C. Haanen

A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.

Flow cytometry of apoptotic cell death

The term apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from necrosis or accidental cell death. The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events which is an integral part of physiological homeostasis. Techniques designed to identify, quantitate and characterize apoptosis are numerous, but flow cytometry (FCM) remains the methodology of choice to study the apoptotic cascade in relation to cell type, trigger and time. This review outlines the main stages of the apoptotic cascade together with current FCM methods. All FCM apoptosis assays described have a solid experimental basis and have been used successfully in basic research on molecular and biochemical mechanisms of apoptosis. In various clinical settings the ability to follow the apoptotic process in patient samples may offer the rationale for optimal treatment schedules.

Relative eosinophilla and functional adrenal insufficiency in critically ill patients

Albertus Beishuizen, Istvan Vermes, Bonno S Hylkema, Clemens Haanen The number of circulating eosinophils was noted to be a marker for adrenocortical function by Thorn 50 years ago, but then relegated to oblivion. Present day electronic cell counters provide accurate data on eosinophil counts, which suggests a practical method to estimate the biological effect of glucocorticoids. The concept of occult or relative adrenal insufficiency in critically ill patients has replaced the notion of complete adrenal insufficiency. However, at present no

Intensive antileukemic treatment of patients younger than 65 years with myelodysplastic syndromes and secondary acute myelogenous leukemia

Intensive antileukemic treatment was evaluated in 22 patients with secondary acute myelogenous leukemia (sAML) and 14 patients with myelodysplastic syndrome (MDS). Results of combination remission‐induction chemotherapy were compared with 126 patients contemporarily treated for primary AML. The duration of hypoplasia, induced by remission induction chemotherapy, tended to be longer in the sAML and MDS patients when compared to de novo AML, but reached significance only for the duration of thrombocytopenia: 26 days versus 18 days (P <0.01). The number of hypoplastic deaths during remission‐induction chemotherapy of patients with sAML and MDS was low. Four of the 36 patients treated for sAML or MDS died during hypoplastic phases induced by remission‐induction chemotherapy. The complete remission (CR) rates were similar in primary AML (67%), sAML (62%), and MDS (64%). The CR rates of patients younger than 45 years were 75% for de novo AML, 75% for sAML, and 71% for MDS. Remission rates in patients older than 45 years were identical in the three subgroups but significantly (P < 0.005) inferior to those obtained in younger patients: 56%, 50%, and 57%, in de novo AML, sAML, and MDS, respectively. The remission duration without bone marrow transplant (BMT) was significantly shorter (P < 0.01) in MDS and sAML when compared with primary AML. Long‐lasting CR in MDS and sAML was only obtained in three of the six patients treated with allogeneic BMT. Intensive antileukemic therapy could be considered in young patients with MDS and life‐threatening cytopenias or patients with sAML.

Apoptosis and Secondary Necrosis of Lymphocytes in Culture

It has been reported that cultured peripheral B lymphocytes of chronic lymphocytic leukemia (B-CLL) patients show a high degree of apoptosis (programmed cell death). Till now, no data exist about the occurrence of in vitro apoptosis of normal B and T cells. We measured the amount of apoptosis and secondary necrosis (type 2 necrosis) in B-CLL lymphocytes and in normal peripheral B and T lymphocytes in culture. Observations were made on B-CLL lymphocytes and on normal B and T cells purified by immunomagnetic cell sorting. Apoptosis and secondary necrosis were measured using a recently described sensitive flow-cytometric assay, probing simultaneously for cell surface exposure of phosphatidylserine with the use of FITC-labeled annexin-V and for cell membrane integrity as demonstrated by the exclusion of propidium iodide. The degree of in vitro apoptosis and secondary necrosis of normal B cells appears to be higher than that of normal T cells, and even higher than that of B-CLL cells. The results indicate that cultured mature circulating normal B lymphocytes exhibit a higher cell death rate than normal T cells and B-CLL lymphocytes.

Hageman factor, a novel sialoglycoprotein with esterase activity

A novel esterase has been isolated from bovine plasma in a highly purified form. According to electrophoresis and ultracentrifugal sedimentation patterns it represents a homogeneous 7.1 S sialoglycoprotein being composed of 5.9% neutral carbohydrates, 4.8% aminosugars and 4.4% sialic acid. In catalytic amounts it normalizes the prolonged clotting time of plasma deficient in Hageman factor and exhibits a strong esterase activity towards N-benzoyl-l-arginine ethyl ester.

Dose intensity of MOPP chemotherapy and survival in Hodgkin's disease.

The association between dose intensity of chemotherapy with the rate of complete remission (CR), the duration of disease-free survival (DFS), and overall survival (OS) was separately analyzed for 67 patients initially treated with mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), and for 75 patients in relapse following radiotherapy who had received MOPP as a salvage regimen. In both groups of patients, the fraction of the total dose of mechlorethamine delivered in all cycles divided by the planned dose for six cycles was strongly associated with OS (P = .002 for patients receiving initial MOPP and P = .02 for the salvage group, respectively). B symptoms were independent of drug-derived variables associated with OS (corresponding P values .03 for initial MOPP and .004 for the salvage group). The predictive value of mechlorethamine dosage with regard to OS was retained in an analysis restricted to the patients receiving greater than or equal to six cycles of chemotherapy. In the initial chemotherapy group, mechlorethamine dosage was associated with attainment of CR but none of the variables tested was predictive of DFS. In the salvage chemotherapy group, mechlorethamine dosage was associated with attainment of CR and duration of DFS as well. The results emphasize that, besides tumor characteristics, optimal dosage of chemotherapy is of great importance for survival.

Familial Thrombopathic Thrombocytopenia

A family with hereditary thrombopathic thrombocytopenia is described, with eight affected members in three generations. The haemorrhagic diathesis manifests itself by bleeding from mucous membranes and after operative procedures. The platelet count varies from 20,000 to 80,000 per cu.mm. The number of megakaryocytes in the bone marrow has been examined in two affected members, and the platelet survival in one, and both are normal.

Apoptosis- and necrosis-inducing potential of cladribine, cytarabine, cisplatin, and 5-fluorouracil in vitro: a quantitative pharmacodynamic model.

Purpose: The purpose of this study was to characterize the concentration-dependent induction of apoptosis by anticancer drugs in vitro. Methods: The apoptosis- and necrosis-inducing potential of the anticancer drugs cladribine (CDA), cytarabine (ARA-C), cisplatin (CDDP), and 5-fluorouracil (5FU) were studied in vitro in the human leukemia cell lines HSB2 and Jurkat using a flow-cytometry assay that permits the simultaneous quantification of vital, apoptotic, and necrotic cells by double-staining with fluorescein isothiocyanate (FITC)-labeled Annexin-V and propidium iodide. The results were fit to different multicompartmental models and the sensitivity of the cell lines to apoptosis and necrosis was estimated. Results: A time- and dose-dependent decrease in vital cells as well as an increase in apoptotic and necrotic cells was observed in HSB2 cells upon continuous incubation with 10−5–10−7M CDA, 10−5–10−8M ARA-C, 5 × 10−5–5 × 10−6M CDDP, and 10−4–10−5M 5FU, whereas no effect was observed relative to controls upon incubation with 10−8–10−9M CDA, 10−9M ARA-C, 10−7–10−8M CDDP, or 10−6–10−9M 5FU. In Jurkat cells, apoptosis- and necrosis-inducing effects were observed at 10−4–5 × 10−6M CDA, 10−5–10−7M ARA-C, 5 × 10−5–5 × 10−6M CDDP, and 10−4–10−5M 5FU. In all experiments, apoptotic cells reached a peak after 6–48 h of drug exposure. These data were best fit by a model in which vital cells became irreversibly apoptotic by a direct pathway and necrotic by an irreversible indirect pathway following the apoptotic state (mean R=0.9876; range 0.9510–0.9993; mean modified Akaikes information criterion 3.88; range 1.86–5.82) and the rate constants of either pathway (Kva and Kan, respectively) were assessed. The sensitivity of both cell lines to apoptosis and necrosis (expressed as EC50 and Emax values) induced by the anticancer drugs could be calculated from the sigmoidal concentration-effect curves. Furthermore, it was shown that drug treatment (10−6M CDA or 10−6M ARA-C) potentiated the apoptosis-inducing effects of irradiation (6 Gy) but not its necrosis-inducing potential. Conclusion: This study demonstrates that CDA, ARA-C, CDDP, and 5FU possess concentration-dependent apoptosis-inducing potential in the cell lines studied. The cytotoxic mechanism and cell-killing potential of these drugs is different, which is reflected by different EC50 and Emax values. Furthermore, a method for pharmacodynamic modeling is introduced that permits a quantitative approach for the assessment of the sensitivity of tumor cells to anticancer drugs and combined treatments.

Cellular and plasma adriamycin concentrations in long-term infusion therapy of leukemia patients

SummaryTo determine whether long-term adriamycin (ADM) infusions resulted in cellular ADM concentrations at least comparable to those observed after bolus injections, ADM cellular and plasma concentrations were measured in 18 patients with leukemia. ADM was administered at 30 mg/m2 per day for 3 days, either as bolus injections or as 4-, 8-, or 72-h infusions. Negligible accumulation of plasma ADM was observed. Peak plasma ADM concentrations after bolus injections were 1640±470 ng/ml (n=7). Maximum levels were 176±34 ng/ml during 4-h infusion (n=5); 85±50 ng/ml during 8-h infusion (n=4); and 47±5 ng/ml (n=2) after 72-h infusion. ADM concentrations in nucleated blood and bone marrow cells correlated well (r=0.82, n=47). ADM accumulated in leukemic cells up to 30–100 times the plasma concentrations. The shorter the administration time-span, the higher the peak leukemic cell concentration and the greater the loss of drug immediately after the end of the administration. The final cellular ADM half-life was approximately 85–110 h. After long-term infusion and bolus injection of the same dose, similar areas under the curve for plasma or leukemic blast cell ADM concentrations were attained. Since comparable therapeutic efficacy was observed in all regimens, the antileukemic effect appeared not to be related to the peak plasma concentrations, while acute toxicity phenomena decreased with increasing duration of the infusion. Long-term ADM infusion deserves more attention in the treatment of patients with anthracyclines.