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H Ipsen

Publications and source records attributed to H Ipsen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crossreactivity and T-cell epitope specificity of Bet v 1-specific T cells suggest the involvement of multiple isoallergens in sensitization to birch pollen.

BACKGROUND: Allergen-specific T lymphocytes play an important role in the pathophysiology of atopic disease. Detailed studies of their epitope-specificity and crossreactivity are required for the development of novel approaches for specific immunotherapy. OBJECTIVES: The aim of the study was to characterize the fine specificity of Bet v 1-specific T cells from allergic donors. METHODS: Polyclonal T-cell lines (TCL) and T-cell clones (TCC), specific for Bet v 1, the major birch (Betula verrucosa) pollen allergen, were isolated from the peripheral blood of three birch-allergic patients. Their epitope-specificity was studied using overlapping synthetic peptides, and crossreactivity with other tree pollen allergens of the Fagales order was evaluated. In addition, the Bet v 1-specific TCC were studied for their phenotype and cytokine production. RESULTS: All isolated Bet v 1-specific TCC (19/21 CD4+, 2/21 CD8+) reacted with affinity purified Bet v 1, but showed different reactivities with recombinant Bet v 1 (rBet v 1), and with group 1 allergens from other Fagales species. Epitope mapping of rBet v 1-reactive TCC with synthetic peptides of Bet v 1 showed the presence of four T-cell epitopes. Polyclonal T-cell lines reacted with 13 different peptides, and displayed even broader crossreactivity with group 1 pollen allergens from other Fagales members. CONCLUSION: This study demonstrates that apart from T-cell epitopes of rBet v 1, many other crossreactive or Bet v 1 isoallergen-specific epitopes exist. This indicates that isoallergenic variation plays an important role in the induction of Bet v 1-specific and crossreactive T-cell responsiveness to allergens.

Allergens↗
PubMed1996

[Clinical consequences of intranasal insulin therapy in insulin-dependent diabetes mellitus].

Metabolic control, hypoglycaemia frequency and nasal mucosal physiology were evaluated in 31 insulin-dependent diabetics treated with intranasal insulin at mealtimes for one month and with subcutaneous fast-acting insulin for another month in a randomized crossover trial. During both periods the patients were treated with intermediate-acting insulin at bedtime. Six of the patients were withdrawn from the study during intranasal insulin therapy due to metabolic dysregulation. Insulin concentrations increased more rapidly and decreased more quickly during intranasal as compared with subcutaneous insulin administration. Metabolic control, assessed by haemoglobin A1c concentrations, deteriorated after intranasal as compared with subcutaneous insulin therapy. The bioavailability of intranasally applied insulin was low, since intranasal insulin doses were approximately 20 times higher than subcutaneous doses. The frequency of hypoglycemia was similar during intranasal and subcutaneous insulin therapy, and nasal mucosal physiology was unaffected after intranasal insulin. We conclude that due to low bioavailability and to a high rate of therapeutic failure, intranasal insulin treatment is not a realistic alternative to subcutaneous insulin injections at the present time.

Administration, Intranasal↗
PubMed1996

Characterization of purified recombinant Bet v 1 with authentic N-terminus, cloned in fusion with maltose-binding protein.

A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.

ATP-Binding Cassette Transporters↗
PubMed1996

Characterisation of recombinant isoforms of birch pollen allergen Bet v 1.

Three isoforms of the major birch pollen allergen, Bet v, 1 from Betula verrucosa have been expressed as recombinant proteins in E. coli and purified. The immunochemical properties of recombinant isoforms (rBet v 1) differed on immunoblots when compared using Mabs and birch pollen allergic patients serum IgE. 2-D gel analysis showed that recombinant isoforms with different epitope structure can focus under the same protein spot after electrophoresis. The structure of conformational epitopes can be distorted by amino acid substitutions even when T-cell epitopes are not affected as judged by T-cell proliferation studies.

Allergens↗
PubMed1996

X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy.

The three-dimensional structure of the major birch pollen allergen, the 17,500 M(r) acidic protein Bet v 1 (from the birch, Betula verrucosa), is presented as determined both in the crystalline state by X-ray diffraction and in solution by nuclear magnetic resonance (NMR) spectroscopy. This is the first experimentally determined structure of a clinically important inhalant major allergen, estimated to cause allergy in 5-10 million individuals worldwide. The structure shows three regions on the molecular surface predicted to harbour cross-reactive B-cell epitopes which provide a structural basis for the allergic symptoms that birch pollen allergic patients show when they encounter pollens from related trees such as hazel, alder and hornbeam. The structure also shows an unusual feature, a 30 A-long forked cavity that penetrates the entire protein.

Allergens↗
PubMed1995

Intranasal insulin therapy: the clinical realities.

To evaluate metabolic control and safety parameters (hypoglycaemia frequency and nasal mucosa physiology), 31 insulin-dependent diabetic patients were treated with intranasal insulin at mealtimes for 1 month and with subcutaneous fast-acting insulin at meals for another month in an open, crossover randomized trial. During both treatment periods the patients were treated with intermediate-acting insulin at bedtime. Six of the patients were withdrawn from the study during intranasal insulin therapy due to metabolic dysregulation. Serum insulin concentrations increased more rapidly and decreased more quickly during intranasal as compared with subcutaneous insulin administration. Metabolic control deteriorated, as assessed by haemoglobin A1c concentrations, slightly but significantly after intranasal as compared with subcutaneous insulin therapy. The bioavailability of intranasally applied insulin was low, since intranasal insulin doses were approximately 20 times higher than subcutaneous doses. The frequency of hypoglycaemia was similar during intranasal and subcutaneous insulin therapy, and nasal mucosa physiology was unaffected after intranasal insulin. We conclude that due to low bioavailability and to a high rate of therapeutic failure, intranasal insulin treatment is not a realistic alternative to subcutaneous insulin injections at the present time.

Administration, Intranasal↗
PubMed1995

Characterization of Chenopodiales (Amaranthus retroflexus, Chenopodium album, Kochia scoparia, Salsola pestifer) pollen allergens.

Pollen extracts of the four taxonomically related weeds, Amaranthus retroflexus (Ama r), Chenopodium album (Che a), Kochia scoparia (Koc s), and Salsola pestifer (S. kali) (Sal p), were characterized by various methods including crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), and SDS-PAGE immunoblotting. The allergen profiles were determined by CRIE and SDS-PAGE IgE immunoblotting. CRIE detected from one to four important allergens, while SDS showed up to four bands that bound IgE from a number of patient sera. CRIE and SDS-PAGE immunoblotting did not recognize the same number of important allergens in the individual weeds, and the number of allergens detected by the two methods differed considerably, suggesting that IgE-binding epitopes may be denatured during SDS-PAGE. However, it was possible to correlate the identity of some of the important allergens detected by CRIE and SDS-PAGE immunoblotting in all four weeds.

Allergens↗
PubMed1993

Characterization of recombinant bet vI, the major pollen allergen of Betula verrucosa (white birch), produced by fed-batch fermentation.

The gene encoding the major allergen, Bet v I, from Betula verrucosa (white birch) pollen was cloned by application of the polymerase chain reaction with double-stranded cDNA as template and specific primers based on the published nucleotide sequence of the gene. The gene was inserted into plasmid pKK223-3 and expressed in Escherichia coli K-12 strain JM105 grown to high cell density in a fermenter using a fed-batch procedure. Ample material was provided for a thorough characterization of the epitope structure of recombinant Bet v I contained in an unpurified soluble lysate. The antibody-binding characteristics of recombinant Bet v I was compared with that of the natural allergen using polyclonal rabbit antibodies raised against Bet v I in crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and Western blotting. IgE from a pool of 16 allergic patients' serum was applied in crossed radioimmunoelectrophoresis, Western blotting, and a quantitative luminescence inhibition immunoassay. Well-defined epitopes were assayed by the application of a panel of six murine monoclonal antibodies in a quantitative radio inhibition immunoassay. In all assays the activity of recombinant Bet v I was comparable to that of natural Bet v I, and it is concluded that the epitope structure of recombinant Bet v I closely resembles that of natural Bet v I. This result has important implications for the future use of recombinant tree pollen allergens as a model system for the study of allergenic B and T cell epitopes aiming at improvements in reagents used for the management of allergic disease.

Allergens↗
PubMed1992

PCR based cloning and sequencing of isogenes encoding the tree pollen major allergen Car b I from Carpinus betulus, hornbeam.

Cloning of the gene encoding the major allergen, Car b I, from Carpinus betulus (hornbeam) pollen was performed using the Polymerase Chain Reaction (PCR) to specifically amplify the gene of interest using single stranded cDNA as template. Specific primers, deduced from the aminoterminal sequence of the purified protein, were tailored to facilitate direct expression of plasmic clones, and the large fraction of positive clones obtained, revealed the presence of isogenic variation. Three clones were characterized in detail by antibody based assays and nucleotide sequencing. The recombinant allergens were shown by crossed immunoelectrophoresis (CIE) to precipitate with monospecific polyclonal rabbit antibodies raised against purified Bet v I, by crossed radioimmunoelectrophoresis (CRIE) to bind tree pollen allergic patient serum IgE, and by immunoblotting to bind murine monoclonal antibodies, raised against purified Car b I from pollen. Car b I is encoded by a 159-triplets open reading frame. The molecular masses (M(r) = 17272, 17355 and 17217 Da, respectively), the amino acid composition, and the aminoterminal sequence of the predicted polypeptides agree well with data obtained by analysis of the protein purified from pollen. The deduced amino acid sequences show pronounced homology (73, 75 and 74% identities respectively) to Bet v I, the major allergen from Betula verrucosa (white birch) pollen. Soluble recombinant Car b I, without a fusion partner, was produced in Escherichia coli with an immunochemical reactivity closely resembling that of the native pollen allergen. The tree pollen major allergens therefore constitute an ideal system for the study of allergenic epitopes.

Allergens↗
PubMed1992

Specificity mapping of patients IgE response towards the tree pollen major allergens Aln g I, Bet v I and Cor a I.

The specificity pattern of IgE from non-treated tree pollen allergic patients (n = 38) were evaluated by solid phase absorption of serum samples followed by CRIE on alder, birch and hazel CIE precipitation profiles. The majority of the serum samples seemed to contain IgE antibodies with the following characteristics: specific towards Bet v I alone and common between Aln g I, Bet v I and/or Cor a I, 'II'. The IgE specificity profiles observed for 95% of the sera tested are compatible with birch pollen allergens being the only sensitizing allergens, indicating that the patients react to allergens from other tree pollens of the Fagales order due to IgE cross-reaction with the major allergens of birch and alder and/or hazel pollens.

Allergens↗
PubMed1991

The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of Alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a I pollens.

The tree pollen major allergens (Aln g I), Bet v I, Car b I and Que a I, were purified to near homogeneity. Rabbit antibodies raised towards the major allergen Bet v I react with the major allergens of alder, hazel, hornbeam and oak, although with distinct variation in affinity. Immunochemically the major allergens seem to resemble one another more within than between the botanically established families of the fagale order. The physico-chemical parameters (molecular weight, pI values, amino acid composition and NH2-terminal amino acid sequence) of the major allergens exhibit a higher degree of consistency than do the immunochemical parameters, indicating that nuances in the allergens' secondary and tertiary structures are likely to govern the immunochemical differences.

Allergens↗
PubMed1988

Immunotherapy with partially purified and standardized tree pollen extracts. I. Clinical results from a three-year double-blind study of patients treated with pollen extracts either of birch or combinations of alder, birch and hazel.

Fifty-four adult patients with tree pollen-induced rhinitis (28), asthma (1), or rhinitis and asthma (25) were selected for immunotherapy with standardized and partly purified tree pollen extracts using a double blind protocol. The selection was based on clinical history, results of nasal or bronchial challenge, skin prick tests and RAST. Further, based on crossed radio-immunoelectrophoresis, sex, age and severity of symptoms, the patients were allocated in matched pairs and the treatment alternatives were randomly distributed within the pairs. Twenty-three patients treated with extracts composed of any combination of alder, birch and hazel pollen which matched their IgE response in CRIE (Group 1 (ABC)) and 22 patients receiving birch pollen extracts (Group 2 (B)) completed all 3 years of treatment. The in vivo results comprising symptom and medicine consumption scores are given here. Changes in specific skin and nasal reactivity as well as in immunological parameters are presented separately. No significant differences were demonstrated between the treatment groups in the two parameters. Both extracts were effective and reduced in general the symptom scores to one tenth of the starting level. Expressed another way, at the end of the study, the patients tolerated 30 times more pollen until symptoms of the same severity were elicited, compared to before. In the Nordic countries, spring-time asthma and rhino-conjunctivitis caused by pollen from deciduous trees can be effectively treated with an extract of birch pollen alone.

Adolescent↗
PubMed1988

Immunotherapy with partially purified and standardized tree pollen extracts. II. Results of skin prick tests and nasal provocation tests from a three-year double-blind study of patients treated with pollen extracts either of birch or combinations of alder, birch and hazel.

Patients allergic to tree pollen entered a 3-year course of immunotherapy (1980-83) with either birch pollen extracts alone (n = 26) or patient-tailored extracts of birch, alder and hazel pollen (n = 27). The clinical and immunological results of this study are published elsewhere. This paper contains an evaluation of skin prick test and nasal provocation test results. There were no significant differences between the two treatment groups concerning these two parameters. In both groups the allergen-specific sensitivity in the skin showed seasonal variations but a significant decrease. During the years of treatment there was also a significant decrease in the specific sensitivity of the nasal mucosa. With the present demands for purification and standardization of allergen extracts it is of practical and economic interest to know that tree pollen-allergic patients showing positive reactions to birch, alder and hazel extracts can be effectively treated using birch pollen extract alone.

Asthma↗
PubMed1988

Immunotherapy with partially purified and standardized tree pollen extracts. III. Specific IgE response to the major allergens of alder, birch and hazel pollen during immunotherapy.

Patients allergic to pollen from alder, birch and hazel were hyposensitized during a 3-year period with either birch pollen extract alone (n = 24) or a mixture of one or more of alder, birch and hazel pollen extracts (n = 27). The effect of the treatment was evaluated by RAST and tandem crossed-radioimmunoelectrophoresis (tandem-CRIE). The patient' specific IgE response to the major allergens of alder (Aln g I), birch (Bet v I) and hazel (Cor a I and Cor a II), as measured by tandem-CRIE, and the total specific IgE response, measured by RAST, decreased significantly (Pc less than 0.05) during immunotherapy, irrespective of the extract used during the treatment. There was no significant difference (Pc less than 0.05) between the two treatment groups. The results obtained indicate either that birch pollen extract alone is adequate in the treatment of the studied patient group or the patients had been sensitized towards birch pollen alone.

Clinical Trials as Topic↗
PubMed1985

The use of a reference allergenic extract in the evaluation of allergen products.

Two commercially available mugwort (Artemisia vulgaris (Av] pollen extracts Av-ALK and Av-PS were compared with a dialysed reference mugwort pollen extract (Av-REF) by means of crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE), RAST-inhibition, RAST-screening and skin prick test. Av-ALK and Av-PS both contained approximately half the number of precipitation arcs detectable in Av-REF, and Av-PS seemed to lack two intermediate allergens and two minor allergens, while Av-ALK seemed to lack one minor allergen. The RAST-inhibition curve obtained for Av-PS did not parallel the curves obtained for Av-ALK and Av-REF, indicating that the allergen composition of Av-PS differs from that of the others. Further, RAST-screening experiments support the CIE observation that Av-PS seemed to lack two of the intermediate allergens. The HEP values determined for Av-ALK and Av-PS diverge considerably from those stated by the manufacturers. The results obtained demonstrate that the outcome of a comparison between a reference allergenic extract and allergenic products depends very much on the method employed.

Allergens↗
PubMed1985

Immunochemical and biological characterization of a mugwort (Artemisia vulgaris) pollen extract.

A reference extract of mugwort pollen (Artemisia vulgaris) was characterized by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE) and quantitative skin prick test (QSPT). CIE revealed that the extract contained at least 42 distinct antigens of which 24 migrated towards the anode and 18 towards the cathode at pH 8.6. A CRIE analysis of the crude mugwort pollen extract, performed with sera from 29 mugwort-allergic patients, showed that 10 antigens may be considered allergens; one was classified as a major allergen, five as intermediate allergens, and four as minor allergens. The QSPT performed on the same 29 allergic patients established that 17.4 micrograms lyophilised reference mugwort pollen extract per ml had a biological potency of 1 HEP (histamine equivalent by prick test).

Adolescent↗