Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives.

BACKGROUND: Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. OBJECTIVE: To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen-induced allergic symptoms. METHODS: A three-arm double-blind placebo-controlled immunotherapy study was conducted with one pre-seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol-treated population (n=84). In addition, skin and nasal provocation responses and allergen-specific antibodies were assessed. RESULTS: There were trends towards improvement in the subjects' well-being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end-point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen-specific antibody responses. Local injection-site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side-effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. CONCLUSION: Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well-being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.

Different allergenic activity of grass pollen allergens revealed by skin testing.

BACKGROUND: Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. MATERIAL AND METHODS: Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. RESULTS: Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. CONCLUSION: Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy.

BACKGROUND: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. METHODS: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. RESULTS: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. CONCLUSIONS: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils.

Platelet-activating factor (PAF-acether), an inflammatory mediator with a wide range of biological activities including neutrophil aggregation and chemotaxis, was studied for its effect on human eosinophil locomotion (chemotaxis and chemokinesis). Human eosinophils (25-95% purity) were obtained from donors with a variety of diseases associated with hypereosinophilia. PAF-acether elicited directional locomotion of eosinophils, in a time- and dose-dependent fashion, at concentrations from 10(-5) to 10(-8) M; lyso-PAF had minimal activity over the same dose range. Compared with PAF-acether, the eosinophil locomotory responsiveness of leukotriene B4 (LTB4), histamine, and the valyl- and alanyl-eosinophil chemotactic factor of anaphylaxis (ECF-A) tetrapeptides was negligible. Conversely, neutrophil responsiveness to PAF-acether (optimum 10(-6) M) was comparable in effect to LTB4 (optimum dose 10(-8) M). It was shown that PAF-acether elicited both chemotaxis and chemokinesis of eosinophils. Comparison of normal density and light density eosinophils revealed no qualitative difference in the response to PAF-acether and the other chemoattractants, although the light density cells seemed to demonstrate a greater degree of locomotion to PAF-acether and LTB4. Thus, PAF-acether appears to be a potent eosinophilotactic agent which may play a role in inflammatory reactions characterized by eosinophil infiltration.

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter.

Rapid and efficient purification of Phleum pratense major allergens Phl p 1 and group Phl p 2/3 using a two-step procedure.

The standardization of natural allergenic extracts and the characterization of recombinant allergens ensures a continuing requirement for highly purified natural allergens. The extraction and purification methods have to be reproducible and also preserve the biological and immunological activity of the allergen. A simple two-step purification system has been established in order to provide milligram amounts of purified natural Phl p 1 and Phl p 2/3. Both major allergens were separated from other proteins of timothy grass pollen extract in one step by hydrophobic interaction chromatography (HIC) under mild conditions. The allergens elute in the flow-through fraction while the rest of the proteins remain bound to the column. The very different molecular weights of Phl p 1 and Phl p 2/3 permitted separation of the allergens by a second step using gel filtration.

Application of reversed-phase high-performance liquid chromatography in the purification of major allergens from grass pollen.

Group 1 and 5 allergens of different grasses possess similar physicochemical parameters (molecular weight, pI) and therefore separation with conventional chromatographic methods (gel filtration, ion exchange chromatography) is difficult or impossible to achieve. In this paper we describe the isolation of biologically active group 1 and 5 allergens from extracts of Lolium perenne and Phleum pratense by means of reverse phase chromatography on HPLC. The chromatograms showed very different retention times for group 1 (Rt 19.1-20.5 min) and group 5 (Rt 24.3-26.3 min) containing fractions. In addition, this technique is suitable for the separation of group 5 allergens into 5a and 5b subgroups and for the estimation of the amounts of allergen (groups 1 and 5) in the different extracts.

The effect of nedocromil sodium on human eosinophil activation.

Nedocromil sodium inhibited the increase in eosinophil activation (measured by IgG- and complement-dependent cytotoxicity assays) induced by platelet activating factor (PAF). Inhibition of the upregulation in eosinophil effector function by nedocromil sodium was dose-dependent (optimal at 10(-7) mol/L) and paralleled that produced by a specific PAF antagonist, BN 52021. In addition, preliminary data suggest that nedocromil sodium can inhibit the increase in IgG-dependent release of LTC4 from human eosinophils after stimulation with the synthetic tripeptide, formyl-methionyl-leucyl-phenylalanine (fMLP). These data support our previous hypothesis that part of the mode of action of nedocromil sodium may be due to its ability to directly block the chemotactic factor-induced enhancement of inflammatory cell activity.

Sensitization to different mite species in German farmers: clinical aspects.

Various mite species referred to collectively as house dust and storage mites are recognized worldwide as a cause of allergic airway disease. Our study aimed to investigate the frequency of sensitization and potential importance of mite species in farmers using a broad mite spectrum. A total of 86 German farmers with rhinitis and/or asthma were studied by skin prick testing and/or enzyme allergosorbent test (EAST) with the following mites: Blomia tjibodas, Blomia tropicalis, Blomia kulagini, Glycyphagus domesticus, Thyreophagus entomophagus, Euroglyphus maynei, Chortoglyphus arcuatus, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, Acarus farris and Cheyletus eruditus. Sensitization to at least one mite species was detected in 51 patients (59%) by skin prick testing, and in 31 patients (36%) by EAST. The most frequent sensitizations determined by skin tests were found for the three Blomia species, E. maynei and G. domesticus. Twelve patients (14%) gave a positive EAST with the predator mite C. eruditus. A total of 22 patients gave positive EAST results with the Dermatophagoides species. We were able to document sensitization to C. arcuatus, E. maynei and T. entomophagus for the first time in Germany. A considerable proportion of the German farmers tested were sensitized to storage mites. The allergological potential of various mite species has been recognized, some for the first time. It was concluded that B. tjibodas, G. domesticus, C. arcuatus and C. eruditus in particular should be included in an allergy diagnosis. Further investigations into the clinical relevance of the sensitizations and possible cross-reactivity between the mite species are necessary.

Generation of a low immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a.

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.

Clinical effects of specific immunotherapy: a two-year double-blind, placebo-controlled study with a one year follow-up.

BACKGROUND: A new depot allergoid of house dust mite (Dermatophagoides pteronyssinus - D.pt) has been created in line with the principles and methodology established in the successful development of pollen allergoids. A two-year double-blind placebo-controlled clinical trial, with one further follow-up year of active treatment, has been conducted to assess clinical efficacy and tolerance. METHODS: 40 patients (20 verum and 20 placebo) with IgE-mediated mite allergy and a history of moderate to severe perennial symptoms of rhinoconjunctivitis with or without asthma participated in a 2-year randomized, double-blind, placebo-controlled trial. Actively treated patients were included in a follow-up year. Active treatment was performed with an aluminium hydroxide adsorbed house dust mite allergoid. Parameters for baseline data and clinical efficacy: nasal challenge, quantitative skin prick testing, Visual Analog Scale (VAS), patients' diaries, physician's assessment of patients? health condition, symptoms and use of anti-allergic medication as well as adverse reactions and changes in specific IgG4 and IgE antibodies. RESULTS: The trial detected superiority (p < 0.05) of mite depot allergoid versus placebo with regard to VAS and symptom intensity sum score in patients who needed anti-allergic medication in the baseline period. Significant differences (p < 0.05) between verum and placebo groups were also seen for patients' reactivities to nasal challenges and prick tests with allergen. The blinded assessment by the physician documented a significant difference (p < 0.05) between the groups in favour of active treatment. After reaching the maximum dose as well as after 12 and 24 months, specific IgG4 antibody concentrations were significantly elevated in the verum group (p < 0.05) by comparison with placebo. Local reactions were less frequent in the verum group and no systemic adverse reactions occurred. A third year of active treatment resulted in further improvement and documented the advantage of booster therapy to stabilize the clinical success. CONCLUSION: Specific immunotherapy with a mite depot allergoid induced significant clinical improvements versus placebo. Safety was assessed as excellent, and no systemic adverse reactions occurred.

Efficacy and safety of preseasonal-specific immunotherapy with an aluminium-adsorbed six-grass pollen allergoid.

BACKGROUND: The clinical efficacy and safety of a six-grass pollen allergoid has been studied. The advent of more exacting clinical guidelines and a better appreciation of the possible mechanisms of treatment prompted this reappraisal. METHODS: A 2-year double-blind multicentre placebo-controlled phase 3 clinical trial was undertaken in 154 patients suffering symptoms of rhinoconjunctivitis with or without asthma (GINA I or II). Therapy comprised two consecutive preseasonal short-courses of subcutaneous injections using a grass pollen allergoid adsorbed to aluminium hydroxide. RESULTS: A combined symptom and medication score (SMS) was used as the primary end-point for clinical efficacy. SMS from the first year showed a significant difference of 26.6% between the two study groups (P=0.026) and this was improved after the second year when there was a 48.4% difference in SMS between active and placebo treatment in favour of the allergoid (P = 0.018). Highly significant increases in grass pollen allergen-specific IgG1 and IgG4 antibody concentrations were measured in association with active treatment. Allergen tolerance was increased as judged by a conjunctival provocation test and significant improvements in quality of life were documented using a standardized questionnaire. The allergoid was well tolerated. CONCLUSIONS: The grass pollen allergoid was shown to be safe and clinically efficacious in the management of hay fever with or without asthma (GINA I or II).

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50-60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13.

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.

The membrane fusion events in degranulating guinea pig eosinophils.

We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTP gamma S induces a 2- to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2-7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances. In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.

Neutrophil chemotactic activity in acute severe asthma (status asthmaticus).

Serum neutrophil chemotactic activity (NCA) was measured in patients with acute severe asthma (status asthmaticus) and compared with that in control subjects (mild asthma, stable chronic irreversible air-flow obstruction, allergic rhinitis, noninfective lung conditions, or asymptomatic). There were 9 subjects in each group. Statistically significant elevations (p less than 0.002) in NCA were detected in acute severe asthma when compared with each control group. Serial measurements of NCA were subsequently undertaken in 12 patients with acute asthma, at the time of admission to hospital, after 3 days of treatment, and on discharge after approximately 7 days. A highly significant (p less than 0.001) reduction in serum NCA activity on Day 7 compared with that on Day 0 was observed, and this correlated inversely with the improvement in lung function (PEFR). Gel filtration by fast protein liquid chromatography (FPLC) using Superose 6 prep grade (6PG) indicated that NCA in acute severe asthma was heterogeneous and consisted of at least 4 peaks of activity associated with proteins with molecular weights of approximately 800, 600, 150, and less than 20 kD. The 800- and 150-kD peaks were also observed in control subjects, but to a lesser degree. The 600- and less than 20-kD activities were virtually confined to the patients with acute severe asthma. FPLC chromatofocusing of the 600-MW peak from the acute asthmatics, using a Mono-P column and a pH gradient from 8.3 to 5.0, revealed considerable activity in fractions eluting between pH 6.0 and 7.0, which was not observed in the normal control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)