Biomedical subjects

J Breton-Gorius

Publications and source records attributed to J Breton-Gorius.

At least 19 recordsLinked to original sources

PubMed2001

Macrothrombocytopenia with abnormal demarcation membranes in megakaryocytes and neutropenia with a complete lack of sialyl-Lewis-X antigen in leukocytes--a new syndrome?

A new megathrombocytopenic syndrome with giant platelets in peripheral blood and severe thrombocytopenia was diagnosed in a 4-month-old boy. His clinical course included repeated hemorrhagic incidents leading to death at age 37 months. Bone marrow ultrastructural analysis revealed numerous dystrophic megakaryocytes with giant membrane complexes. Although these features were similar to those described for megakaryocytes in mice lacking the gene for transcription factor p45-NF-E2, no abnormalities in the p45-NF-E2 gene could be documented. Platelet membrane analysis showed a reduction in glycoprotein (GP) Ib, but normal content of GPIIb and GPIIIa. Analysis of genes encoding for GPIb alpha and beta, GPV, and GPIX ruled out the possibility that the observed platelet abnormality is a variant of Bernard-Soulier syndrome. A moderate neutropenia was associated with a complete lack of expression of sialyl-Lewis-X on the surface of polymorphonuclear neutrophils. A common defect in posttranslational modification of glycoproteins could account for the diverse cellular abnormalities.

Blood Platelets↗

PubMed1999

Erythroblastic synartesis: an auto-immune dyserythropoiesis.

Erythroblastic synartesis is a rare form of acquired dyserythropoiesis, first described by Breton-Gorius et al in 1973. This syndrome is characterized by the presence of septate-like membrane junctions and "glove finger" invaginations between erythroblasts, which are very tightly linked together. This phenomenon, responsible for ineffective erythropoiesis, leads to an isolated severe anemia with reticulocytopenia. In the following report, we describe 3 new cases of erythroblastic synartesis associated with dysimmunity and monoclonal gammapathy. In all cases, the diagnosis was suggested by characteristic morphological appearance of bone marrow smears, and further confirmed by electron microscopy. Ultrastructural examination of abnormal erythroblast clusters showed that these cells were closely approximated with characteristic intercellular membrane junctions. The pathogenesis of the dyserythropoiesis was modeled in vitro using crossed erythroblast cultures and immunoelectron microscopy: when cultured in the presence of autologous serum, the erythroblasts from the patients displayed synartesis, whereas these disappeared when cultured in normal serum. Moreover, synartesis of normal erythroblasts were induced by the patient IgG fraction. Immunogold labeling showed that the monoclonal IgG were detected in, and restricted to, the synartesis. A discrete monoclonal plasmacytosis was also found in the patient bone marrow. The adhesion receptor CD36 appeared to be concentrated in the junctions, suggesting that it might be involved in the synartesis. These experiments indicated that a monoclonal serum immunoglobulin (IgG in the present cases) directed at erythroblast membrane antigen was responsible for the erythroblast abnormalities. Specific therapy of the underlying lymphoproliferation was followed by complete remission of the anemia in these cases.

Adult↗

PubMed1999

IL-3-induced coexpression of histidine decarboxylase, IL-4 and IL-6 mRNA by murine basophil precursors.

Murine low-density bone marrow cells sorted from the blast cell window on the basis of high rhodamine-123 retention (Rh-bright), are highly enriched in histamine-, IL-4-, and IL-6-producing cells. We established by in situ hybridization that up to 50% of this population (around 0.25% of the whole bone marrow) coexpressed the transcripts for these molecules upon stimulation with 1L-3. Rh-bright cells were also positive for mRNA encoding the alpha, beta, and gamma chains of the Fc(epsilon)RI which was functional since aggregated IgE induced the same percentage of cells hybridizing with the HDC probe as IL-3. Clonogenic progenitors and histamine- and cytokine-producing cells copurified in the Rh-bright population, but could be distinguished by their c-kit expression, CFU-C being more frequent in the c-kit(high) fraction, while histamine and IL-6 producers were enriched in the kit(low) counterpart. Ultrastructural analysis of Rh-bright cells revealed essentially two subsets, namely undifferentiated blast cells and basophil precursors. No other lineage-committed population was enriched by this sorting procedure, and it can therefore be concluded that coexpression of HDC, IL-6, and IL-4 transcripts in response to IL-3 or aggregated IgE takes place mainly in hematopoietic precursors belonging to the basophil lineage.

Animals↗

PubMed1999

Hematologic involvement in mitochondrial cytopathies in childhood: a retrospective study of bone marrow smears.

We retrospectively analyzed the bone marrow (BM) smears of 10 children with mitochondrial cytopathies. Light microscopic examination showed large and coalescent cytoplasmic vacuolization of some BM precursors in nine cases, including two children with normal peripheral blood counts and four with sideroblastic anemia. BM ultrastructural study showed abnormal mitochondria in the erythroid lineage in all three children studied. Ultrastructural studies in two cases revealed a population of giant mitochondria with abnormal ultrastructure coexisting with a population of normal mitochondria in proerythroblasts, basophil erythroblasts, and less commonly in more mature erythroblasts. In a third child, mitochondria were normal in size with cristae either absent or exhibiting abnormal longitudinal orientation. Heteroplasmic segregation of mitochondria during cell division could account for the finding of a double population of cells on ultrastructural examination. These features suggest that cytologic and ultrastructural BM examination could be useful for the diagnosis of mitochondrial disorders. That is, when large and coalescent cytoplasmic vacuoles of BM precursor cells are present, the clinician should search for mitochondrial cytopathy in a child with unexplained cytopenia(s).

Bone Marrow↗

PubMed1998

Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules.

We have used ultrathin cryosectioning and immunogold cytochemistry to study the position of alpha-granules in the endocytic and biosynthetic pathways in megakaryocytes and platelets. Morphologically, we distinguished three types of granules; so-called multivesicular bodies type I (MVB I) with internal vesicles only, granules with internal vesicles and an electron dense matrix (MVB II), and the alpha-granules with mainly a dense content and often internal membrane vesicles at their periphery. The MVBs were prominent in cultured megakaryocytes and the megakaryoblastic cell line CHRF-288, but were less numerous in bone marrow megakaryocytes and platelets, whereas alpha-granules were most prominent in mature bone marrow megakaryocytes and in platelets. The internalization kinetics of bovine serum albumin-gold particles and of fibrinogen positioned the MVB subtypes and alpha-granules sequentially in the endocytic pathway. MVBs contained the secretory proteins von Willebrand factor (vWF) and beta-thromboglobulin (beta-TG), the platelet-specific membrane protein P-selectin, and the lysosomal membrane protein CD63. Within the MVBs, endocytosed fibrinogen and endogenous beta-TG were restricted to the matrix, while vWF was predominantly associated with internal vesicles. CD63 was also observed in association with internal membrane vesicles in the alpha-granules. These observations, and the gradual morphologic transition from granules containing vesicles to granules containing predominantly dense material, suggest that MVBs represent a developmental stage in alpha-granule maturation.

Animals↗

PubMed1998

The first pathogenic mitochondrial methionine tRNA point mutation is discovered in splenic lymphoma.

We describe a novel point mutation in the mitochondrial DNA transfer RNA methionine gene, a G-to-A transition at position 4450, in a patient with a splenic lymphoma with villous lymphocytes. The patient's lymphocytes were remarkable by the presence of large cytoplasmic inclusions demonstrated as abnormal mitochondria by electron microscopy and led to the discovery of the mutation using denaturing gradient gel electrophoresis as a screening procedure. The pathogenic potential of the mutation was clearly established by the following criteria. It was absent in a control population. It involves a nucleotide that is highly conserved along the phylogenetic tree. The mutation was heteroplasmic and, when present in a high proportion, was associated with morphological alterations of the mitochondria, with defects of respiratory chain complexes activities and with a decrease in the mitochondrially encoded cytochrome c oxidase subunit II. Transfer of the mutation in Rho0 cells allowed to demonstrate its association with a severe respiratory chain dysfunction. However, although the pathogenicity of the mutation was clearly demonstrated, its link with the patient disease remained disputable.

Aged↗

PubMed1997

Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.

The site and mechanism of platelet production by bone marrow megakaryocytes (MKs) has been the subject of extensive studies, but is still a matter of controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also called megakaryocyte growth and development factor (MGDF) or thrombopoietin, has resulted in considerable progress in the understanding of the maturation of the MK lineage. To better understand the mechanism of platelet production, we examined the late stage of MK maturation by electron microscopy in cells cultured in the presence of Mpl-l. Human bone marrow CD34+ CD38+ cells, which contain late MK progenitors, were purified by flow cytometry and cultured in a serum-free liquid medium containing recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the majority of cultured cells were large MKs with lobulated polyploid nuclei. The MKs displayed a smooth surface with harmonious cytoplasmic maturation and abundant, regularly distributed demarcation membranes and alpha-granules, and even some dense granules. Interestingly, approximately 30% of the MKs observed displayed morphologic evidence of platelet production: at optical microscopy, MKs formed long filamentous cytoplasmic extensions (proplatelets) that fragmented into platelet-sized particles. Moreover, flow cytometric analysis of this cultured cell population showed GPIIb-positive particles of the size of platelets. Electron microscopic observation showed that MKs producing platelets displayed thin pseudopods on the surface, and that the channels of the demarcation membrane system were dilated, allowing long strands of cytoplasm to extend from the cell periphery. These cytoplasmic strands displayed beading with constrictions separating platelet-sized segments; the more distal to the cell core, the smaller the fragments were. They eventually detached from the cell core into the culture medium either occasionally still elongated or, more often, separated into individual platelets. Parallel longitudinal and perpendicular microtubules were visualized in the constricted regions of these cytoplasmic strips; immunogold study of tubulin localization confirmed this subcellular distribution. On both sides of the constricted areas, vacuoles were noted, the fusion of which might have led to the detachment of individual platelets. Finally, in close proximity to the platelet-forming MKs, numerous microparticles were shed. Although some of these particles might correspond to transverse sections of pseudopods, this did not seem to be the case, since they were rarely seen around thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow cytometry showed that apart from shed cytoplasmic fragments of platelet size, numerous smaller particles strongly labeled for CD41 were also released by mature MKs. In conclusion, this study describes the ultrastructure of human platelet production in cultured MKs, involving the formation of proplatelets and the shedding of microparticles.

Blood Platelets↗

PubMed1996

Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.

The aim of the present study was to determine if the human erythroid (E) and megakaryocytic (MK) lineages were closely linked to the existence of a bipotent burst-forming unit (BFU) E/MK progenitor. In methylcellulose cultures, BFU-E/MK colonies were observed at day 12 and closely resembled mature BFU-E with the exception that the erythroid component was surrounded by MK. These colonies were quite different from the colony forming unit (CFU)-GEMM-derived colonies, which were composed of a larger number of erythroblasts and which developed later in culture. The existence of these bilineage colonies composed of 100 to 1,000 erythroblasts intermingled with a few MK and without granulocytic cells was confirmed by the plasma clot technique and immunoalkaline phosphatase labeling of the MK. To investigate if this bipotent progenitor belonged to the compartment of primitive progenitors, CD34+ marrow cells were subfractionated according to expression of the CD38 antigen. The bipotent BFU-E/MK progenitor as well as a large fraction of MK progenitors were found in the CD34+ CD38+/- or in the CD34+ CD38- cell fractions. Growth of this bipotent BFU-E/MK progenitor required the combination of stem cell factor (SCF), Interleukin-3 (IL-3), and Epo in serum free conditions. Addition of IL-6 had only a marginal effect, whereas megakaryocyte growth and development factor (MGDF) was not an absolute requirement, but slightly increased the plating efficiency of CFU-MK and of BFU-E/MK progenitors when combined with SCF, IL-3, and Epo. In contrast, when these cultures were performed in the presence of 30% fetal calf serum, no BFU-E/MK colonies were observed irrespective of the combination of growth factors used, including the presence of MGDF; however, inclusion of the MS-5 cell line restored the growth of this bipotent progenitor. In contrast, in cultures performed in the presence of human normal or aplastic plasma, MS-5 had only a slight effect on the cloning efficiency but improved MK cytoplasmic maturation and MK size, suggesting that the main effect of MS-5 is to diminish the inhibitory effect of the fetal calf serum on the MK differentiation. The clonal origin of bipotent BFU-E/MK colonies was demonstrated in liquid culture of single CD34+ CD38low cells by immunophenotyping individual clones. At day 12, 30% of the clones contained erythroblasts (glycophorin A+) and some MK (CD41+) without granulocytes (G) or macrophages (M) (CD14+ and CD15+). At day 20, clones containing erythroblasts and MK were rare (5%). In contrast multilineage clones could be frequently detected at this time without passage from BFU-E/MK clones at day 12 to GEMM at day 20. These results suggest that a bipotent BFU-E/MK progenitor may be a nonrandom step in the hierarchical development of stem cells.

ADP-ribosyl Cyclase↗

PubMed1995

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets.

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P-selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.

Antigens, CD34↗

PubMed1995

The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin-6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗

PubMed1995

A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23.

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.

Adult↗

PubMed1994

Growth and differentiation of the human megakaryoblastic cell line (ELF-153): a model for early stages of megakaryocytopoiesis.

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Acute Disease↗

PubMed1994

cMpl ligand is a humoral regulator of megakaryocytopoiesis.

Megakaryocytopoiesis is the cellular developmental process that leads to platelet production. At least two humoral growth factors may be necessary for megakaryocyte proliferation and maturation. One is a megakaryocyte-colony stimulating factor (MK-CSF) which induces the proliferation and differentiation of megakaryocyte progenitors, and the second, thrombopoietin, is a megakaryocyte maturation factor. Neither of these factors has been fully characterized. The proto-oncogene c-mpl, an orphan member of the haematopoietin receptor family, is specifically involved in megakaryocyte regulation. Here we present evidence that the c-mpl-encoded receptor binds a ligand (c-Mpl ligand) which is a humoral factor implicated in platelet homeostasis. Our results suggest that c-Mpl ligand, thrombopoietin and MK-CSF might be the same molecule.

Animals↗

PubMed1994

The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein.

Azurophil granules of myeloid cells form in promyelocytes. They store cytotoxic and digestive agents which when released are involved in the defense against infection. In order to characterize the intragranular distribution of these agents, ultrastructural methods using immunogold were used on promyelocytes. Azurophil granules were divided into nucleated, large spherical (large azurophil) and small electron-dense (small azurophil) granules. Myeloperoxidase showed a peripheral distribution of large azurophils and a uniform distribution of small and nucleated azurophils, consistent with previous findings. Likewise, the major neutral proteases of azurophils, cathepsin G, granulocyte elastase, and proteinase 3, displayed a similar distribution, with a peripheral localization in large azurophils and a uniform distribution in small and nucleated azurophils, except for proteinase 3, which was associated with the crystalloid structure in nucleated azurophils. In contrast, the bactericidal/permeability increasing protein, which is bacteristatic and bactericidal for Gram-negative bacteria, was localized to the membrane area in all types of azurophil granules, consistent with a suggested association of this protein with the granule membrane. The observed differences in intragranular distribution of the proteins investigated may reflect variations in binding to matrix structures and granule membranes.

Antimicrobial Cationic Peptides↗

PubMed1993

Characterization and functional analysis of adult human bone marrow cell subsets in relation to B-lymphoid development.

To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.

Adult↗

PubMed1993

Effects of the recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells.

Using a liquid culture system and human CD34+ marrow cells, we examined the effects of recombinant interleukin (IL)-3, IL-6, stem cell factor (SCF), and leukemia inhibitory factor (LIF) on megakaryocyte (MK) growth, endoreplication, and maturation. MK proliferation, ploidy distribution, and volume were studied by flow cytometry. IL-3 was the only cytokine that, alone, induced a marked increase in MK proliferation. At a high CD34+ cell concentration, addition of IL-6, SCF, and LIF to IL-3--containing medium increased the number of MK (approximately 20%). At a low CD34+ cell concentration, IL-3 alone was a less potent inducer of MK growth, but IL-6, SCF, and their combination had a marked effect, increasing the number of MK by a factor 1.7, 2.9, and 4.4, respectively. These differences may be related to the endogenous release of cytokines in the culture. The effects of these cytokines were subsequently tested on a more mature type of MK progenitor (CD34+ cells isolated after 6 days of incubation in liquid culture). IL-3 remained the most potent cytokine, but IL-6 or SCF alone also increased MK number in comparison to unstimulated cultures. The ploidy distribution of MKs grown with IL-3 was not markedly changed by the addition of the other cytokines, with the exception of SCF, which induced a significant increase in the mean ploidy. However, in all cultures, glycoprotein (GP)IIIa+ 2N and 4N cells were present in large but variable numbers (35% to 75%). The number of these low-ploidy MKs directly correlated with MK proliferation. Therefore, we subsequently explored the absolute number of polyploid MK produced in culture. SCF, IL-6, or their combination, in association with IL-3, increased the number of polyploid MK up to fourfold. In addition, they improved the maturation of MK grown in the presence of IL-3, leading to the synthesis of demarcation membranes and platelet shedding. A similar effect of growth factors on the maturation of day 6 CD34+ cells was observed. We conclude that IL-6 and SCF have a broad range of activities on megakaryocytopoiesis, acting both on the early and late stages. However, the proliferative properties of these cytokines largely predominate in our cultures. Therefore, in the absence of a specific MK regulator, this study further extends the need for a combination of growth factors to maximize megakaryocytopoiesis.

Adult↗

PubMed1993

Prenatal diagnosis of syndromes associating albinism and immune deficiencies (Chediak-Higashi syndrome and variant).

We have successfully undertaken the prenatal diagnosis of two hereditary syndromes associating albinism and immune defects. Because the genes responsible for these diseases have not yet been mapped and the immune abnormalities are too subtle to be diagnosed in utero, the prenatal diagnosis was made using a morphological approach. In the case of Chediak-Higashi syndrome, it was based on light microscopic examination of the hair shaft and on light and electron microscopic study of polymorphonuclear cells. In the syndrome associating immune deficiency and partial albinism, the Griscelli syndrome, only examination of the hair was feasible. The diagnosis was negative in 12 fetuses at risk and positive in four.

Albinism↗

PubMed1993

Effect of thrombin on maturing human megakaryocytes.

Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis formation and for megakaryopoiesis autocrine regulation.

Cell Count↗