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Biomedical subjects

S H Sparholt

Publications and source records attributed to S H Sparholt.

15 recordsLinked to original sources
PubMed2004

A double-blind, placebo-controlled birch allergy vaccination study: inhibition of CD23-mediated serum-immunoglobulin E-facilitated allergen presentation.

BACKGROUND: The clinical efficacy of specific allergy vaccination (SAV), previously called specific immunotherapy is well documented. The working mechanism of this treatment is not completely known at present. Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo possibly as a result of serum IgE-facilitated allergen presentation (S-FAP). Previously, we have shown that this process can be inhibited by long-term birch SAV sera. METHODS: In the present study, we have analysed sera from birch-allergic patients in a randomized double-blind, placebo-controlled clinical trial for their ability to mediate S-FAP. Birch-specific IgE levels were not changed after SAV. Bet v 1-specific IgE levels, however, were significantly decreased (P<0.05) and Bet v 1-specific IgG4 levels were strongly increased after SAV (P<0.001). None of these changes were observed in the placebo group. When the sera were tested for their ability to induce S-FAP, a complete abrogation of this effect was noted in the sera from patients receiving active treatment (P<0.001), but not in the control group. This inhibition of S-FAP seemed to be associated with the reduction in the ratio between Bet v 1-specific IgE and IgG4 antibodies in serum, but a clear correlation could not be demonstrated. CONCLUSION: In conclusion, the present study clearly shows that SAV leads to an inhibition of the S-FAP needed to obtain optimal T cell activation at the low allergen concentrations present in vivo. This novel mechanism may explain the increased allergen threshold levels found in allergen provocation tests and the reduction of late-phase reactions observed after SAV.

Allergens↗
PubMed2001

Standardization of food allergen extracts for skin prick test.

The aim of the study was to standardize and evaluate technically optimized food allergen extracts for use in skin prick test (SPT). The standardization procedure comprised 36 allergic histories in 32 food allergic patients with 21 healthy, non-atopic individuals serving as controls. The patients had a history of allergic symptoms upon ingestion of either cow's milk (n=3), hen's egg (n=9), wheat (n=4), hazelnut (n=14) or cod (n=6). They also had specific IgE in serum to the food in question and a positive SPT with a fresh preparation of the food. The diagnosis had been confirmed by a double-blind, placebo-controlled food challenge, except for the hazelnut-allergic patients. The controls were subjected to an open food challenge with all the foods to ensure tolerance. The standardization was performed by means of titrated SPT in accordance with the guidelines on biological standardization from the Nordic Council on Medicine. Regression analysis of the skin wheal areas was performed for each patient and the median protein concentration of allergen preparation (median Ch10) eliciting a wheal area of the same size as histamine 10 mg/ml was calculated. The median Ch10 was 0.56 mg/ml for milk, 0.88 mg/ml for egg, 5.4 mg/ml for wheat, 2.1 mg/ml for hazelnut and 0.017 mg/ml for the cod extract. The sensitivity of the median Ch10 estimated from the SPT data was 1 for milk, 0.98 for egg, 1 for wheat, 1 for hazelnut and 0.87 for the cod extract. The allergenic activity of the hazelnut extract was further investigated by leukocyte histamine release (HR) and immunoblotting experiments using sera from 27 hazelnut allergic patients. The clinical sensitivity of the optimized hazelnut extract evaluated by HR was 0.78 compared to 0.30 for a commercially available hazelnut extract (Soluprick). Immunoblotting results showed a stronger IgE binding capacity and additional IgE-binding bands of the optimized hazelnut extract compared with the Soluprick extract.

Allergens↗
PubMed1999

The polymorphisms S503P and Q576R in the interleukin-4 receptor alpha gene are associated with atopy and influence the signal transduction.

Interleukin-4 (IL-4) plays a major role in immunoglobulin E (IgE) production. Its signal is conferred to effector cells through binding to the alpha chain of the IL-4 receptor (IL-4Ralpha). We present further evidence for polymorphisms in the IL-4Ralpha gene having an effect on IgE regulation. For two of four common polymorphisms, S503P and Q576R, we found an association with lowered total IgE concentrations (P=0.0008 if occurring together). The polymorphism S503P has not yet been described and is located within the I4R motif of the receptor. In vitro analyses using synthetic peptides of this region showed that the tyrosine kinase Janus kinase 1 (JAK1), as well as IRS-1 and IRS-2 bind to the I4R motif irrespective of the polymorphism or a tyrosine phosphorylation. In vivo immunoassays using T cells of four different groups of individuals (S503/Q576; P503/Q576; S503/R576; P503/R576) revealed that only in case of both polymorphisms the phosphorylation of IRS-1 and IRS-2, but not JAK1 was increased. We found no binding of STAT6 to the I4R synthetic peptides; however, the phosphorylation was reduced in the presence of any of the two polymorphisms, including P503 alone. We discuss possible conformational changes of the receptor leading to the observed effects on the phosphorylation status of IRS-1, IRS-2 and STAT6, in addition to previous findings that Q576R alters STAT6 binding. We conclude that P503 and R576 influence the signal transduction pathways through the IL-4Ralpha, an effect that is magnified by the presence of both polymorphisms. This could explain the observed association effects with lowered total IgE concentrations.

Adolescent↗
PubMed1999

Linkage and association studies of atopy and the chromosome 11q13 region.

The clinical syndrome atopy is largely determined by genetic factors. In 1989, the first linkage of markers within and flanking the chromosomal region 11q13 and atopy was reported. In the following years, the gene coding for the beta chain of the high affinity IgE receptor was localised to this region and two polymorphisms in this gene have been shown to be associated with the atopic phenotype. We investigated two independent populations (population based and outpatient department) with different degrees of clinical symptoms. Using highly polymorphic markers we could find no evidence for linkage or allelic association of this particular genomic region to the atopic phenotype defined by enhanced IgE responsiveness (p>0.05). Neither did we succeed in finding either of the two polymorphisms described, nor could we identify any other polymorphisms within the gene. However, we found weak evidence for linkage in asthmatic sib pairs regarding maternal alleles (p=0.03). We conclude from our data that in our populations the gene for the beta chain of the high affinity IgE receptor is of minor importance for enhanced IgE responsiveness, and that it might influence atopy with clinical signs like asthma through maternally derived alleles.

Adolescent↗
PubMed1998

Dissection of the grass allergen-specific immune response in patients with allergies and control subjects: T-cell proliferation in patients does not correlate with specific serum IgE and skin reactivity.

BACKGROUND: Pollinosis, caused by grasses of the Poaceae family, is a problem worldwide. The relative importance of grass groups 1 and 5 major allergens is well established. However, not much is known about the recognition of these allergens by T cells and whether this T-cell reactivity correlates with skin reactivity and serum IgE levels. OBJECTIVES: The aim of this study was to characterize the cross-reactive, grass allergen-specific T-cell responses from patients allergic to grass and nonatopic individuals and to investigate whether these responses correlate with grass-specific IgE and skin reactivity. METHODS: Skin prick test wheal areas and grass-specific serum IgE levels were determined in all patients (n = 21) and nonallergic control donors (n = 20). Peripheral blood mononuclear cells were isolated and stimulated with grass allergen extracts (Phleum pratense, Poa pratensis, Lolium perenne) and immunoaffinity-purified group 5 allergens, and the production of type 1 and type 2 cytokines was determined in the patient group. RESULTS: Donors allergic to grass showed increased T-cell-proliferative responses to grass allergens compared with nonatopic control subjects. We find it interesting that the magnitude of the patients' T-cell responses could not be correlated with the individual skin prick test areas and specific serum IgE levels, and several patients with allergies to grass had group 5-specific T-cell responses in the absence of group 5-specific IgE. The absence of a correlation between T-cell proliferation and IgE levels or skin prick test results may in part be explained by the finding that patients predominantly produced IL-5 in response to Phl p 5, the major allergen, and predominantly IFN-gamma in response to Phl p extract. In general, all donors responded equally well to all three grasses. Additional experiments with Phl p 5-specific T-cell lines indicated that the equal proliferation of peripheral blood mononuclear cells to all three species is the direct result of cross-reactivity. CONCLUSIONS: Grass allergen-specific T-cell responses are highly cross-reactive, and patients with allergies exhibit higher responses than nonallergic donors, suggesting that T cells are involved in the allergic reaction to grass group 5 allergens. However, group 5-specific T-cell responses are also found in donors without group 5-specific IgE, and the patients' grass-specific T-cell responses and cytokine production do not correlate to skin reactivity or to concentrations of grass-specific IgE.

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

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↗
PubMed1994

Association between HLA-DRB3*0101 and immunoglobulin-E responsiveness to Bet v I.

The relationship between HLA-DRB3 antigens and specific IgE responsiveness to highly purified major allergen from white birch (Betula verrucosa), Bet v I, was studied in 43 patients, who were skin-prick test positive to birch pollen extract. Specific IgE to Bet v I was measured using an assay based on the principle of the Magic Lite method. HLA-DR and -DQ typings were performed by standard restriction fragment-length polymorphism techniques and HLA-DRB3 typing by the use of polymerase chain reaction and sequence-specific oligonucleotide. Of the 43 patients, 41 (95%) exhibited Bet v I-specific IgE. The data showed a significant association between HLA-DRB3*0101 and specific IgE to Bet v I (pc = 0.02). Furthermore, it was shown that this association was linked to the overall IgE responsiveness and not to a limited area of IgE sensitivity. The present study confirms and further identifies the association of DRB3*0101/0301 earlier described in an Austrian population to be present also in a Danish study group. This underlines the importance of this particular allele in the allergic immune response to birch pollen and emphasizes the importance of investigating the molecular basis of human allergic immune responsiveness.

Allergens↗
PubMed1992

The allergen specific B-cell response during immunotherapy.

The paper examines the allergen specific B-cell response in peripheral blood from patients undergoing immunotherapy with house dust mite extract. The 12 patients were part of a double blind placebo controlled study, and they were treated with either Dermatophagoides pteronyssinus (n = 4), Dermatophagoides farinae extract (n = 3) (Alutard SQ, ALK, Denmark) or placebo (n = 5). Blood was taken every fortnight on day seven after hyposensitization and tested for IgM, IgG, IgA and IgE antibody secreting cells (AbSC) to D. pteronyssinus and D. farinae allergens and for the total number of immunoglobulin secreting cells (IgSC). The data showed a maximum of approximately 120 Der f I+II specific AbSC/10(6) mononuclear cells (MNC). A comparison of specific AbSC to the major allergens of the two house dust mites demonstrated that there was no measurable species specificity in the B-cell response that could be correlated to immunotherapy with either of the two extracts. The specific IgM, IgG, and IgA response to Der f I+II was examined in the placebo (39 measurements) and the actively treated (56 measurements) groups, and the results demonstrated a significant rise in specific IgM and IgA AbSC following immunotherapy. The number of specific IgG AbSC did not change. There was a mean of less than one specific IgE AbSC/10(6) MNC, and no detectable change following the treatment. It is speculated that immunotherapy to inhalant allergens causes the induction of specific IgA AbSC. It would then be these partly differentiated plasma cells that are detected on their way to the bronchial or gut mucosa to exert their protective function mediated by allergen specific secretory IgA.

Allergens↗
PubMed1991

Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens.

An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen demonstrated that spot formation was specific. Addition of cycloheximide showed that spot formation was due to antibodies actively produced and secreted and not caused by preformed surface-associated Ig. An analogous method was used to count immunoglobulin-secreting cells (IgSC) of any specificity, and the number of circulating IgSC was measured in young healthy subjects. The results (means) were 331 IgM-SC, 532 IgG-SC, 850 IgA-SC and less than 1 IgE-SC per 10(6) mononuclear cells (MNC). Initial experiments performed with blood from patients undergoing immunotherapy with Dermatophagoides pteronyssinus Alutard, showed that 1 week after injection, circulating allergen-specific antibody-secreting cells (AbSC) were detected with this assay. The ranges were between 1 and 13 IgM-, 2 and 20 IgG- and 20 and 55 IgA allergen-specific AbSC per 10(6) MNC. It is expected that this assay will help to understand more thoroughly the basic immunological mechanisms behind the efficacy of immunotherapy.

Adult↗
PubMedDate not supplied

The significance of isoallergenic variations in present and future specific immunotherapy.

The isoallergenic variation of the tree pollen major allergens has been studied by 2D gel electrophoresis, and by analysis of several recombinant clones. The studies have included both antibody-based and T cell stimulation assays. Bet v 1, the major allergen of birch, forms at least 24 spots when conventional extracts are analyzed by 2D gel electrophoresis. Comparison of Bet v 1-encoding DNA sequences reveals a considerable number of amino acid substitutions. This sequence variation can theoretically account for the number of spots observed in 2D gels. Whereas pools of serum from allergic individuals and monospecific antibodies raised in rabbits bind to most if not all spots in 2D gels, analyses of individual serum and/or murine monoclonal antibodies show individual patterns of reactivity with various subsets of spots. These observations point to a model in which amino acid substitutions induce local perturbations of the allergen surface, causing differences in epitope structure. Furthermore, analysis of pollen from individual trees shows that each tree produces individual subsets of Bet v 1 spots. When analyzed in stimulation assays, T cell clones also display differences in reactivity to different isoallergens. In conclusion, we have shown that Bet v 1 is heterogeneous, and that individual trees produce various subsets of isoallergens which display differences in reactivity both towards antibodies and T cells. A careful selection of isoform may therefore be of major importance if recombinant allergens or synthetic peptides are to be used for conventional immunotherapy.

Allergens↗