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J Breton-Gorius

Publications and source records attributed to J Breton-Gorius.

At least 19 recordsLinked to original sources
PubMed1992

Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

Adult↗
PubMed1992

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.

Blood Platelets↗
PubMed1992

Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.

Antigens, CD↗
PubMed1992

c-jun and c-fos are expressed by human megakaryocytes.

Expression of the main nuclear protooncogenes during terminal megakaryocyte (MK) differentiation is poorly understood. Because previous results have suggested that c-fos and c-jun protooncogenes are expressed in human leukemic cell lines induced to undergo megakaryocytic differentiation, we have analyzed the expression of these two protooncogenes in normal MK. Studies were performed, by in situ hybridization and immunofluorescence, on human MK obtained either directly from bone marrow or from culture of MK progenitors. c-fos and c-jun transcripts were detected in most cultured or fresh marrow MK from adult donors. Expression was much higher in cytologically immature than in mature MK whereas no expression was detected in the most mature MK. c-fos and c-jun expression increased dramatically with MK size. In cultured fetal MK, which all remained small in size, c-fos mRNA was present but at a low level. The c-fos-encoded protein (P62fos) was easily detectable in the great majority of MK. We directly demonstrated that the level of P62fos expression was correlated to MK ploidy by flow cytometry using a three-color staining technique. The involvement of serum and growth factors in the induction of P62fos in MK was studied. Whereas a 3-h serum deprivation resulted in the disappearance of P62fos in MK, several growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), and transforming growth factor beta (TGF-beta), as well as normal or aplastic serum, were able to reinduce its expression within 2 h. In conclusion, our results suggest that c-jun and c-fos may play a role in the transduction of signals by several growth factors during terminal MK differentiation.

DNA-Binding Proteins↗
PubMed1991

A familial occurrence of natural killer cell--T-lymphocyte proliferation disease in two children.

Several reports describe the association of hyperlymphocytosis with neutropenia. This syndrome, named lymphoproliferative disease, is characterized by a chronic indolent clinical course, bone marrow lymphocyte infiltration, and granulopenia of central origin. The proliferating lymphocytes share large granular lymphocyte natural killer cell and T-lymphocyte characteristics. They are either of monoclonal or polyclonal origin. In this report the familial occurrence of a similar syndrome observed in two children is described. Lymphocyte morphologic abnormalities including nuclear pockets, were noted, a feature usually present in leukemic cells. Lymphocyte proliferation was distinct in each case as shown by the presence of a predominant CD4+ cell population in one and a predominant CD8+ population in the other. Monoclonal gene rearrangements of T-cell receptor beta-chain gene were found although clonal variations occurred with time in one patient. The cause of this unique familial occurrence of monoclonal lymphoproliferation associated with neutropenia is unknown.

Adolescent↗
PubMed1991

Osteonectin is an alpha-granule component involved with thrombospondin in platelet aggregation.

We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1991

Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes.

The ultrastructural localization of the CD68 antigen, a 110-kd intracellular glycoprotein associated with myeloid cells and with monocytes/macrophages, was investigated in human neutrophil granulocytes by postembedding immunogold staining, using monoclonal antibody KP1. The antigen was found in the primary granules of neutrophils, although not all primary granules were labeled. It was absent from the plasma membrane. In monocytes, it was also detected within cytoplasmic granules, colocalized with lysozyme and myeloperoxidase. This observation confirms and completes results obtained by immunofluorescence and other light-microscopic methods. Moreover this study shows that the CD68 epitope recognized by antibody KP1 is able to resist fixation and embedment and therefore emphasizes the value of using KP1 as a marker for this macrophage-associated molecule.

Antibodies, Monoclonal↗
PubMed1991

Alpha 1-antitrypsin is present within the primary granules of human polymorphonuclear leukocytes.

Elastase is a potent proteolytic enzyme found within human neutrophil primary granules. Its major inhibitor in the serum is alpha 1-antitrypsin, a protein that is synthesized by hepatocytes but which has recently also been shown to be synthesized by circulating neutrophils. The authors have therefore carried out an immunocytochemical study at the light microscopic and ultrastructural level to determine the intracellular localization of alpha 1-antitrypsin. Double labeling with colloidal gold showed that alpha 1-antitrypsin is localized at the same site as neutrophil elastase, i.e., within primary granules. Secondary granules (detected by labeling for lactoferrin) were unstained for alpha 1-antitrypsin. Elastase and its major inhibitor therefore coexist within the same granule population within human neutrophils. Some difference in their intraorganelle distribution existed at the ultrastructural level (in that elastase tended to be localized at the periphery of the granules whereas alpha 1-antitrypsin was usually diffusely present in the matrix of the granules), but further studies are required to determine whether the two molecules are already complexed with each other within the neutrophil.

Colloids↗
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

Fetal to adult hemoglobin switch in cultures of early erythroid precursors from human fetuses and neonates.

Erythroid burst colonies derived from the cord blood of nine neonates and from the blood and liver of three fetuses aborted after 20 weeks of gestation were grown in plasma clot culture. Their quantitative study revealed a higher proportion of burst-forming units (BFU-Es) in cord blood than in cord blood of normal adults. In addition, colony-forming units (CFU-Es) were present in cord blood but absent from adult blood. Study of haemoglobin synthesis in 14-day cultures of cord blood BFU-Es showed a significantly higher degree of Hb A synthesis than was found in reticulocytes from fresh cord blood; this proportion was, however, similar to that expected in vivo about three weeks after birth. These data suggest that the hemoglobin switch is already programmed in most of the early erythroid precursors present in cord blood or full-term neonates and indicate that the differentiation time is probably of the same order of magnitude in vivo and in vitro. The proportion of Hb A and F synthesis in erythroid bursts was not influenced by the concentration of erythropoietin in the range studied--ie, from 0.5 to 12 international units. Low but identical proportions of Hb A synthesis were found both in erythroid cells from liver after two hours of incubation with [3H]-leucine, and in 14-day liver bursts from fetuses aborted at 20 weeks of gestation.

Adult↗
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

Ultrastructural localization of peroxidases in 'undifferentiated' blasts during the blast crisis of chronic granulocytic leukaemia.

Twelve cases of Philadelphia chromosome positive chronic granulocytic leukaemia (CGL) in blast transformation have been investigated using ultrastructural peroxidase detection. In all cases, the leukaemic blasts were negative for myeloperoxidase on the basis of standard cytochemistry. In nine cases a variable proportion of blasts contained peroxidase activity detectable only by electron microscopy, permitting definition of their myeloid nature. By their distinct characteristics and localization, different peroxidase activities were recognized. Thus, several types of blasts were identified: megakaryoblasts (MKB), basophil promyelocytes (BPM), myeloid blasts with small granules containing peroxidase (MyB), and proerythroblasts (ProE). MKB were predominant in two cases and present in four cases, mixed with other myeloid blasts. BPM were abundant in one case and present in seven cases. MyB were identified as a majority in four cases. Three cases remained without any peroxidase. It is concluded that ultrastructural detection of peroxidases is of value for the identification of early myeloid blasts. Their high incidence and the simultaneous presence of several myeloid precursors suggest that during the blast crisis the target cell is frequently a pluripotent myeloid stem cell.

Bone Marrow↗
PubMed1979

Three types of Auer bodies in acute leukemia. Visualization of their protein by negative contrast after peroxidase cytochemistry.

Ten cases of acute granulocytic leukemia with blast cells containing Auer bodies (ABs) have been studied by electron microscopy after cytochemical demonstration of myeloperoxidase. The cytochemical dense reaction product has been used as a dark field to visualize unreactive protein of ABs which may then be easily identified by its negative contrast. This method has allowed us to identify three types of ABs which differ in their substructure. In type I, (five patients with promyelocytic leukemia), our study confirmed that all of the ABs consisted of a hexagonal arrangement of hollow tubes. Cells from four cases of acute myeloblastic leukemia displayed type II ABs, in which a unique pattern of protein associated in a regular linear arrangement with well defined periodicity was identified. Type III appeared characteristic of a subclass of acute myeloblastic leukemia in which large inclusions with Chediak-Higashi-like granules containing numerous micro-ABs were seen. The configuration, size, and organization of the protein in the crystal were distinct from those seen in the two other types of ABs. These features suggest that the nature of the protein in ABs may be heterogeneous.

Cytoplasmic Granules↗
PubMed1979

Pseudo-Chediak-Higashi anomaly in a case of acute myeloid leukemia: electron microscopic studies.

The formation and fine structure of giant granules in neutrophil promyelocytes of a patient with a variant of acute myelogenous leukemia were investigated by electron microscopy. The patient presented with large lymph nodes and disseminated intravascular coagulation (DIC). By light microscopy, numerous giant granules, resembling those of Chediak-Higashi syndrome (CHS), were present, but Auer bodies could not be found. By electron microscopy, these giant granules were seen to be formed by fusion of azurophilic granules, as in CHS; however, they were different from the large granules of CHS, since they contained numerous microcrystalline structures like those of Auer bodies. However, the crystalline cores of these granules exhibited a periodicity different from that of Auer bodies of acute promyelocytic leukemia. This clinical and hematologic syndrome (giant granules, enlarged lymph nodes, and DIC may represent a variant of acute promyelocytic leukemia.

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↗