Predictable mini-epidemics of spontaneous pneumothorax: haemoptysis too?
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Cluster Analysis↗
Biomedical subjects
F Boulay
Publications and source records attributed to F Boulay.
Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells.
The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.
Antigens↗
[Data interchange in blood transfusion: the impact of a new standard on the evolution of health electronic data exchange in France].
The sanitary and social data interchange within care establishments or networks is today the subject of many national or international considerations. Electronic data interchange in the health field has characteristics linked to ethical and deontological principles of care staff. Used daily, this tool contributes to the quality of care, to the optimization of patient treatment and to the organization of the system care. In the transfusion field, the standardization of messages related to the traceability of blood products in now required by the No. 2 instruction of French Blood Agency, which rules the using of national norms elaborated by the French Agency of Normalization. If the technicality is the greater part of these regulated and formalized messages, this standardization systematizes and justifies the nominative and ciphered data interchange in an open environment, opening a new dimension in the interoperability of data system between care establishments. This article analyzes the characteristics and the potential impact of this normalization on the evolution of the electronic data interchange in the health field.
Blood Transfusion↗
[A method for auditing medical records quality: audit of 467 medical records within the framework of the medical information systems project quality control].
Future hospital accreditation could take into account the quality of medical files. The objectives of this study is to test a method for auditing and evaluating the quality of the handing of medical files. We conducted a retrospective regional audit based on the frame of reference the National Agency for Medical Development and Evaluation, by using a sample of cases, stratified by establishment. In our region, the global budgets of 47 public and private hospitals participating in the public hospital service, are adjusted while keeping in mind the medicalised activity data (PMSI). This audit was proposed to the doctors of the Department of Medical Information on the occasion of the regulatory PMSI quality control. A total of 467 questionnaires were given by 39 of the 47 sollicited hospitals (83%). The methodological aspects (questionnaire, cooperative approach...) are discussed. The make-up of medical files can alos be improved by raising the percentage of the presence of important data or documents such as the reason for admission (74.1%), the surgery report (83.2%), and the hospitalisation report (66.6%). A system for classifying the paraclinical results is shared and systematic throughout the service or hospital in only 73.2% of cases. The quality of the handing of medical files seems problematic in our hospitals and actions for improving the quality should be undertaken as a priority.
Accreditation↗
[Can medical files be used to audit hospital health care? Results of a regional audit of hospital file quality].
OBJECTIVES: The clinical audit method based on a retrospective analysis of medical files can be used to assess hospital health care. The reliability of the results obtained depends on the validity of the data in the file and its completeness. The aim of this work was to assess the quality of this information source. METHODS: The simplified ANDEM/ANAES from was proposed to 47 medical information departments of public and private hospitals participating in the public health care service in the Provence-Alples-Côte d'Azur region. The audit was conducted on a sample of hospital stays during a regular 6-month quality control assessment of hospital health care activity. RESULTS: Analysis of the 467 forms returned by 39 hospitals, showed that the quality of medical file recordings should be improved as a large amount of data or important documents were missing: reason for hospitalization (recorded on 74.1% of files), operation report (found in 83.2% of files) and discharge summary (found in 66.6% of files). CONCLUSION: Clinical audits would be compromised in certain hospitals by the use of medical files. Efforts to improve the quality of hospital medical files should be a priority.
Evaluation Studies as Topic↗
Phagocyte chemoattractant receptors.
Myeloid cells are attracted and activated by a variety of chemoattractants that bind to G protein-coupled receptors. In the past few years, the receptors for the classical chemoattractants (fMLF, C5a, PAF) and the chemotactic cytokines, known as C-X-C and C-C chemokines, have been cloned from myeloid cells. This review briefly describes recent advances in structure-function relationships of chemotactic receptors in human leukocytes as well as activation of signaling pathways and regulation of receptor function. In neutrophils, the binding of chemoattractants mainly activates the Gi2 protein inducing PIP2 hydrolysis and activation of the MAP kinase pathway. The C-C chemokine receptor, CC CKR5, and a chemokine receptor homologue, named fusin, have been shown to be the major cofactors for HIV-1 entry in macrophages and T cells. Recent studies suggest that the phosphorylation of chemoattractant receptors is a key event that regulates their biological function.
Amino Acid Sequence↗
Structure and function of leukocyte chemoattractant receptors.
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Amino Acid Sequence↗
Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain.
The C5a anaphylatoxin receptor is a member of the G protein-coupled receptor family involved in chemoattraction and activation of myeloid cells, as well as in host defence against infection by Pseudomonas aeruginosa. Upon challenge by C5a, the C5a receptor undergoes a rapid phosphorylation on serine residues in the carboxyl-terminal region. In this study, we used cells stably transfected with either the wild-type C5a receptor, or mutants affected in their capacity to be phosphorylated, to examine the role played by phosphorylation in the intracellular trafficking of the C5a receptor. Upon agonist binding, the wild-type receptor was rapidly internalized into endosomes that cluster near the nucleus after 10 minutes. Internalization of a non-phosphorylable mutant was severely impaired relative to wild-type receptor, whereas a mutant phos-phorylated on serine 327 and/or serine 338, showed a rate of internalization intermediate between that of wild-type receptor and that of the non-phosphorylable mutant. Under continuous exposure to C5a and in the absence of protein synthesis, the C5a receptor was maintained in a highly phosphorylated state but was not degraded. Confocal microscopy and ligand-binding studies indicated that internalized receptors were recycled to the plasma membrane. During this process, receptors were dephosphorylated with kinetics that correlated with the kinetics of receptor recovery on the cell surface. Altogether, our data suggest that phosphorylation plays a key role in the intracellular trafficking of the C5a receptor. Phosphoryl-ated receptors might be recognized by an adaptor protein that interacts with the endocytic machinery.
Amino Acid Sequence↗
[Public health education integrated in hospital. An internship proposal, "Medical information and pharmacology"].
According to a recent circular reforming french medical studies, we propose a teaching of medical information and pharmacology in situ within hospital instructions. Students could acquire an investigation methodology on the medicine economy. It will cover in four sessions the succeeding stages of medical information processing and be subject to an assessment: case studies and appreciation on student's, instruction record. By combining public health teaching with clinical practice, our project promotes its development in contact with other learnings and activities such as clinical research.
Electronic Data Processing↗
Direct or C5a-induced activation of heterotrimeric Gi2 proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton.
The binding of ligands to N-formyl peptide chemoattractant receptors in human neutrophils results in a rapid association of these receptors with a cytoskeletal fraction and a specific activation and release of Gi2 alpha-subunits from this fraction. In the present study we could show that pretreating neutrophils with GDPbetaS prevented the fMet-Leu-Phe-induced association of its receptor with a cytoskeletal fraction and also blocked the release of Gi2 alpha-subunits from the same cytoskeletal fraction. In contrast, direct activation of Gi2 proteins by addition of GTPgammaS or AlF4- not only caused a release of Gi2 alpha-subunits from the cytoskeleton but also an association of formyl peptide receptors with the cytoskeleton. The receptor for complement fragment 5a, which transduces its signaling through the same Gi2 protein, triggers both a release of Gi2 alpha-subunits from the cytoskeleton fraction and, of even greater interest, an association between formyl peptide receptors and the cytoskeleton. The close relationship between the activation and release of Gi2 alpha-subunits from the cytoskeleton and the association of formyl peptide receptors with the cytoskeleton might, however, not be a matter of protein-protein exchange, since the increased binding of formyl peptide receptors to the cytoskeleton occurs more rapidly than the release of Gi2 alpha-subunits from the cytoskeleton. The present findings suggest a possible mechanism for the initiation of formyl peptide receptor desensitization during neutrophil locomotion.
Aluminum Compounds↗
Site-directed mutagenesis of conserved charged residues in the helical region of the human C5a receptor. Arg2O6 determines high-affinity binding sites of C5a receptor.
The human C5a receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg2O6, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282-->Ala) or partial loss of ligand-binding capacity (Arg175-->Ala, Arg2O6-->Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln2O6]C5aR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (<10%) of high-affinity sites (Kd approximately 2 nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of G alpha-16. The number of high-affinity sites of wild-type and [Gln2O6]C5aR in 293 cells could be up-regulated by coexpression of Gi alpha-2 and down-regulated by GTP[gamma S]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg2O6 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human C5aR by affecting the intracellular G-protein coupling.
Animals↗
Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo.
Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 12-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser314, Ser317, Ser327, Ser332, Ser334, and Ser338. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser332 to Ser338 is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.
Amino Acid Sequence↗
Phosphorylation, dephosphorylation, and recycling of the C5a receptor in differentiated HL60 cells.
Binding of activation peptide from the fifth component of C (C5a) to its receptor triggers events leading to both stimulation of cellular responses and receptor desensitization in myeloid cells. However, although transmission of a signal to pertussis toxin-sensitive G proteins is a prerequisite to neutrophil activation, we show that the process of receptor phosphorylation is mainly independent from activation of this pathway. Treatment of cells with pertussis toxin did not modify the incorporation of phosphate mediated by a saturating concentration of C5a, indicating that agonist-occupied C5aR can be fully phosphorylated, presumably by a specific G protein-coupled receptor kinase, in the absence of activation of the Gi protein. Receptor phosphorylation was transient, with a half-life of 30 to 40 min, which suggested a role for protein phosphatases in the regulation of the state of phosphorylation of C5aR in dHL60 cells. Pretreatment of cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, increased the basal phosphorylation of unoccupied receptor and extended the phosphorylation mediated by C5a binding. Okadaic acid delayed, but did not suppress, the dephosphorylation process, which suggests either the involvement of additional phosphatase(s) or the degradation of nondephosphorylated receptors in the endocytic pathway. The data strongly suggest that internalized C5aR are recycled to the plasma membrane with a time course consistent with the kinetics of dephosphorylation. Dephosphorylation of C5aR might be essential to receptor recycling and resensitization during chemotaxis.
Cell Line↗
Regulation of cytosolic calcium and insulin secretion by galanin and ATP receptors: interactions of pertussis-toxin-sensitive and -insensitive signalling pathways.
In a previous study it was found that the expression of the exogenous fMet-Leu-Phe-receptor (NFPR) in the insulin-secreting cell line RINm5F mediates inhibition of hormone release and additionally raises cytosolic calcium concentration ([Ca2+]i) by activating phospholipase C (PLC) in a pertussis-toxin (PTX)-sensitive manner. We investigated whether an endogenous receptor could elicit similar effects and examined the interaction with PTX-insensitive signalling pathways. The hormone galanin inhibited insulin release at subnanomolar concentrations and increased [Ca2+]i, mainly by a PTX-sensitive mechanism with an EC50 (50 nM) comparable with that for hyperpolarization of membrane potential. The effect of galanin or fMet-Leu-Phe on [Ca2+]i was inhibited by pre-activation of the P2-receptor by ATP, which mobilizes calcium in a PTX-insensitive fashion. Simultaneous activation of the P2- and peptide receptors caused additive increases in [Ca2+]i saturating at a calcium concentration corresponding to the optimal ATP response. This suggests a specific convergence of PTX-sensitive and -insensitive pathways. In contrast, galanin and FMLP inhibited the insulin secretion induced by ATP (1-100 microM), but only when added prior to the nucleotide. In permeabilized cells, FMLP added after the calcium stimulus still inhibited secretion, indicating that the inefficacy observed in intact cells was not due to the rapid ATP-evoked rise in [Ca2+]i. Thus, (i) insulin-secreting cells possess an endogenous PTX-sensitive pathway mobilizing [Ca2+]i, (ii) inhibitory hormones preferentially activate different effectors depending on the agonist concentration and (iii) activation of NFPR or galanin receptor reveals an unusual dissociation between [Ca2+]i rises and insulin secretion, pointing towards an overriding inhibitory control of exocytosis.
Animals↗
Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.
The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.
Cell Fractionation↗
The NH2-terminal region of C5aR but not that of FPR is critical for both protein transport and ligand binding.
The N-formylated tripeptide, formylmethionylleucyl-phenylalanine (fMLP), and the 74-amino-acid long human C5a anaphylatoxin activate phagocytic cells via two structurally related G protein-coupled receptors (FPR and C5aR), which are 34% identical in amino acid sequence. C5aR chimeras were constructed in which the entire NH2-terminal extracellular sequence or part of it was replaced by the counterparts from FPR or FPRH. Although the NH2-terminal region of C5aR presents an extremely high interspecies variability, substitution of the entire NH2-terminal sequence of C5aR by that of FPR or FPRH surprisingly resulted in chimeras that were apparently retained in the endoplasmic reticulum. In contrast, when the NH2-terminal domain of FPR was replaced by the corresponding region from C5aR or FPRH normal expression to the plasma membrane and high affinity binding of N-formylated peptides were observed. Thus, the NH2-terminal region of C5aR, in contradistinction to that of FPR, seems to be required either for the translocation of C5aR through the ER membrane or for correct folding and sorting of C5aR to the plasma membrane. Replacement of the first 8 residues of C5aR by the corresponding region of FPR did not alter the cellular transport and the C5a binding capacity, whereas the exchange of the first 13 residues resulted in a chimera that was readily transported to the plasma membrane but showed no capability to bind C5a. Mutations of Asp into Asn in the NH2-terminal segment of C5aR further indicated that negative charges are required to endow the receptor with a C5a binding capacity. The residues critical for binding are either involved directly by interacting with cationic residues of C5a, or indirectly by influencing the overall structure of the ligand-binding site.
Amino Acid Sequence↗
Expression of transcripts for two interleukin 8 receptors in human phagocytes, lymphocytes and melanoma cells.
Two cDNAs coding for distinct interleukin 8 (IL-8) receptors, IL-8R1 [Murphy and Tiffany (1991) Science 253, 1280-1283] and IL-8R2 [Holmes, Lee, Kuang, Rice and Wood (1991) Science 253, 1278-1280] have been reported, and biochemical studies on human neutrophils have revealed two proteins (p70 and p44) that bind IL-8 with high affinity [Moser, Schumacher, von Tscharner, Clark-Lewis and Baggiolini (1991), J. Biol. Chem. 266, 10666-10671]. We have cloned the cDNA coding for IL-8R1 from a library of differentiated HL-60 cells. Transfection of this cDNA into Jurkat cells resulted in the expression of high-affinity binding for IL-8 and two related cytokines, GRO alpha and neutrophil-activating peptide 2 (Kd 0.5-1.0 nM). Northern-blot analysis with the IL-8R1 cDNA as probe revealed abundant expression of transcripts of different size in human neutrophils and low-level expression of a single RNA species in HL-60 cells differentiated with dimethyl sulphoxide and retinoic acid. Because of the extensive nucleotide sequence similarity of the cDNAs for IL-8R1 and IL-8R2, the reverse-transcription PCR method was used for analysis of RNA expression in myeloid and lymphoid cells, 19 cell lines established from human primary melanomas or metastases, two melanocyte and one fibroblast cell lines. IL-8R1 mRNA transcripts were expressed at high levels in neutrophils, and to a lesser extent in blood monocytes and the myeloid cell lines, HL-60 and AML 193, but were not found in THP-1 cells, lymphocytes and Jurkat cells. IL-8R2 mRNA transcripts, by contrast, were found in all blood cells and related cell lines, as well as in all melanoma, melanocyte and fibroblast cell lines tested. As for IL-8R1, IL-8R2 mRNA expression was highest in neutrophils. These results suggest that IL-8R1 and IL-8R2 may both be involved in neutrophil activation by IL-8 and related cytokines, and presumably correspond to p70 and p44, the receptors that were identified biochemically. Possible IL-8 functions on lymphocytes and melanoma cells, e.g. chemotaxis and proliferation, must be independent of IL-8R1 and may be mediated by IL-8R2.
Base Sequence↗
Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells.
Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.
Amino Acid Sequence↗