Mart M. A. C. Langenhuijsen

A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with 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.

Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay: A methodological study

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.

Does iron-deficient erythropoiesis in pregnancy influence fetal iron supply?

Background. It was investigated whether the iron status in newborns is negatively influenced by iron‐deficient erythropoiesis of the mother during pregnancy.

Influence of iron deficiency anaemia on haemoglobin A2 levels: possible consequences for ß?thalassaemia screening

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.

Imipenem‐cilastatin for empirical therapy in neutropenic patients with fever: An open study in patients with hematologic malignancies

Abstract: 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.

Influence of aspirin on human megakaryocyte prostaglandin synthesis

Abstract: 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 × 103 (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.

Monocytic differentiation induction of HL-60 cells by MC 903, a novel vitamin D analogue.

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.

Radiotherapy versus radiotherapy plus chemotherapy in stages I and II non‐Hodgkin's lymphoma of the upper digestive and respiratory tract

From 1975 through 1985 88 patients with a primary manifestation of non‐Hodgkins lymphoma in the upper digestive and respiratory tract were referred to our hospital. Fifty‐seven of them were in Stage I or II. The results of therapy in 48 evaluable patients are the subject of this retrospective study. Low, intermediate, and high grade malignant histologic features were encountered in 6, 27, and 15, respectively. The authors compared retrospectively the patients treated with radiotherapy (RT) with those who received additionally chemotherapy (RT + CT). Patients have been followed for a minimum of 1 year. Four of 29 patients with intermediate‐grade or high‐grade malignant lymphoma treated with CT and RT relapsed whereas eight of 13 patients treated with RT alone relapsed. Actuarial survival curves differed significantly in favour of the CT and RT group. These figures were also significant when the high‐grade lymphomas plus centroblastic lymphomas were considered. It is recommended to add CT to RT in both Stage I and II NHL of high‐grade and intermediate‐grade malignancy.

Maturation Index: a Contribution to Quantification in the FAB Cla.ssification of Acute Leukaemia

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).

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets

Abstract: 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 (α subunit VLA2), Cdw49e (α subunit VLA5), Cdw49f (α subunit VLA6) and CD62. Circulating megakaryocytes, although >75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogenous (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 heterogenous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.