl inhibition.

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydryl blockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.

Effect of phagocytosis on the reduced soluble sulfhydryl content of human granulocytes.

The role of reduced glutathione in relation to hexose monophosphate shunt activity and peroxide detoxification has been well established in human erythrocytes. Less is known about the content of reduced glutathione in phagocytic leukocytes and the changes that occur during functional activity. We have measured the reduced sulfhydryl content of normal resting human granulocytes and of cells isolated from a patient with chronic granulomatous disease. Normal cells and those from the patient with chronic granulomatous disease contained similar concentrations of reduced sulfhydryls. Stimulation of a phagocytic response by incubation with opsonized zymosan particles resulted in prompt and nearly complete depletion of intracellular glutathione from normal granulocytes. This fall in reduced glutathione concentration was dependent on the phagocytic load. Exposure of chronic granulomatous disease granulocytes to a similar phagocytic load resulted in a slower and less complete fall in reduced glutathione. In normal cells, those from the chronic granulomatous disease patient, and those from an obligate carrier of the disease, the decrement in reduced glutathione during phagocytosis was correlated with oxidation of 14C-1-glucose and 14C-formate, nitroblue tetrazolium reduction, and the chemiluminescence phenomenon.