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C Lacombe

Publications and source records attributed to C Lacombe.

At least 73 records · Page 4Linked to original sources
PubMed1995

Tyrosine phosphorylation of the erythropoietin receptor: role for differentiation and mitogenic signal transduction.

The erythropoietin (Epo) receptor belongs to the cytokine receptor superfamily. Although the cytokine receptors do not possess a tyrosine kinase consensus sequence in the intracellular domain, rapid stimulation of a tyrosine kinase activity occurs after activation by the ligand. We and others have shown that Epo induces the tyrosine phosphorylation of its cognate receptor as well as phosphorylation of other proteins. In this report, we examined the role of the receptor tyrosine residues in signal transduction. Eight tyrosine residues are located within the intracellular domain of the murine Epo receptor. A single tyrosine residue is present in the region previously shown to be sufficient for proliferative signal transduction. This tyrosine (Tyr 343) was mutated to phenylalanine. Moreover, mutant receptors were also generated with either a tyrosine residue or a phenylalanine residue at position 343 and with a COOH terminal truncation that removed the 7 other tyrosine residues. Expression vectors carrying these mutated receptors were transfected into the interleukin-3-dependent murine cell line Ba/F3. Epo-induced growth was sustained efficiently by all these receptors, although receptors without any tyrosine residues conferred a significantly reduced mitogenic activity. Moreover, all receptors were able to mediate Epo-dependant accumulation of beta-globin mRNA. The mutated receptors all induced the tyrosine phosphorylation of several cellular proteins after Epo stimulation. However, the truncated receptors induced the phosphorylation of a reduced number of proteins, suggesting that phosphorylated tyrosines of the receptor could have a role in the recruitment either of a tyrosine kinase or of tyrosine kinase substrate proteins. The receptors were all able to mediate Epo-induced activation of phosphatidylinositol 3-kinase, although truncated receptors no longer bound phosphatidylinositol 3-kinase.

Cell Differentiation↗
PubMed1995

A translocated erythropoietin receptor gene in a human erythroleukemia cell line (TF-1) expresses an abnormal transcript and a truncated protein.

We previously identified a translocation breakpoint in exon 8 of the erythropoietin receptor (EpoR) gene in TF-1 cells, a cell line derived from a human erythroleukemia. To investigate the potential pathogenetic significance of this abnormality, we more precisely mapped the breakpoint within exon 8 and studied the expression of the translocated gene by S1 nuclease mapping of EpoR transcripts and chemical crosslinking of labeled erythropoietin (Epo) to TF-1 cell surface receptors. Transcripts from the abnormal gene were found to be highly expressed in relation to normal EpoR transcripts in TF-1 cells. The breakpoint predicted by S1 mapping of abnormal EpoR transcripts agreed closely with that determined by Southern analysis. Chemical cross-linking of 125I-Epo to TF-1 cells showed an abnormal, low-molecular-weight cross-linked species directly recognized by anti-EpoR antibodies and present in considerable excess over the normal EpoR. Karyotype analysis showed that each of 10 TF-1 cell metaphases had, in addition to multiple other alterations, one chromosome 19 with additional chromosomal material translocated onto the short arm at 19p13.3, the location of the EpoR gene. We conclude that the structurally abnormal EpoR gene in TF-1 cells is highly expressed and produces an abnormal protein. We speculate that the chromosomal material brought into the EpoR locus by translocation is responsible for the high level of expression. We hypothesize that this translocation participated in the evolution of the erythroleukemia from which TF-1 cells were derived.

Base Sequence↗
PubMed1995

[A study of leukocyte chemotaxis in a glass micropipette].

Chemotaxis, the directional locomotion of a cell toward a source of a chemical gradient, is an important phenomenon occurring for mobilizing immune cells at sites of infection and injury. This phenomenon has been simulated in analyzing the movement in vitro of a chemoattracted cell inside a glass micropipette. A microneedle filled with fMLP, N-formyl-methionyl-leucyl-phenylalanine, at a concentration of 9.10(-7) M in 1% gelatin, is inserted in a glass micropipette containing Hanks buffer solution. After diffusion of fMLP in the glass micropipette until a constant gradient is established, the tip of the glass micropipette is moved near a polymorphonuclear neutrophil. Its spontaneous movement inside the micropipette towards the chemotactic source can be observed and quantified. Without any counter-pressure (positive pressure), the cell spontaneously advances with an average velocity of 0.14 +/- 0.04 micron/s. Corresponding to the maximal strength developed by the cell during its motion, the required strength to stop the chemotactic migration has been estimated to be 39 +/- 4 nN. Giving both qualitative and quantitative information on the dynamics of cell motility, this experiment will be helpful in the understanding of some aspects of cell motility in the tissues.

Chemotaxis, Leukocyte↗
PubMed1995

Changes in microrheology of acute promyelocytic leukemia cells during all-trans retinoic acid (ATRA) differentiation therapy: a mechanism for ATRA-induced hyperleukocytosis?

According to French and European experience, hyperleukocytosis occurs during ATRA differentiation therapy in about 70% of de novo and 25% of relapsed APL cases. The most frequently suggested cause for this side-effect is an ATRA-induced proliferation of APL cells. However, no definite explanation for such a proliferative effect has been clearly established. Another mechanism directly related to the differentiation of marrow leukemic cells could be a change in their microrheology, allowing their release from the bone marrow and their transfer toward peripheral blood (PB) and tissues. Using a single cell aspiration assay into a glass restrictive channel, we measured APL cell viscosity values in five de novo APL patients. A deformability index (DI) was defined as the ratio of mean normal neutrophil viscosity x 100/mean APL cell viscosity. Results were the following: (1) at diagnosis, two patients had high marrow DI (96 and 250%) and three patients had low marrow DI (16, 17, and 40%); (2) when PB and marrow APL cells were simultaneously tested, PB APL cells display higher DI than marrow APL-cells; (3) the two patients with high initial marrow DI experienced an ATRA-induced hyperleukocytosis after only 1 day of treatment; (4) in the three patients with low initial marrow DI, the DI was increasing during ATRA therapy and hyperleukocytosis seemed to occur when a large amount of maturing APL cells reached a viscosity value similar to that of mature neutrophils. These results suggest that an asynchronism between rheological and morphological maturation in each APL cell might explain the occurrence of hyperleukocytosis in some patients during ATRA differentiation therapy.

Adult↗
PubMed1994

Putative oncogenic role of the erythropoietin receptor in murine and human erythroleukemia cells.

To determine whether the erythropoietin receptor (Epo-R) plays a role in the course of malignant erythropoietic disorders, this gene was studied in murine and human erythroleukemia cells. An altered Epo-R gene was found in a murine Friend erythroleukemia cell line, FCL1, due to a spleen focus-forming virus (SFFV) long terminal repeat insertion within the noncoding region of the first exon, leading to Epo-R mRNA overexpression. A similar mechanism of Epo-R activation has previously been described in the T3CL-2 Friend erythroleukemia cell line. An elevated number of Epo-binding sites has been observed in two human erythroleukemia cell lines, TF-1 and UT7. In UT7 cells, homogeneously staining region of the short arm of chromosome 19 [hsr (19)] was evidenced, which contained an amplification of the Epo-R gene. This Epo-R gene amplification was confirmed by the quantification of Southern blots in which the intensity of the Epo-R signal was compared in UT7 DNA and in DNA from normal cells. The Epo-R gene was present in UT7 at a mean number of seven to eight copies per cell. Interestingly, the Epo-R gene was rearranged; the breakpoint region was located near the 3' end of the gene, 3 kb downstream from the end of the last exon. Taken together, these results suggest that, in both murine and human systems, genetic alterations of the Epo-R gene are not rare events and could be involved in the occurrence of the erythroleukemic process.

Animals↗
PubMed1994

Effects of two low-osmolar contrast media, ioxaglate and iopamidol, on erythrocyte membrane elasticity.

RATIONALE AND OBJECTIVES: The effect of ioxaglate and iopamidol, two recently developed low-osmolality contrast media, on the shear elasticity of erythrocyte membranes was studied at an iodine concentration of 300 mg/mL and at a 20% volume concentration and compared with that obtained for control solutions matched in osmolality. METHODS: The authors used a micromanipulation technique, which consists of visualizing deformations of individual erythrocytes when gently aspirated into the tip of a glass micropipette by an accurately controlled pressure. An erythrocyte membrane shear elasticity modulus mu was then deduced. An increase in mu corresponded to an increase in erythrocyte membrane rigidity. RESULTS: In all cases, the erythrocytes remained discocytic. The shear elasticity modulus of the erythrocyte membrane is found to be (1) in the presence of ioxaglate (4.3 +/- 0.9 microN/m) lower (P < .001) than in the presence of hyperosmolar saline (6.2 +/- 1.3 microN/m) and lower (P < .02) than in the presence of iso-osmolar control (4.7 +/- 0.7 microN/m); (2) in the presence of iopamidol (5.4 +/- 0.7 microN/m) lower (P < .001) than in the presence of hyperosmolar sucrose (6.4 +/- 1.2 microN/m) and higher (P < .001) than in the presence of iso-osmolar control (4.7 +/- 0.7 microN/m); and (3) lower (P < .001) in the presence of ioxaglate (4.3 +/- 0.9 microN/m) than in the presence of iopamidol (5.4 +/- 0.7 microN/m). CONCLUSIONS: Under the experimental conditions used, both contrast media (ioxaglate and iopamidol) modify the erythrocyte membrane shear elasticity modulus less than do the matched hyperosmolar controls. Moreover, ioxaglate makes the erythrocyte membrane less rigid, and iopamidol makes the erythrocyte membrane more rigid than does the iso-osmolar control.

Elasticity↗
PubMed1994

Structure of the erythropoietin receptor.

Despite extensive studies, the structure of the erythropoietin receptor remains little understood. cDNAs encoding the human and murine erythropoietin receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in erythropoietin binding and in erythropoietin signal transduction, increasing evidence strongly suggests that the erythropoietin receptor is a multimeric complex. The murine erythropoietin receptor has been solubilized under mild conditions and the molecular mass of the native receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other erythropoietin receptor subunits.

Cloning, Molecular↗
PubMed1994

Evaluation of the Coulter MAXM in a children's hospital.

An evaluation of the WBC differential given by the analyzer Coulter MAXM was performed in a children's hospital, in order to establish optimal interpretation of the automated MAXM report in pediatrics. The patient group comprised 419 children of age up to 15 years. Assessment was carried out by comparison of the MAXM results with microscopic examination of blood smears, good correlation being observed between the two methods in non flagged samples and those flagged only with an isolated "Imm Grans/Bands 1" message indicating the presence of band cells. Comparison of the MAXM and optical differentials in normal and abnormal samples was performed according to a standard protocol taking into account both suspect flags and quantitative abnormalities defined in relation to age. In this group of 419 children, there were 20 (4.8%) false positive and 8 (1.9%) false negative reports, the 8 false negative results corresponding to either minor morphological (4) or borderline quantitative abnormalities (4). Overall, the instrument was capable of detecting significant abnormalities in blood samples with a sensitivity of 95.5% and a specificity of 91.7%. Although the review rate was 45.3%, the high efficiency (93.3%) suggested that most blood smear reviews led to the discovery of a morphological and/or quantitative abnormality. Furthermore, the review rate was greater in neonates (< 7 days) and infants (7 days-1 year) but closer to those of adult populations in older children. The Coulter MAXM was thus shown to be appropriate for use in pediatrics, despite a higher frequency of blood smear review than in adults, particularly among patients less than one year old.

Adolescent↗
PubMed1994

[Microrheology of marrow promyelocytes from patients suffering from acute myeloblastic leukemia type 3].

Viscosity values of marrow cells from patients suffering from acute promyelocytic leukemia (acute myeloid leukemia of type 3) have been determined before and during a treatment with all-trans retinoïc acid (active metabolite of the vitamin A, inducing cell differentiation and maturation). Evaluated from aspiration tests into a glass micropipette, the marrow cell viscosity has been compared to that of normal neutrophils. Results seem to point out that the induced hyperleucocytosis is favoured by both a low standard deviation of pathological cell viscosity and a cell viscosity value close to that of normal neutrophils. The systematic study of cell viscosity could therefore help the clinicians to individualize the treatment.

Granulocytes↗
PubMed1994

Serum IgG subclass level determination: methodological difficulties and practical aspects.

During the last decade, the availability of monoclonal antibodies against human IgG subclasses led to a marked increase in serum IgG subclass determinations, including in routine laboratories. Although several drawbacks of polyclonal antisera (poor specificity except in certain precipitation assays, inter-batch variability, etc) are avoided by the use of monoclonal antibodies, a number of methodological difficulties and pitfalls still remain, especially for measurement of IgG2 and with commercial kits. Moreover, most of them are hardly practicable, because of arbitrary standardization and absence of appropriate normal reference values. This increase in routine serum IgG subclass level determination led to a considerable widening of patient recruitment and a significant evolution of patterns and frequencies of subclass deficiencies was observed in patients with unexplained infection and no characterized immunodeficiency syndrome, as compared with initial studies performed in specialized clinical immunology departments. IgG3 deficiency is more frequent in mildly than in severely infected patients, and a marked decrease in the incidence of IgG2 deficiency was found in our recent survey. We also present the results of a recent retrospective study on hypogammaglobulinemia. In this condition, IgG subclass imbalances are more frequent than an overall IgG subclass decrease, and IgG2 deficiency appears to be a marker of severity. Other observations are: 1) the high frequency of selective IgG1 deficiency, especially in adults over 40 years; and 2) that of IgG2 deficiency in children of less than 4 years of age.

Adolescent↗
PubMed1993

Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the erythropoietin receptor.

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.

Cell Line↗
PubMed1993

[Erythroblastopenia: chronic idiopathic or associated with chronic lymphoid leukemia. Value of cultures of erythroblastic progenitors and therapeutic strategy].

Fifty cases of chronic pure red cell aplasia in adults (idiopathic in 32 cases, associated with chronic lymphocytic leukaemia in 18) were followed up after studying their erythroblastic precursors in vitro. Analysis of response to immunomodulators confirmed the existence, in patients with idiopathic chronic pure red cell aplasia, of a relationship between in vitro behaviour and obtention of a remission: patients with normal differentiation of erythroblasts consistently responded to treatments, whereas treatments were usually ineffective when the erythroblasts did not differentiate in vitro. When in vitro differentiation was below normal, responses to treatments were varied. In patients with chronic lymphocytic leukaemia, the erythroblastic precursors were normal in 15/18 cases, and response to immunomodulators was observed in 13/18 cases. A second objective of this study was to determine, on a large series, the most adequate immunomodulator treatment. In idiopathic chronic pure red cell aplasia the most efficient therapy was corticosteroids and cyclophosphamide administered separately or together. The antilymphocyte serum gave disappointing results. In pure red cell aplasia associated with leukaemia cyclophosphamide was the most frequently effective drug, with corticosteroids taking second rank; however, infections were frequent. The treatments to be considered when both corticosteroids and cyclophosphamide fail are discussed.

Adrenal Cortex Hormones↗
PubMed1993

Erythropoietin synthesis by tumor cells in a case of meningioma associated with erythrocytosis.

While secondary erythrocytosis is often associated with tumors arising from the kidney, other tumors have been described to originate in the liver, uterus, ovary, adrenal gland, and central nervous system, among which cerebellar hemangioblastomas are involved in most instances. Two cases of meningioma associated with erythrocytosis have already been reported. We observed a 59-year-old female patient who had developed a frontal meningioma associated with erythrocytosis. Before surgery, she had a significantly elevated total red blood cell volume with a normal plasma volume. Serum erythropoietin (Epo) dosage assessed by radioimmunoassay was within the normal range. The tumor was removed and the pathologic study found a meningotheliomatous meningioma. Total RNA from the tumor was hybridized to a monkey cDNA Epo probe. A strong 1.6-kb messenger RNA (mRNA) signal was observed, which is the expected size of human Epo mRNA. In situ hybridization with the 35S-labeled Epo probe was performed on frozen tumor tissue sections. A significant hybridization was observed in all the tumor cells, whereas the stroma was negative. Therefore, in this meningioma associated with erythrocytosis, Epo was produced by the tumor cells themselves.

Erythropoietin↗
PubMed1993

Effects of ultrasound energy on thrombi in vitro.

Ultrasonic energy may be used for dissolution of venous or arterial thrombi. However, its effects may depend on the mode of ultrasonic vibration and on the length of the probe. We investigated the in vitro effects of an ultrasonic angioplasty device coupled with a 130 cm-long flexible titanium probe, with an incorporated automatic optimal frequency of resonance scanning function and continuous mode of emission. Sixteen clots were treated of which eight were whole blood and eight cell-free. In each of these groups, four were treated in association with streptokinase and four by ultrasound alone. The ages of the clots in these subgroups of four were 1, 3, 7, and 15 days. All thrombi were dissolved in 6 min or less (3'15" +/- 1'35") at a mean optimal frequency of resonance of 19,444 Hz. Ninety-six percent of the debris were less than 10 mu. Fewer than 1% of the particulates were larger than 100 mu. These large particulates were observed in disrupted whole blood clots and were almost non-existent in disrupted cell-free clots. They were very fragile. Clot dissolution was not speeded by adding streptokinase to ultrasound. Ultrasound did not induce D-Dimer production, and its effect was most likely to be due to cavitation. Ultrasound energy could represent an advance for thrombotic vascular occlusion therapy, provided that more flexible probes can be devised.

Angioplasty↗
PubMed1993

Identification of alpha 2-adrenoceptors and of non-adrenergic idazoxan binding sites in pancreatic islets from young and adult hamsters.

1. The current study was undertaken to investigate the characteristics of alpha 2-adrenoceptors and to search for the presence of NAIBS in hamster pancreatic islets. 2. Pancreatic islets were isolated from young (6-7 weeks) and adult (14-15 weeks) animals. 3. The identification of alpha 2-adrenoceptors using [3H]RX821002 indicated that adults exhibited higher number of alpha 2-adrenoceptors than the young animals (194 +/- 20 vs 105 +/- 16 fmol/mg protein) while the Kd value was unchanged. 4. Glucose-evoked insulin release was completely inhibited by the alpha 2-agonist clonidine (0.1 microM) whatever the age of the animals. Agonist inhibition curves showed the following rank order of potency: clonidine > UK14304 > adrenaline. 5. Blockade of UK14304-elicited inhibition by various antagonists indicated that yohimbine has a low affinity for the receptor supporting the conclusion that the receptor is of the alpha 2-D subtype. 6. Binding experiments with [3H]idazoxan under conditions allowing to discriminate between alpha 2-adrenoceptors and NAIBS showed that hamster pancreatic islets express a high number of NAIBS. The density of NAIBS was similar in young and adult hamsters (1550 +/- 245 and 1342 +/- 332 fmol/mg protein).

Adrenergic alpha-Agonists↗
PubMed1993

[Effects of ultrasonic energy on blood clots in vitro].

Ultrasound may be used to dissolve arterial and venous thrombi. Its effects depends on the mode of ultrasonic vibration and on the length of the guide wire. The authors studied the in vitro effects of an ultrasonic angioplasty device coupled with a 130 cm long titanium flexible guide wire. The system comprises an automatic scanning function to determine the optimal frequency of resonance and works in the continuous mode of emission. Sixteen thrombi were studied of which 8 were acellular and 8 whole blood. In each group, 4 were treated in association with streptokinase and 4 by ultrasound alone. The ages of the thrombi in each subgroup were 1, 3, 7 and 15 days. All the thrombi were dissolved in 6 minutes or less (3'15" +/- 1'35") at an average optimal frequency of resonance of 19,444 Hz. Ninety six per cent of the debris had a diameter less than 10 microns. Less than 1% of the debris had a diameter larger than 100 microns. These large particles were observed in cellular thrombi and were almost completely absent in dissolved acellular thrombi. They were very fragile. The dissolution of the thrombi was not accelerated by the association of streptokinase. The ultrasonic energy did not induce D-dimer production and its action was probably due to cavitation. Ultrasonic energy could provide an alternative treatment for thrombotic vascular occlusion provided that more flexible guide wires could be designed.

Humans↗