Proceedings of the National Academy of Sciences of the United States of America, Sep 1, 1993
Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseas... more Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identical TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-ceil clones. The analysis of TCR genes used by the panel of clones reveals oligoclonality, with the variable (V) region gene segments Va8 and V.83 being dominant, although there is minimal conservation of junctional sequences. The results have implications for understanding the TCR recognition of an environmental aeroallergen and the life span of T-cell clones in vivo during a chronic immune response.
Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor... more Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor ligands.
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1990
The exotoxins produced by certain strains of Staphylococcus aureus are able to stimulate powerful... more The exotoxins produced by certain strains of Staphylococcus aureus are able to stimulate powerful polyclonal proliferative responses and to induce nonresponsiveness by clonal deletion of T lymphocytes expressing the appropriate T-cell antigen receptor VP gene products. This paper examines the ability of S. aureus enterotoxins to modulate the respon
Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute ... more Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute to both the specific and non-specific effector mechanisms of the allergic immune response. The chemokine family of cytokines and tumour necrosis factor (TNF)-alpha are leucocyte regulatory and proinflammatory molecules. The chemokines include interleukin (IL)-8 and the RANTES/SIS cytokines. There has been no systematic survey of chemokine production in T-cell subtypes. Because of their wide range of biological properties, it might be expected that they would be closely regulated by T cells. This paper illustrates one way (through the characterization of T-cell clones) these questions might be addressed. Northern blot analysis was used to quantitate steady state transcription of selected cytokine genes and enzyme linked immunosorbent assay (ELISA) was used to quantitate soluble product. mRNA expression of the chemokines (IL-8, HuMIP-1 alpha and HuMIP-1 beta) and TNF alpha is upregulated in TH2-like cloned house dust mite reactive human CD4+ T cells under conditions of activation and during the induction phase of anergy. Although the development of anergy superinduces mRNA for both IL-8 and TNF alpha, protein production is low compared with that released during activation. In contrast, RANTES, a chemoattractant for CD4+/CD45RO+ memory T cells, eosinophils and basophils, is constitutively expressed at the RNA level by the T cells and not modulated by signals of activation and anergy induction. The production of IL-2, IL-4 and IL-5 mRNA and proteins during the induction of anergy peaks at 2 h after stimulation, whereas the kinetics following activation of the T cells is delayed in comparison. These data show that the induction of the anergic state coincides with post-transcriptional regulation of selected cytokine genes. Further study of these phenomena will impact on our understanding of the mechanisms of induction of anergy and the regulation of allergic immune responses in desensitization.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial ... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
RationaleTo gain an understanding as to why Hev b 5 is such a potent allergen, we characterized t... more RationaleTo gain an understanding as to why Hev b 5 is such a potent allergen, we characterized the epitopes for three monoclonal antibodies to Hev b 5.
T cells are pivotal in the elicitation of allergic diseases. Analogues of T-cell epitope peptides... more T cells are pivotal in the elicitation of allergic diseases. Analogues of T-cell epitope peptides with a modification at a T-cell receptor (TCR) contact site can alter selected T-cell effector functions. Thus the ability to modulate allergen-specific T-cell responses towards TH1 -like by stimulation with peptide analogues may downregulate allergic inflammation. The purpose of this study was to characterize the minimal epitope recognized by cloned T cells of a dominant Lol p 5 epitope, p105-116, and identify the critical residues involved in TCR and MHC contact. Using peptides with progressive truncation of N- and C-terminal residues in T-cell proliferation assays, we identified the core epitope recognized by cloned CD4(+) T cells. An additional series of peptides with single amino acid substitutions were used in T-cell proliferation and live-cell MHC binding assays. Taken together, these results allowed identification of MHC binding and TCR contact residues of p105-116. The core epitope of p105-116 was identified as residues 107-114. Within this core epitope, 3 residues were found to be important for MHC binding, positions 107, 110, and 112, whereas those at positions 108, 109, 110, 111, and 113 were putative TCR contact residues. The identification of the TCR and MHC contact residues of a dominant Lol p 5 T-cell epitope and analogues of this peptide capable of modulating T-cell responses will allow the evaluation of these peptides' potential as immunotherapeutic agents for rye grass pollen allergic disease.
International Archives of Allergy and Immunology, 2004
Background: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell ... more Background: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. Methods: The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. Results: All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63–68), PAEGEK (120–125), and PAEEEK (126–131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K...
SummaryBackground Activin A is a member of the transforming growth factor‐β superfamily which is ... more SummaryBackground Activin A is a member of the transforming growth factor‐β superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin.Objective To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2‐driven mucosal inflammation in a murine model of allergic asthma.Methods Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin‐sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes.Results Follistatin was released concurrently...
Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupat... more Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4 1 T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. Objective To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. Methods Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4 1 T cell intracellular cytokines, IL-4 and IFN-g were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. Results All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-g was evident from intracellular cytokine staining. Conclusion Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.
Allergen-specific CD4 + T cells producing Th2-type cytokines play a major role in the elicitation... more Allergen-specific CD4 + T cells producing Th2-type cytokines play a major role in the elicitation and progression of allergic diseases (1). Upon appropriate presentation of allergen peptide these T cells produce a cocktail of cytokines, most importantly IL-4, which drives IgE class switching by allergen-stimulated B cells, and IL-5 which plays an important role in the maturation and activation of eosinophils. Allergen immunotherapy has been successfully used to specifically treat allergic disease (2), yet the precise mechanisms involved are as yet not fully understood. Moreover, efficacy for treatment of common allergic diseases, such as those induced by grass pollens and house dust mite, is only 60-80% and there is a risk of IgE-mediated side-effects from injection of the current crude extracts (2). Several studies have shown that clinically effective immunotherapy induces an alteration in allergen-specific T cell cytokine phenotype from a predominant Th2/Th0-type to a more Th1/Th0-type (reviewed in (3)). Since the nature of the T cell response is pivotal in determining whether clinical tolerance or allergy follows allergen challenge, T cells are obvious targets for new immunotherapy strategies. Based on a knowledge of dominant T cell epitopes of allergens, short peptides or recombinant hypoallergenic forms of allergens are exciting effective alternatives to current immunotherapy allergen extracts. Since these new preparations cannot cross-link mast-cell-bound IgE, they should also be associated with improved safety of immunotherapy treatment. Peptide immunotherapy has already been successfully clinically trialled for the major cat allergen Fel d 1 (4-6) and the major bee venom allergen phospholipase A2 (PLA 2) (7). In addition, strategies for the generation of hypoallergenic recombinant allergens by site-directed mutagenesis have been developed for birch pollen, Timothy grass pollen and house dust mite allergens (8-10). Rye grass pollen (RGP) is an important aeroallergen source in cool temperate climates during the grass flowering season. A soluble extract of RGP contains at least 17 allergenic proteins as identified by immunoblotting with patient serum IgE, ranging in size from 11 to 89 kDa (11, 12). Lol p 1 and Lol p 5 are the major allergens of RGP, which together are recognized by Background: Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals. Methods: Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3 Hthymidine uptake and IL-5 and IFN-c in culture supernatants analysed by ELISA. Results: Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found.
The impact of allergic rhinitis (AR) on asthma and the abnormal lung function in children with al... more The impact of allergic rhinitis (AR) on asthma and the abnormal lung function in children with allergic rhinitis is an important issue. In Ciprandi et al. (1) study, the authors demonstrated that FEV1 increased after bronchodilation test in comparison to basal values and to controls’ levels. Over 20% of the patients had a positive reversibility (‡12% basal levels) test. The patients group in the study comprised 78 patients with perennial AR and 122 patients with seasonal AR (according to skin prick test resultsTable 3). Positive bronchodilation test was mainly found in patients with perennial AR 48.7% (38/78) compared to only 4% (5/122) in patients with seasonal AR. Owing to this big difference, we suggest that the authors cannot relate them as one group in relation to bronchodilation test results. Indeed, in ARIA 2008, it was recommended that specifically all patients with persistent allergic rhinitis, asthma should be routinely investigated by history and, if needed, using pulmonary function tests assessing the reversibility of airflow obstruction under inhaled short-acting ß2-agonists (2). Second, it is important to assess the relationship between the severity of the AR (mild/moderate to severe) and the bronchodilation test results. This might help us to better define a group of patients with a high risk of developing asthma, thus being a better candidate for a more aggressive therapy and close clinical follow-up. Another important question is weather nasal treatment, as in patients with asthma can improve lung function in children with allergic rhinitis. In order to answer this question, we conducted a study that demonstrated that children with persistent AR to dust mite without underlying asthma had impaired lung function especially diminished FEF25–75. After treating these patients with nasal corticosteroids for 3 months and loratidine once a day for 10 days, their lung function improved including their FEF25–75, FEV1 and FEV1/FVC levels (3). Thus, decreasing the inflammation in the nose might prevent the development of inflammation of the lower airways presumably by reducing the systemic allergic reaction (4). It seems important to better define the group of patients with AR that will have abnormal lung function and eventually will develop asthma. Defining such a group in an early stage might offer us an opportunity to try and change their natural history to develop asthma maybe by allergen immunotherapy.
The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate pote... more The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulat...
Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to sp... more Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to specific allergen extracts over several months to years can successfully induce clinical tolerance in patients with allergy to insect venoms, pollen, house dust mite, and domestic animals. Different regimens and routes for immunotherapy include subcutaneous, sublingual, oral, and intralymphatic. Food allergies have been difficult to treat in this way due to high anaphylactic potential and only recently the first immunotherapy for peanut allergy has received regulatory approval. Several clinical trials have indicated high efficacy in desensitisation of peanut-allergic individuals using oral immunotherapy, which allows for safer administration of relatively high allergen concentrations. Still, the risk of adverse events including serious allergic reactions and high anxiety levels for patients remains, demonstrating the need for further optimisation of treatment protocols. Here we discuss the...
Despite recent technological advances, novel allergenic protein discovery is limited by their low... more Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins ...
BackgroundAnnual influenza vaccination is recommended to all individuals over 6 months of age, in... more BackgroundAnnual influenza vaccination is recommended to all individuals over 6 months of age, including predominantly antibody deficiency (PAD) patients. Vaccination responses are typically evaluated by serology, and because PAD patients are by definition impaired in generating IgG and receive immunoglobulin replacement therapy (IgRT), it remains unclear whether they can mount an antigen‐specific response.ObjectiveTo quantify and characterise the antigen‐specific memory B (Bmem) cell compartment in healthy controls and PAD patients following an influenza booster vaccination.MethodsRecombinant hemagglutinin (HA) from the A/Michigan/2015 H1N1 (AM15) strain with an AviTag was generated in a mammalian cell line, and following targeted biotinylation, was tetramerised with BUV395 or BUV737 streptavidin conjugates. Multicolour flow cytometry was applied on blood samples before and 28 days after booster influenza vaccination in 16 healthy controls and five PAD patients with circulating Bme...
The Journal of Allergy and Clinical Immunology: In Practice, 2020
What is already known about this topic? Clinical relevance of fish collagen for fish-allergic pat... more What is already known about this topic? Clinical relevance of fish collagen for fish-allergic patients is poorly understood, likely due to its low abundance in commercial diagnostic tests. Patients may be exposed to such collagens via pharmaceutical products, food, beverages, and cosmetics. What does this article add to our knowledge? We demonstrated the potential clinical relevance of sensitization to fish collagen in fish-allergic patients, some of whom were not sensitized to the major fish allergen parvalbumin. How does this study impact current management guidelines? Current diagnostic tests for fish allergy contain low quantities of collagen due to its insolubility in aqueous solutions. Inclusion of collagen in diagnostic tests is indicated to improve patients' safety. BACKGROUND: Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. OBJECTIVE: To investigate the relevance of fish collagen as an allergen in a large patient population (n [ 101). METHODS: Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. RESULTS: Purified fish collagen contained type I a1 and a2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I a chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. CONCLUSIONS: IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy.
The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate pote... more The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulat...
Proceedings of the National Academy of Sciences of the United States of America, Sep 1, 1993
Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseas... more Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identical TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-ceil clones. The analysis of TCR genes used by the panel of clones reveals oligoclonality, with the variable (V) region gene segments Va8 and V.83 being dominant, although there is minimal conservation of junctional sequences. The results have implications for understanding the TCR recognition of an environmental aeroallergen and the life span of T-cell clones in vivo during a chronic immune response.
Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor... more Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor ligands.
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1990
The exotoxins produced by certain strains of Staphylococcus aureus are able to stimulate powerful... more The exotoxins produced by certain strains of Staphylococcus aureus are able to stimulate powerful polyclonal proliferative responses and to induce nonresponsiveness by clonal deletion of T lymphocytes expressing the appropriate T-cell antigen receptor VP gene products. This paper examines the ability of S. aureus enterotoxins to modulate the respon
Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute ... more Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute to both the specific and non-specific effector mechanisms of the allergic immune response. The chemokine family of cytokines and tumour necrosis factor (TNF)-alpha are leucocyte regulatory and proinflammatory molecules. The chemokines include interleukin (IL)-8 and the RANTES/SIS cytokines. There has been no systematic survey of chemokine production in T-cell subtypes. Because of their wide range of biological properties, it might be expected that they would be closely regulated by T cells. This paper illustrates one way (through the characterization of T-cell clones) these questions might be addressed. Northern blot analysis was used to quantitate steady state transcription of selected cytokine genes and enzyme linked immunosorbent assay (ELISA) was used to quantitate soluble product. mRNA expression of the chemokines (IL-8, HuMIP-1 alpha and HuMIP-1 beta) and TNF alpha is upregulated in TH2-like cloned house dust mite reactive human CD4+ T cells under conditions of activation and during the induction phase of anergy. Although the development of anergy superinduces mRNA for both IL-8 and TNF alpha, protein production is low compared with that released during activation. In contrast, RANTES, a chemoattractant for CD4+/CD45RO+ memory T cells, eosinophils and basophils, is constitutively expressed at the RNA level by the T cells and not modulated by signals of activation and anergy induction. The production of IL-2, IL-4 and IL-5 mRNA and proteins during the induction of anergy peaks at 2 h after stimulation, whereas the kinetics following activation of the T cells is delayed in comparison. These data show that the induction of the anergic state coincides with post-transcriptional regulation of selected cytokine genes. Further study of these phenomena will impact on our understanding of the mechanisms of induction of anergy and the regulation of allergic immune responses in desensitization.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial ... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
RationaleTo gain an understanding as to why Hev b 5 is such a potent allergen, we characterized t... more RationaleTo gain an understanding as to why Hev b 5 is such a potent allergen, we characterized the epitopes for three monoclonal antibodies to Hev b 5.
T cells are pivotal in the elicitation of allergic diseases. Analogues of T-cell epitope peptides... more T cells are pivotal in the elicitation of allergic diseases. Analogues of T-cell epitope peptides with a modification at a T-cell receptor (TCR) contact site can alter selected T-cell effector functions. Thus the ability to modulate allergen-specific T-cell responses towards TH1 -like by stimulation with peptide analogues may downregulate allergic inflammation. The purpose of this study was to characterize the minimal epitope recognized by cloned T cells of a dominant Lol p 5 epitope, p105-116, and identify the critical residues involved in TCR and MHC contact. Using peptides with progressive truncation of N- and C-terminal residues in T-cell proliferation assays, we identified the core epitope recognized by cloned CD4(+) T cells. An additional series of peptides with single amino acid substitutions were used in T-cell proliferation and live-cell MHC binding assays. Taken together, these results allowed identification of MHC binding and TCR contact residues of p105-116. The core epitope of p105-116 was identified as residues 107-114. Within this core epitope, 3 residues were found to be important for MHC binding, positions 107, 110, and 112, whereas those at positions 108, 109, 110, 111, and 113 were putative TCR contact residues. The identification of the TCR and MHC contact residues of a dominant Lol p 5 T-cell epitope and analogues of this peptide capable of modulating T-cell responses will allow the evaluation of these peptides' potential as immunotherapeutic agents for rye grass pollen allergic disease.
International Archives of Allergy and Immunology, 2004
Background: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell ... more Background: Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. Methods: The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. Results: All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63–68), PAEGEK (120–125), and PAEEEK (126–131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K...
SummaryBackground Activin A is a member of the transforming growth factor‐β superfamily which is ... more SummaryBackground Activin A is a member of the transforming growth factor‐β superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin.Objective To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2‐driven mucosal inflammation in a murine model of allergic asthma.Methods Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin‐sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes.Results Follistatin was released concurrently...
Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupat... more Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4 1 T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. Objective To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. Methods Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4 1 T cell intracellular cytokines, IL-4 and IFN-g were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. Results All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-g was evident from intracellular cytokine staining. Conclusion Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.
Allergen-specific CD4 + T cells producing Th2-type cytokines play a major role in the elicitation... more Allergen-specific CD4 + T cells producing Th2-type cytokines play a major role in the elicitation and progression of allergic diseases (1). Upon appropriate presentation of allergen peptide these T cells produce a cocktail of cytokines, most importantly IL-4, which drives IgE class switching by allergen-stimulated B cells, and IL-5 which plays an important role in the maturation and activation of eosinophils. Allergen immunotherapy has been successfully used to specifically treat allergic disease (2), yet the precise mechanisms involved are as yet not fully understood. Moreover, efficacy for treatment of common allergic diseases, such as those induced by grass pollens and house dust mite, is only 60-80% and there is a risk of IgE-mediated side-effects from injection of the current crude extracts (2). Several studies have shown that clinically effective immunotherapy induces an alteration in allergen-specific T cell cytokine phenotype from a predominant Th2/Th0-type to a more Th1/Th0-type (reviewed in (3)). Since the nature of the T cell response is pivotal in determining whether clinical tolerance or allergy follows allergen challenge, T cells are obvious targets for new immunotherapy strategies. Based on a knowledge of dominant T cell epitopes of allergens, short peptides or recombinant hypoallergenic forms of allergens are exciting effective alternatives to current immunotherapy allergen extracts. Since these new preparations cannot cross-link mast-cell-bound IgE, they should also be associated with improved safety of immunotherapy treatment. Peptide immunotherapy has already been successfully clinically trialled for the major cat allergen Fel d 1 (4-6) and the major bee venom allergen phospholipase A2 (PLA 2) (7). In addition, strategies for the generation of hypoallergenic recombinant allergens by site-directed mutagenesis have been developed for birch pollen, Timothy grass pollen and house dust mite allergens (8-10). Rye grass pollen (RGP) is an important aeroallergen source in cool temperate climates during the grass flowering season. A soluble extract of RGP contains at least 17 allergenic proteins as identified by immunoblotting with patient serum IgE, ranging in size from 11 to 89 kDa (11, 12). Lol p 1 and Lol p 5 are the major allergens of RGP, which together are recognized by Background: Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals. Methods: Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3 Hthymidine uptake and IL-5 and IFN-c in culture supernatants analysed by ELISA. Results: Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found.
The impact of allergic rhinitis (AR) on asthma and the abnormal lung function in children with al... more The impact of allergic rhinitis (AR) on asthma and the abnormal lung function in children with allergic rhinitis is an important issue. In Ciprandi et al. (1) study, the authors demonstrated that FEV1 increased after bronchodilation test in comparison to basal values and to controls’ levels. Over 20% of the patients had a positive reversibility (‡12% basal levels) test. The patients group in the study comprised 78 patients with perennial AR and 122 patients with seasonal AR (according to skin prick test resultsTable 3). Positive bronchodilation test was mainly found in patients with perennial AR 48.7% (38/78) compared to only 4% (5/122) in patients with seasonal AR. Owing to this big difference, we suggest that the authors cannot relate them as one group in relation to bronchodilation test results. Indeed, in ARIA 2008, it was recommended that specifically all patients with persistent allergic rhinitis, asthma should be routinely investigated by history and, if needed, using pulmonary function tests assessing the reversibility of airflow obstruction under inhaled short-acting ß2-agonists (2). Second, it is important to assess the relationship between the severity of the AR (mild/moderate to severe) and the bronchodilation test results. This might help us to better define a group of patients with a high risk of developing asthma, thus being a better candidate for a more aggressive therapy and close clinical follow-up. Another important question is weather nasal treatment, as in patients with asthma can improve lung function in children with allergic rhinitis. In order to answer this question, we conducted a study that demonstrated that children with persistent AR to dust mite without underlying asthma had impaired lung function especially diminished FEF25–75. After treating these patients with nasal corticosteroids for 3 months and loratidine once a day for 10 days, their lung function improved including their FEF25–75, FEV1 and FEV1/FVC levels (3). Thus, decreasing the inflammation in the nose might prevent the development of inflammation of the lower airways presumably by reducing the systemic allergic reaction (4). It seems important to better define the group of patients with AR that will have abnormal lung function and eventually will develop asthma. Defining such a group in an early stage might offer us an opportunity to try and change their natural history to develop asthma maybe by allergen immunotherapy.
The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate pote... more The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulat...
Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to sp... more Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to specific allergen extracts over several months to years can successfully induce clinical tolerance in patients with allergy to insect venoms, pollen, house dust mite, and domestic animals. Different regimens and routes for immunotherapy include subcutaneous, sublingual, oral, and intralymphatic. Food allergies have been difficult to treat in this way due to high anaphylactic potential and only recently the first immunotherapy for peanut allergy has received regulatory approval. Several clinical trials have indicated high efficacy in desensitisation of peanut-allergic individuals using oral immunotherapy, which allows for safer administration of relatively high allergen concentrations. Still, the risk of adverse events including serious allergic reactions and high anxiety levels for patients remains, demonstrating the need for further optimisation of treatment protocols. Here we discuss the...
Despite recent technological advances, novel allergenic protein discovery is limited by their low... more Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins ...
BackgroundAnnual influenza vaccination is recommended to all individuals over 6 months of age, in... more BackgroundAnnual influenza vaccination is recommended to all individuals over 6 months of age, including predominantly antibody deficiency (PAD) patients. Vaccination responses are typically evaluated by serology, and because PAD patients are by definition impaired in generating IgG and receive immunoglobulin replacement therapy (IgRT), it remains unclear whether they can mount an antigen‐specific response.ObjectiveTo quantify and characterise the antigen‐specific memory B (Bmem) cell compartment in healthy controls and PAD patients following an influenza booster vaccination.MethodsRecombinant hemagglutinin (HA) from the A/Michigan/2015 H1N1 (AM15) strain with an AviTag was generated in a mammalian cell line, and following targeted biotinylation, was tetramerised with BUV395 or BUV737 streptavidin conjugates. Multicolour flow cytometry was applied on blood samples before and 28 days after booster influenza vaccination in 16 healthy controls and five PAD patients with circulating Bme...
The Journal of Allergy and Clinical Immunology: In Practice, 2020
What is already known about this topic? Clinical relevance of fish collagen for fish-allergic pat... more What is already known about this topic? Clinical relevance of fish collagen for fish-allergic patients is poorly understood, likely due to its low abundance in commercial diagnostic tests. Patients may be exposed to such collagens via pharmaceutical products, food, beverages, and cosmetics. What does this article add to our knowledge? We demonstrated the potential clinical relevance of sensitization to fish collagen in fish-allergic patients, some of whom were not sensitized to the major fish allergen parvalbumin. How does this study impact current management guidelines? Current diagnostic tests for fish allergy contain low quantities of collagen due to its insolubility in aqueous solutions. Inclusion of collagen in diagnostic tests is indicated to improve patients' safety. BACKGROUND: Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. OBJECTIVE: To investigate the relevance of fish collagen as an allergen in a large patient population (n [ 101). METHODS: Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. RESULTS: Purified fish collagen contained type I a1 and a2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I a chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. CONCLUSIONS: IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy.
The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate pote... more The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulat...
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Papers by Robyn O'Hehir