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Background: It was investigated whether the iron status in newborns is negatively influenced by iron-deficient erythropoiesis of the mother during pregnancy., Methods: The iron status is characterized by hemoglobin, erythrocyte zinc protoporphyrin and serum ferritin values. Iron-deficient erythropoiesis of the mother is characterized by erythrocyte zinc protoporphyrin values. Measurement of erythrocyte zinc protoporphyrin was performed in 103 non-anemic females within 24 hours after delivery. The iron status of their newborns was measured in cord blood., Results: Erythrocyte zinc protoporphyrin concentrations were normal in 62% and elevated in 38% of the females, hence 39 women had iron-deficient erythropoiesis. There were no significant differences in mean values of the iron status parameters between neonates born to females with iron-deficient erythropoiesis and neonates born to females not having iron-deficient erythropoiesis. No correlation was found between maternal erythrocyte zinc protoporphyrin values and any of the neonatal parameters., Conclusion: It is concluded that fetal iron supply is not negatively influenced by iron-deficient erythropoiesis in the mother.

Background: To compare the usefulness of serum ferritin, hemoglobin and erythrocyte zinc protoporphyrin estimations as indicators of the need of iron supplementation in pregnancy., Methods: In 97 healthy pregnant women serum ferritin, hemoglobin and erythrocyte zinc protoporphyrin concentrations were measured at each trimester period. Iron supplementation was prescribed at hemoglobin values below 113 g/L., Results: Neither in the first nor in the second trimester differences in serum ferritin and erythrocyte zinc protoporphyrin values have been found between women who got iron therapy prescribed and women who did not get iron therapy, whereas already in the first trimester hemoglobin values were lower in the group that got iron therapy. These results indicate that hemodilution rather than iron deficiency determined which women got iron therapy. In the third trimester 14 out of 80 women without iron supplementation had depleted iron stores and a significant erythrocyte zinc protoporphyrin increase, indicating evolving iron deficiency anemia. However, only five of them showed hemoglobin levels below 113 g/L and got iron therapy. Low serum ferritin levels were found in 23% of the women at booking and in 92% in the third trimester. Serum ferritin levels neither predicted the development of iron deficient erythropoiesis nor of anemia., Conclusions: Iron deficiency can neither be diagnosed nor predicted by serum ferritin or hemoglobin measurements at booking or later on in pregnancy. Erythrocyte zinc protoporphyrin measurements can be of help in determining which women have iron deficient erythropoiesis and may benefit by iron therapy.

Iron deficiency modulates the synthesis of HbA2, resulting in reduced HbA2 levels in patients with iron deficiency anaemia. The diagnosis heterozygous beta-thalassaemia is based on a raised HbA2 level. Patients with beta-thalassaemia and concomitant iron deficiency can show normal HbA2 levels. It is of clinical importance to know the quantitative effect of iron-deficient erythropoiesis on the levels of HbA2 in order to be able to determine which iron-deficient patients with normal HbA2 levels have to be retested after iron therapy in thalassaemia screening programmes. In this study, HbA2 levels in 150 patients with iron-deficiency anaemia and 71 healthy controls have been measured. A linear correlation is found in the patient group between HbA2 and Hb, HbA2 and MCV, and HbA2 and erythrocyte zinc protoporphyrin (ZPP). In future studies, the correlation between HbA2 and erythrocyte parameters in patients with heterozygous beta-thalassaemia and concomitant iron deficiency has to be examined. We recommend that ZPP be measured in these studies too, as ZPP levels may be a better indicator of concomitant iron deficiency than Hb or MCV in thalassaemic patients.

In a retrospective study the diagnostic value of erythrocyte zinc protoporphyrin (ZPP) measurement as a means of distinguishing iron deficiency anemia from thalassemia syndromes in patients with microcytosis was explored. ZPP values were increased in all patients with iron deficiency and in part of the patients with thalassemia. The combined measurement of erythrocyte mean corpuscular volume (MCV) and ZPP resulted in a correct classification of patients with iron deficiency and with thalassemia in more than 95%. The predictive value of this method is better than the results obtained by using formulae derived from red cell indices. In population screening programs for thalassemia syndromes, in which MCV determination is used as the initial test, the ZPP test is recommended as a second test, in order to discriminate between patients with microcytosis due to iron deficiency and patients with microcytosis due to thalassemia syndromes.

Erythrocyte zinc protoporphyrin (ZPP) was measured in 102 women blood donors to evaluate its usefulness in screening for evolving iron deficiency anemia, a reason for the deferral of donors. The results were compared with serum ferritin determinations. Five women were deferred before their first donation and eight women were deferred after one or two donations. Women with increased ZPP values all had low serum ferritin concentrations, indicating iron-deficient erythropoiesis that was caused by iron depletion. The positive predictive value of an increased ZPP in predicting deferral of the donor after one or two donations was 75%, whereas a serum ferritin concentration < or = 12 microg/L predicted deferral in 26% of the donors. The results indicate that the ZPP test can be recommended as a feasible and inexpensive predonation test to determine a subset of donors with iron-deficient erythropoiesis at risk of developing iron deficiency anemia.

In the present study the migration of human monocytes towards the supernatants of five different human myeloid leukemic cell lines, four different human lymphatic leukemic cell lines and blasts derived from three different patients with acute myeloid leukemia (AML) was studied and the role of monocyte chemoattractant protein (MCP)-1 was established with an ELISA assay. Large differences in migration of monocytes towards the leukemic cell supernatants were shown (variation of approximately 10 to 150% compared to positive control), but high amounts of monocyte migration was always restricted to myeloid leukemic cells (cell lines or patient blasts). MCP-1 turned out to play a major role in the migration, firstly since there was a direct correlation between the amount of migration and the concentration of MCP-1 in the supernatants, and secondly since the addition of anti-hMCP-1 was able to inhibit migration to background level in all cases. Cytotoxicity experiments with a MTT test using MCP-1-stimulated monocytes against two human myeloid leukemic cell lines showed no increase in cell death compared to unstimulated monocytes. It is concluded that monocyte migration towards leukemic cells is restricted to the myeloid lineage and is regulated by MCP-1, which is produced in different amounts by the leukemic cells. Besides, MCP-1 does not increase the direct toxic effects of monocytes on leukemic cells.

In this study, we evaluated the effect of hyperthermia on hematopoietic progenitors from six chronic myeloid leukemia (CML) bone marrow (BM) samples at diagnosis and four peripheral blood stem cell (PBSC) samples from CML patients after stem cell mobilisation. CD34-positive cells, isolated from these samples, were incubated for 2 h at 37, 42 or 43 degrees C and were plated in the colony-forming unit granulocyte-macrophage (CFU-GM) and the long-term culture initiating cell (LTCIC) assay. To evaluate purging, individual colonies from these assays were analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and/or RT-PCR. BM samples showed a significant higher sensitivity both at the CFU-GM and LTCIC level, after treatment at 42 degrees C, as compared to the control BM samples obtained from healthy volunteers. The four BM samples of CML patients with a low leukocyte number at diagnosis harbored a mixture of bcr-abl-negative and positive colonies and an increase in the percentage of bcr-abl-negative colonies was observed in all cases. CML patients with a high leukocyte count at diagnosis, however, showed only bcr-abl-positive progenitors even after hyperthermia. PBSCs showed a significant higher sensitivity at the LTCIC level but not at the CFU-GM level, after treatment at 42 degrees C, as compared to the control PBSC samples obtained from nonhematologic cancer patients. Molecular analysis of individual colonies demonstrated an increase of bcr-abl-negative progenitors after thermic treatment in two out of three samples. When comparing both stem cell sources, PBSCs showed a decreased thermic sensitivity as compared to the BM samples at the CFU-GM level, whereas at the LTCIC level an increased thermic sensitivity was observed, both for the controls and the CML samples. In conclusion, both for BM and PBSCs samples, CML progenitors are more sensitive to hyperthermia than control cells, especially at the LTCIC level. In agreement with these results, an increase of bcr-abl-negative progenitors in six out of seven samples could be demonstrated either at the CFU-GM level, LTCIC level or both. Hyperthermia should be explored further as a possible purging modality in CML.

In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte-macrophage (CFU-GM) colonies. One half was analyzed with I-FISH, for the presence of bcr-abl fusion gene. The other half was analyzed with RT-PCR for the presence of the bcr-abl mRNA. We wanted to address the following questions: (1) is the bcr-abl gene always expressed in CFU-GM colonies and (2) which technique has to be preferred to analyze individual CFU-GM colonies? In total, 133 colonies, derived from six CML patients, could be analyzed both with I-FISH and RT-PCR. We found a positive correlation in 89% of the cases: 118 colonies showed the same results with both techniques. However, 15 of the 106 I-FISH-positive colonies were negative in the RT-PCR. Serial analysis of the cDNA derived from 22 colonies showed in each round of amplification 21-29% RT-PCR-negative but I-FISH-positive colonies. However, all I-FISH-positive colonies showed at least one positive RT-PCR, either in the first, second or third round of amplification. These results indicate that the bcr-abl gene is probably always transcriptionally active in CFU-GM colonies. Reliable analysis with RT-PCR is possible but likely to generate false negative results. We conclude that: (1) I-FISH offers a reliable alternative to RT-PCR for analyzing individual hematopoietic colonies and (2) results obtained with RT-PCR should only be interpreted with caution.

Human bone marrow endothelial cells (HBMEC) are intimately involved in the homing of hematopoietic progenitor cells (HPC) to the bone marrow and in the regulation of proliferation and differentiation of these cells. Because availability of primary HBMEC and their capacity to be cultured in vitro are limited, we used isolated HBMEC to establish a cloned cell line by microinjection of a recombinant plasmid expressing simian virus 40 early genes under the control of a deletion mutant of the human vimentin promoter. Serum requirements for growth of a transformed HBMEC line (TrHBMEC) were markedly decreased compared with those of primary cells, and added growth factors were not required for proliferation. Cells took up acetylated low-density lipoprotein normally, bound to Ulex europaeus lectin, and stained positively for von Willebrand factor, P-selectin, CD31, CD34, CD44, very late antigen-5, and intercellular adhesion molecule-2 (ICAM-2). After treatment with TNF-alpha or lipopolysaccharide, TrHBMEC increased surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 in a manner similar to primary HBMEC. In contrast, IL-1 beta elicited much less up-regulation of these adhesion molecules than in primary cells. In previous work, we reported that, in a flow adhesion model, rolling of peripheral blood CD34+ cells on primary HBMEC was E-selectin-dependent, whereas VCAM-1 and ICAM-1 contributed to firm adhesion. In the present study, we show that HPC adhere in a similar way to TrHBMEC. A less-pronounced role for VCAM-1 and ICAM-1 was found in the adhesion of HPC to human umbilical vein endothelial cells. Furthermore, significantly more CD34+ cells adhered to TNF-alpha-stimulated HBMEC and TrHBMEC than to similarly stimulated human umbilical vein endothelial cells. These data emphasize the importance of using microvessel HBMEC for studying the homing of HPC to the bone marrow, and indicate the usefulness of the above-described bone marrow endothelial cell line.

Monocytes or monocyte-derived supernatants are able to kill leukemic cells via apoptosis, thereby preferentially effecting more mature leukemic cells. In the present study, the relationship between apoptosis and the apoptosis related proteins, bcl-2 and bax, was investigated in a number of human leukemic cell lines. Monocyte-derived supernatant induces extensive apoptosis in U937 myeloid leukemia cells and minor apoptosis in HL60 cells. No apoptosis was seen in four other cell lines (THP1, HL60-D3, KG1, and K562). The expression of bcl-2 and bax protein was determined in both groups of leukemic cell lines by flow cytometry (bcl-2 and bax) and Western blotting (bcl-2) at baseline level and after incubation with monocyte supernatant after different time periods. No clear relation was found between baseline bcl-2 or bax protein expression and the occurrence of apoptosis after incubation with monocyte supernatant. After different incubation time periods, no change was found in bcl-2 protein expression in U937 and K562 cells, whereas in KG1, HL60, and especially in THP1 cells, a significant decrease could be noticed. On the other hand, there was an increase in bcl-2 expression in HL60-D3 cells. Bax protein expression, measured at the same time points, remained essentially unchanged in HL60-D3 cells, decreased significantly in U937, HL60, and THP1 cells and slightly in K562 cells, and increased significantly in KG1 cells. Also, the ratio bax/bcl-2 decreased in HL60D3, but especially in U937 and HL60 cells, increased slightly in THP1 and KG1 cells, and remained essentially unchanged in K562 cells. Rh-tumor necrosis factor-alpha (TNF-alpha), the main mediator of monocyte mediated cytotoxicity, induced apoptosis in U937, HL60, and THP1 cells, thereby showing changes in bcl-2 expression similar to those found for monocyte derived supernatants. We concluded that in human leukemic cell lines, there is no relation between either bcl-2 or bax protein expression or the ratio of both, and apoptosis mediated by monocyte derived supernatant or TNF-alpha.

The first step in the homing of hematopoietic progenitor cells (HPCs) from the peripheral blood to the bone marrow involves an adhesion molecule-dependent contact with human bone marrow endothelial cells (HBMECs). In the present study we describe the constitutive expression of two of these molecules, E-selectin and vascular cell adhesion molecule-1 (VCAM-1), on endothelial cells of hematopoietic tissues. Immunophenotypic analysis of tissue sections of hematopoietically active (human adult and fetal bone marrow, fetal spleen, fetal liver, and adult spleen with extramedullary hematopoiesis) and inactive tissues (human adult spleen, lymph node, appendix, and liver; and fetal lung and fetal intestine) revealed that E-selectin and VCAM-1 are selectively expressed on endothelial cells of adult and fetal hematopoietic organs. These results were validated by means of reverse-transcriptase polymerase chain reaction. Adhesion studies revealed that binding of normal mobilized peripheral blood HPCs to HBMECs was completely inhibited by preincubation of HBMECs with anti-E-selectin (ENA2), whereas no effect of anti-VCAM-1 (1G11B1) was detected. These results suggest that E-selectin plays a role in the homing of HPCs and that its constitutive expression on endothelial cells of hematopoietic organs may be essential in the initial step of the homing process.

Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor cells, we studied several methods of isolating endothelial cells from human bone marrow, including fluorescence activated cell sorting (FACS) and separation by immunomagnetic beads. FACS sorting gave the best results as contamination with other cells did not occur. After density-gradient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells was achieved by FACS sorting using BNH9 or S-Endo1 monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell reactive MAbs, were found to react highly specifically with sinus endothelial cells as tested by immunohistochemistry on bone marrow tissue sections and cell culture preparations and by double-colored FACS analysis on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor positivity. Ultrastructural analysis revealed cells with endothelial characteristics. Cells were cultured in fibronectin-coated, 24-well culture plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle-shaped cells developed expressing endothelial cell antigens. Cells could be kept in culture for 4 to 6 weeks. In conclusion, the method described provides highly purified preparations of human bone marrow endothelium that may permit in vitro adhesion experiments with normal and leukemic hematopoietic progenitor cells.

In this study we explored the effects of bryostatin-5 on the clonogenic response of normal bone marrow mononuclear (BM) cells and HL60 myeloid leukemia cells. Leukemic HL60 colony formation was strongly inhibited by bryostatin-5 depending on dose and schedule. An inhibitory effect on HL60 colony formation was readily demonstrated after 1 h of exposure, reaching a maximal inhibitory effect at 96 h. Normal BM cells differed in their clonogenic response: short-term exposure to bryostatin-5 resulted in increased clonogenicity while longstanding exposure to bryostatin-5 permitted the survival of a substantial fraction of committed progenitors. This differential modulation of normal and leukemic myeloid clonogenicity by bryostatin-5 suggests a possible role for bryostatin-5 in the treatment of acute myeloid leukemia.

The MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into formazan, was evaluated using leukemic cells from five representative human leukemic cell lines and from 28 patients with acute myeloid leukemia (AML). An excellent linearity between absorbance and leukemic cell number was observed up to 5 x 10(4) cells/well and 50 x 10(4) cells/well for all cell lines and patients samples tested, respectively, in a 96-wells microtiter culture system. A huge variability in the susceptibility of leukemic cells to purified and IFN-gamma-activated human monocytes could be observed at effector-to-target cell (E:T) ratios of 1. The mean signal-to-noise ratio of the MTT assay for monocyte-leukemic cell mixtures from patients was 2.69 +/- 0.39 at E:T 1. In conclusion, the MTT based monocyte mediated cytotoxicity assay should be useful for studying the susceptibility of a variety of leukemic cells from cell lines and from patients with AML to monocytes in a rapid, sensitive and semi-automated manner.

In view of cellular immunotherapy with cytotoxic monocytes in minimal residual leukemia we have studied the effects of monocytes on the growth and survival of leukemic cells from cell lines and from patients with acute myeloid leukemia (AML). Using highly purified and interferon-gamma (IFN gamma) activated human monocytes, monocyte-mediated cytotoxicity (MMC) was evaluated in an MTT-based colorimetric cytotoxicity assay against six human leukemic cell lines (U937, THP1, KG1, K562, HL60, and 1,25(OH)2D3 differentiated HL60 cells) and cells from AML patients. Leukemic cells from cell lines with an immature phenotype were found to be resistant to MMC, whereas leukemic cells with a more mature and monocytic phenotype were sensitive. This paralleled the sensitivity to tumor necrosis factor-alpha (TNF-alpha). AML cells from patients with an immature phenotype (FAB-M1/M5A) were significantly less sensitive to MMC as compared to more mature AML cells (FAB-M2/M4/M5B). The growth stimulatory effects of non-activated monocytes on immature AML cells could be abrogated in the presence of IFN gamma or IL-3 and GM-CSF. In addition, these cytokines further potentiated MMC, preferentially affecting cells with a more mature phenotype. AML cells with an immunologically immature phenotype (CD34(high), HLA-Dr(low), CD13(low), CD14(low)) were revealed as the least sensitive cells to MMC. The growth stimulatory effects of IL-3/GM-CSF with or without TNF-alpha on AML cells correlated with resistance to MMC. In addition, the cytolytic effects of TNF-alpha in the presence of IFN gamma correlated with an increased susceptibility of AML cells to MMC. In conclusion, our data strongly indicate that MMC is related to maturation in AML, which is correlated to the differential stimulatory and cytolytic effects of monocyte-derived cytokines such as IL-3, GM-CSF, and TNF-alpha.

With a view to the immunologically mediated purging of autologous bone marrow transplants in acute myeloid leukaemia, the efficacy of cytotoxic monocytes to eradicate leukaemic cells has been studied using clonogenic assays. U937 cells were found to be sensitive to highly purified and interferon-gamma-activated human monocytes whereas HL60 cells were rather resistant as measured in an MTT-based cytotoxicity assay under liquid conditions. A spectrophotometric clonogenic assay measured almost complete inhibition of clonogenic activity for U937 cells at low effector-to-target cell (E/T) ratios of at least 0.1. Limiting dilution analysis detected a 2-3 log10 unit reduction in clonogenic activity. In an experimental mixture of U937 cells with a 20-fold excess of normal bone marrow nuclear cells a maximum 2-log10-unit killing could be measured at E/T = 10. Only at high E/T ratios could a reduction in granulocyte/macrophage-colony-forming units (cfu) be observed with only marginal effects on erythroid cfu and erythroid burst-forming. In conclusion, cytotoxic monocytes are highly potent anti-leukaemic effector cells, as measured in clonogenic assays, that do not compromise normal human progenitors.

Both bone marrow and circulating megakaryocytes may produce platelets. Changes in number and properties of the circulating megakaryocytes in cardiac diseases may provide information about thrombopoiesis in these disease states. Circulating megakaryocytes were obtained by retrograde aspiration through wedged pulmonary artery catheters from patients without cardiac abnormalities, patients with cardiac diseases and patients with disorders known to affect the megakaryocyte-platelet axis or marrow-blood barrier. Their number, ploidy distribution and expression of the beta 1-integrin adhesion antigens were estimated. We found evidence for an increased number of circulating megakaryocytes during acute myocardial infarction and in patients with triple vessel disease. A significantly higher proportion of circulating megakaryocytes obtained within 60 h after acute myocardial infarction, expressed CDw49b (alpha-chain of VLA2), CDw49e (alpha-chain of VLA5), and CDw49f (alpha-chain of VLA6) compared to megakaryocytes of patients without cardiac diseases. In myocardial infarction the increase in number of circulating megakaryocytes together with the higher proportion that expressed the beta 1-integrin adhesion antigens favour the hypothesis that these megakaryocytes are acutely released from the bone marrow. This could reflect either a selective response to an increased demand for circulating platelets or be the result of an a selective mechanism resulting from damage to the marrow-blood barrier following the acute infarction or thrombolytic therapy administered.

Bryostatin 5 is a macrocyclic lactone which activates protein kinase C (PKC). PKC activation has been implicated in leukemic cell differentiation. We have examined the effect of PKC activation by bryostatin 5 on human acute myeloid cell differentiation in the presence and absence of vitamin D3. In vitro treatment of 20 patient samples of acute myeloid leukemias in a 4 days culture system with 10 nM bryostatin 5 induced strongly adherent macrophage-like cells in all cases. Bryostatin 5 induced a significant (p = 0.00006) increment in esterase activity in a majority of the samples, which was further enhanced by vitamin D3. CD14 expression was significantly (p = 0.035) enhanced with the combination of bryostatin 5 and vitamin D3. Nitroblue tetrazolium (NBT) reducing ability was, however, nearly abolished (p = 0.0007). A loss of CD34 expression occurred during cell culture; this loss was enhanced by vitamin D3, but prevented partly by bryostatin 5. Together these findings indicate that exposure to bryostatin 5 leads to a strong macrophage-like cell differentiation in human myeloid leukemia and that VD3 has an additional effect. These findings strengthen the potential role of bryostatins as possible antileukemic agents.

Growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) is effective at progenitor release into the peripheral blood. After high-dose chemotherapy haematopoietic reconstitution occurs after reinfusion of these peripheral blood progenitor cells (PBPC). However, the collection by leukapheresis and further processing of PBPC are very time consuming and expensive. We have studied the transplantation potential of a small volume of unprocessed autologous whole blood after G-CSF mobilisation. Six patients with plasma cell disorders received G-CSF 10 micrograms/kg sc during 6 days. Subsequently 11 of whole blood was collected by phlebotomy, kept unprocessed at room temperature and reinfused 24 h after high-dose melphalan 140 mg/m2. CFU-GM content was 845 per ml blood (median, range 320-3472) and CD34+ cells rose to a median percentage of 0.9 (range 0.4-2.0). Haematological recovery was significantly faster in the study group compared with the control group of 20 patients who received the same dose of melphalan without reinfusion of PBPC. The neutrophil count reached 0.5 x 10(9)/l at a median of 12.5 days after infusion of PBPC vs 38 days in the control group (p = 0.0003). The platelet count reached 20 x 10(9)/l after a median of 23.5 days vs 38 days (p = 0.0218). The shortened recovery was reflected by less transfusions, less antibiotic use and shortening of hospital stay (19 days vs 43 days, p = 0.0003). We conclude that this easy technique of mobilisation and collection of PBPC is very effective for hastening haematologic recovery after high-dose chemotherapy.

Little is known about the precise ways in which monocytes and macrophages recognize tumor cells and how they exert their cytolytic and/or cytostatic effects. By a functional, morphologic, and flow-cytometric approach, we have studied monocyte/macrophage- and cytokine-mediated cytotoxicity against U937 cells, a human histiocytic lymphoma cell line. A rapid decrease in cell viability of U937 cells (MTT assay) could be observed at an effector-to-target cell (E:T) ratio of 10 in the presence of interferon (IFN)-gamma-activated monocytes. Light and electron microscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either monocytes or tumor necrosis factor (TNF)-alpha. TNF-alpha-induced apoptosis (10(4) U/mL) as measured by multiparameter flow cytometry (propidium iodide [PI]) paralleled the functional decrease in cell viability (MTT assay) of 20 +/- 3% after 24 hours up to a maximum of 50 +/- 4% after 48 hours. Apoptosis could be confirmed by the detection of DNA degradation into multiples of 200-bp subunits by agarose gel electrophoresis. After prolonged incubation times, monocyte-mediated leukemic cell death could be quantified as apoptosis by flow cytometry, whereas no decrease in net cell viability of tumor cells relative to the initial cell number could be observed by MTT spectrophotometry. In conclusion, our data provide evidence that apoptosis is the major mode of TNF-alpha-dependent monocyte-mediated cytotoxicity against U937 cells. Furthermore, multiparameter flow-cytometric analysis offers a sensitive method to quantify cytokine- and cell-induced apoptosis in leukemia.

Human monocytes are involved in host defense against neoplastic cells. In view of cellular immunotherapy with cytotoxic monocytes in minimal residual disease of acute myeloid leukemia we have studied the role of monocytes in cell cycle dependent leukemic cell death of U937, THP-1, and HL-60 cells in vitro. Leukemic cells separated in G1 of the cell cycle by countercurrent centrifugal elutriation were highly susceptible to monocyte mediated cytotoxicity, whereas cells in S and G2-M were less sensitive or completely resistant as compared to unfractionated control cells. HL-60 cells resistant to cytotoxic monocytes became sensitive to monocyte mediated cytotoxicity upon differentiation induction with 1,25-dihydroxyvitamin D3 which paralleled an accumulation of cells in G1 of the cell cycle. The differences in susceptibility of cell phase separated populations to monocyte mediated cytotoxicity paralleled differences in sensitivity to the cytotoxic effects of tumor necrosis factor alpha, as secreted by gamma-interferon activated monocytes. Furthermore, monocyte mediated cytotoxicity was markedly inhibited in the presence of anti-CD11/CD18 monoclonal antibodies recognizing the alpha and beta chains of the beta 2-integrin adhesion proteins. By fluorescence activated cell sorter immunofluorescence a marked increase in mean fluorescence density of the beta 2-integrins could be demonstrated on cells in G1 of the cell cycle as compared to unseparated leukemic cells. A decrease in mean fluorescence density was shown for cells in G2-M. By blocking experiments with anti-CD11/CD18 monoclonal antibodies, the differences in mean fluorescence density were functionally relevant since cells in G1 were shown to be the most sensitive cells to beta 2-integrin dependent monocyte mediated cytotoxicity. In conclusion these data show that differences in sensitivity to tumor necrosis factor and in the expression of beta 2-integrins may play a central role in cell cycle dependent monocyte mediated antileukemic activity.

To evaluate whether currently popular aspirin regimens have an effect on the prostaglandin synthesis in human megakaryocytes we measured thromboxane B2 (TXB2) synthesis in response to thrombin stimulation in human megakaryocytes ex vivo. Human megakaryocytes were purified by Counterflow Centrifugal Elutriation from bone marrow punctures, taken from volunteers before and 2 hours after ingestion of one dose of 500 mg (n = 4), 80 mg (n = 4) or 40 mg (n = 2) aspirin. Subsequently, megakaryocytes were purified before and 12 h after ingestion of 80 mg (n = 3) aspirin twice daily for 1 week and 12 h after 500 mg (n = 3) aspirin. On average, 140 +/- 102 x 10(3) (mean +/- 1 SD) megakaryocytes were recovered. We found that aspirin inhibits megakaryocyte cyclooxygenase in a dose-dependent manner. Two hours after 500 mg of aspirin, TXB2 synthesis in megakaryocytes was inhibited by 96.8 +/- 2%, whereas one dose of 80 and 40 mg aspirin showed an inhibition of 79.4 +/- 13.7% and 80 +/- 6.2% respectively. However, the inhibition of TXB2 synthesis seems not to be long-lasting since, 12 h after the ingestion of aspirin, an increase of megakaryocyte TXB2 production could be observed which reached significance after the 500 mg aspirin dosage (p < 0.048). We conclude that human megakaryocyte cyclooxygenase is sensitive to aspirin inhibition and that low doses of aspirin (40 and 80 mg) enter the systemic circulation and are able to inhibit megakaryocyte cyclooxygenase, but this inhibition is incomplete and megakaryocyte cyclooxygenase seems to recover within 12 h after ingestion of aspirin.

In this study we describe a semiautomated clonogenic assay in which human leukemic cell lines (U937 and HL60) are cultured in a semisolid 96-well microtiter culture system and clonogenicity is measured spectrophotometrically in a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT)-assay and sulforhodamine (SRB)-assay. Culture medium with 0.6% (wt/vol) methylcellulose and 10% (vol/vol) fetal calf serum (FCS) appeared to provide optimal culture conditions with a low background absorbance in both colorimetric assays and an optimal linearity between cell number and optical density (OD). An excellent correlation between clonogenic growth of U937 and HL60 cells in the conventional colony-forming unit assay (CFU-assay) and the microtiter CFU-assay was observed. The value of this microtiter CFU-assay was assessed by modulating U937 and HL60 cells with either 1,25(OH)2D3 or cytosine arabinoside (Ara-C). Additionally, the number of living cells was quantitated spectrophotometrically in both MTT- and SRB-assays. Results obtained by either method did not differ significantly (p < 0.001). In liquid culture, however, significantly (p < 0.001) less reduction of cellular growth was observed with 1,25(OH)2D3-modified cells. No significant differences between the liquid and semisolid assay systems could be observed in the presence of Ara-C. In conclusion, the microtiter clonogenic assay provides a rapid and objective way of measuring clonogenic capacity of (leukemic) cell lines. The semiautomated clonogenic assay will be useful to assess large series of immune modulations and/or cytostatic drug screening.

Activated human monocytes and macrophages are involved in host defense against neoplastic cells. In view of cellular adoptive immunotherapy, we have studied the role of tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) in monocyte-mediated cytotoxicity on the level of both effector and leukemic target cells. Highly purified and IFN-gamma-activated monocytes were cytolytic to U937 cells up to 81.9 +/- 5.3% (mean +/- SEM) in a 24-hour MTT cytotoxicity assay at an effector-to-target-cell ratio of 10. Upon IFN-gamma activation these monocytes showed a 20-fold increase in TNF-alpha secretion of 663 +/- 122 pg/mL. Comparable concentrations of recombinant human TNF-alpha showed only cytostatic effects on U937 cells of approximately 20% after 24 hours, similar to the cytostatic effects of IFN-gamma-activated monocyte culture supernatants. These effects could be fully reversed by anti-TNF-alpha antibodies. U937 cells pretreated with TNF-alpha were almost completely resistant to monocyte-mediated cytotoxicity, supernatant-mediated cytostasis and to TNF-alpha up to 10(4) U/mL. IFN-gamma-activated monocytes were able to lyse TNF-alpha-modified U937 cells whereas IFN-gamma-activated monocyte supernatants showed only cytostatic activity after prolonged incubation. Additionally, target cell modulation by IFN-gamma potentiated the TNF-alpha-dependent cytolytic and cytostatic effects of monocytes, monocyte culture supernatants and TNF-alpha. We conclude that monocytes as a cellular component in monocyte-mediated cytotoxicity are far more potent in lysis of leukemic target cells than are secreted monokines. Furthermore, IFN-gamma and TNF-alpha are involved in the regulation of the susceptibility of leukemic cells for lysis by interactions with monocytes.

In order to study the adhesive interactions of the human bone marrow microenvironment and acute myeloid leukaemic cells, we investigated the binding capacity of KG-1 cells upon human long-term bone marrow cultures derived from 17 healthy volunteers and 12 patients with acute myeloid leukemia. Adhesion was measured using a 51-chromium labelling assay. Adhesion of KG-1 cells upon 'normal' stromal layers: 33% +/- 4.0, n = 17 (mean +/- SEM) was higher as compared to the binding to 'leukaemic' stromas: 24% +/- 3.7, n = 12 (p < 0.05). Blocking monoclonal antibodies against adhesion molecules reduced the binding of KG-1 cells upon 'normal' stroma, when anti-VLA4 (p < 0.03), anti-Mac1 (p < 0.03) and anti-p150/95 (p < 0.04) were used. Binding of KG-1 cells on 'leukaemic' stromas was partly inhibited by anti-VCAM1 (p < 0.03). Blocking achieved by single or combined antibodies was never complete, suggesting that the adhesion is a multifactorial process, including a variety of adhesion molecules and/or adhesion mechanisms.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.

Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow samples collected from 15 patients with acute myeloid leukemia (AML) and compared with HLTBMCs from eight healthy volunteers. During 6 weeks of culture, the cellular composition of HLTBMCs was quantitatively studied. The cells of the HLTBMCs were divided into three main categories: fibroblasts, macrophages, and 'other cells' (endothelial cells, hematopoietic cells and undefined cells). HLTBMCs derived from healthy volunteers demonstrated a very consistent development. The number of fibroblasts increased during culture and the number of macrophages decreased, resulting in a steady state after 3 weeks of culture. In contrast, HLTBMCs derived from patients with AML showed a strikingly different pattern of irregular development and a steady state was not reached under our conditions. The APAAP technique was used to demonstrate expression of adhesion molecules. VLA2, VLA5, VLA6, LFA1, Mac1, p150/95, beta 2-chain, HCAM, ICAM1, NCAM, and VCAM1 were more expressed on 'normal' as compared with 'leukemic' bone marrow stromal cells, although this reached significance only for beta 2-chain and NCAM. VLA1, 3, and 4 were expressed in a higher percentage on 'leukemic' stroma (not significant). More expression was seen on 'normal' as opposed to 'leukemic' macrophages for the adhesion molecules tested, except for VLA5. The differences reached significance for the majority of molecules tested. It is concluded that striking differences exist in cellular composition and adhesion molecule expression between HLTBMCs from healthy individuals and those from patients with AML. This may have an impact on the pathogenesis of AML.

In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocyte-derived macrophages were activated with interferon gamma (IFN) or tumour necrosis factor alpha (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0 +/- 0.7% (mean +/- SEM) (conventional [3H]dT uptake assay) and 81.9 +/- 5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.

1.25 (OH)2D3 is a potent inducer of differentiation of leukaemic cells into a monocytic direction. However, therapeutic application is difficult because of the development of hypercalcaemia. We examined a novel vitamin D analogue, MC 903, which is at least 100 times less effective on calcium metabolism in rats than 1.25 (OH)2D3. Using the HL-60 cell line, differentiation was measured with a comprehensive panel of qualitative and quantitative parameters. Development of monocytic cells was shown morphologically, immunophenotypically and functionally by increased capability of reducing NBT (vs cultures without MC 903, p less than 0.0001) and by qualitatively and quantitatively increased non-specific esterase activity. Furthermore, a concomitant decreased activity of myeloperoxidase and lactate dehydrogenase was noticed. In conclusion, MC 903 is a potent inducer of monocytic differentiation, comparable with 1.25 (OH)2D3 and will therefore be an interesting and potential therapeutic agent for studies in human acute leukaemia.

Acute myeloid leukaemia (AML) cells have a variable capacity to egress from bone marrow into peripheral blood. This may be due to a variable lack of adhesion molecules on leukaemic cells. The expression of VLA1, 3, 4, 5, 6, beta 1-chain, LFA1, beta 2-chain, ICAM1 and NCAM appeared to be higher in bone marrow as compared to peripheral blood leukaemic cells, although this only reached significance for beta 1-chain (p less than 0.01). The number of cases with more than 20% positive cells in bone marrow leukaemic cells was lower in immature FAB-subtypes (M1, M5a) as opposed to more mature subtypes (M2, M3, M4, M5b) for the adhesion molecules tested. This reached significance for VLA5 (p less than 0.05) and beta 1-chain (p less than 0.007), while there was trend for VLA4. It is discussed that VLA4 and 5 may play a role in the release of leukaemic cells from the bone marrow.

To estimate the efficacy of human megakaryocyte purification techniques, mixtures of known numbers of megakaryocytes with a known ploidy range and of bone marrow or peripheral blood mononuclear cells were made. These artificial bone marrow samples were submitted to either a counterflow centrifugal elutriation or Percoll density separation followed by the glycoprotein Ib-dependent agglutination procedure. Also crude bone marrow samples were submitted to counterflow centrifugal elutriation directly followed by a glycoprotein Ib-dependent agglutination. The counterflow centrifugal elutriation resulted in a mean megakaryocyte recovery of 81% (mean 81% +/- 2.3). Purification by glycoprotein Ib-dependent agglutination after either a Percoll density separation or counterflow centrifugal elutriation resulted in a recovery of 61% (mean 61% +/- 15%) and 81% (mean 81% +/- 6) respectively. Purity of the resulting material was 87% (mean 87% +/- 11) and 83% (mean 83% +/- 5) respectively. The various isolation procedures did not affect the ploidy distribution of megakaryocytes greater than or equal to 8N. Counterflow centrifugal elutriation was preferred as the preparing step before glycoprotein Ib-dependent agglutination because of the lower variability in recovery and purity of megakaryocyte populations. When large numbers of rather pure and mature megakaryocytes are required, counterflow centrifugal elutriation followed by the glycoprotein Ib-dependent agglutination is a relatively simple method to purify human megakaryocytes without an appreciable loss in ploidy class greater than or equal to 8N.

The colorimetric MTT assay based on the selective ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide into formazan was adapted for measuring antibody independent monocyte mediated cytotoxicity. In view of the suggested application of adoptive immunotherapy we studied the anti leukaemic effects of activated human monocytes and macrophages against U 937 cells in vitro. Purified monocytes (greater than 98%) isolated by centrifugal elutriation and activated with interferon-gamma (IFN) were incubated with U 937 cells for 24 h at effector-to-target cell ratios (E/T) of 0.1-10. We assayed cytotoxicity by relating the optical density (OD) of residual metabolically active U 937 cells after exposure to effector cells to the OD of the initially inoculated U 937 cells. MTT reduction of effector cells was dependent on monocyte activation and differentiation into macrophages but did not interfere with the target cell signal up to an E/T ratio of 10. Improved signals could be obtained by dissolving formazan in DMSO with the addition of glycine instead of using propanol as solvent. Maximum cytostasis (95%, conventional [3H] incorporation assay) and cytotoxicity (80%, modified MTT assay) was reached with IFN activated monocytes at an E/T ratio of 10. In conclusion these data show that the modified MTT assay is a useful method of measuring monocyte mediated cytotoxicity in a sensitive, rapid, semi-automatic manner without the use of radioactive isotopes.

14 patients with acute myeloid leukaemia (AML) and 7 with myelodysplastic syndrome (MDS) were treated with cytarabine in low dosage. In AML a complete remission rate of 43% was found and in all patients profound cytopenia was noticed without any sign of maturation induction. In MDS no effect of low-dose cytarabine could be detected. We also studied the effect of low-dose cytarabine in vitro in freshly isolated leukaemic cells of 10 patients with AML. Maturation induction was measured by a comprehensive panel of quantitative and qualitative markers of maturation. No differentiation inducing effect of low-dose cytarabine could be found. We conclude on the basis of our own results and after reviewing the literature that low-dose cytarabine exerts its effect by cytotoxicity instead of maturation induction.

In adult neutropenic patients with hematological malignancies, we explored imipenem-cilastatin as empirical antibiotic therapy in a dose of 500 mg four times daily. Changing to second-line treatment was only resorted to if clinical deterioration, new infections or recurrence of fever occurred. A clinically or microbiologically documented infection was apparent in 115 of 150 episodes studied (76.7%). Imipenem-cilastatin was successful in 70.7% of all episodes and in 67.8% of all proven infections. Modification was necessary and successful in 22.0% of all episodes. 11 patients died while still febrile, 6 of them by infection (4%) and 5 because of progressive disease (3.3%). Imipenem-cilastatin is safe initial therapy in neutropenic febrile patients.

Freshly obtained leukemic cells from 64 patients with acute myeloid leukemia were investigated. Intracellular protein and enzyme content (myeloperoxidase (MPO), acid esterase with alpha naphthyl acetate (ANAE) and alpha naphthyl butyrate (ANBE) and lactate dehydrogenase) were measured together with the cell diameter and compared with FAB-classification, cytochemical staining and clinical features. Mean values of MPO and acid esterase discriminate between the pure myeloid leukemias, although overlap between various subgroups is considerable. No correlation between cytochemical and quantitative enzyme determination was found. The logistic regression analysis revealed a dependency of the occurrence of complete remission on protein and ANBE (p less than 0.01) content. We concluded that quantitative enzyme determination is an easily measurable parameter of maturation and may have prognostic significance.

Bone marrow stromal cells are critical for the proliferation and differentiation of hemopoietic stem cells. The hemopoietic microenvironment is reproduced in long term bone marrow culture (LTBMC). Normal LTBMC versus leukemic LTBMC and their stroma conditioned medium were compared with respect to their proliferative and differentiation-inducing capacities. Myeloid leukemic cells (HL60) were layered onto LTBMCs derived from normal volunteers and patients with AML. Differentiation was measured with a comprehensive panel of maturation parameters, i.e. morphology, cytochemistry, quantitative enzyme determination, NBT test and immunophenotyping. Inhibition of proliferation occurred in all cocultures. Clear maturation in monocytic direction was obvious in one culture of HL60 cells layered onto a leukemic stroma. As stroma-derived conditioned medium has no effect, a cellular interaction seems involved. These observations support not only the concept that normal stroma influences leukemic cell growth but also that leukemic stroma can modulate cell growth and maturation.

In order to study normal and abnormal human megakaryocytopoiesis we modified a glycoprotein Ib dependent megakaryocyte purification technique originally described in guinea pigs. After Percoll density centrifugation, 2 ml bone marrow aspirates were subjected to 3 steps of bovine plasma induced cell clumping, which should selectively agglutinate glycoprotein Ib positive cells. In 13 experiments the resulting samples contained 28-102 x 10(3) megakaryocytes (median 70) with a median purity of 79%. Megakaryocyte recovery ranged from 30-86% (median 51%). The DNA profile showed median values of 10% for 8 N, 44% for 16 N and 43% for 32 N megakaryocytes. The described method is relatively simple, inexpensive and gives results comparable with more elaborate techniques.

In a retrospective study of 96 patients with a myelodysplastic syndrome, the reproducibility of the French-American-British (FAB) classification was determined. Morphological abnormalities in peripheral blood and bone marrow were studied. Slides were reviewed by 3 examiners. All 3 observers agreed on morphological classification in 61% of cases, pairs of 2 in 64, 76 and 76%. The final diagnosis was refractory anaemia (RA) in 7 patients, acquired sideroblastic anaemia (AISA) in 2, chronic myelomonocytic leukaemia (CMML) in 31, refractory anaemia with excess of blasts (RAEB) in 34 and RAEB in transformation (RAEB-t) in 22. Dyserythropoiesis, dysgranulopoiesis and dysmegakaryopoiesis were found in all FAB subgroups. Dyserythropoiesis was significantly more frequently encountered in RAEB than in RAEB-t and CMML.

In healthy persons, a daily dose of 2,000 IU of vitamin E given for 10 days does not prolong the bleeding time and has no influence on collagen- and ADP-induced platelet aggregation nor on platelet prostaglandin synthesis. Therefore, even high doses of vitamin E have no place in platelet function suppressing therapy.

The French--American--British co-operative study group (FAB) criteria were used to classify acute leukaemia in 194 adult patients. The dividing lines between FAB subgroups M1--M2 and M5A--M5B appeared to be less well defined. In an effort to improve subtyping we introduce the maturation index (MI). (formula: see text). Clearly distinct subgroups emerged after arrangement of cases according to MI values. Immature and rather mature cases in both myeloblastic and monoblastic leukaemia were divided at a MI of 4%. Assignment to M3, which is based essentially on morphological grounds, could be made on numerical grounds too, MI being at least 85% in all of our cases. A few cases showing M2 morphology and a positive alpha-naphthylacetate esterase (ANE) staining should probably be categorized as M4. Application of the original FAB criteria resulted in disagreement of 14% between three investigators, while with our additional criteria the discrepancy was only 2%.