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Biomedical subjects

A Barbier

Publications and source records attributed to A Barbier.

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

Characterization of three human apolipoprotein E isoforms (E2, E3 and E4) expressed in Escherichia coli.

Apolipoprotein E is one of the apolipoproteins involved in cholesterol metabolism. Three major isoforms are present in men: E2, E3, E4 corresponding to the products of three alleles. They have different affinities for receptors and the epsilon 4 allele is a risk factor for cardiovascular diseases and more recently for Alzheimer's disease. We describe here the production, by heterologous expression in Escherichia coli, of the three apolipoprotein E isoforms for use in both research and clinical laboratories. By Surface Plasmon Resonance, the purified recombinant apolipoprotein E isoforms were able to recognize three monoclonal anti-human apolipoprotein E antibodies with affinity constants close to those of purified human apolipoprotein E. For receptor binding studies, the recombinant apolipoprotein E isoforms were associated with VLDL isolated from apolipoprotein E knockout mice. Although the association of the recombinant apolipoproteins E with the mouse VLDL was less efficient than that of human plasma apolipoprotein E3, the recombinant apolipoprotein E3 and apolipoprotein E4 complexes competed efficiently with 125I-labelled LDL for binding to the LDL receptor in J774 macrophages, whereas the recombinant apolipoprotein E2-VLDL complexes did not. These results suggest that the recombinant apolipoprotein E isoforms have biological properties similar to the human apolipoprotein E isoforms.

Animals↗
PubMed1996

The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase.

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

Alendronate↗
PubMed1996

Single-step purification of two functional human apolipoprotein E variants hyperexpressed in Escherichia coli.

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH2-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into the Escherichia coli strain BL21(DE3). Recombinant strains were grown at 37 degrees C in a Luria and Bertani medium and the addition of isopropyl beta-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.

Antibodies, Monoclonal↗
PubMed1996

Effect of ovariectomy on intraosseous vascularization and bone remodelling in rats: action of tiludronate.

In order to study the action of tiludronate on the changes in intraosseous vascularization induced by ovariectomy, and to link these effects to those observed in bone remodelling, 30 female Sprague-Dawley rats (age 40 weeks) were studied. Ten rats were shamoperated and treated by vehicle, 10 rats were ovariectomized and treated by vehicle, and 10 rats were ovariectomized and treated orally with tiludronate, 0.16 mmol/kg/per day, 3 days a week for 16 weeks, from the day following ovariectomy. The rats were killed after 4 months, and a histomorphometric study and quantification of intraosseous vessels carried out on the sixth lumbar vertebra. The area of the intraosseous sinusoidal capillaries increased after ovariectomy, which also induced a moderate increase in resorption surfaces and osteoid surfaces leading to a decrease of 40% in the trabecular bone volume at the lumbar spine level. This bone mineral loss was completely prevented by tiludronate, which normalized the bone turnover. However, tiludronate was without any effect on intraosseous vascularization. These results indicate that the surface area of the intraosseous sinusoidal capillaries was correlated positively with resorption surfaces and negatively with trabecular bone volume and the number of bone trabeculae. In these experimental conditions, an inhibitor of bone resorption can exert its positive effect on bone mass without normalization of vascularization.

Animals↗
PubMed1995

Expression of type V adenylyl cyclase is required for epidermal growth factor-mediated stimulation of cAMP accumulation.

Previously, this laboratory has demonstrated that epidermal growth factor (EGF) increases adenylyl cyclase activity in cardiac membranes and elevates cAMP accumulation in hearts and cardiac myocytes. Since EGF does not increase cAMP accumulation in all tissues, we investigated the possibility that the expression of a specific isoform of adenylyl cyclase (AC) was necessary to observe EGF-elicited stimulation of cAMP accumulation. HEK 293 cells were transfected with different isoforms of AC, and the ability of EGF to increase AC activity as well as elevate cAMP accumulation was determined. In cells transfected with AC I, II, V, and VI cDNAs, neither the expression nor the amount of the two isoforms of Gs alpha (45 and 52 kDa) were altered. Similarly, EGF-elicited phosphorylation of cellular proteins on tyrosine residues in various transfectants was unaltered. However, EGF increased AC activity and elevated cAMP accumulation only in cells expressing the rat and canine ACV. EGF did not alter either AC activity or cAMP accumulation in cells overexpressing types I, II, and VI isozymes. As assessed by the ability of an anti-Gs alpha antibody to obliterate the effect, stimulation of AC activity in AC V transfectants involved the participation of Gs alpha, a finding consistent with previous data concerning EGF effects on cardiac AC (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B. (1990) J. Biol. Chem. 265, 21317-21322). Thus we conclude that the expression of AC V isoform confers specificity to the ability of EGF to stimulate AC activity.

Adenosine Triphosphate↗
PubMed1995

Tiludronate: bone pharmacology and safety.

The pharmacological properties of tiludronate (4-chlorophenyl)thiomethylene bisphosphonate), a sulfured bisphosphonate, have been characterized in a series of preclinical in vivo and in vitro studies. In vivo, tiludronate exerts a dose-dependent inhibitory activity on bone resorption. This property was demonstrated in several animal models, including rats, ewes, and dogs, when bone resorption was induced by administration of retinoid acid or parathyroid hormone, or by immobilization, ovariectomy or orchidectomy. By uncoupling bone resorption from bone formation, tiludronate can induce a positive calcium and phosphate balance. When administered either continuously or intermittently to ovariectomized osteoporotic rats, tiludronate promotes a significant increase in bone mass. This positive effect is associated with an increase in mechanical resistance. Bone tolerance studies indicate that tiludronate is a safe compound with an appreciable therapeutic margin since it can effectively inhibit bone resorption without reducing bone mineralization and strength. In vitro, tiludronate added to bone tissue culture inhibits calcium release, lysosomal enzyme secretion and collagen matrix degradation when induced by various stimulators of bone resorption. At the cellular level, tiludronate does not appear to exert its inhibitory effect on bone resorption by impairing either the recruitment, the migration or the fusion of osteoclast precursors. Tiludronate could act on mature osteoclasts by reducing their capacity to secrete proton into the resorption space and also by favoring their detachment from the bone matrix. The available preclinical data indicate that tiludronate should be an efficacious bisphosphonate in the management of clinical conditions characterized by excessive bone resorption.

Animals↗
PubMed1995

A possible mechanism of the specific action of bisphosphonates on osteoclasts: tiludronate preferentially affects polarized osteoclasts having ruffled borders.

The mechanism of action of tiludronate [(4-chlorophenyl)-thiomethylene bisphosphonate] on osteoclastic bone resorption was examined in mouse culture systems. Tiludronate did not inhibit the formation of osteoclast-like multinucleated cells (OCLs) induced by 1 alpha,25-dihydroxyvitamin D3 in cocultures of mouse osteoblastic cells and bone marrow cells. OCLs obtained from cocultures on collagen gel-coated dishes, treated with tiludronate, formed as many resorption pits on dentine slices as those obtained from the control cocultures. However, pit formation by OCLs was dose-dependently inhibited when tiludronate was added directly to the pit formation assay. Other bisphosphonates such as alendronate and etidronate dose-dependently inhibited pit formation according to the in vivo potencies of the respective bisphosphonates to inhibit bone resorption. However, they had no inhibitory effect on the recruitment of OCLs induced by 1 alpha,25-dihydroxyvitamin D3 in the cocultures. When OCLs were placed on dentine slices, they formed the ringed structure of F-actin-containing podosomes and ruffled borders (polarized OCLs) even in the presence of tiludronate. However, the actin rings in OCLs were disrupted by the addition of tiludronate soon after they began to resorb dentine. In contrast, OCLs placed on collagen gel formed neither actin rings nor ruffled borders (nonpolarized OCLs), and showed no response to tiludronate. OCLs formed from the spleen cells of osteosclerotic (oc/oc) mice developed the ringed structure of podosomes, but not ruffled borders, on dentine slices. The actin ring in the oc/oc spleen cell-derived OCLs placed on dentine slices was not disrupted by the addition of tiludronate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Comparative study of 2 culture methods by seeding, in hemoculture bottles, the dialysis fluid from patients in continuous ambulatory peritoneal dialysis].

BACKGROUND: Peritonitis remains a major complication of continuous ambulatory peritoneal dialysis (CAPD). The accurate diagnosis of peritonitis is a requirement for a successful CAPD program. A prospective study was performed to evaluate two culture methods. METHODS: 1. Culture of 10 ml of uncentrifuged peritoneal fluid and 2. Culture of the sediment of 50 ml centrifuged and resuspended in 20 ml of distilled water, into aerobe/anaerobe hemoculture bottles (Hemoline, BioMérieux). We processed 162 PF from 138 CAPD peritonitis episodes, 33 of whom were in antimicrobial drug therapy. RESULTS: The sensibility/specificity of both methods (87.0/70.8 method 1 and 81.9/79.2) were similar. Both methods were more sensitive (p = 0.001) when the patient was no in antimicrobial drug therapy (60.6/88.6 method 1 and 69.7/92.4 method 2). We isolated 132 microorganisms, 64.4% Gram positive, 25.0% Gram negative, 4.6% anaerobes and 6.1% levures. CONCLUSION: Both methods were similar and the more important factor to increase the yield of culture is to remove the antibiotic presents in centrifuged fluid.

Bacterial Infections↗
PubMed1994

[Venous hemodynamics of the lower limbs].

Low pressure venous return results from several different pump systems. Blood flow and velocity depend on sympathetic tone and contraction of the veins modulating distensibility. In patients with chronic venous insufficiency, different components including hyperdistensibility, hyperpressure, valve failure, back-flow and stasis, lead to a vicious circle. These elements modify the venous wall and lead to hyperdistension producing modified distensibility and/or formation of varicose veins. Endothelial and smooth muscle cells participate in these changes. Smooth muscle cells play a major role: some hypertrophy and/or lose their contractility, synthesizing and sometimes rapidly destroying tissue. These changes in the circulation, sometimes increased by inflammation and thrombosis, affect microcirculatory haemodynamics with a variable delay. The venous system, even when pathological (as long as it is not totally invaded by fibrosis) responds to a large number of relaxing or constricting pharmacological agents, justifying their therapeutic value.

Hemodynamics↗
PubMed1994

[Microcirculation and rheology].

Microvessels, especially in the skin and muscles are organized in functional units. These units are controlled by the adrenergic system and hormones but also have autonomous metabolic and myogenic regulatory systems independent of vasomotion. Microcirculatory blood flow is affected by special rheologic properties: a low arteriolar haematocrit rises to the systemic level in the venules. The flow rate in the venules is low, and together with the raised haematocrit, explains the microvenular sensitivity to hyperviscosity. Capillarovenular microangiopathy, recently described by standard and fluorescent capillaroscopy, develops during chronic venous insufficiency. The capillary loops appear dilated and knotted together with fibrous deposits and pericapillary oedema. Venous hyperpressure is the cause of this microangiopathy. Together these phenomena disrupt normal haemodynamics and physiology of the microcirculatory unit: baseline hyperhaemia, lowered vasomotor and vasomotion reactivity, development of rheologic disorders (haemoconcentration, hyperfibrinogenaemia, erythrocyte agregation) and decreased fibrinolysis. Modifications in the transcapillary exchange is related to hypoxia and is aggravated by depressed lymphatic drainage. The main consequence is oedema. Inflammation, a characteristic of these microangiopathies could occur when the endothelium is activated by the hypoxia. The classical mediators of inflammation would activate interactions between the different cells: endothelium, granulocytes, monocytes and platelets. Several pharmacological models have been developed for the analysis of these data including exploration of the permeability and capillary resistance and rheological analysis. Objective observation of the microangiopathy with capillaroscopy, together with modern haemodynamical, biological and pharmacological methods are essential for a better understanding of microvascular disorders in chronic venous insufficiency.

Capillary Permeability↗
PubMed1994

Effects of tiludronate on bone mass, structure, and turnover at the epiphyseal, primary, and secondary spongiosa in the proximal tibia of growing rats after sciatic neurectomy.

To evaluate the effects of tiludronate on the mass, structure, and turnover of cancellous bone regions in immobilized rat tibiae, we performed a 4 week dosing experiment. The right hindlimbs of 84 Sprague-Dawley rats (5 weeks old) wee neurectomized or sham operated. Animals were assigned to seven groups (n = 12 each); group 1 was sham operated, and groups 2-7 were neurectomized. Groups 1 and 2 were given vehicle only (distilled water), and groups 3, 4, and 5 were given tiludronate orally at doses of 25, 50, and 100 mg/kg body weight (BW)/day, respectively, throughout the experimental period. Group 6 was given 100 mg/kg BW/day of the agent for the first 2 weeks only, and group 7 received vehicle only for the first 2 weeks and then 100 mg/kg BW/day of the agent for the last 2 weeks. After tetracycline labeling was performed, the right tibiae were removed from the animals and processed to yield undecalcified sections. Histomorphometry was performed in the epiphyseal, primary, and secondary spongiosa of the proximal tibia. In group 2, trabecular bone volume (BV/TV) and trabecular number (Tb.N) were significantly decreased in the primary and secondary spongiosae, but this did not occur in the epiphyseal spongiosa. Osteoid surface (OS/BS) was decreased and osteoclast surface (Oc.S/BS) was increased in the secondary spongiosa. Tiludronate increased BV/TV and Tb.N in the primary spongiosa by reducing the values for the parameters of osteoclast surface (Oc.S/BS) and osteoclast number (Oc.N/BS). Osteoid surface in this region was not decreased by the agent. In groups 4 and 5, tiludronate prevented bone loss in the secondary spongiosa by reducing both OS/BS and Oc.S/BS. In group 6, BV/TV in the primary spongiosa was maintained at the level of group 1, but Oc.S/BS and Oc.N/BS were elevated. In the secondary spongiosa, bone mass was preserved and the reduction in these parameters was maintained. In group 7, however, BV/TV was increased in the primary spongiosa as a result of a reduction in osteoclastic resorption; in the secondary spongiosa, however, BV/TV was decreased and trabecular turnover was not reduced at the end of the experiment in these growing animals. Mineral apposition rates were not reduced by tiludronate. This study clearly demonstrated that this agent prevented immobilization bone loss by inhibiting resorption.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1994

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy.

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4+ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluorescence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37 degrees C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37 degrees C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1) T = 0, continuous peripheral staining; (2) T = 15 min, patchy peripheral staining; (3) T = 30 min, patches or intracellular vesicular staining; (4) T = 45 min, intracellular vesicular staining; (5) T = 60 min, diffuse intracellular staining; (6) T = 90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1992

Longitudinal effect of tiludronate on bone mineral density, resonant frequency, and strength in monkeys.

The effect of Tiludronate on bone was studied in 72 growing monkeys (Papio papio), 36 males and 36 females, aged 4-7 years. They were randomly allocated into four groups (18 animals per group, 9 males and 9 females): group I, controls; group II, 10 mg/kg/day; group III, 20 mg/kg/day; and group IV, 40 mg/kg/day of Tiludronate. A total of 12 animals (6 males and 6 females) in each group were sacrificed at the end of treatment (1 year) and 6 animals (3 males and 3 females) per group 1 year later. Bone mineral density (BMD) was measured by dual-photon absorptiometry. Biomechanical properties were evaluated by an impact torsion test and by resonant frequency analysis. Bone mineral measurements indicated that at the end of 1 year of treatment BMD was significantly higher, especially at the distal epiphysis of the radius, than in controls. No significant differences between groups were found in BMD 1 year after stopping treatment. Biomechanical analyses indicated that torsional stiffness increased after treatment. No differences between groups were found 1 year after stopping treatment. Results of resonant frequencies indicated an increased calculated transversal stiffness after treatment and 1 year later and an increased buckling strength 1 year after stopping treatment. In conclusion, the results on the effect of Tiludronate in growing monkeys indicate a profound effect of this drug on bone density and biomechanical properties. The biomechanical results indicate that this drug is safe, with conservation of bone strength despite a change in intrinsic mechanical properties of the bone.

Animals↗
PubMed1992

Effect of substance P and Sar9Met(O2)11-substance P on cutaneous capillary permeability in guinea pig skin.

The purpose of this study was to assess the effect of several drugs on the increase in cutaneous capillary permeability induced by intradermal injection of substance P (SP) and Sar9Met(O2)11-SP in guinea pig skin. On the one hand, the increase in cutaneous capillary permeability was partly reduced by spantide, promethazine, atropine or SR 40037, an inhibitor of the angiotensin-converting enzyme. On the other hand, norepinephrine and B3824, a B2-antagonist of bradykinin, showed an enhancing effect. Our results suggest that the effect of SP and Sar9Met(O2)11-SP in guinea pigs is partly mediated by histamine and acetylcholine, and that there is a relationship between tachykinins and bradykinin.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1991

Effect of Endotelon (procyanidolic oligomers) on experimental acute lymphedema of the rat hindlimb.

Endotelon (Procyanidolic Oligomers, Sanofi-Labaz Laboratories) are recognized "angioprotectors" for human venous insufficiency. Using an operative experimental model of lymphedema, we sought pharmacological evidence for potential lymphagogue activity of Endotelon. After surgical interruption of hindlimb lymphatics, rats developed for 7 days, peripheral edema that represented a mean 24% increase in volume compared to the nonoperated hindlimb (control). In this experimental counterpart of acute lymphedema, Endotelon (400mg/kg/day p.o.) administered "prophylactically" (i.e., before, during, and 7 days after development of lymphedema) decreased rat hindlimb lymphedema by approximately 4.7%. When administered just before lymphedema became established (i.e., curatively), Endotelon and Coumarin (another "angioprotector") failed to affect hindlimb edema. These results suggest that Endotelon may reduce or prevent postoperative acute edema.

Acute Disease↗
PubMed1990

Selective vulnerability in the gerbil hippocampus: morphological changes after 5-min ischemia and long survival times.

The morphology of the hippocampus of Mongolian gerbils was investigated by light and electron microscopy after 5-min forebrain ischemia and survival times of up to 10 months. After 3 weeks recirculation only 5.8% of pyramidal neurons of the CA1 (cornu ammonis 1) sector had survived but the thickness of the inner and outer hippocampal layers did not change. After recirculation times of 6 and 10 months the number of surviving neurons declined no further but all layers of the CA1 subfield shrank markedly. Ultrastructurally, many but not all surviving CA1 neurons were altered. After 3 weeks both "dark" and "pale" type neurons were present, while after 6 and 10 months only the "pale" type of injury persisted. Axonal enlargements and myelin breakdown were observed at all survival times up to 10 months of recirculation. The astrocytes of CA1 sector contained numerous glial fibrils which were most pronounced after the longer recirculation times. The stratum radiatum presented intact presynaptic terminals densely packed with an abundance of clear vesicles even after survival of 10 months. Initially, morphologically damaged postsynaptic structures were still attached to these terminals but they disappeared after longer recirculation times. However, even after 10 months some intact synapses were observed involving dendrites which probably originated from surviving CA1 neurons. In CA3 sector and dentate gyrus no ultrastructural changes occurred at any survival time.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effect of (chloro-4-phenyl) thiomethylene bisphosphonic acid (SR 41319) on the autoimmune disease activity in MRL/l mice.

MRL - lpr/lpr (MRL/l) mice spontaneously develop an autoimmune pathology including arthritic lesions. SR 41319, a bisphosphonate, having previously shown to be active in in vitro and in vivo models of arthritis, the aim of this study was to search for its possible effect on the pathology of the MRL/l mice. Results showed that SR 41319 reduced the severity of the disease in its early stages and increased mean life span. Further investigation would be necessary to define the effect of the drug during later stages when major changes in immune status occurred.

Animals↗