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Biomedical subjects

M Berard

Publications and source records attributed to M Berard.

At least 19 recordsLinked to original sources
PubMed2003

Shear stress modulates tumour cell adhesion to the endothelium.

The adhesion of breast adenocarcinoma cells (MDA-MB-231) to human umbilical vein endothelial cells (HUVEC) was studied in whole blood and under varying flow conditions. This study was done on HUVEC either kept under static conditions or pre-conditioned in flow for 2 hours at a shear stress of 5 or 13 dyn/cm(2). Coverslips coated by HUVEC were placed in a parallel plate perfusion chamber and perfused at a shear rate of 300 or 1500 sec(-1) with heparin-anticoagulated blood containing 111In labelled MDA-MB-231 cells. We report here the optimal conditions for studying the adhesion of MDA-MB-231 to endothelial cells under shear constraints corresponding to those observed into small and medium sized arteries.

Adenocarcinoma↗
PubMed2001

[An appreciation of maternal smoking with high-performance liquid chromatographic determination of maternal and neonatal urinary cotinine].

OBJECTIVE: To study the correlation of urinary cotinine levels in mothers and newborns with the number of cigarettes smoked at the end of pregnancy. Population and methods. We recorded the smoking habits of 123 mothers attending a university maternity clinic and measured urinary cotitine levels in mothers and their newborns. All mothers were Europeans and gave birth to a normal full-term (37 weeks gestation) infant. Cotinine levels were measured with high-performance liquid chromatography from urine samples taken during the 6-hour period prior to or after delivery for the mothers and 24-h after birth for the newborns. RESULTS: The average cotinine level for non-smoking mothers, for those who smoked one to nine cigarettes a day and heavy smokers (ten or more cigarettes per day) were 0.21, 2.17 and 4.28 mol/l respectively (p<0.001). The average levels in their newborns were 0.04, 0.39 and 1.36 mol/l respectively (p<0.001). Thirteen percent of the mothers who claimed they did not smoke had cotinine levels higher than the significance cut-off (0.3 mol/l). There was a significant correlation 1) between the number of cigarettes the mothers stated they smoked at the end of pregnancy and their urinary cotinine concentrations (cotinine level=0.213 + 0.349 cigarettes, r=0.78, p<0.001); 2) between the number of cigarettes smoked and newborn's urinary cotinine concentration (cotinine level=0.002 + 0.104 cigarettes/day, r=0.81, p<0.001); and 3) between the mother's and the newborn's urinary cotinine concentrations (newborn cotinine=0.027 + 0.219 maternal cotinine, r=0.77, p<0.001). CONCLUSION: The number of cigarettes smoked at the end of pregnancy accounts for roughly 50% of the variance in the mother's urinary cotinine level and that in her newborn at birth. The urinary cotinine concentration in newborns is 3 to 5 times lower than that of their mothers. A woman smoking 3 cigarettes per day has a urinary cotinine concentration of 1 mol/l. The urinary cotinine level in newborns is 1 mol/l for mothers smoking 10 cigarettes per day.

Chromatography, High Pressure Liquid↗
PubMed2000

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells.

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.

Apoptosis↗
PubMed2000

On the assessment of drug metabolism by assays of codeine and its main metabolites.

Codeine and its main metabolites appear to have advantages for assessing drug metabolic phenotypes. The authors have further developed a high-performance liquid chromatography (HPLC) method for the quantification of codeine and six of its metabolites in urine. Quantification was performed by electrochemical detection for morphine, normorphine, morphine-6-glucuronide, and the internal standard 4-O-methyldopamine; and by ultraviolet detection for codeine, norcodeine, and morphine-3-glucuronide. The method had a detection limit of 2 nmol/L(-1) for morphine and normorphine, 4 nmol/L(-1) for morphine-6-glucuronide, 3 nmol/L for the internal standard, 20 nmol/L(-1) for morphine-3-glucuronide, and 60 nmol/L(-1) for codeine and norcodeine. The coefficients of variations were <9% for intraday and <10% for interday analyses. The recovery of codeine and its metabolites ranged from 55% (for morphine-3-glucuronide) to 90% (for codeine, norcodeine, morphine, and morphine-6-glucuronide). Eleven healthy volunteers were phenotyped for CYP2D6 using codeine as well as debrisoquine and dextromethorphan. Ten subjects were extensive metabolizers (EM) and one a poor metabolizer (PM) of codeine, debrisoquine, and dextromethorphan. Significant correlations between the metabolic ratios (MRs) of the different probe drugs were obtained (r2 > 0.95, p < 0.001). This HPLC method is simple, sensitive, accurate, and reproducible for assessing the CYP2D6 phenotype.

Adult↗
PubMed1999

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.

Apoptosis↗
PubMed1999

Activation sensitizes human memory B cells to B-cell receptor-induced apoptosis.

The outcome of antigen receptor (B-cell receptor; BCR) ligation on B-cell survival can be influenced by multiple parameters. They are linked to the physical properties of the antigen itself, the maturational stage of the cells and the costimuli provided by different components of the innate and acquired immunity. Here we report that apoptosis prevails over stimulation when a BCR agonist is applied to human memory B cells which have been preactivated by CD40 ligand or anti-immunoglobulin antibodies. The susceptibility of activated memory B cells to BCR-induced killing is correlated with their enhanced expression of the transcripts encoding the pro-apoptotic molecules Bax, c-Myc and p53. The BCR-mediated apoptosis of activated memory B cells does not require extensive cross-linking of the antigen receptors and relies neither on engagement of the FcgammaRII nor on the Fas/Fas ligand (Fas-L) system. Our findings suggest that activation stimuli open the BCR-induced apoptotic pathway in memory B cells. Therefore we propose that the concept of activation-induced cell death (AICD), originally described for T cells, also applies to mature B lymphocytes. The functions fulfilled by the AICD of mature B cells in the regulation of B-cell responses are discussed.

Antibodies, Anti-Idiotypic↗
PubMed1998

Human neutrophil elastase in temporal (giant cell) arteritis: plasma and immunohistochemical studies.

OBJECTIVE: Few enzymes are able to attack the internal elastic lamina, which is destroyed in temporal arteritis (TA). Because human neutrophil elastase (HNE) is one of these, its role in the pathogenesis of TA was examined in patients undergoing temporal artery biopsy for suspected TA. METHODS: Over a 6 month period, 33 patients undergoing temporal artery biopsy were prospectively included in the study. TA was diagnosed in 15 patients; 9 of them had positive temporal artery biopsy. The other 18 patients made up the non-TA group. Nineteen healthy age matched subjects (mean age 74 +/- 9 yrs) served as controls. Levels of plasma HNE bound to alpha1-antitrypsin (pHNE-alpha1AT) were measured by ELISA. The presence of HNE in the temporal artery wall of 7 TA and 7 non-TA patients was evaluated immunohistochemically. RESULTS: Age, neutrophil counts, and erythrocyte sedimentation rates were similar in TA and non-TA patients. The mean pHNE-alpha1AT concentration in the TA group (84 +/- 20 microg/l) was significantly higher (p < 0.001) than in the non-TA group (51 +/- 26 microg/l) or in healthy controls (52 +/- 23 microg/l). The diagnostic sensitivity of pHNE-alpha1AT > 50 microg/l was 100%. Immunohistochemistry detected no HNE within the temporal artery wall of any patient. CONCLUSION: High levels of pHNE-alpha1AT were associated with TA. Our preliminary results indicate this could be a diagnostic marker for TA. Further studies are needed to confirm its reliability. Because HNE was not detected locally, no conclusions can be drawn as to its pathogenic role in TA.

Aged↗
PubMed1997

Antigen receptor-induced apoptosis of human germinal center B cells is targeted to a centrocytic subset.

The outcome of the signals transduced through the B cell antigen receptor (BCR) depends both on their maturational stage and on the extent of receptor cross-linking. It is established that the BCR-mediated apoptosis of immature B cells represents an important mechanism for tolerance induction in the pre-immune B cell compartment. We show here that mature germinal center (GC) B cells can re-acquire sensitivity to BCR-induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor-triggered apoptosis upon CD40 stimulation, suggesting that this phenomenon may play a role in the shaping of the mature B cell repertoire in GC. Our data reveal that the death signal evoked through the BCR does not involve the Fcgamma receptors, does not operate through the Fas/Fas ligand system, and can be blocked by interleukin-4. Finally, we found that the acquisition of sensitivity to the death-promoting effect of anti-Ig antibodies in CD40-stimulated GC B cell cultures correlates with the induction of a centrocytic phenotype. We propose that negative regulatory signals via the BCR may delete somatically mutated centrocytes with self-reactivity.

Antibodies, Monoclonal↗
PubMed1997

Thrombomodulin, a functional surface protein on human keratinocytes, is regulated by retinoic acid.

Thrombomodulin, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a cofactor for protein C activation. It has previously been shown that thrombomodulin, present in human epidermis and in lysates of cultured keratinocytes, is implicated in cellular differentiation during mouse fetal development. The role of retinoic acid in keratinocyte differentiation prompted us to study retinoic acid regulation of thrombomodulin expression in primary cultures of keratinocytes isolated from adult human skin, grown at low (undifferentiated keratinocytes) and normal calcium levels (differentiated keratinocytes). Thrombomodulin antigen levels and total and surface activities were measured in cultures without and with retinoic acid. Thrombomodulin mRNA visualized by in situ hybridization was quantified by computer-based image analysis. Functional thrombomodulin was expressed on the surface and in the cytoplasm of cultured human keratinocytes regardless of the calcium concentration. In contrast, retinoic acid induced significant increases in the total antigen level and in surface and intracellular thrombomodulin activities only in keratinocytes grown in a low-calcium medium. In these undifferentiated keratinocytes, quantification of mRNA transcripts showed a threefold increase after retinoic acid stimulation. Thus, functional thrombomodulin is a human keratinocyte surface protein whose expression is controlled through the keratinocyte differentiation program and is modulated in vitro by retinoic acid.

Adult↗
PubMed1997

Thrombomodulin is synthesized by human mesangial cells.

Thrombomodulin (TM), an endothelial receptor for thrombin, endowed with a powerful anticoagulant activity, plays an important role in the antithrombogenicity of the vascular endothelium. Its presence within the human renal glomerulus is already known but was thought to be only endothelial. We looked for TM expression in human mesangial cells (MC), both in situ, in freshly prepared glomeruli, and in primary culture. Both fresh and cultured MC were strongly reactive for TM by immunocytochemical methods. Total TM antigen measured on MC lysates and surface TM activity on MC were 0.292 +/- 0.075 ng/mg of cellular proteins and 1.20 +/- 0.02 pmole of activated protein C/min/mg of cellular proteins, respectively. As shown by the presence of numerous transcripts detected by in situ hybridization, TM was shown to be synthesized by MC in vivo and in culture. The synthesis of active TM by both endothelial and mesangial cells within the renal glomerulus stresses the importance of its role in maintaining renal hemostatic equilibrium, and sheds some light on the conflicting reports of TM over- and underexpression in glomerulopathies to open a new field for investigation.

Antigens↗
PubMed1997

Vascular origin determines angiotensin I-converting enzyme expression in endothelial cells.

Previous observations on the heterogeneous distribution of von Willebrand factor in the vascular endothelium led us to examine the expression of angiotensin I-converting enzyme (ACE) in function of the vascular origin of endothelial cells (EC). EC from pig thoracic aorta, pulmonary artery, inferior vena cava and brain capillaries were cultured and assayed for ACE by enzymatic radiochemical determination and by western-blot and immunofluorescence using an antiACE polyclonal antibody. EC from the various vascular levels secreted ACE in the culture medium; western-blot analysis showed its presence at cellular level and immunofluorescence confirmed its location on the plasma membrane. But quantification revealed that EC from pulmonary artery contain more ACE than EC from the other vessels, especially from brain capillaries; immunofluorescence correlated well with the functional data. In contrast, secretion of ACE by brain capillaries EC was faster than that of arteries and of vena cava, the latter being the less effective. This differential ACE expression along the vascular tree could have a pharmacological implication since ACE inhibitors, used in the treatment of arterial hypertension, may act more at the vascular level than on the plasma renin-angiotensin system. On the other hand, endothelial distribution of ACE was different from that of von Willebrand factor; in particular we showed that EC cultured from vessels of pigs homozygous for the von Willebrand disease, in which von Willebrand factor synthesis was completely abolished, normally express ACE.

Animals↗
PubMed1997

Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas.

Neonatal hemangioma is a common benign proliferation of unorganized structures containing stromal and capillary endothelial cells. We tested the hypothesis that such cell proliferation might result from the release by stromal cells of endothelial cell mitogens. Stromal cells cultured from biopsies of surgically removed life-threatening hemangiomas released an endothelial cell mitogen in vitro that was indistinguishable from vascular endothelial growth factor (VEGF) based on independent criteria such as affinity chromatography for heparin or anti-VEGF IgG and radioreceptor assay. A functional product of the KDR gene encoding a cognate VEGF receptor was also expressed by these stromal cells. Transient transfection with antisense oligonucleotides targeted on the translation initiation codon of KDR abolished its tyrosine phosphorylation and mitogenic response of neonatal hemangioma cells to VEGF, confirming the existence of an autocrine loop of proliferation. When grafted in nude mice, these stromal cells elicited an angiogenic response that was blocked by neutralizing anti-VEGF IgG. These results might provide a clue to the importance of stromal cells in the pathogeny of neonatal hemangiomas.

Animals↗
PubMed1996

Advantage of using both anionic and zwitterionic phospholipid antigens for the detection of antiphospholipid antibodies.

To investigate the benefit of assaying for antiphospholipid antibodies (aPA) with different antigenic specificities, sera from 141 patients suspected of having aPA were tested by ELISA for IgG and IgM antibodies directed against the following phospholipids (PL) coated individually or together: cardiolipin, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine. Nonspecific background optical density (OD) was systematically subtracted from the test OD value. Positive reactions were defined as having an OD greater than the 97th percentile OD distribution obtained with sera from 100 healthy individuals. Although the majority of the 79 detected aPA (89% IgG and 77% IgM) were polyspecific, 11 reacted with a single PL and, moreover, belonged to only one isotype. Seven of these 11 patients presented recurrent fetal losses or thrombotic events. These results suggest that routine use of a mixture of both anionic and zwitterionic PL antigens to coat ELISA plates would better detect aPA involved in suggestive pathologies and enhance the ability to identify patients with these mono- or polyspecific antibodies directed or not against cardiolipin, the current standard.

Antibodies, Antiphospholipid↗
PubMed1996

Antiphosphatidylethanolamine antibodies as the only antiphospholipid antibodies. I. Association with thrombosis and vascular cutaneous diseases.

OBJECTIVE: To detect antiphosphatidylethanolamine antibodies (aPE) as the only antiphospholipid antibodies (aPL) in 122 patients we investigated for aPL and to correlate the presence of aPL with clinical manifestations. METHODS: Patients with autoimmune diseases (n = 41), thromboembolic episodes (TEE) (n = 34), livedo reticularis (LR) without (n = 17) or with (n = 14) thrombosis or recurrent fetal losses (RFL), systemic vasculitides (n = 10), and miscellaneous disorders (n = 6) were investigated for antibodies directed against 4 anionic phospholipids (PL) (cardiolipin, phosphatidylserine, inositol, phosphatidic acid) and lupus anticoagulant (LAC) and then for aPE by modified ELISA. RESULTS: 15 patients had aPE and no antibodies to anionic PL including LAC. 7 had IgM, 4 had IgG plus IgM, and 4 had IgG. These aPE were significantly more often associated with TEE alone, with TEE and LR, or with LR alone (p = 0.004) than with autoimmune diseases. CONCLUSION: The detection of aPE as the sole aPL in one patient with mesenteric infarcts and RFL led to the diagnosis of primary antiphospholipid syndrome. Followup of 3 patients showed that aPE cannot be considered as transient autoantibodies. Therefore, patients whose clinical symptoms suggest antiphospholipid syndrome but whose sera are negative for antibodies to cardiolipin or another anionic PL should be screened for aPE, particularly patients with thrombosis, RFL, and/or LR.

Adolescent↗
PubMed1996

Antiphosphatidylethanolamine antibodies as the only antiphospholipid antibodies detected by ELISA. II. Kininogen reactivity.

OBJECTIVE: To study the requirement for serum and for low (LMWK) and high molecular weight kininogen (HMWK) and/or HMWK binding proteins to detect antiphosphatidylethanolamine antibodies (aPE) in ELISA. METHODS: Eighteen patients with aPE (9 IgG and 13 IgM) as the only antiphospholipid antibody (aPL) detected by ELISA were assigned to 4 groups: thromboembolic episodes (TEE) (Group I, n = 6); livedo reticularis (LR) without TEE, (Group II, n = 4); both LR and thrombosis (Group III, n = 4); and systemic lupus erythematosus (SLE) or primary antiphospholipid syndrome (APS) (Group IV, n = 4). All sera were analyzed in ELISA with and without bovine serum and with a purified chromatographic fraction containing LMWK, HMWK, and HMWK binding proteins. RESULTS: Eleven aPE were serum dependent: mostly IgG (7/9) and some IgM (4/13). Among the 11 serum dependent aPE, all the 7 IgG and 2 IgM were kininogen reactive. Some serum independent IgM were better detected in the absence than in the presence of serum in the ELISA. CONCLUSION: In the 18 patients, kininogens and/or HMWK binding proteins served as a "cofactor" significantly more often for aPE IgG than for aPE IgM (p = 0.007). Kininogen dependent aPE Ig were observed more often in patients with LR with or without TEE (6/8) than in those with SLE or primary APS (0/4) but this difference merely tended to significance (p = 0.06). In 2 patients, one with TEE, the other with primary APS, the IgM aPE was dependent on a serum "cofactor" that was not kininogen.

Antibodies, Antiphospholipid↗