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Biomedical subjects

F Wendling

Publications and source records attributed to F Wendling.

At least 55 records · Page 3Linked to original sources

A method to quantify invariant information in depth-recorded epileptic seizures.

In the field of epilepsy, the analysis of stereoelectroencephalographic (SEEG) signals recorded with depth electrodes provides major information on interactions between brain structures during seizures. A methodology of comparing SEEG seizure recordings is applied in 4 patients suffering from temporal lobe epilepsy. It proceeds in 3 steps: (i) segmentation of SEEG signals, (ii) characterization and labeling of segments and (iii) comparison of observations coded as sequences of symbol vectors. The third step is based on a vectorial extension of Wagner and Fischer's algorithm to first, quantify similarities between observations and second, extract invariant information, referred to as spatio-temporal signatures. These are automatically extracted by the algorithm without the need to make a priori assumptions on the 'patterns' to be searched for. Theoretical results show that two observations of non-equal duration can be matched by deforming the first one (using insertion/deletion operations on vectors) to optimally fit the second, under a minimal cost constraint. Clinical results show that the study brings objective results on reproducible mechanisms occurring during seizures: for a given patient, quantified descriptions of seizure periods are compared and similar ictal patterns, or signatures, are extracted from SEEG signals. Some of these signatures (particularly those containing spikes, spike-and-waves, slow waves and rapid discharges) are relevant: they seem to reflect reproducible propagation schemes whose analysis may help in the understanding of epileptogenic networks.

Electroencephalography↗

Positive and negative regulation of megakaryocytopoiesis.

The recently cloned physiological regulator of megakaryocytopoiesis, known as Mpl ligand, thrombopoietin (TPO), megakaryocyte growth and development factor (MGDF) or megapoietin, is undergoing preclinical and clinical trials. This factor is an extremely potent thrombocytopoietic agent in vivo in normal animals, and accelerates platelet recovery in some but not all models of myelosuppression. Together with its apparent lack of adverse effects, the preclinical data suggest that TPO might permit the use of higher doses of chemotherapy in dose-intensive regimens and be useful in patients with ineffective platelet production or production abnormalities. The direct effects of TPO on primitive and various myeloid committed haematopoietic progenitor cells predict that TPO may be used in combination with other cytokines in a variety of clinical disorders. Along with the progress made in the understanding of the positive regulation of megakaryocytopoiesis, accumulating data demonstrate that platelet production is also controlled by negative regulators with potential clinical applications. Some of these regulators are effective in the treatment of essential thrombocythaemia and myeloproliferative disorders, while others seem capable of protecting progenitor cells from the cytotoxicity of chemotherapeutic drugs.

Animals↗

Thrombopoietin (Mpl-ligand) and the regulation of platelet production.

Thrombopoietin is the primary physiological regulator of platelet production. TPO stimulates both early and late megakaryocyte (MK) progenitors, and acts additively or synergistically with several cytokines on various progenitors including the most primitive stem cells. Much has been learn about the physiology of the endogenous TPO production and its regulation. TPO is constantly produced by the liver and kidney; its plasmatic clearance occurs by binding to the Mpl receptor expressed on MKs and platelets. TPO binding is followed by internalization and catabolism. The factor is a potent thrombopoietic agent in vivo and accelerates platelet recovery in several models of marrow suppression. Phase I clinical trials have begun. The lack of adverse effects suggests that TPO may join other cytokines in the clinical armamentarium. Nevertheless, several important areas of basic or clinical haematology remain to be explored to fully understand the biology of this new cytokine. For example, is TPO needed for the terminal stage of platelet formation? What genetic events are involved in the process of endomitosis? What are the physiopathological mechanisms underlying thrombocytopenia or thrombocytemia in human?

Blood Platelets↗

Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis.

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.

Animals↗

Extraction of spatio-temporal signatures from depth EEG seizure signals based on objective matching in warped vectorial observations.

In the field of epilepsy, the analysis of stereoelectroencephalographic (SEEG) signals recorded with depth electrodes provides major information on interactions between brain structures during seizures. A comprehensive methodology of comparing SEEG seizure recordings is presented. It proceeds in three steps: 1) segmentation of SEEG signals; 2) characterization and labeling of segments; and 3) comparison of observations coded as sequences of symbol vectors. The third step reports a vectorial extension of the Wagner and Fischer's algorithm to first, quantify similarities between observations and second, extract invariant sequences of events, referred to as spatiotemporal signatures. The study shows that two observations of nonequal duration can be matched by deforming the first one to optimally fit the second, under cost constraints. Results show that the methodology allows to exhibit signatures occurring during epileptic seizures and to point out different types of seizure patterns. The study brings objective results on reproducible interactions between brain structures during ictal periods and may help in the understanding of epileptogenic networks.

Algorithms↗

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Animals↗

[Mpl ligand (thrombopoietin) and platelet regulation].

After 35 years of research, the physiological regulator of platelet production has been isolated and its gene cloned. This discovery originates from studies performed with the myeloproliferative leukemia virus (MPLV), a murine retrovirus which induces an acute myeloproliferative syndrome in adult mice. MPLV carries in its genome the v-mpl oncogene which corresponds to a truncated form the c-mpl proto-oncogene. c-mpl encodes a cytokine receptor (Mpl-R) belonging to the hematopoietin receptor superfamily. Among the hematopoietic cell lineages, Mpl-R is preferentially expressed on late megakaryocyte progenitors, megakaryocytes and platelets. The ligand for Mpl-R, called Mpl-L or TPO or MGDF or megapoietin, is a glycosylated hormone of 352 amino acids in human which comprises two domains: the N-terminus domain shares 50% similarity with erythropoietin and is responsible for the biological activity; the C-terminus part is required for secretion. Notwithstanding its major action on megakaryocytopoiesis and thrombocytopoiesis, Mpl-L also potentiates the action of other cytokines on several hematopoietic lineages. Mpl-L/TPO/MGDF, the homeostatic regulator of platelet production, might be a useful therapeutical cytokine to treat thrombocytopenia induced in patients by chemotherapy.

Animals↗

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↗

The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.

The Mpl receptor (Mpl-R) is a cytokine receptor belonging to the hematopoietin receptor superfamily for which a ligand has been recently characterized. To study the lineage distribution of Mpl-R in normal hematopoietic cells, we developed a monoclonal antibody (designated M1 MoAb) by immunizing mice with a soluble form of the human Mpl-R protein. With few exceptions, Mpl-R was detected by indirect immunofluorescent analysis on all human leukemic hematopoietic cell lines with pluripotential and megakaryocytic phenotypes, but not on other cell lines. By immunoprecipitation and immunoblotting, M1 MoAb recognized a band at 82 to 84 kD corresponding to the expected size of the glycosylated receptor. Among normal hematopoietic cells, M1 MoAb strongly stained megakaryocytes (MK) and Mpl-R was detected on platelets by indirect immunofluorescence staining or immunoblotting. On purified CD34+ cells, less than 2% of the population was stained, but the labeling was weak and just above the threshold of detection. However, dual-labeling with the M1 and antiplatelet glycoprotein MoAbs showed that most Mpl-R+/CD34+ cells coexpressed CD41a, CD61, or CD42a, suggesting that cell surface appearance of Mpl-R and platelet glycoproteins could be coordinated. M1-positive and M1-negative subsets were sorted from purified CD34+ cell populations. Colony assays showed that the absolute number of hematopoietic progenitors was extremely low and no primitive progenitors were present in the CD34+/Mpl-R+ fraction. However, this cell fraction was significantly enriched in low proliferative colony-forming units-MK. When the CD34+/Mpl-R+ fraction was grown in liquid culture containing human aplastic serum and a combination of growth factors, mature MK were seen as early as day 4, whereas the predominant cell population was erythroblasts on day 8. Similar data were also obtained with the CD34+/Mpl-R- fraction with, however, a delay in the time of appearance of both MK and erythroblasts. In conclusion, Mpl-R is a cytokine receptor restricted to the MK cell lineage. Its expression is low on CD34+ cells and these cells mainly correspond to late MK progenitors and transitional cells. These data indicate that the action of the Mpl-R ligand might predominate during the late stages of human MK differentiation.

Amino Acid Sequence↗

From the v-mpl oncogene to thrombopoietin.

v-mpl is a mouse oncogene that is part of the genome of the myeloproliferative leukemia virus (MPLV). The cellular homolog gene c-mpl encodes an hematopoietic cytokine receptor restricted to the megakaryocytic lineage of differentiation. Using the Mpl receptor protein, cDNAs corresponding to the receptor ligand were isolated in humans, pigs, dogs and mice. In vivo and in vitro, the recombinant molecule has both the thrombopoietin (TPO) and megakaryocyte-colony stimulating factor (MK-CSF) activities. This molecule (Mpl ligand/TPO/MK-CSF) is the humoral physiological regulator of platelet production.

Animals↗

c-mpl, the thrombopoietin receptor.

v-mpl is a mouse oncogene that is part of the genome of the myeloproliferative leukemia virus (MPLV). The cellular homolog gene c-mpl encodes an hematopoietic cytokine receptor mainly restricted to the megakaryocytic lineage of differentiation. Using the Mpl receptor protein, cDNAs corresponding to the receptor ligand were isolated in humans, pigs, dogs and mice. In vivo and in vitro, the recombinant molecule has both the thrombopoietin (TPO) and megakaryocyte-colony stimulating factor (MK-CSF) activities. This molecule (Mpl ligand/TPO/MK-CSF) seems to be the humoral physiological regulator of platelet production.

Animals↗

Autocrine stimulation by erythropoietin (Epo) requires Epo secretion.

Erythropoietin (Epo) autocrine stimulation has been implicated in erythroblastic leukemia. To examine whether this stimulation could occur intracellularly, we developed Epo autocrine models of stimulation in the human pluripotent UT-7 cell line. Retroviral expression of Epo totally abolished the growth factor requirement of UT-7 cells. Autonomous proliferation was not cell density-dependent and occurred at a unicellular level, showing a genuine autocrine mode of stimulation. Total blockage of Epo secretion induced by the endoplasmic reticulum-retention amino acids Lys-Asp-Glu-Leu (KDEL) signals in 11 lines prevented autonomous proliferation, whereas a leaky retention system, observed in 3 other lines, resulted in limited autocrine stimulation without true long-term autonomous proliferation. Production of Epo, in contrast to KDEL-modified Epo, induced reductions in Epo binding, Epo receptor (EpoR) mRNA, and phosphorylation levels similar to those induced by the addition of exogenous Epo to the parental cell line. In addition, autonomous growth and survival were inhibited by the addition of Epo-neutralizing antibodies, affording evidence that autocrine stimulation through EpoR activation takes place on the cell surface. Finally, phenotypic analysis of the virus-infected clones indicated that Epo production did not change the differentiative capacities of UT-7 cells. All these data show that Epo autocrine stimulation is dependent on Epo secretion and takes place on the cell surface. From all analyzed parameters, the effects of Epo autocrine stimulation and those of exogenously added Epo appear to be identical.

Antibodies↗

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↗

[Hematopoiesis and its regulation. Comparison between erythropoiesis and megakaryocytopoiesis].

Hematopoiesis is the cellular system which leads to the continuous production of blood cells. This highly complex cellular system is organized into three main compartments: (i) stem cells which are both pluripotent and theoretically capable of self renewal; (ii) hematopoietic progenitors which are committed to (only) one cell lineage and are able to proliferate along each particular differentiation pathway; (iii) a maturation compartment in which cells become morphologically identifiable since they synthesize lineage specific proteins. The maturation cell compartment represents the majority of marrow cells. At the present time, the regulation of true stem cells remains poorly understood since these cells are difficult to assay in vitro. In contrast, the regulation of each hematopoietic lineages becomes to be well known. These knowledges are mainly due to two reasons: (i) hematopoietic progenitors can be purified and assayed in culture. Their proliferation and differentiation are strictly dependent upon the presence of hematopoietic growth factors; (ii) these different hematopoietic growth factors have been isolated and their cDNA cloned. Erythropoiesis and megakaryocytopoiesis are two branches of hematopoiesis which lead to the production of RBC and platelets, respectively. These two cell lineages have several common features. However, they markedly differ by their regulation since RBC production depends upon one main stimulus (hypoxia) and, therefore, the terminal erythroid differentiation is regulated by a single growth factor. In contrast, regulation of platelet production may depend on several stimuli such as the platelet mass (homeostasis), inflammation, infection and hypoxia. Therefore, several cytokines are involved in the regulation of megakaryocytopoiesis. In addition, the mechanisms of platelet production are highly complex and, in contrast to all the other hematopoietic lineages where the production of mature cells depends on a single parameter (the proliferation during differentiation), three independent parameters modify thrombopoiesis: a) the number of marrow megakaryocytes (MK) (proliferation of the precursor cells). b) the megakaryocyte volume which directly depends on the MK ploidy. During MK differentiation, MK precursors switch from a mitotic process (DNA duplication followed by cytokinesis) to an endomitotic process (DNA duplication without cytokinesis). Endomitosis is a specific process of the megakaryocytic differentiation and differs from all the other cellular models of polyploidization by the existence of a single polyploid and polylobulated nucleus in each cell. This polyploidization induces a major amplification of the platelet production since it is associated with a parallel increase in the cytoplasmic mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Erythropoiesis↗