Influence of zinc on iron uptake by monolayer cultures of rat hepatocytes and the hepatocellular ferritin.

Monolayer cultures of isolated rat hepatocytes on collagen gels were used to study the effect of zinc on (a) 59Fe uptake by the hepatocytes; (b) 59Fe uptake by the hepatocellular ferritin and (c) biosynthesis of ferritin and total proteins. While deposition of iron into the cellular ferritin was inhibited by zinc in a dose-dependent manner, iron uptake by the hepatocytes was inhibited significantly only at high concentrations of zinc. Biosynthesis of total proteins or ferritin was not affected by zinc. Since only a small fraction of the cellular 65Zn was found to be associated with ferritin, it was concluded that zinc was not deposited in ferritin. Intracellular distribution of 59Fe revealed that newly accumulated iron went into an iron pool the size of which could be increased by the presence of zinc.

Non-enzymic glycosylation of horse spleen and rat liver ferritins.

Incubation of horse spleen ferritin and rat liver ferritin with [14C]glucose, [14C]mannose or [14C]fucose resulted in the covalent incorporation of the sugar into ferritin. The rate of reaction depended on the concentrations of ferritin and sugar and time of incubation. The order of this nonenzymic incorporation was glucose greater than mannose greater than or equal to fucose. Glucosylated or mannosylated ferritin was not retained by concanavalin A. The plasma half-life of rat liver ferritin and apoferritin was found to be 20 min. This value remained unaffected by in vitro glucosylation or mannosylation of ferritin. It is suggested that varying degrees of glycosylation might account for the occurrence of isoferritins.

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver.

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.

Studies on the mechanism of transferrin iron uptake by rat reticulocytes.

Mechanism of transferrin iron uptake by rat reticulocytes was studied using 59Fe- and 125I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound 59Fe was located in the cytoplasmic fraction, only 25-30% of 125I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10-15% of the cytoplasmic 125I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound 125I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering 59Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.

The myelodysplastic syndromes.

The myelodysplastic syndromes constitute a fascinating model for monoclonal premalignant disorders. Haemopoiesis is 'dysplastic' with inefficient maturation of a slowly expanding or sometimes of a stable population, of blood cell precursors. About one third of the patients evolve into acute leukaemia, the result of either a progressive expansion of the original clone or a new mutation producing a more malignant subclone. The majority of patients suffer from the results of bone-marrow insufficiency, with pancytopenia and possibly immune deficiency. Characteristic karyotype anomalies involving mainly chromosomes 5, 7 and 8 are seen in half the patients. These same chromosomes are known to carry different oncogenes. The myelodysplastic syndrome occurs mainly in the aged and there is a moderate male preponderance. The incidence is still unknown but is probably similar to that of acute leukaemia. The etiology is also unknown; however, a secondary myelodysplastic syndrome precedes acute myeloid leukaemia, as a late consequence of chemo- and radio-therapy in treated Hodgkin's disease. This suggests that environmental mutagens might also be involved in primary myelodysplastic syndromes. Treatment remains highly unsatisfactory but a few recent developments improve prognosis, at least in the younger patient.

Protective effect of vitamin E on immune triggered, granulocyte mediated endothelial injury.

The effect of alfa-tocopherol on the cell-cell interactions at the vessel wall were studied, using an in vitro model of human umbilical vein endothelial cell cultures (HUEC). Immune triggered granulocytes (PMN) will adhere to and damage HUEC and platelets enhance this PMN mediated endothelial injury. When HUEC are cultured in the presence of vitamin E, 51Cr-leakage induced by complement stimulated PMN is significantly decreased and the enhanced cytotoxicity by platelets is completely abolished (p less than 0.001). The inhibition of PMN induced endothelial injury is directly correlated to a diminished adherence of PMN to vitamin E-cultured HUEC (p less than 0.001), which may be mediated by an increase of both basal and stimulated endogenous prostacyclin (PGI2) from alfa-tocopherol-treated HUEC (p less than 0.025). The vitamin E-effect is abolished by incubation of HUEC with the irreversible cyclo-oxygenase inhibitor, acetylsalicylic acid, but the addition of exogenous PGI2 could not reproduce the vitamin E-mediated effects. We conclude that vitamin E exerts a protective effect on immune triggered endothelial damage, partly by increasing the endogenous anti-oxidant potential, partly by modulating intrinsic endothelial prostaglandin production. The failure to reproduce vitamin E-protection by exogenously added PGI2 may suggest additional, not yet elucidated vitamin E-effects on endothelial metabolism.

Mechanisms of vascular damage in gout and oxalosis: crystal induced, granulocyte mediated, endothelial injury.

Immune triggered granulocyte (PMN)-endothelial interactions have been implicated in the pathogenesis of vascular diseases. While hyperuricemia and gout are associated with an increased risk of atherogenesis, we studied the modulation by monosodium-urate (MSU) crystals of PMN-endothelial interactions in vitro. The relationship between calcium oxalate (COX) crystals - implicated in the vasculitis of primary oxalosis - and immunologically mediated endothelial injury was also explored. Both MSU- and COX-crystal treated sera stimulate PMN to adhere to and induce significant 51Cr-release from endothelial cells in vitro. Platelets significantly increase crystal-triggered PMN endothelial cell adherence and 51Cr-release. This platelet augmenting effect depends on the release of platelet constituents (e.g. serotonin). Microcrystalline material present in vessel walls, thus may cause C-activation and may trigger PMN and platelets to damage endothelium in vitro and in vivo. These findings may have relevance to the understanding of the accelerated atherogenesis of hyperuricemia and the fulminant vasculitis of oxalosis or ethylene glycol poisoning.

Enkephalins modify granulocyte-endothelial interactions by stimulating prostacyclin production.

Granulocyte (PMN)-endothelial interactions have been implicated in the primary events of vascular injury and atherogenesis. We now present data which show that endogenous opioid peptides, e.g. enkephalins (ENK), dampen immune-triggered, granulocyte-induced endothelial damage by enhancing prostacyclin production. Concurrent exposure of human umbilical vein endothelial cells (HUEC) to Met5-enkephalin increased arachidonic acid (AA, 20 muM) and thrombin (T, 10 U/ml) induced 6-keto-PGF1 alpha-release to respectively 197.2 +/- 28.1% and 204.1 +/- 17.8% (mean +/- SEM) of base line stimulation (p less than 0.025). The increases noted were significant at ENK-concentrations as low as 10(-12)M. Simultaneous addition of naloxone with ENK completely abolished the enhanced 6-keto-PGF1 alpha-release. Addition of a protease resistant enkephalin analogue significantly (p less than 0.01 over several different concentrations) reduced PMN adherence to HUEC; concomitantly 51Cr-leakage from HUEC that had been exposed to PMN plus activated serum complement was decreased. The even further enhanced 51Cr-leakage that occurs when platelet release products (e.g. serotonin) are included is also decreased by added enkephalin. These data suggest that endogenous neurotransmitters may affect endothelial prostaglandin metabolism, and by so doing provide a protective effect during in vitro, and perhaps in vivo, PMN mediated endothelial injury. This link between neurohumoral and inflammatory systems might enhance our understanding of stress-related phenomena in inflammation and vascular diseases.

Bone marrow transplantation performed as first-time treatment in two cases of secondary acute myeloid leukemia.

Two young patients with secondary acute myeloid leukemia were treated with allogeneic bone marrow transplantation as first-line treatment for their disease. High dose cytosine arabinoside was added to the conventional preparative regiment of cyclophosphamide and total body irradiation. No major problems were encountered to the immediate post-transplantation period. Complete remission of the acute myeloid leukemia was noted in both patients, with no evidence of recurrent disease at 15+ and 13 months. One patient developed severe pancytopenia 10 months after grafting with the presence of antibodies against platelets and granulocytes from the bone marrow donor. She died 3 months later from a generalised Aspergillus septicemia, probably precipitated by therapy with high doses of methylprednisolone. The other patient is alive in excellent clinical condition and in hematologic remission. Bone marrow transplantation must be taken into consideration as first-line therapy in any young patient who is suffering from a refractory type of leukemia and who has a suitable donor.

Adult T-cell leukemia: a report on two white patients.

Two white European males are reported with adult T-cell leukemia (ATL), a disease first described in Japan, but recently also in the U.K. and U.S.A. Both patients presented with lymphadenopathy, but without a mediastinal mass. In addition, one patient had skin infiltrates and the other had hepatosplenomegaly. Morphologic and ultrastructural examination of the blasts in bone marrow and lymph node biopsy revealed a predominance of polymorphic lymphoid cells with pronounced nuclear irregularities and a semi-mature chromatine pattern. Histopathology of the lymph nodes showed a diffuse infiltration with medium-sized lymphoblasts with irregular nuclei. The blasts in the bone marrow formed E rosettes with sheep erythrocytes, lacked terminal deoxynucleotidyl transferase (Tdt) activity but expressed the Ia-like antigen; although the majority of the cells reacted with a polyclonal anti-T-cell serum, they were negative for OKT3. In one patient a helper/inducer phenotype (OKT4+) was found in the lymphoblasts of bone marrow and lymph node, while in the other only in the lymph node. The difference between bone marrow and lymph node phenotype is discussed. To our knowledge, these are the first two European patients reported with ATL, a disease clearly different from convoluted T-cell acute lymphocytic leukemia.

Monocyte counting: discrepancies in results obtained with different automated instruments.

To determine the accuracy of several methods for measuring the monocyte count, the results obtained by a number of different automated cell counters were analysed. Considerable discrepancies occurred for monocyte counts obtained in normal blood among the counters. The results of a visual monocyte count on a total of 800 leucocytes were used as the reference method. The technique of measuring the monocyte count by using dual staining with monoclonal antibodies CD45 and CD14 provided the closest agreement with the reference method. Six other automated counting systems were assessed. Two of these systems (Coulter VCS and Technicon H1) gave results, which, although under-estimating monocytosis, correlated well with the results obtained by the reference technique. A third system (Toa Sysmex NE-8000) gave unreliable results. Three of the automated systems evaluated measured a "third population"--that is, monocytes together with other leucocytes. One of these systems (Ortho ELT 1500), overestimated the count, as expected, but correlated well with the reference method. The second of these "third population counters" (Coulter S Plus IV) correlated moderately well with the reference monocytosis, while the Toa Sysmex E-5000 correlated poorly. It is clear that problems exist in the evaluation of different instruments for counting monocytes. An accurate and reliable reference method is a pre-requisite to evaluate this aspect of cell counters. As the visual method is too cumbersome a different reference method would be useful. Based on the results of this study, it is suggested that the technique using fluorescence labelled monoclonal antibodies should be regarded as an acceptable alternative.

The histological characterization of ALIP in the myelodysplastic syndromes.

Bone marrow biopsies of patients with a myelodysplastic syndrome (MDS) may, in the absence of an increased number of blasts in the bone marrow smears, contain small clusters of immature precursors. The presence of these cell nests, previously described as "abnormal localized immature precursors" or ALIP, bears a strong prognostic value predisposing patients to early death with an increased risk to develop myeloid leukaemia. In order to describe and delineate this histological characteristic more precisely, we compared bone biopsies of patients with MDS, used in these previous studies, with bone marrow biopsies performed for staging procedures in patients with Hodgkin's disease, non-Hodgkin's lymphoma and carcinoma. From this comparison we conclude that immature precursors are readily differentiated from proerythroblasts, myeloblasts and small-sized megakaryocytes, and that they most probably represent precursors of the myelo-monocytic-erythroid series. A clearcut increase in their number, mostly resulting in the formation of small clusters, is only found in biopsies from patients with MDS.

The role of standardization in quality assurance.

The main organizations involved in standardization of laboratory medicine are described. At the international level these are mainly: ISO, IEC, WHO, ICSH, IFCC and COWS/WASP, while at the European level CEN, CENELEC and BCR are involved in standardization activities in Europe. At the present the European Commission is preparing a directive on in vitro diagnostic. This document will rely, for its implementation, on standards prepared by CEN/CENELEC.

Intravascular haemolysis and renal failure caused by intermittent rifampicin treatment.

Rifampicin is a widely used anti-tuberculous drug. Administered daily, only minimal side-effects occur. With intermittent therapy and, as happened in the present case, when the drug is administered after an interruption of treatment, severe adverse reactions [5] (thrombocytopenia, renal failure, and haemolysis) may occur.