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D Meyer

Publications and source records attributed to D Meyer.

At least 199 records · Page 11Linked to original sources
PubMed1997

Relative importance of the glycoprotein Ib-binding domain and the RGD sequence of von Willebrand factor for its interaction with endothelial cells.

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the alpha vbeta3 integrin and the RGD sequence of von Willebrand factor (vWF). To define the potential involvement of glycoprotein Ib alpha (GPIb alpha) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet alphaIIb beta3, and deltaA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIb alpha. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to delta A1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 microg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-alpha (TNF alpha), reported to upregulate the expression of the putative endothelial GPIb alpha, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIb alpha, blocking vWF interaction with platelet GPIb alpha, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the alpha vbeta3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to deltaA1-rvWF (50% inhibition at a concentration of 11 and 15 microg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIb alpha-binding domain, we were unable to detect endothelial surface expression of GPIb alpha by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIb alpha mRNA was undetectable in endothelial cells, even after stimulation by TNF alpha. These studies indicate that GPIb alpha is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an alpha vbeta3-dependent, GPIb alpha-independent mechanism.

Antibodies, Monoclonal↗
PubMed1997

A patient with type 2N von Willebrand disease is heterozygous for a new mutation: Gly22Glu. Demonstration of a defective expression of the second allele by the use of monoclonal antibodies.

We report the case of a Chinese patient who has subnormal von Willebrand factor (vWF) level and normal vWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patient's vWF gene were analyzed by DGGE and a G2354 --> A substitution which changes the encoded amino acid sequence from Gly22 to Glu was identified. The patient is heterozygous for this substitution, creating a unique Sac I restriction site. Recombinant vWF (rvWF) containing the candidate mutation was transiently expressed in COS-7 cells. It was processed and secreted normally but failed to bind FVIII. FVIII binding ability of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between patient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antibodies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 418 did not recognize mutated rvWF. Because MoAb 418 also failed to capture the plasma vWF from propositus, it means that his second nonmutated allele is not expressed or expressed at a very low level.

Adult↗
PubMed1997

Primary structure of the propeptide and factor VIII-binding domain of bovine von Willebrand factor.

A 2811 base-pair cDNA, encoding the amino-terminal part of the bovine pre-pro-von Willebrand factor, was characterized and sequenced. The deduced amino acid sequence shares significant homology with the human von Willebrand antigen II and the amino-terminal part of the factor VIII-binding domain of von Willebrand factor. In contrast to human, there is no RGD motif in the bovine von Willebrand antigen II. High levels of Cys, characteristic of D domains, are also found in bovine and the Cys position is markedly conserved between the two species.

Amino Acid Sequence↗
PubMed1997

Protein C, protein S and antithrombin III in children with portal vein obstruction.

BACKGROUND/AIMS: Portal vein obstruction is the most common single cause of portal hypertension in children and its cause is unknown in most instances. The aim of the present study was to evaluate the incidence of protein C, protein S and antithrombin III deficiencies and to screen for possible genetic deficiencies of these proteins. METHODS: A prospective study was undertaken in 20 consecutive children with portal vein obstruction, their parents and 20 matched healthy children. RESULTS: Children with portal vein obstruction displayed a reduction in protein C (p<0.05), protein S (p<0.0001), and antithrombin III (p<0.001) activities as compared with controls. Protein C and protein S activities were below 60% in nine and eleven of the children with portal vein obstruction, respectively, and antithrombin III activities were below or equal to 85% in ten of them. Repeated assay of protein C activity in seven children following a surgical portosystemic shunt showed a decrease as compared with preoperative values. Protein C, protein S and antithrombin III were normal in all parents, except two mothers in whom the levels of protein S activity and protein S antigen were found to be either below or at the lower limit of the normal range. CONCLUSIONS: These results indicate that protein C, protein S and/or antithrombin III deficiencies are frequently found in children with portal vein obstruction but are not likely to be of genetic origin in most cases.

Adolescent↗
PubMed1997

MHC-I antigen expression determines sensitivity of hematopoetic progenitor cells as targets for NK cells.

Hybrid resistance is suggested to be mediated by NK lymphocytes as effector cells. NK cytotoxicity is triggered by specific NK cells receptors. One group of receptors recognizing MHC-I antigens is predominantly transmitting an inhibitory signal into the cell. However, NK cells have not been shown to recognize hematopoetic progenitor cells directly. In these studies we demonstrate that hematopoetic progenitor cells are sensitive targets for NK recognition. NK cytotoxicity is shown to depend on MHC-I antigen expression on these target cells. Bone marrow-derived hematopoetic progenitor cells from a beta 2-microglobulin-deficient mouse strain exhibit a significant increase of cytotoxic susceptibility compared with the wild type control. CFU-assays reveal an almost complete loss of proliferation after coincubating MHC-I-deficient bone marrow cells with NK cells from the wildtype mouse strain. The examination of H-2K in allorecognition of hematopoetic progenitor cells reveals an increased cytotoxicity after treatment of resistant syngeneic hematopoetic progenitor cells with H-2Kb F(ab)2-antibodies. Also masking of allogeneic hematopoetic progenitor cells with anti-H-2Kd F (ab)2-antibodies results in enhanced NK killing. Thus, hematopoetic progenitor cells are sensitive targets for NK cells and MHC-I antigen complex is the critical structure in NK recognition of hematopoetic progenitor cells. This complex mediates resistance of NK-specific lysis of hematopoetic progenitor cells.

Animals↗
PubMed1997

Study of the complex between the contrast agent Iobitridol (Xenetix) and Elastase (PPE): a model for hydrophobic site protection in drug-protein interactions.

PURPOSE: The concept of Hydrophilic Sphere Stabilization, or Hydrophobic Shielding, has been postulated in the synthesis of biocompatible contrast agents in vascular imaging. To improve the safety of these polyiodinated agents, interactions with protein hydrophobic sites in biomacromolecules should be kept as low as possible. In order to evaluate the level of interactions with proteins, we have selected the serine proteinase Elastase, in presence of Iobitridol (Xenetix), as a model. METHODS: The complex between Iobitridol and Pancreatic Porcine Elastase was investigated by X-ray diffraction techniques, on saturated monocrystals, using the synchrotron radiation at 0.98A. RESULTS: In contrast to Iohexol, which displays several interactions including one in the active site, Iobitridol is unable to interact directly with elastase. Only one partially occupied site is found in between two molecules of the crystal packing. CONCLUSIONS: The validation of the "hydrophobic shielding" concept, which was at the origin of the design of the Iobitridol molecule, has been proven to be an essential feature in minimizing in vivo protein interactions.

Animals↗
PubMed1997

Requirement of prestimulated THP-1 monocytic cells for endothelial cell activation. Involvement of TNF alpha.

Blood monocytes spontaneously activate endothelial cells in culture, leading to adhesion of monocytic cells onto the endothelial surface and overproduction of endothelial proteins such as von Willebrand factor (vWf) and plasminogen activator inhibitor type 1 (PAI-1). To overcome the difficulty in obtaining quiescent monocytes, we studied the ability of promonocytic THP-1 cells to activate endothelial cells. Lipopolysaccharide (LPS)-prestimulated and untreated THP-1 cells were cocultured with resting human umbilical vein endothelial cells (HUVEC) for 3 and 24 h in the presence of colimycin to neutralize LPS traces. Addition of untreated THP-1 cells had little effect on HUVEC adhesiveness. Addition of prestimulated THP-1 cells was followed by a noticeable adhesion after 3 h which reversed to basal values within 24 h. Under these conditions HUVEC adhesion molecules, E-selectin, VCAM-1 and ICAM-1, were increased at 3 h with only ICAM-1 remaining overexpressed at 24 h. Diffusible endothelial proteins such as soluble E-selectin, PAI-1 and vWf to a minimal extent, increased in supernatants from HUVEC cocultured for 24 h with prestimulated THP-1 cells. In those cocultures, TNF alpha concentrations peaked at 3 h whereas IL-1 beta levels progressively rose until 24 h. Addition of an anti-TNF alpha antibody decreased by 40% E-selectin and ICAM-1 induction and suppressed PAI-1 overproduction with a weak effect on vWf. An anti-IL-1 beta antibody had negligible effects on HUVEC adhesion molecules, PAI-1 or vWf production. These results provide evidence that promonocytic THP-1 cells require prestimulation in order to activate HUVEC and that TNF alpha contributes to this phenomenon.

Cell Adhesion↗
PubMed1997

Absence of cross-reactivity of SR90107A/ORG31540 pentasaccharide with antibodies to heparin-PF4 complexes developed in heparin-induced thrombocytopenia.

New carbohydrate-based anticoagulants devoid of the side effects of unfractionated heparin are currently under development and show a major potential for patients with heparin-induced thrombocytopenia (HIT) who still require efficient antithrombotic therapy. As HIT is usually associated with antibodies to heparin-platelet factor 4 (H-PF4) complexes, cross-reactivity of the heparin pentasaccharide SR90107A/ORG31540 was tested in the presence of PF4 with the plasma from 49 patients with HIT. No cross-reactivity was observed whatever the pentasaccharide concentrations. Although more extensive studies are required for excluding its total absence of immunogenicity and pathogenicity, this pentasaccharide is a candidate for use in emergency situations in patients with HIT.

Autoantibodies↗
PubMed1997

Experimental evaluation of the vascular effects and transport of an iodinated macromolecular contrast medium.

RATIONALE AND OBJECTIVES: For assessment of the tissue blood pool and overall vascularity, macromolecular contrast media have significant advantages over low molecular weight contrast agents. The authors evaluated the vascular effect and transport of a new macromolecular contrast media (MMCM), an iodinated dextran polymer of 32 kDa. METHODS: The new MMCM was obtained from dextran activated by carboxy methylation, followed by linkage with triiodinated aminophtalamid conjugates. To detect whether the tracer induces vascular leakage, MMCM (350 mg I/kg) was administered intravenously in 10 mice, or applied on the cremaster muscle of 26 mice previously injected with carbon particles; after 30 or 45 minutes, the cremaster was fixed and examined by optical microscopy. For investigation of the vascular transport 3, 5, and 15 minutes after MMCM administration, various tissue fragments were processed and examined by electron microscopy. RESULTS: In all vascular examined, MMCM does not induce plasma extravasation and the probe was detected mostly within the vascular lumen. At the ultrastructural level, a small fraction of MMCM was found in endothelial plasmalemmal vesicles (endosome-like structures) and, in time, transcytosed to the subendothelial space. No intercellular junctions were permeated by MMCM. CONCLUSIONS: The MMCM induces no vascular leakage and it is retained mainly in the plasma. Transport of MMCM is restricted to endothelial vesicles, which may explain, in part, its prolonged vascular space retention.

Angiography↗
PubMed1997

A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins.

An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.

Adult↗
PubMed1997

Molecular characterization of glucokinase from Escherichia coli K-12.

glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its Vmax was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.

Amino Acid Sequence↗
PubMed1997

The hypothalamic-pituitary-adrenocortical axis in severe falciparum malaria: effects of cytokines.

Patients with malaria can have features of adrenal insufficiency. Because of the pathophysiological and clinical implications of an Addisonian state, the hypothalamic-pituitary-adrenocortical axis was assessed in nine Vietnamese adults with complicated malaria. A CRH test was performed on admission (in convalescence in five cases) and in six healthy controls. Basal plasma ACTH concentrations in the patients and controls were similar [median (range): 2.9 (0.2-9.7) vs. 3.5 (1.9-13.4) pmol/L, respectively; P > 0.1]. Serum cortisol levels were greater in the patients [882 (294-1682) vs. 190 (110-676) nmol/L; P < 0.01], but three (33%) had values within the control range. Basal serum corticosteroid-binding globulin concentrations were similar in patients and controls (P = 0.23). The post-CRH rise in plasma ACTH was attenuated in the patients [peak: 6.1 (0.9-23.2) vs. 14.5 (6.2-21.5) pmol/L in controls; P < 0.05]; basal and peak plasma ACTH correlated with plasma interleukin-6 in this group (rs > or = 0.60; P < or = 0.04). Serum cortisol responses to CRH were depressed in acute illness [peak 990 (394-1, 805) nmol/L or 10 (0-50%) above baseline vs. 500 (429-703) nmol/L or 160 (10-380%) in controls; P < 0.05]. The median estimated serum cortisol t1/2 was 4.6 h in the patients and 1.6 h in the controls. These data suggest that, relative to a normal stress response, primary and secondary adrenal insufficiency can occur in severe malaria but may be attenuated by increased circulating interleukin-6 concentrations and impaired cortisol metabolism. The benefits of stress-dose corticosteroid replacement are unknown but could be considered in hypoglycemic patients or those with a serum cortisol within or below the reference range.

Adrenocorticotropic Hormone↗
PubMed1997

Isoform-specific expression and function of neuregulin.

Neuregulin (also known as NDF, heregulin, ARIA, GGF or SMDF), induces cell growth and differentiation. Biological effects of neuregulin are mediated by members of the erbB family of tyrosine kinase receptors. Three major neuregulin isoforms are produced from the gene, which differ substantially in sequence and in overall structure. Here we use in situ hybridization with isoform-specific probes to illustrate the spatially distinct patterns of expression of the isoforms during mouse development. Ablation of the neuregulin gene in the mouse has demonstrated multiple and independent functions of this factor in development of both the nervous system and the heart. We show here that targeted mutations that affect different isoforms result in distinct phenotypes, demonstrating that isoforms can take over specific functions in vivo. Type I neuregulin is required for generation of neural crest-derived neurons in cranial ganglia and for trabeculation of the heart ventricle, whereas type III neuregulin plays an important role in the early development of Schwann cells. The complexity of neuregulin functions in development is therefore due to independent roles played by distinct isoforms.

Animals↗
PubMed1997

Proposal for a New Staging Scheme for Intraocular and Extraocular Retinoblastoma Based on an Analysis of 103 Globes.

Management of retinoblastoma is highly individualized and depends heavily on grouping or staging. In view of evolving methods of imaging and treating retinoblastoma, we have developed and evaluated a revised staging scheme. We analyzed the survival data of 73 patients treated at St. Jude Children's Research Hospital to compare the ability of the Reese-Ellsworth grouping system, the original St. Jude, and the modified St. Jude staging schemes to predict progression-free survival. None of the staging schemes significantly correlated with progression-free survival. This modified staging scheme provides an instrument for assessing the natural history of retinoblastoma based on ophthalmolgic, other clinical, and imaging findings. Because it can identify patients at higher risk, the modified St. Jude scheme may be useful in selecting appropriate therapy regimens for children with retinoblastoma.

Journal Article↗
PubMed1997

Abnormal proteolytic processing of von Willebrand factor Arg611 Cys and Arg611His.

The structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient's samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients' plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.

Arginine↗
PubMed1997

Impaired prothrombin consumption in Bernard-Soulier syndrome is corrected in vitro by human factor VIII.

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein Ib (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII:C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

Antibodies, Monoclonal↗
PubMed1997

Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: from the patient to the gene. INSERM Network on Molecular Abnormalities in von Willebrand Disease.

Type 2 vWD is defined by qualitative defects of vWF and is subdivided into four subtypes: 2N, 2B, 2A and 2M. The characterization of 150 unrelated French cases with type 2 vWD emphasizes the heterogeneity of this group. In 51 cases of type 2N vWD, new mutations were found not only in the D' domain (Cys25Tyr and Cys95Phe) but also in the D3 domain (Asp116Asn and Cys297Arg). In 42 cases of type 2B vWD, no new mutation was detected. In 45 cases with type 2A phenotype, three new candidate mutations were found in the A2 domain: Gln793Arg, Val841Phe and Leu876Pro. In addition, four new candidate mutations were detected in the A1 domain: Cys509Gly, Arg545His, Arg552Cys and Cys695Tyr. Finally, five new candidate mutations were identified in 12 patients with 2M (or unclassified) phenotype: Leu513Pro, Gly561A1a, Glu596Lys, Arg611Leu and IIe662Phe. For all candidate mutations, expression studies are in progress. This study of a large number of French variants of vWD brings further insight into the relationship between phenotype and genotype.

Amino Acid Sequence↗