Introduction: Horse allergens are less studied than allergens from other furry animals and these ... more Introduction: Horse allergens are less studied than allergens from other furry animals and these allergens must be evaluated to understand the complexity of allergy to horses. The aims of this study were to develop assays for the horse allergens Equ c 1 and Equ c 2 in dander and saliva and to determine their levels in ten horse breeds. The study also included a comparison of these findings with previous results on the levels of Equ c 4 performed on the same study population. Method: The study population included 170 horses from 10 horse breeds including American Curly and Russian Bashkir horse, which have been suggested to be hypoallergenic. Competitive ELISA assays were developed, with polyclonal antibodies as capture antibodies, for the detection of Equ c 1 and Equ c 2 in dander and saliva samples. Results: The horse allergens Equ c 1 and Equ c 2 were found in all dander and saliva samples from the ten horse breeds. The GM level (ng/µg protein) of Equ c 1 in dander was 470 (range ...
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which ... more Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid–binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+and human leukocyte antigen–DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search ...
Allergy to dogs affects around ten percent of the population in developed countries. Immune thera... more Allergy to dogs affects around ten percent of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. Herein, we established a mouse model of dog allergy and tested the efficacy of a recombinant protein containing Can f 1, f 2, f 4 and f 6 as a sublingual immune therapy (SLIT). Repeated inhalation of dog extracts induced infiltration of the airways by TH2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. However, dog allergen extracts also induced robust TH17 cell responses, which was associated with a high neutrophilic infiltration of the airways and promoted airway hyperresponsiveness more potently than HDM allergens. scRNA-Seq analysis of T helper cells responding to dog allergens identified several unique clusters with TH17 cells being hallmarked by the expression of several receptors including IL-17RE. Analysis of T cell receptors also depicted a hig...
The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in ... more The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in several systems and has rendered interest as a candidate adjuvant in vaccine formulations. The mechanisms underlying this effect are not completely understood at the cellular and molecular level, but activation of inflammasome and caspase-1 in antigen presenting cells (APC) was recently suggested to be vital for immune activation. In this study, chitosan-based viscoelastic hydrogel particles of two sizes, 10 µm and 200 µm, were evaluated in regard to cellular uptake and activation of APCs. Macrophages derived from the human cell line THP-1 were shown by flow cytometry and confocal laser scanning microscopy to take up FITC-labelled chitosan particles of both sizes via an active process that could be inhibited by cytochalasin D. To investigate if the viscoelastic chitosan particles cause inflammasome activation, NFκB and IL-1β was measured in THP-1 derived macrophages after 24 h incubation with the chitosan particles with or without priming of the cells with LPS. We found that chitosan particles of both sizes stimulated upregulation of NFκB and IL-1β in the absence of LPS. Finally the dependence of this effect on inflammasome-mediated activation of caspase-1 was assessed. Active caspase-1 was not detected in THP-1derived macrophages stimulated with chitosan-based viscoelastic hydrogel particles, neither alone nor in combination with LPS. In conclusion, we show that viscoelastic chitosan particles in the size range of 10-200 µm, are taken up by human APCs. Moreover, our study suggests that the chitosan particles stimulate NFκB upregulation and IL-1β secretion through inflammasome activation via a caspase-1 independent pathway.
To the Editor, Exposure to dog allergens is almost impossible to avoid, as dogs are part of our s... more To the Editor, Exposure to dog allergens is almost impossible to avoid, as dogs are part of our society and frequently encountered both outdoors and indoors. This poses problems to individuals allergic to dogs, a common condition with reported sensitization rates around 20%.
Disclosure of potential conflict of interest: J. R. Konradsen has received payment for lectures b... more Disclosure of potential conflict of interest: J. R. Konradsen has received payment for lectures by MEDA AB and Novartis. M. H. Shamji serves as a consultant for ASIT Biotech.sa through Imperial College London, and has received lecture fees from ASIT Biotech.sa, ALK, and Allergopharma. M. van Hage serves as a consultant for Biomay AG and Hycor Biomedical LLC, and has received lecture fees from Thermo Fisher Scientific and ALK. The rest of the authors declare that they have no relevant conflicts of interest.
BackgroundFive to ten percent of the population in affluent countries are allergic to dog. Diagno... more BackgroundFive to ten percent of the population in affluent countries are allergic to dog. Diagnosis and treatment is based on allergen extracts from natural sources where composition and concentration are poorly defined.ObjectiveWe aimed to quantify six dog allergens (Can f 1‐6) in commercial skin prick test (SPT) solutions and to determine individual allergen profiles in dogs.MethodThe allergen content of SPT solutions from five vendors and allergen source material from three anatomical sites were analyzed. Fur and saliva samples were collected from a mixed population of 120 dogs. Can f 1‐6 were quantified by inhibition ELISA using purified recombinant or natural allergens and polyclonal or monoclonal antibodies. Allergenicity was analyzed by basophil activation test.ResultsExtensive variation in allergen composition was observed in commercial SPT vials resulting in a patient‐dependent ability to activate basophils. Extract heterogeneity depended on collection site and allergen co...
We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensit... more We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.
International Archives of Allergy and Immunology, 2016
Background: Selective inhibition of cyclooxygenase-2 (COX-2) reduces the production of prostaglan... more Background: Selective inhibition of cyclooxygenase-2 (COX-2) reduces the production of prostaglandin E2 (PGE2), which can have both pro- and anti-inflammatory effects on allergic inflammation. Moreover, in vitro PGE2 has been shown to affect inflammation through the modulation of lymphocyte responses. Methods: Sixteen subjects with mild allergic asthma were recruited to a two-period cross-over study: one treatment period with the selective COX-2 inhibitor etoricoxib and one without. Each treatment period ended with an airway challenge with the patient's relevant allergen. Antigen-specific proliferation with the major cat allergen, Fel d 1, was analysed in PBMCs. CD4+ T cells were phenotyped using flow cytometry, and mRNA expression of FOXP3 in anti-CD3-stimulated CD4+ cells were analysed. Results: No significant impact of in vivo inhibition of COX-2 was detected on the proportion of Th1, Th2, or Treg cells in peripheral blood. Likewise, the treatment had minor effects on the sti...
MLR. Few CD11c-HLA-DR+ lin-/dim colonic cells expressed markers associated with blood CD11c-HLA-D... more MLR. Few CD11c-HLA-DR+ lin-/dim colonic cells expressed markers associated with blood CD11c-HLA-DR+ lin-/dim plasmacytoid DC (BDCA2, BDCA4 and high levels of CD123) but a major subset expressed high levels of the Natural Killer (NK) marker CD56. Intestinal inflammation in UC is associated with the presence of a population of cells that share phenotypic features of both antigen presenting cells and NK cells. This novel population of human colonic cells may function in immune regulation or tissue repair. In addition, their increased presence in quiescent UC may be a marker of sub-clinical inflammatory activity.
Proceedings of the National Academy of Sciences, 2004
IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated cont... more IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients ( n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen de...
Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allerg... more Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wildtype allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.
SummaryInflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor... more SummaryInflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor (TNF) therapy. We set out to investigate the unclear immunoregulatory mechanisms of the treatment. Thirty-four patients with IBD treated with anti-TNF were included. Lymphocytes from peripheral blood and intestinal biopsies were analysed by flow cytometry. Regulation of antigen-stimulated proliferation was analysed by blocking of interleukin (IL)-10, transforming growth factor (TGF)-β or depletion of CD25+ cells in peripheral blood mononuclear cell cultures. No changes in CD4+CD25+, CD25+TNF-RII+ or CD4+CD25+forkhead box protein 3 (FoxP3+) T cells could be observed in peripheral blood after, in comparison to before, 6 weeks of treatment. The suppressive ability of CD4+CD25+ cells did not change. There was an initial decrease of CD4+CD25+ cells in intestinal mucosa after 2 weeks of treatment, followed by an increase of these cells from weeks 2 to 6 of treatment (P < 0·05). This was ac...
Background Treating allergies with modified allergens is an approach to make the treatment safer ... more Background Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. Methods The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. Results Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4 1 cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. Conclusions Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.
SummaryBackgroundAtopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exace... more SummaryBackgroundAtopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exacerbations. Many patients experience no obvious seasonal variation, although a majority of patients are allergic to common airborne allergens.ObjectiveTo investigate the allergic reaction, to conjunctival provocation with airborne allergens, in patients with AKC.MethodsEleven patients with AKC and birch and/or grass pollen allergy participated in the study, which was performed outside the pollen season. Five patients with seasonal allergic conjunctivitis (SAC) and five healthy subjects were included for validation purposes. The challenge was performed in one eye with the allergen, to which the patient was reactive, and with dilution buffer in the other eye. Signs and symptoms from both eyes were graded at baseline and at 10 min, 8 and 48 h after provocation. Tear fluid was collected from both eyes for cytokine analyses at baseline and at 8 and 48 h.ResultsA significant change in clinical s...
Introduction: Horse allergens are less studied than allergens from other furry animals and these ... more Introduction: Horse allergens are less studied than allergens from other furry animals and these allergens must be evaluated to understand the complexity of allergy to horses. The aims of this study were to develop assays for the horse allergens Equ c 1 and Equ c 2 in dander and saliva and to determine their levels in ten horse breeds. The study also included a comparison of these findings with previous results on the levels of Equ c 4 performed on the same study population. Method: The study population included 170 horses from 10 horse breeds including American Curly and Russian Bashkir horse, which have been suggested to be hypoallergenic. Competitive ELISA assays were developed, with polyclonal antibodies as capture antibodies, for the detection of Equ c 1 and Equ c 2 in dander and saliva samples. Results: The horse allergens Equ c 1 and Equ c 2 were found in all dander and saliva samples from the ten horse breeds. The GM level (ng/µg protein) of Equ c 1 in dander was 470 (range ...
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which ... more Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid–binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+and human leukocyte antigen–DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search ...
Allergy to dogs affects around ten percent of the population in developed countries. Immune thera... more Allergy to dogs affects around ten percent of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. Herein, we established a mouse model of dog allergy and tested the efficacy of a recombinant protein containing Can f 1, f 2, f 4 and f 6 as a sublingual immune therapy (SLIT). Repeated inhalation of dog extracts induced infiltration of the airways by TH2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. However, dog allergen extracts also induced robust TH17 cell responses, which was associated with a high neutrophilic infiltration of the airways and promoted airway hyperresponsiveness more potently than HDM allergens. scRNA-Seq analysis of T helper cells responding to dog allergens identified several unique clusters with TH17 cells being hallmarked by the expression of several receptors including IL-17RE. Analysis of T cell receptors also depicted a hig...
The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in ... more The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in several systems and has rendered interest as a candidate adjuvant in vaccine formulations. The mechanisms underlying this effect are not completely understood at the cellular and molecular level, but activation of inflammasome and caspase-1 in antigen presenting cells (APC) was recently suggested to be vital for immune activation. In this study, chitosan-based viscoelastic hydrogel particles of two sizes, 10 µm and 200 µm, were evaluated in regard to cellular uptake and activation of APCs. Macrophages derived from the human cell line THP-1 were shown by flow cytometry and confocal laser scanning microscopy to take up FITC-labelled chitosan particles of both sizes via an active process that could be inhibited by cytochalasin D. To investigate if the viscoelastic chitosan particles cause inflammasome activation, NFκB and IL-1β was measured in THP-1 derived macrophages after 24 h incubation with the chitosan particles with or without priming of the cells with LPS. We found that chitosan particles of both sizes stimulated upregulation of NFκB and IL-1β in the absence of LPS. Finally the dependence of this effect on inflammasome-mediated activation of caspase-1 was assessed. Active caspase-1 was not detected in THP-1derived macrophages stimulated with chitosan-based viscoelastic hydrogel particles, neither alone nor in combination with LPS. In conclusion, we show that viscoelastic chitosan particles in the size range of 10-200 µm, are taken up by human APCs. Moreover, our study suggests that the chitosan particles stimulate NFκB upregulation and IL-1β secretion through inflammasome activation via a caspase-1 independent pathway.
To the Editor, Exposure to dog allergens is almost impossible to avoid, as dogs are part of our s... more To the Editor, Exposure to dog allergens is almost impossible to avoid, as dogs are part of our society and frequently encountered both outdoors and indoors. This poses problems to individuals allergic to dogs, a common condition with reported sensitization rates around 20%.
Disclosure of potential conflict of interest: J. R. Konradsen has received payment for lectures b... more Disclosure of potential conflict of interest: J. R. Konradsen has received payment for lectures by MEDA AB and Novartis. M. H. Shamji serves as a consultant for ASIT Biotech.sa through Imperial College London, and has received lecture fees from ASIT Biotech.sa, ALK, and Allergopharma. M. van Hage serves as a consultant for Biomay AG and Hycor Biomedical LLC, and has received lecture fees from Thermo Fisher Scientific and ALK. The rest of the authors declare that they have no relevant conflicts of interest.
BackgroundFive to ten percent of the population in affluent countries are allergic to dog. Diagno... more BackgroundFive to ten percent of the population in affluent countries are allergic to dog. Diagnosis and treatment is based on allergen extracts from natural sources where composition and concentration are poorly defined.ObjectiveWe aimed to quantify six dog allergens (Can f 1‐6) in commercial skin prick test (SPT) solutions and to determine individual allergen profiles in dogs.MethodThe allergen content of SPT solutions from five vendors and allergen source material from three anatomical sites were analyzed. Fur and saliva samples were collected from a mixed population of 120 dogs. Can f 1‐6 were quantified by inhibition ELISA using purified recombinant or natural allergens and polyclonal or monoclonal antibodies. Allergenicity was analyzed by basophil activation test.ResultsExtensive variation in allergen composition was observed in commercial SPT vials resulting in a patient‐dependent ability to activate basophils. Extract heterogeneity depended on collection site and allergen co...
We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensit... more We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.
International Archives of Allergy and Immunology, 2016
Background: Selective inhibition of cyclooxygenase-2 (COX-2) reduces the production of prostaglan... more Background: Selective inhibition of cyclooxygenase-2 (COX-2) reduces the production of prostaglandin E2 (PGE2), which can have both pro- and anti-inflammatory effects on allergic inflammation. Moreover, in vitro PGE2 has been shown to affect inflammation through the modulation of lymphocyte responses. Methods: Sixteen subjects with mild allergic asthma were recruited to a two-period cross-over study: one treatment period with the selective COX-2 inhibitor etoricoxib and one without. Each treatment period ended with an airway challenge with the patient's relevant allergen. Antigen-specific proliferation with the major cat allergen, Fel d 1, was analysed in PBMCs. CD4+ T cells were phenotyped using flow cytometry, and mRNA expression of FOXP3 in anti-CD3-stimulated CD4+ cells were analysed. Results: No significant impact of in vivo inhibition of COX-2 was detected on the proportion of Th1, Th2, or Treg cells in peripheral blood. Likewise, the treatment had minor effects on the sti...
MLR. Few CD11c-HLA-DR+ lin-/dim colonic cells expressed markers associated with blood CD11c-HLA-D... more MLR. Few CD11c-HLA-DR+ lin-/dim colonic cells expressed markers associated with blood CD11c-HLA-DR+ lin-/dim plasmacytoid DC (BDCA2, BDCA4 and high levels of CD123) but a major subset expressed high levels of the Natural Killer (NK) marker CD56. Intestinal inflammation in UC is associated with the presence of a population of cells that share phenotypic features of both antigen presenting cells and NK cells. This novel population of human colonic cells may function in immune regulation or tissue repair. In addition, their increased presence in quiescent UC may be a marker of sub-clinical inflammatory activity.
Proceedings of the National Academy of Sciences, 2004
IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated cont... more IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients ( n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen de...
Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allerg... more Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wildtype allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.
SummaryInflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor... more SummaryInflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor (TNF) therapy. We set out to investigate the unclear immunoregulatory mechanisms of the treatment. Thirty-four patients with IBD treated with anti-TNF were included. Lymphocytes from peripheral blood and intestinal biopsies were analysed by flow cytometry. Regulation of antigen-stimulated proliferation was analysed by blocking of interleukin (IL)-10, transforming growth factor (TGF)-β or depletion of CD25+ cells in peripheral blood mononuclear cell cultures. No changes in CD4+CD25+, CD25+TNF-RII+ or CD4+CD25+forkhead box protein 3 (FoxP3+) T cells could be observed in peripheral blood after, in comparison to before, 6 weeks of treatment. The suppressive ability of CD4+CD25+ cells did not change. There was an initial decrease of CD4+CD25+ cells in intestinal mucosa after 2 weeks of treatment, followed by an increase of these cells from weeks 2 to 6 of treatment (P < 0·05). This was ac...
Background Treating allergies with modified allergens is an approach to make the treatment safer ... more Background Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. Methods The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. Results Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4 1 cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. Conclusions Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.
SummaryBackgroundAtopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exace... more SummaryBackgroundAtopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exacerbations. Many patients experience no obvious seasonal variation, although a majority of patients are allergic to common airborne allergens.ObjectiveTo investigate the allergic reaction, to conjunctival provocation with airborne allergens, in patients with AKC.MethodsEleven patients with AKC and birch and/or grass pollen allergy participated in the study, which was performed outside the pollen season. Five patients with seasonal allergic conjunctivitis (SAC) and five healthy subjects were included for validation purposes. The challenge was performed in one eye with the allergen, to which the patient was reactive, and with dilution buffer in the other eye. Signs and symptoms from both eyes were graded at baseline and at 10 min, 8 and 48 h after provocation. Tear fluid was collected from both eyes for cytokine analyses at baseline and at 8 and 48 h.ResultsA significant change in clinical s...
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