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Biomedical subjects

J Maclouf

Publications and source records attributed to J Maclouf.

At least 91 records · Page 5Linked to original sources
PubMed1994

Fate of etiproston, a synthetic analogue of PGF2 alpha, in cows.

The pharmacokinetics and metabolic fate of labelled compounds were investigated after intramuscular administration of 3H-radiolabelled etiproston to nine cows. Elimination was rapid (t 1/2 beta = 2.8 h). Forty-eight h after administration 92.6% of the radioactivity had been eliminated, mainly via the urinary (66% at 48 h) and faecal routes (26% at 48 h). In comparison, little elimination in milk occurred (less than 0.034% dose/l by 24 h). Radioactivity at the injection site 48 h after administration was seen in one cow (< 4.68 x 10(-5%) dose/g). No radioactivity was detected in the tissues. Urinary metabolites were purified and isolated using XAD-2 extraction and preparative HPLC in reverse and normal phases. The main urinary metabolite, identified by mass spectrometry, was the tetranor acid derivative in equilibrium with its lactone form.

Animals↗
PubMed1994

Strain-dependency of leukotriene C4 generation from isolated lungs of immunized mice.

1. The antigen-induced leukotriene C4 (LTC4)-like-material release from isolated perfused lungs of actively sensitized Swiss, Balb/C and CBA/J mice was compared. The intra-tracheal (i.t.) instillation of 1 and 100 micrograms ovalbumin to lungs from Swiss mice was followed by a dose-dependent generation of LTC4-like material into the effluent, as detected by radio-immunoassay and h.p.l.c., followed by an enzyme-immunoassay. In contrast, lungs from sensitized Balb/C and CBA/J mice failed to exhibit LTC4-like-material release in amounts above the basal values. No histamine secretion was observed when lungs of the three strains of mice were challenged with ovalbumin. 2. The i.t. instillation of 1 or 10 micrograms platelet-activating factor (PAF) or of 100 micrograms arachidonic acid to lungs from non-sensitized mice, induced the release of comparable amounts of LTC4-like-material in the effluent, irrespective to the strain. However, N-formyl-L-methionyl-L-leucyl-L- phenylalanine (fMLP, 0.1, 10 micrograms), was more effective in inducing the release of LTC4-like-material from lung from Swiss and CBA, than from Balb/C, mice. 3. The intraplantar injection of 0.01 microgram ovalbumin to sensitized Swiss mice induced an intense oedema formation, as measured plethysmographically, while Balb/C mice required a dose of antigen at least 10 fold higher for a similar response. CBA/J mice did not respond to antigen challenge in terms of oedema formation. The intraplantar injection of PAF or fMLP to non-immunized mice induced an oedema of similar intensity in all the strains considered. Accordingly, the different responses to ovalbumin of the three strains of mice is not accounted for by different paw responsiveness to inflammatory mediators.4. Swiss and CBA/J mice exhibited higher titers of circulating IgG antibodies, as measured by passive cutaneous anaphylaxis (PCA), than Balb/C mice. Conversely, lower IgE titers were measured in the serum of sensitized Swiss and CBA/J mice, as compared to Balb/C.5. Our results demonstrate a strain-dependency of antigen-induced LTC4 release from lungs from sensitized mice. This difference is related to the ability of sensitized animals to develop immediate hypersensitivity responses, such as paw oedema formation, but not to the antibody subclass involved in the immunization. Strain-dependent factors may influence the intensity of the response to antigen stimulation. It is thus essential to characterize the different components of the immune response when mouse models for studying hypersensitivity reactions are developed.

Animals↗
PubMed1994

Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells.

Activation of the gene for inducible cyclooxygenase (cyclooxygenase-2 [Cox-2], prostaglandin endoperoxide synthase) is an early response to injury in vascular smooth muscle cells. We used in vitro and in vivo models to demonstrate that activation of quiescent smooth muscle cells by mitogens leads to a rapid, short-term rise in mRNA for Cox-2, followed by synthesis of new Cox-2 enzyme protein and a marked increase in prostaglandin production that depends on new enzyme synthesis. Moreover, the Cox-2 mRNA response observed in smooth muscle cells differs in both time and degree, depending on the particular mitogenic stimulus. Serum, platelet-derived growth factor, epidermal growth factor, and thrombin are strong inducers of Cox-2 mRNA, whereas acidic and basic fibroblast growth factors and interleukin-1 alpha are weak inducers. In contrast to the transient activation of Cox-2 in vitro after introduction of a mitogenic stimulus, the Cox-2 response after in vivo vascular injury extends over many days and may represent protracted cellular activation. During induction of Cox-2 message and protein, expression of constitutive cyclooxygenase (Cox-1) remains unchanged, however. These data suggest a pathophysiological role for Cox-2 in the early modulation of vascular responses to injury.

Angioplasty, Balloon↗
PubMed1994

Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases.

The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2.

Cyclooxygenase (Cox) exists in two forms in human endothelial cells (HUVEC). We have raised antibodies that recognize the sequence of the carboxyl-terminal portion of the human Cox-2 (C)-NASSSRSGLD-DINPTVLLK. Cyclooxygenase activity of HUVEC challenged with interleukin 1 alpha or a phorbol ester increased in parallel with the mass of a protein doublet analyzed by Western blot using antibodies directed against the Cox-2 peptide; a monoclonal antibody directed against Cox-1 showed a small change in protein mass. A 35S-labeled protein doublet with a molecular mass of approximately 70,000 daltons was immunoprecipitated with the anti-Cox-2 antiserum in L-[35S] methionine-labeled cells stimulated with interleukin 1 alpha. This protein was not recovered by pretreating the antiserum with the Cox-2 peptide before immunoprecipitation. A minor variation in 35S-immunoprecipitated protein was obtained with the polyclonal anti-Cox-1 antibody. Both immunoprecipitated Cox-1 and Cox-2 possessed cyclooxygenase activity that was inhibited by flurbiprofen. Endoglycosidase H treatment of immunoprecipitated Cox-2 proteins caused a decline in the apparent molecular size similar to that observed with immunoprecipitated Cox-1 or sheep cyclooxygenase but did not suppress the doublet. These results show by direct protein measurement that HUVEC synthesize the novel Cox-2 under appropriate stimulation, with little changes of Cox-1.

Amino Acid Sequence↗
PubMed1993

Antiphospholipid antibodies enhance thrombin-induced platelet activation and thromboxane formation.

In a group of 6 patients with lupus anticoagulant (LA) and antiphospholipid (aPL) antibodies detected by ELISA overnight urine and blood were simultaneously collected. A significantly increased urinary excretion of the platelet-derived thromboxane (TX) metabolite 11-dehydro-TXB2 was found in this group, as compared to 12 healthy individuals. In contrast, a small but significant reduction of the vascular prostacyclin (PGI2) metabolite 2,3-dinor-6-keto-prostaglandin F1 alpha was observed. To further elucidate the effect of these antibodies on platelet activation we isolated the F(ab')2 fragments from IgG of the 6 patients and 5 controls, and we evaluated the effect of these fragments on the responses of isolated normal platelets to thrombin. Patients' F(ab')2 increased platelet aggregation and serotonin release of platelets stimulated by low dose thrombin (0.01 U/ml). At threshold thrombin concentration (0.05 U/ml) an enhanced TXB2 production was also observed. In summary, our results show, in addition to the altered TXA2/PGI2 balance observed in vivo, a direct stimulatory effect of aPL antibodies on platelet activation in vitro. This effect is related to recognition of phospholipid epitopes on platelets as shown by its neutralization upon preincubation with phospholipids. This phenomenon may be relevant for the thrombotic tendency of these patients.

Adult↗
PubMed1993

Enzyme immunometric assay for endothelin using tandem monoclonal antibodies.

Seven distinct mouse monoclonal antibodies (mAbs) directed against human endothelin-1 (ET-1) have been obtained. On the basis of specificity studies performed with competitive immunoassays and of complementary binding studies, these mAbs were classified in two groups. mAbs of group A (Endo-4, -5, -6 and -10) were shown to be directed against the N terminal loop while those of group B (Endo-2, -8 and -18) recognized the C terminal part of the peptide. A pair of monoclonal antibodies with optimal properties for a two-site immunometric assay were selected and the test was performed in 96-well microtiter plates coated with one mAb (Endo-18), while another mAb (Endo-4) covalently labeled with enzyme acetylcholinesterase was used as tracer. Under optimal conditions, the assay appeared to be very sensitive since concentrations as low as 1 pg/ml could be significantly detected. The precision was also very good with a coefficient of variation below 10% from 3 to 250 pg/ml. The assay was specific for mature endothelin presenting no cross-reactivity with the precursor Big ET-1. On the other hand, strong cross-reactivity was observed with other ET-1-related peptides, including ET-2, ET-3, VIC peptide and sarafotoxin 6-b. The assay permitted specific determination of ET-1 in supernatants of cultured endothelial cells and the validity of the test was demonstrated by HPLC fractionation experiments. In addition, the assay also appeared to be suitable for direct determination of ET-1 in plasma. Studies performed with plasma from healthy subjects revealed that circulating levels of ET-1 are below or close to the detection limit of the method (< 8 pg/ml).

Amino Acid Sequence↗
PubMed1993

Transcellular biosynthesis of arachidonic acid metabolites: from in vitro investigations to in vivo reality.

The discovery that arachidonic acid metabolism in a multicellular environment could be different from that expected from the sum of individual cell types has led to the concept of transcellular metabolism. In this process, several cells can contribute to the formation of a novel compound with potent biological action. The study of this mode of synthesis is important in the context of the current appraisal of thrombotic diseases as part of an inflammatory reaction. In this context, blood cell-vessel wall interactions present a regulated expression of adhesive molecules on either type of cell. These complex processes are initiated by signalling molecules such as cytokines that can deeply modify the phenotype of endothelial cells, which may ultimately lead to a change in the vascular tone and to atherosclerotic complications. Such reaction processes are part of the autocrine-endocrine system whereby cells can control and modify their own phenotype through the action of a local network of mediators. In this context, arachidonic acid metabolites may be an important part of unifying signal molecules that participate in these changes. The significance of transcellular biosynthesis where combined cells acquire a different metabolic potential can be viewed as an additional modification of blood cell and vessel cell phenotype in thrombotic diseases.

Animals↗
PubMed1993

Formation of sulphidopeptide-leukotrienes by cell-cell interaction causes coronary vasoconstriction in isolated, cell-perfused heart of rabbit.

1. We have studied the transcellular biosynthesis of bioactive leukotrienes (LTs), generated upon blood cell-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. Rabbit isolated hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) cells of unpurified (buffy coat) or purified human neutrophils (PMNL), and challenged with 0.5 microM A23187 for 30 min. Coronary perfusion pressure (CPP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and left ventricular pressure (LVP) were monitored continuously. Leukotriene formation was measured by specific enzyme-immunoassay and confirmed by reversed phase h.p.l.c. and u.v. spectral analysis. 2. Basal CPP values averaged 44 +/- 1.4 mmHg; A23187 triggered a marked increase in CPP both in the presence of buffy coat cells (+100% above basal) and PMNL (+270% above basal); the latter change in CPP was accompanied by a rise in LVEDP (+138% above basal). 3. The increase in CPP was preceded by a statistically significant rise in iLTC4-D4 concentration in the circulating buffer. Pretreatment with two structurally unrelated LTD4 receptor antagonists, LY171883 and SKF104353 (10 microM), fully prevented the increase in CPP and LVEDP. A similar protection was also observed when the rabbit heart was perfused with PMNL that had been pretreated with MK886 (1 microM), a potent inhibitor of leukotriene biosynthesis. 4. The increased coronary tone was accompanied by a marked release of lactate dehydrogenase (LDH), a marker of ischaemic damage; pretreatment of the heart with the LTD4 receptor antagonists as well as of the PMNL with MK886 resulted in a complete suppression of LDH activity release. 5. Positive identification of LTC4-D4 in the perfusates was obtained and a significant correlation observed between the CPP values and iLTC4-D4 concentrations.6. This study suggests that challenge of PMNL present within the coronary vasculature, causes a LTD4-dependent coronary vasoconstriction, favoured by an efficient uptake of PMNL-derived LTA4 by endothelial cells. The activation of the 5-lipoxygenase pathway in the context of tight interactions between blood cells and coronary vasculature, is suggested to have an important outcome in the alterations of coronary flow and cardiac contractility.

Animals↗
PubMed1993

Increased in vivo production of thromboxane in patients with sickle cell disease is accompanied by an impairment of platelet functions to the thromboxane A2 agonist U46619.

Thrombosis represents an important cause of mortality in patients with sickle cell disease, in addition to the complications caused by the primary defect of inherited abnormal hemoglobin. To study the involvement of platelets in these complications, we assessed the biosynthesis of thromboxane A2 in samples from 49 patients with sickle cell disease and in 33 control subjects. The urinary excretion of the major arachidonic acid metabolite of platelet origin (11-dehydro-thromboxane B2) and of the vascular endothelial cell (2,3-dinor-6-ketoprostaglandin F1 alpha) were very significantly increased (p < 0.0002) in the patients. In a small group of patients (n = 14), we further investigated the ex vivo response of their platelets to U46619, a stable analogue of thromboxane A2. We observed decreased aggregation and [14C]serotonin release compared with control (p < 0.05); similarly, we found impaired p47 protein phosphorylation (p < 0.05). In contrast, platelets from these patients responded normally to thrombin (0.1 unit/mL). In vivo desensitization of platelets from these patients to thromboxane may constitute a form of regulation that may prevent the propagation of aggregation by this potent inducer, as has been hypothesized in in vitro studies. Our results may also provide a rationale for using antiplatelet drugs in the prophylaxis of thrombotic complications in sickle cell patients.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

A chronic hypercoagulable state and life-long platelet activation in beta thalassemia major.

Increased frequency of thromboembolic events has been recently observed in patients with thalassemia major (TM), causing hypoxemia and cor pulmonale. Autopsy findings demonstrated "old" and recent pulmonary and renal infarcts as well as premature atherosclerosis. Studies to determine hypercoagulability showed: impaired platelet aggregation, increased circulating platelet aggregates, shortened platelet survival, enhanced excretion of urinary metabolites of thromboxane A2 (TXA2) and prostacyclin and decreased plasma levels of Protein C, Protein S or anti-thrombin III. Erythrocytes from TM patients enhanced thrombin formation in a "prothrombinase" assay (using a chromogenic substrate). Chronic anti-thrombotic therapy may be indicated in thalassemic patients to prevent the cardiac and pulmonary complications.

Blood Platelet Disorders↗
PubMed1992

Reversible membrane association of neutrophil 5-lipoxygenase is accompanied by retention of activity and a change in substrate specificity.

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.

Arachidonate 5-Lipoxygenase↗
PubMed1992

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations.

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Arachidonate 5-Lipoxygenase↗
PubMed1992

Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation.

The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Increased production of platelet-derived thromboxane in patients with lupus anticoagulant.

The in vivo production of thromboxane A2 and prostacyclin was assessed in 31 samples from 25 patients with lupus anticoagulant and in 32 controls. The urinary excretion of 11-dehydro-thromboxane B2 (a major thromboxane metabolite of platelet origin) was very significantly increased (p less than 0.0003) in the patients contrasting with a lesser increase of urinary 2,3-dinor-6-keto-prostaglandin F1 alpha reflecting the vascular production of prostacyclin (p less than 0.02). Our study shows that in patients with lupus anticoagulant, platelet activation may occur without a compensatory increment in the vascular biosynthesis of prostacyclin suggesting an increased risk for thrombosis.

Adult↗