PubMed Health⌕ Search

Biomedical subjects

L Vogel

Publications and source records attributed to L Vogel.

At least 19 recordsLinked to original sources
PubMed2007

Mediator release assays based on human or murine immunoglobulin E in allergen standardization.

BACKGROUND: The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products. OBJECTIVE: The general performance and application potential of biological in vitro assays in batch control and standardization of allergens and as a tool for verifying activity and stability of allergen standards were analysed. METHODS: Allergen extracts of five clinically relevant allergens from three to five different manufacturers were investigated. A CAP-IgE-inhibition assay was compared with mediator release assay (MRA)s based on murine or human basophils. Rat basophilic leukaemia (RBL) cells were passively sensitized with pooled murine allergen-specific IgE-containing sera. Humanized RBL cells and human-stripped basophils were sensitized with pooled patient's sera, which were also used for the CAP-IgE-inhibition assay. Allergen specificity of the sera was determined by immunoblotting. RESULTS: A good batch-to-batch consistency was found with each assay among all manufacturers and allergens tested. Between different manufacturers, the products showed differences in activity and the various assays indicated an almost identical ranking. However, the biological assays revealed qualitative differences of biological activity or composition of allergen preparations undetectable by IgE-inhibition assay. CONCLUSIONS: MRAs provide refined information on allergen activity, either confirming the results of IgE-inhibition assay, or indicating differences requiring further investigation, and represent a highly sensitive novel tool in allergen standardization. By using permanently cultivated cell lines, repeated venepuncture to obtain human basophils is avoided. As in the RBL assay, the coefficient of variation for the release values were below 15% and for the ED50 below 25%, the assay is suitable to determine differences that are relevant for batch control purposes.

Allergens↗
PubMed2006

Nitration enhances the allergenic potential of proteins.

BACKGROUND: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. METHODS: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. RESULTS: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-gamma production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. CONCLUSIONS: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

Allergens↗
PubMed2006

Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus.

BACKGROUND: Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen-specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp-specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody-binding capacity for specific immunotherapy. AIM: The aim was to clone, express and characterize a full-length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE-mediated mediator release. METHODS: Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5' RACE) was performed to obtain full-length cDNA coding for Pen a 1. Using a gene-specific primer, PCR was performed and full-length cDNA was cloned and sequenced. Recombinant His-tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody-binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE-mediated type I allergy. RESULTS: The deduced amino-acid sequence was 284 residues long and amino-acid sequence identities with allergenic and non-allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had alpha-helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild-type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation. CONCLUSIONS: Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component-resolved diagnosis and the generation of modified shrimp tropomyosin for allergen-specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp extract in combination with cholera toxin as adjuvant may be a suitable model to study shrimp allergy.

Allergens↗
PubMed2005

Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1.

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

Allergens↗
PubMed2005

Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.

BACKGROUND: Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of allergens. METHODS: Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding alpha-chain was detected by FACS analysis and the functional integration of the 'humanized' receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking allergen preparations. RESULTS: Several clones were obtained that were able to bind allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of allergen extracts, including highly diluted products that cannot be analysed by conventional methods. CONCLUSION: A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.

Allergens↗
PubMed2000

Epidemiologic typing of Escherichia coli using RAPD analysis, ribotyping and serotyping.

OBJECTIVE: To compare random amplified polymorphic DNA (RAPD) analysis and ribotyping with serotyping for epidemiologic typing of Escherichia coli. METHODS: Thirty-two epidemiologically unrelated strains, nine cerebrospinal fluid isolates with the O7K1 serotype from nine patients, and nine sets of epidemiologically related E. coli isolates from nine patients were typed by RAPD analysis, ribotyping and serotyping. RESULTS: Among the 32 epidemiologically unrelated E. coli isolates, 29 types were distinguished by RAPD analysis, 25 by ribotyping and 27 by serotyping. Indistinguishable patterns were obtained by RAPD analysis and ribotyping within the collection of nine cerebrospinal fluid isolates. For the epidemiologically related isolates, intrapatient variation was only found by RAPD analysis among the isolates of one set and by ribotyping among the isolates of two sets. No interpatient variation was observed between three sets of isolates. With serotyping, the epidemiologically related isolates yielded similar typing relationships to those obtained by RAPD analysis and ribotyping. CONCLUSIONS: RAPD analysis had the highest discriminatory capacity for typing E. coli isolates. RAPD analysis, ribotyping and serotyping can all be used for assessment of strain relationships.

Adult↗
PubMed2000

Performance of phenotypic and genotypic methods to determine the clinical relevance of serial blood isolates of staphylococcus epidermidis in patients with septicemia.

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2-1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of Staphylococcus epidermidis. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2-1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.

Bacteremia↗
PubMed1998

Do chickens immunized with bacterial immunoglobulin G-binding proteins produce rheumatoid factor-like antibodies?

An association between the production of rheumatoid factor (RF)-like antibodies in animals immunized with bacterial immunoglobulin (Ig)G-binding proteins has been noted. Three potential explanations have been proposed: (1) altered host IgG due to binding of the immunogen; (2) B-cell superantigenic properties of the binding proteins; and (3) idiotype-anti-idiotype response leading to an antibody which acts as an antigen mimic. In order to distinguish among these possibilities, it is necessary to carry out studies in animals whose IgG does not react with the IgG-binding protein immunogen. Consequently, we have determined the effects of immunizing chickens with a purified group C streptococcal IgG-binding protein, FcRc, a bacterium expressing this protein, and appropriate control immunogens. The results of these studies provided evidence for production of specific antibodies to FcRc in groups of chickens immunized with either the pure protein or bacteria expressing the protein. No significant association with production of RF-like antibodies was noted, favouring the altered IgG-binding explanation for the association between RF-like antibodies and immunization with the bacterial IgG-binding proteins.

Animals↗
PubMed1997

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae.

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.

Adult↗
PubMed1997

31P-NMR determinations of cytosolic phosphodiesters in turtle hearts.

As part of our ongoing research on cardiac hypoxia tolerance we have conducted 31P nuclear magnetic resonance (NMR) studies of isolated, perfused, working hearts from freshwater turtles, animals that are well known for their ability to tolerate prolonged periods of anoxia. A striking feature of turtle heart spectra is an extremely high concentration of NMR visible phosphodiesters (PDEs). Cardiac spectra from mammals, on the other hand, typically exhibit only a small resonance in the PDE region. Our aim in this study was to compare myocardial PDE profiles between the highly hypoxia tolerant western painted turtle (Chrysemys picta bellii) and the relatively hypoxia sensitive softshelled turtle (Trionyx spinifer) in order to begin to rest the hypothesis that high constitutive levels of cytosolic PDEs may play a role in conferring hypoxia and ischemia tolerance on the myocardium. We also collected 31P-NMR spectra of PCA extracts of tissue from these species and from Kemp's ridley sea turtles (Lepidochelys kempi), as well as spectra from isolated hearts and PCA extracts of red-eared sliders (Trachemys [formerly Pseudemys] scripta]). Total NMR visible phosphodiesters make up 24 +/- 8.6% of the total NMR visible phosphorus in Chrysemys hearts, 20.7 +/- 5.9% in Trachemys hearts, but only 12.2 +/- 5.1% in Trionyx hearts (P < 0.05). We have identified three distinct PDEs in turtle hearts: glycerophosphorylcholine (GPC); glycerophosphorylethanolamine (GPE); and serine ethanolamine phosphodiester (SEP). SEP is the dominant compound in Chrysemys and Trachemys (79.3 +/- 10.2% and 84.7 +/- 3.7% of total PDE, respectively), while GPC is most abundant in Trionyx (74.0 +/- 4.3% of total PDE) and Lepidochelys (not quantitated). The function of this class of compounds is unclear but it has been suggested that cytosolic PDEs may function as lysophospholipase inhibitors, a role that would decrease the rate of membrane phospholipid turnover. Our comparative data suggest that cytosolic PDEs could play a role in phospholipid sparing during anoxic or ischemic stress in turtles but a direct test of this hypothesis awaits future experimentation.

Animals↗
PubMed1997

Characterization of components of the murine B cell receptor complex and their role in anti-Ig stimulation.

Signal transduction in B cells is mediated by B cell receptor (BCR) complexes that are composed of membrane immunoglobulins (mIg) and additional proteins (e.g. Ig-alpha and Ig-beta) that have been implicated with several aspects of B cell activation. In this paper, experiments are described that have been designed to characterize new components of the BCR and to elucidate their involvement in B cell activation directly after the binding of anti-Ig. The data obtained prove the assumption that IgM is degraded after internalization. Neither immunoglobulin nor the Ig-alpha/beta heterodimer are re-expressed after they have disappeared from the cell surface. The newly detected Ig-alpha associated proteins are neither degradation products of immunoglobulins nor different forms of Ig-alpha or Ig-beta. In addition, they are not recognized by antibodies against any of the kinases known to date. The mIg molecules are linked to the cytoskeleton and internalized. On the other hand, the additional Ig-alpha associated proteins as well as the Ig-alpha/beta heterodimer could not be detected in the detergent-insoluble fraction. The former molecules remain on the B cell surface where they are attached to the unaffected isotype and might interact with downstream members of the signalling cascade. In addition, evidence is presented that the internalization of the receptor complex is not necessary for signal transduction and B cell activation. From these results the authors conclude that the BCR complexes are removed from the cell surface after binding of ligands, probably to prevent further activation of this cell, while the Ig-alpha associated proteins interact with the intracellular components of the signal transduction pathway to promote the further activation of the B cells.

Animals↗
PubMed1997

Iron-binding proteins in sputum of chronic bronchitis patients with Haemophilus influenzae infections.

Airway inflammation during infection is associated with increased transudation of serum proteins and increased production of protein by the airway epithelium. We therefore, assessed whether Haemophilus influenzae infections in patients with chronic bronchitis are associated with increased levels of transferrin and lactoferrin in the sputum compared to uninfected patients. Sputum sol phase and serum samples from 14 infected and 13 uninfected patients with chronic bronchitis and from 12 bronchial asthma patients were included in the study. The median Q-values (the concentration in sputum sol phase/the concentration in serum) x 10(3) of transferrin appeared increased in chronic bronchitis patients with an H. influenzae infection (26.0, n=13) compared to uninfected controls (9.5, n=11) and bronchial asthma patients (4.5, n=6). The ratio of the Q(transferrin)/Q(albumin) was >1 in infected chronic bronchitis patients, indicating local production of transferrin. Growth of H. influenzae was stimulated more in sputum from infected and uninfected patients with chronic bronchitis than in sputum from patients with bronchial asthma. The concentrations of lactoferrin were not significantly different in infected (n=14) and uninfected (n=13) chronic bronchitis patients and bronchial asthma patients (n=12) (median 137.4, 84.6, 87.1 mg x L(-1), respectively). We conclude that in patients with chronic bronchitis with Haemophilus influenzae infections, the levels of transferrin are increased and the levels of lactoferrin are not associated with infections.

Adult↗
PubMed1996

Suc1: cdc2 affinity reagent or essential cdk adaptor protein?

CKS proteins, for which the original member, p13suc1, was identified as a suppressor of cdc2 alleles in S. Pombe, have long served as a reagent for the purification of p34cdc2, whereas their biological function has remained elusive. Apparently conflicting data derived from different model systems may indicate a diversity of function for these proteins. Several new observations in yeast and Xenopus egg extracts together with new structural information tends to enhance the hypothesis that CKS proteins function to alter the activity of cdc2 at several important points in the cell cycle. Here we review previous observations and recent data that suggest CKS proteins serve as adaptor proteins that modify the functions of cdc2 throughout the cell cycle.

Amino Acid Sequence↗
PubMed1996

Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus influenzae.

Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variant-specific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.

Animals↗
PubMed1996

Immune selection for antigenic drift of major outer membrane protein P2 of Haemophilus influenzae during persistence in subcutaneous tissue cages in rabbits.

During persistence of nonencapsulated Haemophilus influenzae in the respiratory tracts of patients with chronic bronchitis, the major outer membrane proteins (MOMPs) P2 and P5 show antigenic drift. The hypothesis that appearance of antigenic variants is the consequence of antibody-dependent selection was tested in a rabbit model. Persistence of H. influenzae d1 was achieved in subcutaneous tissue cages for up to 948 days. During persistence in the rabbits, similar changes in MOMP P2 of H. influenzae occurred, as observed in isolates from chronic bronchitis patients. In rabbits vaccinated with strain d3 and in nonvaccinated rabbits, antigenic drift occurred later than in rabbits vaccinated with strain d1. High titers of antibodies against H. influenzae were measured in tissue cage fluid and serum. Vaccination of the rabbits with H. influenzae d1 or d3, an antigenic variant of strain d1, resulted neither in eradication of H. influenzae d1 nor in increased antibody titers in serum and tissue cage fluid. The sera of nonvaccinated rabbits during persistence had no strain d1-specific bactericidal activity in the presence of complement. Vaccination with H. influenzae d1 induced serum bactericidal activity against strain d1 in the presence of complement. However, a variant of strain d1 appearing in the tissue cages was not killed by this serum bactericidal activity. We conclude that immunological pressure leads to the selection of MOMP variants of H. influenzae and that these variants escape the antibody-mediated strain-specific bactericidal activity against H. influenzae.

Amino Acid Sequence↗