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Biomedical subjects

J Van der Heyden

Publications and source records attributed to J Van der Heyden.

At least 19 recordsLinked to original sources
PubMed2005

Collecting information on the quality of prescribing in primary care using semi-automatic data extraction from GPs' electronic medical records.

OBJECTIVES: To evaluate a semi-automatic data extraction from the electronic medical record (EMR) of general practitioners (GPs) through a comparison with a paper sheets data collection simultaneously used in a primary care research project on the quality of prescribing for osteoarthritis in the elderly. SUBJECTS: One hundred and fifty-two GPs using five different EMR-software systems participated with the semi-automatic data extraction from the EMR and 233 GPs collected data with paper registration sheets. METHODS: The proportion of patients with respectively a drug prescription, paracetamol, a non-steroidal anti-inflammatory drug (NSAID) and ibuprofen were compared between the semi-automatic extraction and the paper data collection and among the EMR-software systems. RESULTS: Using the semi-automatic data extraction, a significantly lower proportion of patients on drugs was obtained compared to the paper data collection (adjusted OR: 0.31; 95% CI 0.25-0.39). However, the proportion of patients on a specific type of drug was comparable. Within the results from the semi-automatic extraction, the results were heterogeneous among the different EMR-software systems. CONCLUSIONS: The semi-automatic data extraction with multiple EMR-software systems proposed in this study seems suitable for quality of prescribing assessment in primary care. However, it may be less reliable when only a single EMR-software is used.

Aged↗
PubMed2003

Enhanced soluble interleukin-5 receptor alpha expression in nasal polyposis.

BACKGROUND: Alternative splicing of the interleukin-5 receptor alpha (IL-5Ralpha)-subunit leads to the generation of a signalling, membrane-anchored (TM) isoform, or a secreted [soluble (SOL)], antagonistic variant. Given the key role of IL-5 in eosinophil function, we investigated SOL IL-5Ralpha expression pattern in an eosinophil-associated disease such as nasal polyposis (NP). METHODS: An SOL IL-5Ralpha enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction (PCR) were established and applied in serum, nasal secretion and nasal tissue of controls (n = 12), and NP patients (n = 42) with or without asthma. RESULTS: Analysis of serum, nasal secretion, and nasal tissue samples revealed that SOL IL-5Ralpha protein concentrations were significantly increased in NP vs control tissue. Within the NP group, there was a significant up-regulation of SOL IL-5Ralpha in patients with systemic airway disease. These findings were confirmed at the mRNA level, using an optimized real-time reverse-transcriptase PCR procedure. CONCLUSIONS: This report demonstrates SOL IL-5Ralpha transcript and protein up-regulation in NP. Soluble IL-5Ralpha differentiates nasal polyps with or without concomitant asthma. As SOL IL-5Ralpha is strongly up-regulated for disease and has antagonistic properties in vitro, our studies shed new light on the mechanisms of specific immunomodulatory therapies, such as anti-IL-5.

Adult↗
PubMed2002

Belgian coca-cola-related outbreak: intoxication, mass sociogenic illness, or both?

An epidemic of health complaints occurred in five Belgian schools in June 1999. A qualitative investigation described the scenario. The role of soft drinks was assessed by using a case-control study. Cases were students complaining of headache, dizziness, nausea, vomiting, abdominal pain, diarrhea, or trembling. Controls were students present at school on the day of the outbreak but not taken ill. An analysis was performed separately for school A, where the outbreak started, and was pooled for schools B-E. In school A, the attack rate (13.2%) was higher than in schools B-E (3.6%, relative risk = 3.6, 95% confidence interval (CI): 2.5, 5.3). Exclusive consumption of regular Coca-Cola (school A: odds ratio (OR) = 29.7, 95% CI: 1.32, 663.6; schools B-E: OR = 7.3, 95% CI: 2.9, 18.0) and low mental health score (school A: OR = 16.1, 95% CI: 1.3, 201.9; schools B-E: OR = 3.1, 95% CI: 1.5, 6.6) were independently associated with the illness. In schools B-E, consumption of Fanta, consumption of Coca-Cola light, and female gender were also associated with the illness. It seems reasonable to attribute the first cases of illness in school A to regular Coca-Cola consumption. However, mass sociogenic illness could explain the majority of the other cases.

Adolescent↗
PubMed2002

MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells.

Identifying novel targets for therapy in allergic disease: protein interactions inside the cell Therapy of allergic disease currently relies on pharmacological manipulation of mediators or immunotherapy. Drugs have been developed to target specific mediators and their receptors: for example antihistamines blocking the H1 receptor have been refined to maximize antagonism and reduce central side-effects or adverse effects of activity on other receptors such as muscarinic cholinergic receptors. Traditional pharmacological approaches identify new surface receptors against which chemists will then design or screen compounds for activity: examples are H3 or H4 histamine receptors. With the advent of the sequenced human genome we are faced with a vast array of genes and proteins that interact to define normal physiology or indeed pathology. A major challenge to biotechnology is to evolve novel techniques to understand the function and interaction of these myriad proteins. One particular area of current interest is the signalling cascades downstream of surface receptors. For many years pathways have appeared overlapping and to offer little chance of specific intervention. However, greater understanding of the complexity and integration of signalling, together with the possibility of directing drugs to specific cells has aroused considerable interest in this area for novel therapeutics. Indeed, targeting events within the cell has been done for many years with steroids. Here, Jan Tavernier and colleagues describe some signalling pathways relevant to allergic disease and potential methods for understanding protein interactions that allow mapping of the cascades. In particular they describe an elegant new system of analysis of protein-protein interactions in a mammalian system, which they have developed, termed MAPPIT. The basis of the system is an engineered receptor with JAK kinase but which lacks STAT activation sites. To the cytoplasmic end of the receptor is added a bait protein of interest, and the cell line can then be transduced with plasmid containing 'prey' cDNA from a library of interest linked to an active STAT binding site. If this cDNA encodes a protein which, upon expression, is activated and recruited to the membrane complex, it will bind to the receptor via the bait, then STAT activation will occur and activate a reporter gene system such as luciferase or puromycin resistance. This novel system allows study of known protein-protein interactions by targeted mutagenesis, or screening for novel interactions. It has the advantage over existing systems such as yeast 2 hybrid that it uses mammalian cells and thus can reproduce the physiological conditions for protein processing or activation. As new genes and proteins are linked to the atopic phenotypes, systems such as this hold promise of rapidly defining their function and interacting proteins and may be important in linking genomics and proteomics with function and pharmacology in the future.

Animals↗
PubMed2001

Neutralizing monoclonal antibodies can potentiate IL-5 signaling.

IL-5 is a major determinant in the survival, differentiation and effector-functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ligand-specific alpha-chain and a beta-chain, shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor. We have generated and mapped the epitopes of three monoclonal antibodies (mAb) directed against this cytokine: the strong neutralizing mAb 5A5 and 1E1, and the very weak neutralizing mAb H30. We found that H30 as well as 5A5 can increase proliferation above the level induced by human (h)IL-5 alone, in a JAK-2-dependent manner, and at every sub-optimal hIL-5 concentration analyzed. This effect is dependent on mAb-mediated cross-linking of IL-5R complexes, and is only observed on cell lines expressing a hybrid human/mouse IL-5Ralpha-chain. We discuss these findings in view of the stoichiometric and topological requirements for an activated IL-5R. Since humanized anti-IL-5 mAb are currently in clinical testing, our findings imply that such mAb should be carefully evaluated for their potentiating effects.

Animals↗
PubMed2000

Interleukin 5 regulates the isoform expression of its own receptor alpha-subunit.

The receptor for interleukin 5 (IL-5) consists of a cytokine-specific alpha chain (IL-5Ralpha) and a signaling beta chain, which is shared with interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). These 3 cytokines can act in eosinophil development and activation in vitro, but gene deletion or antibody blocking of IL-5 largely ablates eosinophilic responses in models of allergic disease or helminth infection. We investigated factors acting in differential IL-5Ralpha gene splicing to generate either the membrane-anchored isoform (TM-IL-5Ralpha) which associates with the common beta chain to allow IL-5 responsiveness, or a secreted, antagonist variant (SOL-IL-5Ralpha). In a murine myeloid cell line (FDC-P1), transfected with minigenes allowing expression of either IL-5Ralpha variant, IL-5 itself, but not IL-3 or GM-CSF, stimulated a reversible switch toward expression of TM-IL-5Ralpha. A switch from predominantly soluble isoform to TM-IL-5Ralpha messenger RNA (mRNA) expression was also seen during IL-5-driven eosinophil development from human umbilical cord blood-derived CD34(+) cells; this was accompanied by surface expression of IL-5Ralpha and acquisition of functional responses to IL-5. IL-3 and GM-CSF also supported eosinophil development and up-regulation of TM-IL-5Ralpha mRNA in this system, but this was preceded by expression of IL-5 mRNA and was inhibited by monoclonal antibody to IL-5. These data suggest IL-5-specific signaling, not shared by IL-3 and GM-CSF, leading to a switch toward up-regulation of functional IL-5Ralpha and, furthermore, that IL-3 and GM-CSF-driven eosinophil development is dependent on IL-5, providing an explanation for the selective requirement of IL-5 for expansion of the eosinophil lineage. (Blood. 2000;95:1600-1607)

Animals↗
PubMed1999

The cell surface expression level of the human interleukin-5 receptor alpha subunit determines the agonistic/antagonistic balance of the human interleukin-5 E13Q mutein.

The human interleukin-5 (IL-5) receptor consists of an alpha-chain that specifically binds the ligand with intermediate affinity, and a beta c-chain, that associates with the IL-5/IL-5R alpha complex, leading to a high-affinity, signal transducing receptor complex. Structure-function studies showed that modification of the putative beta c-chain binding site in IL-5 (E13Q mutein) converted the molecule into an antagonist. However, analysis of the effect of this mutant IL-5 on COS-1 cells transfected with both receptor subunits, did not show reduced interaction with the beta c subunit [Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Van der Heyden, J., Guisez, Y. & Oefner, C. (1995) Proc. Natl Acad. Sci. USA 89, 7041-7045]. To gain more insight into the mechanism of IL-5 antagonism by E13Q, we tested its biological activity on two FDC-P1 subclones that express clearly different numbers of alpha-subunits yet an almost constant number of murine beta c-subunits. Here we show that E13Q has a biological activity comparable to wild-type IL-5 only when a high number of alpha-chains is present on the cells. Confirming the critical role of the IL5R alpha cell-surface expression level, treatment with suboptimal doses of a neutralising anti-IL-5R alpha antibody results in reduced activity of the mutant but not of wild-type IL-5.

Animals↗
PubMed1998

Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization.

The genes encoding the mature forms of mouse (mOB) and human OB (hOB) protein (also called leptin) were fused to the secretion signal coding sequence of the Escherichia coli outer membrane protein A (sOMP A). The hybrid genes were preceded by a ribosome binding site (RBS) and were expressed under transcriptional control of both the lipoprotein promoter (Plpp) and the lac promoter-operator (POlac). The recombinant fusion proteins were efficiently expressed and exported into the periplasmic compartment of E. coli cells from where they were recovered by osmotic shock as soluble mature polypeptides with the sOMP A precisely removed. Recombinant mOB and hOB proteins were also produced in Sf9 insect cells using the baculovirus expression system. Milligram quantities of both proteins were purified to homogeneity using ion-exchange, hydrophobic interaction chromatography and gel filtration and were found to be biologically active and to have antiobesity effects upon testing in genetically obese ob/ob mice.

Animals↗
PubMed1997

Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding.

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.

Amino Acid Sequence↗
PubMed1997

A human leptin mutant induces weight gain in normal mice.

Leptin, a fat secreted hormone, regulates ingestive behaviour and energy balance by binding to a specific receptor. Using site-directed mutagenesis, we screened for single amino acid residues in human leptin which are critical for receptor binding and biological activity. Here we report that one of these mutants has in vivo antagonistic properties. An Arg to Gln substitution at position 128 of human leptin does not affect receptor binding but knocks out biological activity. Repeated injection of R128Q in normal C57BL/6J mice results in a progressive increase in body weight. This demonstrates that R128Q is able to interfere with the negative feedback control of endogenous leptin. This mutant could be of therapeutic use for wasting disorders, such as anorexia and cachexia, where weight gain would be beneficial.

Animals↗
PubMed1995

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Amino Acid Sequence↗
PubMed1995

Characterization of critical residues in the cytoplasmic domain of the human interleukin-5 receptor alpha chain required for growth signal transduction.

Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, alpha and beta, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse beta chain (AIC2B) that were transfected with human IL-5 receptor (R)alpha cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL-3 induce tyrosine phosphorylation of beta chain and JAK 2 kinase. Phosphorylated beta receptor was co-precipitated with anti-JAK 2 antibodies, suggesting that both molecules were physically associated. IL-5 and IL-3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon-gamma responsive region of human Fc gamma 1 gene DNA element. A deletion mutant of hIL-5R alpha lacking the cytoplasmic part could bind hIL-5 normally in association with the beta chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane-proximal proline-rich element of the hIL-5R alpha cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL-5-inducible JAK 2 phosphorylation.

Amino Acid Sequence↗
PubMed1995

Interleukin-5 and its receptor: a drug target for eosinophilia associated with chronic allergic disease.

A characteristic feature of chronic allergic diseases such as asthma is the increase in eosinophil numbers in the inflamed tissue. In light of its specificity for the development of eosinophils, interleukin-5 (IL-5) is considered the most important cytokine involved in the regulation of eosinophilia. Hence, an antagonist for IL-5 activity is a new target for drug discovery programs. We have examined the opportunity for both a random and a rational approach for the identification of such an antagonist. The elucidation of the structure of IL-5 and the initial structure/function analysis of the ligand/receptor complex constitute a first step towards the design of antagonistic compounds. The identification of a small compound by random screening able to inhibit the IL-5/IL-5 receptor interaction indicated an important domain in the receptor. We examine here protein-based IL-5 antagonists, such as IL-5-muteins, soluble IL-5 receptor constructs, and monoclonal antibodies, for their potential as IL-5/IL-5 receptor antagonists, and the use of a murine model of eosinophil airway inflammation for their evaluation.

Animals↗
PubMed1995

Structure/function analysis of human interleukin 5 and its receptor.

We have performed a detailed structure-function analysis of human interleukin 5 (hIL5) and its receptor. By testing a hIL5 mutant panel in a solid phase binding assay and a proliferation assay using hIL5 dependent cell-lines, areas on hIL5 involved in either the receptor alpha-subunit interaction or in receptor activation were identified. Epitope mapping data of a neutralizing and a non-neutralizing monoclonal antibody were in agreement with the mutant analysis. hIL5 binding areas on the IL5R alpha-subunit were identified by interspecies chimaera analysis. Finally, hIL5 mutants with reduced receptor activation potential have antagonistic properties.

Animals↗
PubMed1993

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 A.

Animals↗
PubMed1993

Increased IL-6 production and IL-6-mediated Ig secretion in murine host-vs-graft disease.

BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.

Animals↗