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J Guichard

Publications and source records attributed to J Guichard.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1981

Embryonic and fetal hemoglobin synthesis in K562 cell line.

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.

Butyrates↗
PubMed1981

Heterogeneity in the cellular commitment of a human leukemic cell line: K 562.

The cellular origin of the K 562 cell line, established from a patient in the blast crisis of chronic myeloid leukemia has been investigated. In agreement with previous reports, an erythroid differentiation was observed. A minority of immature, but hemoglobinized erythroblasts were identified both by electron microscopy and by immunofluorescence using an antibody to gamma-globin chains. Embryonic and fetal hemoglobin (Hb) were synthesized. Hemin increased the number of erythroblasts as well as the absolute amount of Hb synthesized: the Hb pattern was also significantly modified. By cytochemical ultrastructural detection of peroxidase activity (PA), a weak PA, distinct from granulocytic peroxidases, was found exclusively in the nuclear envelope and rough endoplasmic reticulum in a small number proportion of cells. In its localization this PA resembled that of normal and leukemic promegakaryoblasts. The addition of sodium butyrate or dimethylformamide markedly increased the number of these cells (up to 30%) but did not modify their cytoplasmic maturation. No modification of Hb synthesis was observed. Cloning of the K 562 line revealed a marked heterogeneity from one clone to another in Hb production, in the phenotype of Hb synthesis, and in the inducibility by butyrate or dimethylformamide. An inverse relationship between the number of cells with PA and Hb production was found in the different clones. Recloning some of these primary clones resulted in secondary clones, which displayed properties similar to those from which they had originated. All attempts to obtain granulocytic differentiation by addition of different inducers failed. These results clearly indicate that the K 562 cell line arises from the proliferation of bipotent stem cells, these cells possessing variable capacities of differentiation toward erythroid and presumably megakaryocytic cell lineages.

Cell Line↗
PubMed1980

Congenital dyserythropoietic anaemia type I: absence of clonal expression in the nuclear abnormalities of cultured erythroblasts.

Erythroid colonies derived from the circulating early erythroid precursor (BFU-E) of a patient with congenital dyserythropoietic anaemia type I (CDA I) have been grown in plasma clot and studied by electron microscopy. The number of circulating BFU-E was in the normal range with a roughly normal appearance at the light microscopic level. However, investigation of individual colonies by electron microscopy has always shown a mixture of normal and abnormal erythroblasts exhibiting the typical nuclear aberrations found in vivo. The proportion of normal erythroblasts varied from one colony to another. After the release of the cells from the clot in order to permit new cellular interactions, macrophages were observed to phagocytose abnormal erythroblasts but also a few erythroblasts with normal nuclei. These findings demonstrate that CDA I is a disorder which results from a defective erythroid stem cell but that the progeny of each BFU-E may vary considerably in the extent to which they express the morphological defects. Based on studies of cultures of BFU-E, similar conclusions were previously made for CDA II.

Adult↗
PubMed1980

Congenital dyserythropoietic anemia type III. studies on erythroid differentiation of blood erythroid progenitor cells (BFUE) in vitro.

In order to investigate whether the morphological abnormalities observed in congenital dyserythropoietic anemia type III (CDA III) have a cellular or an environmental origin; BFUE from the blood of a patient exhibiting a CDA type III were grown in vitro. The progeny derived from these BFUE were subsequently studied at light and electron microscopic level. Giant multinuclear erythroblasts which represent the most prominent finding of CDA III in bone marrow were also found in culture. Nuclear clefts found in vivo were also observed by electron microscopic studies performed on the erythroblasts growing in vitro. In each erythroid colony, morphologically normal and giant multinuclear erythroblasts were intermingled. This finding indicates that the two populations of erythroblasts derive from the same defective stem cell. The studies by indirect immunofluorescence of i antigen was preferentially expressed in the immature erythroblasts as in culture from normal subjects but not in the giant mature erythroblasts. This finding suggests that the excess of i antigen expression of CDA III in vivo is rather the indirect consequence of a stimulation of erythropoiesis than result of the disease.

Adult↗
PubMed1979

Growth of human megakaryocyte colonies in culture from fetal, neonatal, and adult peripheral blood cells: ultrastructural analysis.

Megakaryocyte colonies can be grown in culture from human blood cells and fetal liver cells in plasma clot containing erythropoietin. Megakaryocyte progenitors were found in a fraction of mononuclear cells isolated by Ficoll density gradient centrifugation from adult, neonatal, and fetal blood. Megakaryocytes were identified by their morphology and particularly by their polylobulated nucleus when examined by light microscopy. The megakaryocytic nature of large cells was clearly confirmed by the presence of platelet peroxidase, demarcation membranes, and alpha-granules detected by electron microscopy; in addition mature small megakaryocytes were recognized. Megakaryocyte colonies were seen after 9 days of culture and consisted of 2 to 20 cells. The colonies were pure or mixed with the burst erythroblasts. The mixed colonies were numerous in fetal and neonatal cultures, while pure megakaryocyte colonies were seen three times more frequently in those from adult blood. The total number of colonies was also much lower in adult cultures. In colonies derived from neonatal and fetal cells, megakaryocytes often reached a more complete maturation than in those from the adults, proceeding as far as platelet shedding. This study demonstrates for the first time that a megakaryocyte committed cell present in human blood can develop megakaryocyte colonies in culture.

Blood Cells↗
PubMed1979

Morphological Abnormalities in cultured erythroid colonies (BFU-E) from the blood of two patients with HEMPAS.

The results of cytological and ultrastructural analysis of erythroid burst colonies derived from the peripheral blood of two patients with HEMPAS have been compared to those obtained in normal controls. Using the plasma clot technique, in studies on 10 subjects we confirmed that most of the colonies consisted or erythroblasts with a synchronous and normal maturation involving a wave of nuclear extrusion at day 13. In contrast, the majority of well-haemoglobinized colonies from HEMPAS consisted of numerous bi- or multinucleated erythroblasts displaying the supplementary double membrane beneath their plasma membrane. This excessive membrane may be present as a continuous or fragmented structure in different erythroblasts from the same colony. These findings suggest that the progeny derived from one BFU-E may vary considerably in their morphological defects. Furthermore, one third of the packed colonies appeared to be formed by non-haemoglobinized cells which were clearly identified by electron microscopy as very early erythroblasts. These cells were unable to mature and subsequently lysed. Thus dyserythropoiesis occurred in culture both at early and late stages of maturation. These studies clearly demonstrate that HEMPAS is a disorder resulting from defective erythroid committed cells.

Adult↗
PubMed1979

Megakaryocyte colony formation from human bone marrow precursors.

We report the growth in plasma clot culture of megakaryocyte colonies from adult bone marrow cells with the use of four different sources of erythropoietin (Ep) as the stimulating factor. A major proportion of the megakaryocyte colonies (75%) were pure, while the others were mixed, involving erythroblasts and megakaryocytes. Ultrastructural studies have shown that the maturation of megakaryocytes was essentially normal and that platelet shedding occurred. Megakaryocyte colony formation required a large number of plated cells (greater than 3 X 10(5)/ml). In the absence of erythropoietin, rare spontaneous megakaryocyte colonies could be observed, while no erythroid colonies were present. However, erythropoietin induced a fivefold increase in the total number of colonies. These data suggest that erythropoietin is involved in the differentiation of human megakaryocytes, but that it does not act alone, since another factor related to the number of seeded cells appears essential for the formation of human megakaryocyte colonies.

Animals↗
PubMed1979

Absence of erythroblastic islands in plasma clot culture and their possible reconstitution after clot lysis.

Ultrastructural studies of erythroid colonies derived from human peripheral blood and growing in plasma clot culture have confirmed the absence of a macrophage inside each colony of erythroblasts. However, when macrophages and erythroblasts were liberated from the semisolid media by clot lysis, these two types of cells rapidly acquired intimate contacts, suggesting the reconstitution of any erythroblastic island. The possible significance of this phenomenon is discussed.

Cell Communication↗
PubMed1978

[Differentiation of human megakaryocytes in culture starting from the primordial circulating cells in the newborn].

Human neonatal blood mononuclear cells were seeded in plasma clot containing high dose of a crude erythropietin. Pure megakaryocyte colonies were observed rarely and most of the colonies were mixed, megakaryocytes being located between subcolonies of erythrocytic bursts. The megakaryocytic nature of large cells could be clearly confirmed by the presence of platelet peroxidase, demarcation membranes, and alpha granules detected by electron microscopy; in addition mature micromegakaryocytes were recognized, shedding platelets.

Cell Differentiation↗
PubMed1978

Cytochemical distinction between azurophils and catalase-containing granules in leukocytes. I. Studies in developing neutrophils and monocytes from patients with myeloperoxidase deficiency: comparison with peroxidase-deficient chicken heterophils.

The neutrophils and monocytes of two patients with hereditary myeloperoxidase (MPO) deficiency lacked MPO activity as determined by light and electron microscopic cytochemical staining. With a technique employing neutral 3,3'-diaminobenzidine, azurophils of precursor and mature neutrophils were devoid of MPO whereas eosinophil, basophil, and platelet peroxidases exhibited normal activity. After incubation in alkaline DAB medium, which stains catalase, some small granules were strongly reactive in both immature and mature neutrophils and monocytes. These catalase-containing granules were distinct from all other categories of granules. Their number decreased with maturation. In the presence of cyanide or aminotriazole, peroxidatic activity could also be detected in ellipsoid azurophils, although large spherical granules remained unreactive. This peroxidatic activity is apparently not due to MPO inasmuch as it has been demonstrated that this protein is not synthesized in these patients. Thus, the significance of the last finding is unclear but suggests a heterogeneity of azurophil content. In contrast to human MPO-deficient cells, chicken heterophils naturally devoid of peroxidase are unable to produce hydrogen peroxide upon phagocytosis and were also devoid of catalase-containing particles. This observation suggests that catalase is involved in the control of the intracellular level of hydrogen peroxide in human cells.

Animals↗
PubMed1976

[Improved methods for the cytochemical demonstration of platelet peroxidase (author's transl)].

The variability of results to demonstrate the peroxidase of platelets has been attributed to an inhibition of the enzyme by glutaraldehyde. In order to eliminate this difficulty, two new methods have been employed prior to incubation in the diaminobenzidine medium: 1. Fixation was omitted. 2. Fixation was performed in a mixture of tannic acid-formaldehyde glutaraldehyde. With these two procedures, intense and reproducible peroxidase staining of human platelets and chicken thrombocytes was obtained. Only when the fixation procedure was omitted could the peroxidase of rat platelets be demonstrated histochemically. The importance of the detection the peroxidase as a marker enzyme of the megakaryocyte cell line and the relationships of this peroxidase with those of leukocyte peroxidases are discussed.

3,3'-Diaminobenzidine↗
PubMed1975

[Peroxidase activity of neutrophil granules in two cases of congenital myeloperoxidase deficiency].

The neutrophils and monocytes of two patients with hereditary myeloperoxidase deficiency lacked myeloperoxidase activity as determined by light and electron microscopic cytochemical staining. Using Graham-Karnovsky media, azurophils of neutrophils were devoid of peroxidase whereas all eosinophilic and basophilic granules exhibited normal peroxidase activity. After incubation in alkaline diaminobenzidine media which stains the catalase of microperoxisomes, some small granules were seen to be strongly stained in both immature and mature neutrophils. These small granules were distinct from all other neutrophilic granules which lacked a positive reaction. Only, in the presence of cyanide or aminotriazole, peroxidatic activity was also detected in some ellipsoid azurophils. This observation suggests that these substances activated an oxidase whose nature is discussed.

Basophils↗
PubMed1975

Fine structural and cytochemical identification of microperoxisomes in developing human erythrocytic cells.

An alkaline diaminobenzidine (DAB) medium has been used to identify peroxidase activity in small granules (0.09 to 0.2 mu in diameter) present in all forms of maturing erythrocytic cells with the exception of erythrocytes. These granules, which were more frequent in proerythroblasts (from two to seven by thin section), were distinct from pleomorphic granules present in the close proximity to the Golgi apparatus. They were also distinct from ferritin molecules which were seen as aggregates in siderosomes of polychromatophilic erythroblasts. They often appeared in close association with the smooth membrane of the nuclear envelope. Optimal conditions for the visualization of these granules by incubation in alkaline DAB were obtained when the peroxidase activity of hemoglobin was reduced by addition of low concentrations of potassium cyanide. Lack of hydrogen peroxide in the incubation media completely inhibited the staining reaction of hemoglobin, while the positive reaction persisted in the granules. Aminotriazole in the incubation media prevented the staining of these organelles. These findings suggest that small granules seen in maturing erythroblasts contain catalase and that they correspond to microperoxisomes described in other tissues. The mechanism of their disappearance during reticulocyte maturation is unknown. The relationship between particulate catalase of erythroblasts and soluble erythrocytic catalase has not been elucidated.

3,3'-Diaminobenzidine↗