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.
Doxorubicin (adriamycin) has a very wide antitumour spectrum, compared with other anticancer drugs; however, except for Hodgkin's disease, it is not associated with curative chemotherapy. Doxorubicin has been in clinical use for more than 2 decades, and only recently has it been recognised that the cytotoxic effect is produced at the cellular level by multiple mechanisms which have not yet been conclusively identified. Key factors are a combination of doxorubicin-induced free radical formation due to metabolic activation, deleterious actions at the level of the membrane, and drug-intercalation into DNA. Multiple aspects of the clinical pharmacokinetics of this drug have been described. Wide interpatient variations in plasma pharmacokinetics have been noted, but without firm relation to clinical outcome. An apparent volume of distribution of approximately 25 L/kg points to extensive uptake by tissues. Up to several weeks after administration, significant concentrations of doxorubicin have been found in haematopoietic cells and in several other tissues. The maximum cellular doxorubicin concentrations reached in vivo remain significantly below those at which all clonogenic leukaemic cells are killed in vitro. Doxorubicin has been administered as frequent (weekly) low doses, single high doses, and as a continuous infusion. The optimal schedule with respect to tumour cytotoxicity and dose-limiting side effects such as myelosuppression or cardiotoxicity, has never been investigated in a prospective, randomised manner. Clinical trials large enough to study optimal, and possibly individualised, doxorubicin chemotherapy need to be performed. This review summarises pharmacological and pharmacodynamic data of doxorubicin, and discusses these in relation to possible improvement of its therapeutic index. Furthermore, drug interactions, dose-response relationships, mechanisms of action, multidrug resistance, and treatment scheduling are discussed in the perspective of the development of novel treatment strategies.
The regulatory mechanisms of the hypothalamo-pituitary-adrenal system were studied in critically ill, intensive care unit patients. Serial measurements of immunoreactive ACTH-(1-39) (ACTHi), cortisol, endothelin-1 (ETi), and atrial natriuretic hormone (ANHi) were performed in blood samples of 18 patients with clinically defined sepsis, 12 critically ill patients after multiple trauma, and 15 hospitalized matched control subjects without acute illness for 8 consecutive days after admission. On admission, plasma levels of cortisol and ACTHi were significantly elevated in patients with sepsis (1.32 +/- 0.21 mumol/L and 130.0 +/- 38.2 pmol/L, mean +/- SD) and with multiple trauma (1.23 +/- 0.28 mumol/L and 123.7 +/- 41.3 pmol/L) compared to those in the control subjects (0.37 +/- 0.08 mumol/L and 15.6 +/- 5.8 pmol/L, respectively). The plasma cortisol levels of critically ill patients remained high (> 0.8 mumol/L) during the whole observation period. In contrast, plasma ACTHi levels decreased between days 3-5, reaching significantly lower levels on day 5 compared to those in the control group and remained below 5.0 pmol/L during the rest of the observation period. Plasma levels of ETi and ANHi were significantly elevated during the whole period in both patient groups (ETi, > 10 ng/L; ANHi, > 250 ng/L) compared to those in control subjects (< 5 and < 50 ng/L, respectively). The high plasma concentration of ETi observed in our patients may stimulate the steroid secretion of the adrenal cortex directly or potentiate the adrenal effect of ACTH. On the other hand, the increased concentration of ANHi found in critically ill patients together with the increased plasma cortisol level may explain the inhibition of ACTH secretion. Accordingly, we speculate that the high ET level exerts a positive drive on the adrenocortical level, that the high ANH level has an inhibitory effect on the hypothalamo-pituitary level, and that both mechanisms play a role in regulation of the hypothalamo-pituitary-adrenal axis during critical illness.
During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972) introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.
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 less than 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 less than 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 less than 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.
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