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Y Beuzard

Publications and source records attributed to Y Beuzard.

At least 37 records · Page 2Linked to original sources
PubMed1997

[Erythropoietin gene: regulation and therapeutic concern].

The erythropoietin (Epo) is a model of proteins expressed upon hypoxia, regulated at the transcriptional and post transcriptional levels. The Hypoxia Induced Factor (HIF-I) activates the transcription of genes which exhibit the Hypoxia Regulatory Element (HRE). In addition the mRNA is stabilized upon hypoxia, increasing the hormone synthesis. The Epo gene is a convenient reporter gene to develop gene delivery systems and the in vivo regulation of the transgene. The beta thalassemia and acquired chronic anemias may benefit from the Epo gene transfer and expression, if it becomes safe, tunable and inexpensive.

Animals↗
PubMed1997

[Effect of smoking on blood rheology].

The aim of this study was to compare the effects of cigarette smoking on biologic and rheologic tests, chiefly on the red blood cells (RBC) in measuring the deformability by the Cell Transit Analyser (CTA) and their aggregation by using an ultrasonic interferometry method based on A-mode echography allowed for the measurement of the accumulation rate of particles in a solid plate which is related to their sedimentation rate (Echo-Cell). Nine male smoker subjects with a high nicotine addiction measured by Fagerström questionnaire (> 8) and level of carbon monoxide (CM) in the breathed out air (> 20 ppm), have been compared with ten healthy no-smoker volunteers (CM < 3 ppm). One smoker has been eliminated of statistic evaluations because his glucose level showed a diabetes (10.5 mmol/l). A nailfold capillaroscopy performed in all subjects has eliminated the patterns of latent vasculitis or scleroderma. RBC and platelets counts, hemoglobin, ionogram, gamma GT, ASAT, ALAT, uric acid, total cholesterol and glucose levels were not significantly different between the two groups. On the other hand, in the smoker group, white blood cells count, serum triglycerides and especially fibrinogen values were higher than in the non-smoker's group. RBC sedimentation rate was normal in the two groups but was higher in smoker's group too. Without consumption of alcohol, the mean RBC volume was more important in smokers (91.9 +/- 1.2 versus 87.5 +/- 0.4, p = 0.003). Rheologic tests were more pathologic in smokers. The transit time or RBC by CTA was longer than in control group (1.6 ms +/- 0.02 versus 1.2 +/- 0.05, p = 0.0003). Echo-Cell technic showed a number and size of RBC aggregates more important with a rate of speed of accumulation higher than in the control group. These results demonstrated the toxic effects of smoking alone on blood toward a propensity for thrombotic status.

Adult↗
PubMed1996

Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse.

Prevention of erythrocyte dehydration is a potential therapeutic strategy for sickle cell disease. Increasing erythrocyte magnesium (Mg) could inhibit sickle cell dehydration by increasing chloride (CI) and water content and by inhibiting potassium chloride (K-CI) cotransport. In transgenic SAD 1 and (control) C57BL/6 normal mice, we investigated the effect of 2 weeks of diet with either low Mg (6 +/- 2 mg/kg body weight/d) or high Mg (1,000 +/- 20 mg/kg body weight/ d), in comparison with a diet of standard Mg (400 +/- 20 mg/ kg body weight/d). The high-Mg diet increased SAD 1 erythrocyte Mg and K contents and reduced K-CI cotransport activity, mean corpuscular hemoglobin concentration (MCHC), cell density, and reticulocyte count. SAD 1 mice treated with low-Mg diet showed a significant reduction in erythrocyte Mg and K contents and increases in K-CI cotransport, MCHC, cell density, and reticulocyte counts. In SAD 1 mice, hematocrit (Hct) and hemoglobin (Hb) decreased significantly with low Mg diet and increased significantly with high-Mg diet. The C57BL/6 controls showed significant changes only in erythrocyte Mg and K content, and K-CI cotransport activities, similar to those observed in SAD 1 mice. Thus, in the SAD 1 mouse, changes in dietary Mg modulate K-CI cotransport, modify erythrocyte dehydration, and ultimately affect Hb levels.

Anemia, Sickle Cell↗
PubMed1996

Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Animals↗
PubMed1996

Keratinocytes as a target for gene therapy. Sustained production of erythropoietin in mice by human keratinocytes transduced with an adenoassociated virus vector.

BACKGROUND AND DESIGN: Keratinocytes are ideal targets for somatic gene therapy. Among the viral gene transfer systems, adenoassociated virus vectors have recently gained attention. We studied the feasibility of using adenoassociated virus-transduced human keratinocytes to provide a long-term, high-level production of a therapeutic factor after implantation in mice. RESULTS: Transduction of HeLa cells by an adenoassociated virus vector was ascertained by transfer of the beta-galactosidase reporter gene, which was visualized by the blue staining of infected cells after fixation and coloring by X-Gal (the substrate of the reaction for beta-galactosidase activity). In a second step, 2 HeLa cell lines transduced with an AAV harboring the erythropoietin complementary DNA and producing high amounts of erythropoietin in vitro were isolated. After implantation in nude mice, a high-level and long-term increase in hematocrit (for the 1-month duration of the study) was found, which was correlated to the size of the induced tumor. CONCLUSIONS: Adenoassociated virus-transduced HeLa keratinocytes provide high-level, stable, and long-term production of a therapeutic protein in mice. These results must now be extended to human primary keratinocytes.

Animals↗
PubMed1996

Transgenic mouse models of sickle cell disease.

An array of sickle cell syndromes has been obtained in transgenic mice, expressing HbS or super HbS, from the asymptomatic phenotype similar to the human A/S state to a syndrome more severe than the human homozygous S/S state, inducing 100% fetal death. Anemia was observed in SAD and SAD (beta th/ beta +) neonates and disappeared during postnatal development. In adults, many features of sickle cell disease are found in transgenic mice, especially in SAD and SAD (beta th/ beta +) mice, including abnormal hemolysis, vasoocclusion, microthrombosis, infarct, priapism, chronic organ defects, and death on hypoxia. These mouse models are relevant to the study of the pathophysiology of sickle cell disease and the induction of vasoocclusion and to evaluate new therapeutic approaches in vivo. Clotrimazole and Mg2+ restore hydration of sickle cells and 12 C79 protected SAD mice from lethal acute hypoxia.

Anemia, Sickle Cell↗
PubMed1996

A new type of hereditary persistence of fetal haemoglobin (HPFH): HPFH Tunisia beta + (+C-200)G gamma.

Nd-HPFH are haematological conditions which are natural models to aid understanding of the haemoglobin (Hb) switch. In this paper we describe a new non-deletional hereditary persistence of fetal haemoglobin (nd-HPFH) associated with the highest Hb F level observed to date (up to 49% without haemopoietic stress). Sequence of the G gamma promoter revealed a cytidine insertion within a stretch of four cytidines, located between -200 and -203 bp with respect to the cap site. This insertion is situated within a polypyrimidine-polypurine region which can adopt a triple helix structure, and is therefore of particular interest with respect to the Hb switch mechanism.

Consanguinity↗
PubMed1995

Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.

We have examined whether the secretion of erythropoietin (Epo) from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to Epo. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine Epo cDNA is expressed under the control of the murine phosphoglycerate kinase promoter. "Neo-organs" containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit (> 80%) and elevated serum Epo concentration (ranging from 60 to 408 milliunits/ml) were observed in recipient animals over a 10-month observation period. Hematocrit values measured in recipient mice varied according to the number of implanted Epo-secreting fibroblasts (ranging from 2.5 to 20 x 10(6)). The implantation of neo-organs containing Epo-secreting fibroblasts appeared, therefore, as a convenient method to achieve permanent in vivo delivery of the hormone. We estimated that the biological efficacy of the approach may be relevant for the treatment of human hemoglobinopathies.

Animals↗
PubMed1995

Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse.

The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.

Animals↗
PubMed1995

Organoids direct systemic expression of erythropoietin in mice.

Organoids are adenoviral vector transduced cells embedded ex vivo in a collagen-polytetrafluoroethylene lattice that is saturated with angiogenic factors. Organoids provide an alternative method of cell mediated gene transfer following implantation in the donor/recipient. The feasibility of adenovirally mediated delivery via organoids using the erythropoietin (Epo) cDNA was tested. Fibroblasts were transduced by two recombinant adenoviral vectors encoding the Macaca cynomolgus Epo cDNA, driven by a viral (RSV LTR) or a murine housekeeping gene promoter (PGK-1). A functional in vivo assay was used to monitor Epo production via the rise in hematocrit(s) (hct). The hct remained elevated for as long as 6 weeks after implantation. Subcutaneous implants gave consistently higher hct than intraperitoneal implants, while organoids made with a greater number of cells, or an equal number of cells transduced at higher multiplicities of infection (MOI) also produced a larger increase in hct. AdPGKEpo-organoids produced a greater increase in hct than AdRSVEpo-organoids under comparable conditions, but the duration of expression was similar. A 10- to 50-fold lower input of AdRSVEpo using organoids versus direct intravenous injections resulted in an equal to, or greater than hct response in mice. Explanted organoids caused a rapid decrease in the hct of mice. Organoid supernatant had little or no detectable free viral particles making this method safe from unwanted recombinant adenovirus dissemination.

Animals↗
PubMed1994

Sickle cell disease of transgenic SAD mice.

Erythrocyte sickling on deoxygenation in vitro occurs in transgenic SAD mice, hemizygous for a modified human sickle hemoglobin, HbSAD [alpha 2 beta 2S(beta 6val)Antilles (beta 23 lle)D- Punjab (beta 121Gln)] (SAD-1, 19% HbSAD; beta-thal/SAD-1, 26% HbSAD). The present study examines the cellular defects in vivo and pathologic changes observed in SAD-1 mice at atmospheric oxygenation as well as the effect of acute hypoxia. The transgenic mice showed generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis, and myocardium. The most prevalent chronic organ lesions were congestive splenomegaly (83% of animals) and renal glomerulopathy, which affected 75% of animals by 10 months of age. Further, SAD mice have a mean lifespan that was reduced by 40% when compared with nontransgenic littermates. Premature death of SAD mice was associated with acute vasoocclusive events or severe renal disease. SAD mice developed lethal vasoocclusive processes when exposed to reduced pO2 conditions, whereas control mice survived normally. The sensitivity to hypoxia appears to depend on the cellular level of HbSAD, because death occurred at pO2 of 42 mmHg for SAD mice and 49 mmHg for beta-thal/SAD. Administration of an antisickling agent that increases oxygen affinity (BW12C79) protected SAD and beta-thal/SAD mice from the lethal hypoxic stress. In conclusion, the transgenic SAD and beta-thal/SAD mice developed a pathophysiology that strongly resembles human sickle cell disease. Moreover, this animal model allows studies on the effect of antisickling agents.

Anemia, Sickle Cell↗
PubMed1994

Retrovirus-mediated transfer of the erythropoietin gene in hematopoietic cells improves the erythrocyte phenotype in murine beta-thalassemia.

Repeated injections of large doses of erythropoietin (Epo) have been shown to be of benefit in the treatment of murine and human beta-thalassemia. To determine whether Epo gene therapy could replace this treatment for long-term periods, lethally irradiated beta-thalassemic (Hbbd3th haplotype) and normal DBA/2J (Hbbd haplotype) mice were grafted with syngeneic bone marrow cells infected with a retroviral vector carrying the Epo cDNA. In normal mice, dysregulated Epo production induced elevated serum Epo levels (176 +/- 68 mU/mL), high hematocrit levels (73% +/- 8%), and elevated beta-minor globin chain synthesis. In contrast, in thalassemic mice, moderate increases in the hematocrit levels (from 33% +/- 1% to 43% +/- 9%), associated with limited increases in the initially elevated Epo levels (from 83 +/- 22 to 190 +/- 230 mU/mL), were recorded 2 months after transplantation. In mice in which the hematocrit increased most, from 33% +/- 1% before transplantation to 49% +/- 10%, the retroviral Epo gene expression induced a striking improvement of the beta-thalassemic syndrome. These mice exhibited normal or near-normal beta/alpha-globin chain synthesis ratios, induced by the activation of the beta-minor chain. This led to the elimination of the high amounts of unpaired alpha chains in erythrocytes and finally reduced the reticulocyte count despite the permanent Epo stimulation. These results show that efficient Epo gene expression corrects the erythrocyte phenotype of the mouse beta-thalassemic syndrome. However, the incidence of lethal polycythemia or of transient improvements indicates that the present strategy is only the first step toward such indirect gene therapy.

Animals↗
PubMed1994

A potential regulatory region for the expression of fetal hemoglobin in sickle cell disease.

We describe a 0.5-kb region located 1.65 to 1.15 kb upstream of the G gamma fetal globin gene with three polymorphisms of erythroid and ubiquitous nuclear protein binding motifs (GATA, CRE, and a new protein binding site). These three polymorphisms result in high-affinity and low-affinity motifs for nuclear proteins, and are combined in four arrangements called pre-G gamma frameworks (pG gamma Fs). Each pG gamma F is linked with one of the major haplotypes of the beta-globin gene cluster observed in sickle cell disease (SCD) associated with different mean levels of hemoglobin F (Hb F) expression (P < .001). This strong linkage and the differing affinities suggest that this region may be involved in the modulation of Hb F expression in SCD.

Anemia, Sickle Cell↗
PubMed1994

The involvement of the Ca-dependent K channel and of the KCl co-transport in sickle cell dehydration during cyclic deoxygenation.

We have investigated the mechanisms involved in sickle cell dehydration upon continuous or cyclic deoxygenation: the Ca(2+)-activated K+ channel and the KCl co-transport system. Short-term continuous deoxygenation (1 h) of sickle cells in a Ca(2+)-containing medium promoted a stimulation of the efflux of K+ and cell dehydration. This latter was reduced by the replacement of Ca2+ in the medium by EGTA, but not by addition of [(dihydro-indenyl) oxy] alkanoic acid (DIOA), an inhibitor of the KCl co-transport. During cycles of deoxygenation-reoxygenation, cell dehydration was partly prevented by EGTA and significantly reduced by DIOA only in the presence of Ca2+. The present data support the view that sickle cell dehydration during deoxygenation arises from the stimulation of the Ca(2+)-dependent K+ permeability leading to water loss, whereas during reoxygenation periods, subsequent activation of the KCl co-transport also contributes to cell dehydration.

Anemia, Sickle Cell↗
PubMed1994

[Measurement of immunologic agglutination of erythrocytes by ultrasonic interferometry].

Ultrasound interferometry is a new methodology which has been developed in our laboratories in order to measure precisely and quickly the size of particles sedimenting in liquid on horizontal surface, upon gravity. Applied to red blood cells, this method evaluates the sedimentation of erythrocytes, their aggregation induced by proteins or aggregating compounds as well as their agglutination upon immune reactions. The quantitative assessment of red cell agglutination was applied to the study of blood groups and to the search for red cell antibodies. Preliminary results show that ultrasound interferometry is 1) quantitative, measuring the size of agglutinates; 2) sensitive; 3) specific; 4) fast; 5) able to detect irregular antibodies.

Erythrocytes↗
PubMed1994

Erythropoietin gene transfer and expression in adult normal mice: use of an adenovirus vector.

A hormonal model of erythropoietin (Epo) delivery by use of an adenovirus vector was investigated. We constructed a replication-defective adenovirus carrying the monkey (cynomolgus) Epo cDNA under control of the Rous sarcoma virus long terminal repeat promoter. Fifty 8-week-old mice were injected with escalating doses of the recombinant virus from 10(6) to 10(10) plaque-forming units (pfu). Different modes of administration were studied. Intravenous (i.v.) injection was the most effective mode of administration and exhibited a dose-dependent response. After a single i.v. injection with high doses (5 x 10(9) and 10(10) pfu), a dramatic increase in hematocrit (Hct) and long-term Epo expression (6 months at this time) were observed. Intravenous administration with lower doses and intramuscular (i.m.) administration were inefficient or had a very transient effect. A localized muscle attrition prior to i.m. administration of 10(10) pfu enhanced Hct response. This initial study opens the way for high level and durable Epo therapy by gene transfer. Moreover, this recombinant virus provides a convenient means to study the efficacy, duration, and safety aspects of hormonal delivery by an adenoviral vector.

Adenoviridae↗