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Biomedical subjects

M Prenant

Publications and source records attributed to M Prenant.

33 records · Page 2Linked to original sources

Leukemic cell maturation: variability of the myeloid leukemic cell phenotype.

Leukemia is characterized by a proliferation of cells that exhibit an arrest in the normal differentiation sequence. The HL-60 promyelocytic leukemia is a useful model of the phenomenon of maturation arrest, particularly as modulated by inducers that partially restore myeloid differentiation. In this investigation, we report the development of two sublines of HL-60 and the acquisition of two others that are clonal variants of the parent cell line. Each of the sublines demonstrates an altered pattern of differentiation and resistance to one or more of the drugs that serves as an inducer of the parent line. The two cell lines developed in this study, HL-60S and HL-60I, are resistant to arabinosylcytocine (ARA-c); HL-60I is also resistant to PMA. A study of the phenotype as expressed by the granule-associated cytoplasmic enzymes revealed that each subline had a slightly different pattern of maturation in response to dimethylsulfoxide (DMSO) or ARA-c as inducers. The proliferative rate of all sublines was similar. These data demonstrate that the maturation arrest observed in this model is in part reversible. The maturation arrest observed in myeloid leukemias is due to a reversible block secondary to altered proliferative activity.

Butyrates↗

[Influence of macrophage culture supernatants on erythroblastic islets in rat erythropoiesis].

The isolation of central macrophages from erythroblastic islands (EI) permitted analysis of the role of EI macrophage factors in erythropoiesis. EI macrophage supernatant and peritoneal macrophage supernatant both had a similar effect on erythropoiesis. They exerted an erythropoietin-like effect. We observed changes in the number of erythroblasts per island; their distribution within each island during the maturation process was described. The role of the central macrophage in EI was discussed. We suggested a direct transfer of an erythropoietin-like activity to the erythroblasts which increases the efficacy of stimulation.

Animals↗

Polyploid megakaryocytes develop randomly from a multicompartmental system of committed progenitors.

Cumulative distributions of the number of doublings undergone by mixed megakaryocytic/erythroblastic colonies and by pure megakaryocytic colonies were determined from plasma clot cultures of bone marrow (from C57BL/6 mice) supplemented with erythropoietin. Analysis of these distributions suggests that these colonies are produced by three distinct progenitors. At days 7-14, progenitors of mixed megakaryocytic/erythroblastic colonies (BFU-ME) generate tri-exponential distributions and the mean (+/- SD) fraction of this progenitor pool ceasing to proliferate per doubling (FCP) increases stepwise from 0.07 +/- 0.06 to 0.27 +/- 0.07 and 0.73 +/- 0.07. In this interval, progenitors of pure megakaryocytic colonies (CFU-M) generate bi-exponential slopes whose FCP values are compatible with the two last slopes above. Finally, CFU-M at day 3 express only the last slope. From days 5 to 9, megakaryocytes generated by BFU-ME reach lower ploidy levels than do those generated by CFU-M. It is concluded that, in the culture system used, (i) megakaryocyte progenitors that do not switch to polyploidization mature through the three consecutive compartments indicated, (ii) each progenitor population has a probability of becoming polyploid that reflects the fraction that ceases to proliferate, (iii) the exponentially distributed mitotic reserve of progenitors is determined by the combination of maturing into the next compartment and the probabilistic switch to the pathway of polyploidization, and (iv) the ploidy distribution of megakaryocytes probably depends on the progenitor from which they originate.

Acetylcholinesterase↗

[Technique for isolation and culture of erythroblastic islands and separation of their central macrophage].

Erythroblastic islands (EI) were isolated using the adherence of their central macrophage to glass or plastic. The EI obtained were cultured and erythroblasts achieved a complete maturation within 72 h of incubation. When cultured in serum-free medium, the central macrophages were rapidly isolated, by detachment from their erythroblasts. Isolation and culture of central macrophages allow the study of their physiological characteristics and particularly the effect of their supernatant on erythropoïesis.

Animals↗

Kinetics of platelets, megakaryocytes and their precursors: what to measure?

A sequential exploration of the kinetics of platelets, megakaryocytes, and their progenitors is devised, wherein abnormalities at one level of differentiation are the subject of further analysis at the preceding level. Platelet kinetics yield estimates of mean platelet life span, fraction of platelet mass in circulation and daily production, making it possible to recognize the disorders of hyperdestruction, hypersequestration, hypoproduction, and hyperproduction. Theoretical considerations and regression analysis of a variety of computer-simulated survival tests show that sufficient information is contained in the first four days of the disappearance curve of population labeled platelets to provide an estimate of mean life span with an error of 9%--15%. Identifying the type of destruction disorder depends on developing tests and parameters which will make it possible to integrate into a coherent model such indices as the rate of senescence, the rates of reversible and irreversible aggregation, and the rates of adhesion to and phagocytosis by the reticuloendothelial system. Foremost among the existing models is the multiple-hit theory, although its validity rests on the unproved assumption that platelets keep a permanent memory of their injuries. Identifying the type of production disorder is the purpose of megakaryocyte kinetics. The daily production of megakaryocytes could be derived from the daily platelet production and the number of platelets released per megakaryocyte; determining megakaryocyte number would also yield the mean megakaryocyte maturation time. All these parameters could be obtained by combining a simple radioiron dilution method with cytochemical identification of megakaryocytes and with advanced, automated morphometric techniques. Abnormalities of megakaryocyte number and size can be further analyzed by studying the kinetics of thrombocytic precursors in tissue culture, i.e., by recording the distribution of precursor doublings and megakaryocyte ploidy histograms. The application of these techniques to cultures of bone marrow suggest that endomitosis may be initiated by any megakaryocyte precursor and that the kinetics of this process influence the number and final ploidy level of megakaryocytes.

Blood Platelets↗

Three-dimensional model of bone marrow.

Three-dimensional scale models of bone marrow from a hypertransfused and a normal rat were constructed. The model of marrow from the hypertransfused rat demonstrated the existence of distinct erythroblastic islands in situ in which the erythroblasts underwent sychronous maturation. Macrophages were found in close association with the developing erythroblasts. The immature erythroblasts were tightly grouped, but as they matured they began to move apart. Erythroblasts in individual clusters were found to be at the same stage of morphologic maturation. In contrast, the model of marrow from the normal rat showed a majority of clusters containing erythroblasts at various stages of maturation. Erythropoiesis was not spatially restricted to the area proximal to the sinuses but was found to occur over the entire marrow space. Thrombopoiesis, however, was found to take place exclusively in the immediate vicinity of the marrow sinuses.

Animals↗

Erythropoiesis: comparison of in vivo and in vitro amplification.

1. Amplification is defined as the phase of erythropoiesis that includes all cell divisions of the recognizable erythron. 2. In vivo, amplification always takes place surrounding a central histiocyte (erythroblastic island). It is made up of four to five successive synchronous divisions. 3. In vitro, complete maturation and amplification of erythropoietic cells can be obtained without apparent association with a central histiocyte. 4. The functional significance of the 'erythroblastic island' and disorders of amplification are discussed in relation to clinical disorders of erythropoiesis.

Animals↗

On the proper use of the Soret band for hemoglobin detection in erythrocytic cells.

Intracellular hemoglobin detection by light microscopy in the Soret band (414nm) is a sensitive means of correlating morphology and biochemical function in studies of erythrpoiesis. Correct application of the technique requires a light source with strong emission in the near ultra-violet, a filter with a pass-band at 414nm, a preparation of living cells or an unfixed smear, and a receiver that is sensitive to the Soret wavelength. The human eye is very insensitive to light at 414nm and quite sensitive to stray green light: it is consequently much inferior to a black and white television or camera film for viewing a Soret image. The appearance of hemoglobin during maturation of erythroid cells in human bone marrow can be detected with greater sensitivity by this method than by a peroxidase-benzidine stain and it is the only method applicable to living cells.

Animals↗

Reticulocyte motility and form: studies on maturation and classification.

A new two-stage living cell cytological classification is presented for reticulocytes in the rat and shown on an overall population basis to be the equivalent of the classical new methylene blue staining characterization. Class 1 reticulocytes (R1) are motile and multilobular. They comprise about 25% of normal bone marrow reticulocytes, but none of the reticulocytes in normal blood. Class 2 reticulocytes (R2) are asymmetrically "deep dished" in appearance and contain visible refractile granules, viewed in phase-contrast microscopy. In animals "stimulated" by bleeding, the percentage of motile marrow reticulocytes is found to be a linear function of hematocrit over the range of about 25%-70% total reticulocytes. In the blood, the associated maximum rise of class R1 is only to about 8%. These and related cellular properties are discussed with respect to the maturation and release of normal reticulocytes. Applicability of this work to studies on humans and to pathology is also indicated.

Animals↗

[Isolation of erythroblastic islands. Study by optical and scanning electron microscopy (author's transl)].

1. A simple technique is described for isolation and estimation of number of erythroblastic islands (EBI) in rat bone marrow. The number of EBI decreases during hypertransfusion polycythemia, and increases during experimentally-induced hemolytic anemia. It has also been possible to maintain EBI in vitro, under culture conditions, for up to 36 hours. 2. Each EBI contains a central cell having the character of a macrophage-histiocyte which adheres to glass and spreads, while still retaining its connections with the erythroblasts. 3. In an EBI the erythroblasts are arranged in the form of concentric rings, with the outer cells being more mature than the inner. The outermost layer is frequently made up of reticulocytes even in the isolated island. The erythroblasts of a single ring appear to be at the same stage of maturation which suggests that they are derived from the same stem cell. 4. Using a coordinate reference system, studies of nuclear expulsion were made, by both optical microscopy and scanning electron microscopy.

Animals↗

Origin of stress macroreticulocytes from macronormoblasts.

Stress erythropoiesis was induced in rats and guinea-pigs by graded amounts of bleeding and phenyl-hydrazine administration. Hemoglobin and DNA content was measured by microspectrophometry of single erythroblasts in the process of nuclear extrusion. DNA content was always 2 C. Hemoglobin content was in all cases superior to the normal value, occasionally reaching a value of almost twice normal.

Animals↗

[In vitro studies on amplification of rat erythropoiesis (author's transl)].

From rat bone marrow in which erythropoiesis has been depressed (by suppression of erythropoietin production), liquid culture has yielded a predominantly synchronous amplification of erythroblast development. Groups of 2, 4, 8, 16 and 32 erythrolbasts are observed at 9-15 hour intervals in successive stages of maturation, similar (by their staining characteristics and nuclear size) to that seen in vivo.

Animals↗