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J N Larsen

Publications and source records attributed to J N Larsen.

At least 19 recordsLinked to original sources
PubMed2006

Inhibition of rBet v 1-induced basophil histamine release with specific immunotherapy -induced serum immunoglobulin G: no evidence that FcgammaRIIB signalling is important.

BACKGROUND: Human basophils and mast cells express the low-affinity immunoglobulin (Ig)G receptor FcgammaRIIB. It has previously been shown in artificial model systems that cross-linking of the high-affinity IgE receptor FcepsilonRI and FcgammaRIIB leads to inhibition of FcepsilonRI signalling. OBJECTIVE: The aim of the present study was to investigate whether cross-linking of FcepsilonRI and FcgammaRIIB contributes to IgG-mediated inhibition of histamine release in human basophils in a system using the sera from specific immunotherapy (SIT) patients and the major allergen from birch pollen, Bet v 1. As IgG4 furthermore has been proposed to have special blocking properties, we investigated the significance of IgG subclass specificity for this inhibition. METHODS: Binding of recombinant Bet v 1-IgG complexes to FcgammaRII and IgG-binding activities in the sera from 25 birch pollen-allergic patients treated with SIT were measured using (125)I-rBet v 1. Inhibition of basophil histamine release was assessed by incubating washed leucocytes with complexes of rBet v 1-IgG with or without blocking of FcgammaRII. RESULTS: We observed low binding of rBet v 1-IgG complexes to FcgammaRII, which was negatively correlated with the relative IgG4-binding activities. Blocking of FcgammaRII did not reverse the SIT-IgG-induced inhibition of basophil histamine release. However, IgG-binding activities correlated significantly with the ability of the SIT sera to inhibit basophil histamine release. CONCLUSION: We suggest that at least in birch pollen SIT, the contribution of FcgammaRIIB-mediated inhibitory signalling to SIT-IgG-induced inhibition of human basophil histamine release is of minor importance. The main contributor to the inhibitory effect of SIT-induced IgG seems to be blocking of the allergen-IgE interaction.

Allergens↗
PubMed2001

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.

Allergens↗
PubMed2001

Molecular basis of allergic cross-reactivity between group 1 major allergens from birch and apple.

Patients allergic to birch pollen often also react with fruits and vegetables, such as apple. The major cause of cross-reactivity between birch and apple is biochemical and immunological similarity between the major allergens, Bet v 1 and Mal d 1, as demonstrated by serological and cellular immunoassays. In addition, birch pollen-specific therapeutic allergy vaccination has been shown to improve allergic symptoms caused by oral ingestion of apple. Detailed analysis of molecular surface areas based on the crystal structure of Bet v 1, and primary sequence alignment, identify potential epitopes for cross-reactive antibodies. Two or more conserved patches are identified when comparing Bet v 1 and Mal d 1, thus providing a molecular model for serological cross-reactivity involving more than one IgE-binding epitope. A minimum of two epitopes would be necessary for cross-linking of receptor bound IgE in functional histamine release assays and skin test. Individual amino acid substitutions, as occurring in isoallergenic variation, may, however, have a dramatic effect on epitope integrity if critical residues are affected. Thus, one area large enough to accommodate antibody-binding epitopes shared by all known Mal d 1 isoallergens and variants is identified, as well as areas shared by Bet v 1 and individual Mal d 1 isoallergens or variants. The occurrence of limited epitope coincidence between Bet v 1 and Mal d 1 is in agreement with the observation that some, but not all, birch pollen allergic patients react with apple, and that the epitope repertoire recognised by the IgE of the individual patients determines the degree of cross-reactivity.

Allergens↗
PubMed2001

Recombinant allergens/allergen standardization.

Recombinant allergens are genetically engineered isoforms representing allergen molecules from allergen extracts. Immunologic responses of allergic patients toward allergen extracts define the major allergens. For the average allergic patient, the diagnostic sensitivity and treatment efficacy correlate with the concentration of major allergen. Standardization of allergen products (extracts or genetically engineered allergens) can therefore advantageously be performed using a selected recombinant major allergen as a standard. The standardization will furthermore require reagents for which both monospecific, monoclonal, or preferably, recombinant antibodies can be used. Due to differences in the allergenic activity of individual isoallergens and the naturally occurring mixture of isoallergens found in an allergen extract, and due to additional contribution to the allergenic activity from other molecules in the extract, a biologic potency assessment must always be performed as a supplement. This is also the case for a genetically engineered allergen product.

Allergens↗
PubMed2001

Neoplasia versus hyperplasia of the retinal pigment epithelium. A comparison of two cases.

PURPOSE: To present the clinical and histopathological characteristics of two different tumor-like lesions of the retinal pigment epithelium (RPE). METHODS: Two cases of tumor-like lesions of the RPE were identified in the files of the Eye Pathology Institute. The clinical characteristics and the light- and electron microscopical morphology of the lesions were compared and the diagnoses were re-evaluated applying modern immunostainings. RESULTS: Clinically, both adenoma and tumor-like hyperplasia of the RPE may present with prominent retinal feeder arterioles. The lesions are hypofluorescent in the filling phases and have multiple hyperfluorescent zones in the late phase in fluorescein angiography. They show high internal reflectivity by A-scan and appear as solid tumors by B-scan ultrasonography. Histologically, the two presented lesions of the RPE are different. The first is an adenoma of the vacuolated subtype. The other lesion is a hyperplasia of the RPE disclosing a tubular morphology. The pathologically active cells in both cases were positive for the reaction with antibodies against: cytokeratin, NSE, vimentin, S-100, HMB-45, desmin and SMA. However, only the adenoma was sporadic melan-A positive. CONCLUSION: Adenomas and tumor-like hyperplastic lesions of the RPE are very rare lesions. They share many morphological and immunohistological characteristics. Of the presented cases only the RPE adenoma is sporadic melan-A positive.

Adenoma↗
PubMed2000

Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1.

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.

Allergens↗
PubMed1999

Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab' fragment.

The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of the clinical symptoms of allergy. In order to study the structural basis of allergen-antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab' fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 A, alpha = 105.7, beta = 98.32, gamma = 97.62 degrees, and diffract to 2.9 A resolution when analyzed at 100 K using synchrotron-generated X--rays.

Allergens↗
PubMed1998

Preserved epitope-specific T cell activation by recombinant Bet v 1-MBP fusion proteins.

BACKGROUND: Allergen-specific T cells play an important role in the allergic immune response, and are thought to be the principal target in specific immunotherapy. OBJECTIVE: The aim of the present study was to evaluate if fusion proteins of allergens with bacterial proteins can be used to activate and bias allergen-specific T cells, and to characterize T cell epitopes. METHODS: The complete gene of Bet v 1, the major birch pollen allergen, was amplified by PCR from birch pollen mRNA, and cloned in pKK223-3. The complete gene or truncated sequences were transferred to pMAL-c and expressed in E. coli as fusion proteins with maltose binding protein (MBP). The complete fusion protein, and the truncated fusion proteins were used for studies with Bet v 1-specific T cells. RESULTS: Bet v 1-specific T cells reacted similarly with purified and crude Bet v 1-MBP proteins. Therefore, crude preparations were used to study the epitope-specificity of 11 Bet v 1-specific T cell clones. Six distinct T cell epitopes were determined in this way. Interestingly, the T cell epitope of three T cell clones, that did not react with synthetic peptides in a previous study, was identified. In addition, the presence of MBP as a fusion partner to Bet v 1 was shown to influence TH2/TH1 cytokine production in T cell lines, but not in established T cell clones. CONCLUSION: Using crude preparations of recombinant fusion proteins of Bet v 1 with MBP, multiple T cell epitopes were identified in Bet v 1. As T cell activation is preserved in this system, the generation of recombinant allergens with TH1-inducing proteins as fusion partners might be considered as a T-cell targeted approach for specific immunotherapy.

ATP-Binding Cassette Transporters↗
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

Crystallization and preliminary X-ray investigation at 2.0 A resolution of Bet v 1, a birch pollen protein causing IgE-mediated allergy.

The 17 kDa protein from Betula verrucosa (White Birch) pollen, Bet v 1, is the clinically most important birch pollen allergen causing immediate Type I IgE-mediated allergy. The three-dimensional structure of Bet v 1 and its IgE-binding epitopes are at present not known. In addition, the biological function of Bet v 1 in birch pollen is not fully established. In this work, Bet v 1 has been expressed in Escherichia coli as a recombinant protein, purified and crystallized. The space group of recombinant Bet v 1 crystals is orthorhombic C2221 with unit cell parameters a = 32.13 A, b = 74.22 A, and c = 118.60 A. There is one Bet v 1 molecule per asymmetric unit and the water content is 41%. Crystals diffract to 2.0 A resolution and a complete native data set was collected from a single crystal using CuK alpha X-rays from a rotating anode.

Allergens↗
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

Somatostatin in the retina.

One single gene codes for the peptide somatostatin in mammals. Among species the propeptide shows a high degree of homology as the porcine and human propeptides are identical and only differs in two positions compared with the rat propeptide. Somatostatin and prosomatostatin are therefore very suitable for a comparative investigation of the posttranslatoric processing. Antibodies directed against four putative parts of the processing products of the prosomatostatin molecule were used in order to investigate the comparative biosynthetic processing of the precursor for the neuropeptide somatostatin, in the retina from frog, rat, gerbil, mink and monkey. The antibodies were employed to quantify, characterize, and localize somatostatin and prosomatostatin by biochemical and histochemical techniques. Further, somatostatin producing cells were demonstrated by in-situ hybridization using an antisense probe for somatostatin. Somatostatin and prosomatostatin were demonstrated in retinal extracts from all five species investigated. Retinal posttranslational processing of the prosomatostatin molecule varies between the species. In rat and monkey only one form of the bioactive somatostatin (SS-14) is present. In frog, gerbil and mink two known bioactive forms of somatostatin (SS-14 and SS-28) were present. In all five species prosomatostatin-64 (proSS-64) was recognized. In addition, the fragment somatostatin 1-28(1-12) was detected in rat and monkey retinae. In the rat somatostatin and prosomatostatin were demonstrated in subpopulations of amacrine cells located in the inner part of the retina. Cell bodies were labelled at the proximal boarder of the inner nuclear layer and in the ganglion cell layer. From these cells processes were limited to the plexiform layers of the retina and were never observed leaving the retina. By in-situ hybridization autoradiographic signals were demonstrated according to the cell bodies represented by immunohistochemistry. No seasonal variation was observed in the processing pattern of the peptide, investigated in the frog retina. However, the concentration measured during wintertime was almost two fold the concentration measured during summertime. In conclusion, the findings indicate that somatostatin and prosomatostatin are well conserved peptides in vertebrate retinae. Species differences in posttranslational processing excist. The physiological implication of this fact needs to be solved.

Amino Acid Sequence↗