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Biomedical subjects

W Vainchenker

Publications and source records attributed to W Vainchenker.

At least 217 records · Page 12Linked to original sources
PubMed1983

Glycoprotein I identification during normal and pathological megakaryocytic maturation.

The expression of glycoprotein I (GP I) on normal and pathological megakaryocyte (MK) precursors has been investigated in vivo and in a cell culture system with mouse monoclonal antibody (AN51) using immunofluorescence, immunogold and immunoferritin with electron microscopy. Our results confirmed the observation of Rabellino et al. (1979, 1981), using polyclonal antibodies, that GP I was expressed throughout normal MK maturation. The present result differs from our previous work which failed to detect binding of AN51 on promegakaryoblasts (PMKB). Improvement of immunofluorescent techniques has permitted detection of weak fluorescent labeling on normal PMKB by day 6-7 of in vitro culture from normal CFU-MK. An increased number of labeled PMKB was observed in bone marrow from patients with idiopathic thrombocytopenic purpura (ITP) as compared with normal bone marrow. In 20 selected cases of acute megakaryoblastic leukemia, AN51 labeled the PMKB but at a lower percentage than with J15 (monoclonal anti-glycoprotein IIb/IIIa complex). The MK nature of the blasts was confirmed by the ultrastructural detection of platelet peroxidase (PPO) and by the binding of AN51 demonstrated with immunoferritin-conjugated anti-mouse IgG. The immunogold technique revealed that the density of gold particles on the cell membrane of PMKB was variable, but generally weaker than in platelets or pathological micromegakaryocytes. The specificity of AN51 for MK lineage was shown by the absence of binding of AN51, by all blasts from 60 cases of acute leukemia involving other cell lines. Because of its weak labeling of PMKB due to the small number of antigenic sites, AN51 must be associated with another megakaryocytic marker in the diagnostic assessment of acute megakaryoblastic leukemia.

Acute Disease↗
PubMed1983

Myeloid and megakaryocytic properties of K-562 cell lines.

The expression of myeloid and megakaryocytic markers of differentiation has been studied in one K-562 cell subline, in its clones, and in the original cell line. Cytotoxicity, electron microscopy, immunofluorescence studies with a panel of polyclonal and monoclonal antibodies, and radioimmunoassays were performed on K-562 cells before and after induction with hemin, sodium butyrate, and 12-O-tetradecanoylphorbol-13-acetate. Myeloid membrane markers were present in all K-562 cell lines. Only the early granulopoietic cell surface markers were expressed in 75 to 95% of the cells, while none of the late membrane markers was detected. In contrast, neither the early (myeloperoxidase) nor late (lactoferrin) cytoplasmic markers were present. Thus, K-562 cells showed a membrane phenotype similar to that of a normal or leukemic promyelocyte but lacking myeloperoxidase. Membrane megakaryocytic markers, such as platelet glycoprotein IIIa and platelet peroxidase, were also detected in K-562 cells. However, some other early megakaryocytic markers, such as platelet glycoprotein lb, Factor VIII-R-Ag, and platelet Factor 4, could not be detected by fluorescent labeling. Cloning of the cell line did not result in the selection of a unipotential cell line. These results could be explained by the expression of multilineage markers in a single cell. In all of the cell lines and clones, hemin slightly increased the expression of the myeloid membrane markers without any modification of the megakaryocytic markers. Sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate diminished most of the myeloid markers and very significantly increased the expression of the megakaryocytic markers.

Antibodies, Monoclonal↗
PubMed1983

[Treatment of chronic autoimmune thrombopenic purpura with intravenous immunoglobulins. Study of 18 cases].

The authors reported the effect of high dose intravenous immunoglobulin therapy in 18 patients with chronic autoimmune thrombocytopenic purpura. A good but transitory response was observed in 60% of the cases. The intensity of the response was variable, the duration was between 11 and 26 days. The mechanism of the effect of this therapy is poorly understood. Since this therapy is expensive and the response inconsistent and transitory, the indications are limited.

Adolescent↗
PubMed1982

Inhibition of insulin receptor binding by phorbol esters.

Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.

Animals↗
PubMed1982

Hb switching in neonatal cultures. Increase of Hb A synthesis in presence of an erythroid potentiating activity (EPA).

The role of EPA (erythroid potentiating activity) on the growth and on the pattern of hemoglobin synthesis in erythroid colonies from human neonates was investigated. Conditioned medium from the Mo cell line was used as a source of EPA. The results have shown that the addition of Mo medium to cultures determined a significant enhancement of the number and size of BFU-E and an increase of beta chain synthesis. The acceleration of hemoglobin switching is not related to an amelioration of the maturation of the erythroid colonies when grown in the presence of Mo medium. The enhancement of Hb A synthesis induced by Mo medium can directly be related to its EPA, which may operate by two different mechanisms: (1) the recruitment of early erythroid progenitors already preprogrammed to synthesize prevalently beta chains, or (2) the modulation of beta and gamma gene activity in cord blood BFU-E. Some evidence suggests that the first mechanism does operate.

Cell Differentiation↗
PubMed1982

Peroxidatic activity distinct from myeloperoxidase in human monocytes cultured in vitro and in alveolar macrophages.

Human monocytes develop a peroxidatic activity (PA) in rough endoplasmic reticulum (RER) after adherence or after culture in semi-solid medium. This enzyme activity disappears after three days of culture in the majority of macrophages derived from adult monocytes but persists for one week in macrophages derived from neonatal monocytes. The PA is due to an enzyme distinct from myeloperoxidase (MPO), since monocytes from a patient with MPO deficiency develop the same PA as that of normal monocytes after adherence. By its localization and other characteristics, PA of adherent monocytes resembles that of rodent macrophages. We therefore investigated whether human alveolar macrophages exhibit PA, using a sensitive cytochemical method which prevents inhibition by aldehyde in adherent monocytes. In various pathological cases, four types of macrophages could be identified: the majority were peroxidase-negative, a small percentage was of exudate type exhibiting a PA in granules as blood monocytes, while few macrophages were intermediate, possessing only PA in RER i.e. of type resident and a smaller proportion had PA in RER and in granules i.e. exudate-resident macrophages. These findings demonstrate that human macrophages and adherent monocytes may exhibit PA in RER as has been reported for rodent macrophages. The true nature and function of the enzyme responsible for this PA, which is distinct from MPO, remains unknown, but some arguments seem to suggest its role in prostaglandin synthesis.

Cells, Cultured↗
PubMed1982

Megakaryocyte cultures in the chronic phase and in the blast crisis of chronic myeloid leukaemia: studies on the differentiation of the megakaryocyte progenitors and on the maturation of megakaryocytes in vitro.

Megakaryocyte (MK) colony formation has been studied in the chronic phase and in the blast crisis of chronic myeloid leukaemia (CML). Blood cells were grown in plasma clot for 13 d. MKs were subsequently identified by immunofluorescent techniques using two monoclonal antiplatelet antibodies (AN51 and J15). The maturation process was studied by ultrastructural methods. A marked increase in the number of circulating CFU-MK was observed in all the 10 cases studied prior to chemotherapy (70-fold increase per ml of blood). No significant modification in the regulation of MK colony formation as compared to that of normal subjects was observed. The predominant abnormality in maturation in culture was the occurrence of many hypoploid MKs (microMKs). However, the cytoplasmic maturation of the MKs was identical to that of normal subjects with occasional platelet shedding. Since microMKs predominated in some patients, scoring of MK colonies in CML necessitated immunofluorescent labelling to permit identification of MKs. During the blast crisis, MK colony formation occurred in four out of five patients with an extremely high plating efficiency in the case of promegakaryoblastic transformation. In contrast, MK colonies could not be grown from blood samples of patients with acute leukaemia, including two cases of promegakaryoblastic leukaemia. Maturation of MKs in blast crisis was identical to that of the chronic phase. Furthermore, after short periods of culture in liquid medium, circulating promegakaryoblasts from patients in blast crisis matured with the consequent production of alpha-granules and demarcation membranes. These results confirm the contention that CML represents a pluripotent stem-cell disease, involving the MK lineage, and suggest that the block in maturation during the acute phase can be overcome in vitro.

Cell Differentiation↗
PubMed1982

Hemoglobin expression in clones of K562 cell line.

The K562 cell line was cloned by dilution and the pattern of hemoglobin (Hb) production was analyzed in the clones thus obtained. The pattern of hemoglobin synthesis was different from one clone to another. According to the Hb phenotype the clones were classified in three main groups; group I no detectable Hb; group II Hb Portland; group III Hb Portland + Hb Gower I. The addition of hemin induced a better hemoglobinization and a shift in the pattern of Hb production: clones of group I were induced to produce Hb Portland and clones of group II to synthesize Hb Gower I. A constant order in the sequential expression of the different hemoglobins was observed: no Hb leads to Hb Portland + Hb Gower I. The proportion of the different globin chains varied from one clone to another, but an inverse correlation between the synthesis of G gamma and epsilon chains was observed. The alpha/non-alpha chain ratio was unbalanced in all the clones and the addition of hemin induced only a moderate increase of the synthesis of alpha chains. Recloning of three primary clones increased the homogeneity of the hemoglobin pattern, in particular after hemin induction.

Clone Cells↗
PubMed1982

Clonal expression of the Tn antigen in erythroid and granulocyte colonies and its application to determination of the clonality of the human megakaryocyte colony assay.

To evaluate whether exposure of Tn determinants at the surface of human erythrocytes, platelets, and granulocytes could arise from a somatic mutation in a hemopoietic stem cell, burst-forming unit erythroid (BFU-E) colonies, colony-forming unit granulocyte-macrophage (CFU-GM), and colony-forming unit-eosinophil (CFU-Eo) were grown from a blood group O patient with a typical Tn syndrome displaying two distinct populations (Tn(+) and Tn(-)) of platelets, granulocytes, and erythrocytes. A large number of colonies was observed. Individual colonies were studied with a fluorescent conjugate of Helix pomatia agglutinin (HPA). A sizeable fraction of each of the erythroid and granulocytic colonies appeared to consist exclusively of either HPA-positive or HPA-negative cells, thereby demonstrating the clonal origin of those exhibiting the Tn marker. Similar results were obtained from a second patient. These findings establish that the HPA labeling of Tn cells is an accurate marker permitting assessment of the clonality of the human megakaryocyte (MK) colony assay. For the study of MK cultures a double-staining procedure using the HPA lectin and a monoclonal antiplatelet antibody (J-15) was applied in situ to identify all MK constituting a colony. Our results, obtained in studies of 133 MK colonies, provide definitive evidence that the human MK colony assay is clonal because all MK colonies were exclusively composed of Tn(+) and Tn(-) MK. Furthermore, the distribution of MK within a single colony was shown to be seminormal with a mean at 6 MK, isolated MK typically being absent in culture. Comparison of the proportion of mature Tn(+) cells in blood with their respective Tn(+) progenitors has also shown that no proliferative advantage occurs after the commitment; because Tn polyagglutinability is an acquired disorder, then the expansion of the Tn(+) clone must occur either during the proliferative stage of the pluripotent stem cell or during the commitment itself. This study therefore affords evidence that a blood group antigen plays a role in the differentiation of a pluripotent stem cell.

Adult↗
PubMed1982

Reticulocytosis, hypochromia, and microcytosis: an unusual presentation of the preleukemic syndrome.

Two patients exhibiting a highly unusual preleukemic syndrome with marked reticulocytosis, hypochromia, and microcytosis are reported. This red cell phenotype has been investigated by means of HbF, HbA2, and i-antigen activity dosages, immunofluorescence labeling of F cells, reticulocyte survival, globin chain synthesis, and electron microscopy study. The marked reticulocytosis is explained by a delayed disappearance of the reticulum. Serum iron is normal, and a thalassemic syndrome is excluded because of a balanced alpha/non-alpha globin chain synthesis. Electron microscopy studies are consistent with a defect in iron uptake by erythroid cells. All the hematologic data and investigations are similar to those observed for the Belgrade laboratory rat. It is hypothesized that the low expression of HbF and i-Ag associated with microcytosis are related to a prolongation of erythroid maturation as reflected by abnormal reticulocyte survival.

ABO Blood-Group System↗
PubMed1982

Regulation of i- and I-antigen expression in the K562 cell line.

Expression of i- and I-antigen in K562 cultured under different conditions of culture was investigated. Under standard conditions of culture, i-antigen expression was very high (100% of i-labeled cells) in contrast to I-antigen expression of which was very low (2 to 5% of I-labeled cells). The addition of hemin to K562 cells did not modify the mean antigenic density or the proportion of i- and I-labeled cells. In contrast, sodium butyrate elicited an important increase in the proportion of cells exhibiting I-antigen associated to a decrease of i-antigenic density. The effect of butyrate was reversible and dependent upon de novo protein and messenger RNA synthesis since it was abolished in the presence of cycloheximide or actinomycin D. The stimulation of i-antigen conversion to I-antigen elicited by butyrate cannot be directly related to an induction of differentiation since evidence in this sense is lacking; in fact, butyrate did not increase the hemoglobin content of K562 cells. The passage from exponential to stationary phase of growth (cell density inhibition) was associated with an increase in I-antigen expression and a slight decrease in i-antigen density on the surface of K562 cells.

Antigens↗
PubMed1982

Normal human serum contains a factor(s) capable of inhibiting megakaryocyte colony formation.

Growth of megakaryocyte colonies from human bone marrow progenitors has been achieved in plasma clot culture. Megakaryocyte colonies were identified either by cytological examination or by immunofluorescent labelling using a monoclonal antiplatelet antibody (J 15). No significant differences were observed in the quantitation of the colonies by these two methods. In the absence of a stimulating factor, MK colonies were detectable when high cellular concentrations were seeded. Among the numerous conditioned media tested for their ability to stimulate MK colony formation, conditioned medium from leukocytes stimulated by PHA (PHA-LCM) was the most effective. However, under standard conditions of culture corresponding to 20% normal human sera, colony formation was not related linearly to seeding density even when cultures were stimulated by PHA-LCM. Upon reduction of the concentration of serum (2.5-5%) in the culture medium, colony formation displayed a linear relationship seeding density only when PHA-LCM was used as the stimulating factor. At the same time, the size of the colonies increased. Such inhibition was observed with all the human sera tested but it varied in extent from one batch to another. Replacement of serum by albumin, iron-saturated transferrin, alpha-thioglycerol and low density lipoproteins at physiological concentration but in the presence of bovine plasma provided a culture system whose ability to support colony formation equalled that of low concentrations of whole serum; spontaneous MK colony formation still occurred. Our results provide evidence for the presence of an inhibitor(s) of MK colony formation in normal human sera, and demonstrate the role of cellular factors in stimulating MK colony formation.

Blood↗