Influence of peanut matrix on stability of allergens in gastric-simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion-resistant peptides.

BACKGROUND: Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; <10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. OBJECTIVE: The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. METHODS: Two-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. RESULTS: Ara h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. CONCLUSION AND CLINICAL RELEVANCE: Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut.

Immunoprofile of α-Gal- and B-antigen-specific responses differentiates red meat-allergic patients from healthy individuals.

BACKGROUND: The galactose-α-1,3-galactose (α-Gal) epitope is involved in red meat allergy. As α-Gal is structurally similar to the blood group B-antigen, we explored the relationship between the immune responses to α-Gal- and the B-antigen in red meat-allergic patients compared to healthy A/O or B blood donors. METHODS: Sera from 51 red meat-allergic patients IgE-positive to α-Gal and 102 healthy blood donors (51 blood group A/O; 51 blood group B) were included. α-Gal- and B-antigen-specific IgE (ImmunoCAP) and IgG/IgG1-4 (ELISA) responses were determined. Basophil activation tests were performed. RESULTS: Fifteen healthy donors were IgE positive to α-Gal, of which 3 had blood group B. The allergic patients had significantly higher α-Gal IgE levels compared to the healthy donors. The majority of the allergic patients, but none of the healthy donors, had IgE against the B-antigen. Inhibition studies revealed cross-reactivity between α-Gal and the B-antigen. The biological activity of the B-antigen was confirmed by basophil activation tests. Anti-α-Gal IgG1 and IgG4 levels were significantly higher in the patients compared to the healthy donors. Moreover, the IgG response to the B-antigen was comparable between the allergic patients and healthy A/O donors. CONCLUSION: Red meat-allergic patients showed significantly higher α-Gal IgE, IgG1 , and IgG4 levels, reflecting a Th2 response, compared to healthy blood donors. Blood group B donors had significantly reduced antibody responses to α-Gal, due to similarities with the B-antigen, resulting in a lower risk of sensitization to α-Gal and development of red meat allergy.

Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study.

BACKGROUND: Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients. METHODS: Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics. RESULTS: The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described. CONCLUSION AND CLINICAL RELEVANCE: We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.

Anaphylaxis to foods in a population of adolescents: incidence, characteristics and associated risks.

BACKGROUND: Information about severe reactions to foods in adolescence is limited. OBJECTIVE: To describe reactions to foods, including anaphylaxis, with regard to incidence, characteristics and associated risks, among 16-year-olds (adolescents) in a large, population-based birth cohort. METHODS: Parent-reported questionnaire data from ages 2-3 months, and 1, 2 and 16 years were used (N = 3153). Anaphylaxis at age 16 years was defined per NIAID/FAAN criteria. Immunoglobulin E (IgE) antibodies to 14 common food and inhalant allergens were analysed at ages 4 (n = 2283) and 16 years (n = 2510). Among adolescents with food-related symptoms (FRS) and for whom blood was available (n = 221), 25 additional food allergen extracts or allergen components were analysed. Associations between reactions to foods, and sensitization and allergic multimorbidity were investigated. RESULTS: In the 12 months prior to the 16-year assessment, 8.5% of adolescents had FRS. This included 0.8% (n = 24) adolescents who were classified as having anaphylaxis, yielding an incidence rate of 761/100 000 person-years. One-third of adolescents accessed health care during anaphylaxis. Allergic multimorbidity in infancy, as well as sensitization to foods and airborne allergens at age 4 years, was associated with an increased risk for FRS in adolescence. Peanuts and tree nuts were the most common culprit foods for anaphylaxis, and fruits and vegetables for non-anaphylactic reactions. Adolescents with anaphylaxis were significantly more likely to be sensitized to storage proteins (Ara h 2, Cor a 9, Cor a 14) and to be polysensitized to foods (P < 0.001 vs. non-anaphylactic reactions). CONCLUSIONS AND CLINICAL RELEVANCE: The incidence of food-induced anaphylaxis during adolescence in our population-based birth cohort is higher than previously reported. Adolescents with anaphylaxis differ from adolescents with non-anaphylactic FRS with regard to culprit foods and sensitization. Adolescents with previous anaphylaxis are likely to be polysensitized to foods, particularly tree nut and peanut storage proteins, and which warrants consideration at follow-up.

Rhinovirus-specific antibody responses in preschool children with acute wheeze reflect severity of respiratory symptoms.

BACKGROUND: Some children with rhinovirus (RV) infections wheeze, but it is unknown whether this is due to more virulent strains of virus or differences in host immune responses. The aim of this study was to investigate the RV species-specific antibody responses measured at a follow-up visit in preschool children in relation to reported time with respiratory symptoms and the presence of different RV species during an acute episode of wheeze. METHOD: Nasopharyngeal swabs and blood samples were taken among 120 preschool children (<4 years of age) at an acute episode of wheeze and at a follow-up visit (median 11 weeks later). Nested PCR was used to detect different RV strains, and serum levels of IgG1 against purified recombinant VP1 proteins from representatives of the three RV species (RV-A, RV-B, and RV-C) were measured by ELISA. RESULTS: Rhinovirus was detected in 74% (n = 80/108) of the children at the acute visit, and RV-C was the most common subtype (n = 59/80, 74%). An increase in RV-specific IgG1 was seen in 61% (n = 73) of the children at follow-up, most frequently against RV-A (n = 61/73, 86%) irrespective of the RV strains detected by PCR. Increases in RV-specific IgG1 against RV-A or against RV-A and RV-C were significantly associated with more respiratory symptoms (p = 0.03, p = 0.007). CONCLUSION: Antibody response to recombinant RV VP1 proteins was associated with longer time with respiratory symptoms.

IgE antibodies in relation to prevalence and multimorbidity of eczema, asthma, and rhinitis from birth to adolescence.

BACKGROUND: Eczema, asthma, and rhinitis affect a large proportion of children, but their prevalence varies with age. IgE antibodies are also common in the pediatric population. However, the links between IgE, disease, and trajectories are unclear. OBJECTIVE: To better understand the links between sensitization and disease, we studied IgE sensitization ever in relation to eczema, asthma, and rhinitis, in children followed up to 16 years of age. METHODS: From the Swedish population-based birth cohort BAMSE, 2607 children were included. Parental reports from six time points between 1 and 16 years were used to identify children with eczema, asthma, and rhinitis. Blood was collected at 4, 8, and 16 years, and sensitization ever was defined as allergen-specific IgE ≥0.35 kUA /l to common food and/or inhalant allergens at any time point. Odds ratios for eczema, asthma, rhinitis, and multimorbidity in relation to sensitization ever were calculated using generalized estimating equations. RESULTS: Fifty-one percent were sensitized at least once up to 16 years. Almost a quarter of ever-sensitized children did not have any disease. After adjustment for potential confounders, sensitization ever was significantly associated with the following: (i) eczema throughout childhood, (ii) multimorbidity of eczema, asthma, and rhinitis from 1 to 16 years (OR for multimorbidity: 5.11, 95% CI: 3.99-6.55), (iii) asthma and rhinitis from 4 to 16 years of age. CONCLUSIONS: Specific IgE is strongly associated with eczema and allergic multimorbidity throughout childhood and with asthma and rhinitis from age 4 years. However, 23% of the children with IgE sensitization do not develop any disease in childhood.

Parental smoking and development of allergic sensitization from birth to adolescence.

BACKGROUND: The relation between secondhand tobacco smoke (SHS) exposure and the development of allergic sensitization in children is unclear. The aim of this study was to determine whether maternal smoking during pregnancy and postnatal SHS exposure contributes to the development of allergic sensitization in children and adolescents up to 16 years of age. METHODS: We included 3316 children from a birth cohort followed up for 16 years. SHS exposure and symptoms of allergic disease were assessed using repeated parental questionnaires. Serum immunoglobulin E against eight common inhalant and six food allergens was assessed at ages 4, 8, and 16 years with ImmunoCAP. The association between SHS exposure and sensitization was explored using logistic regression and generalized estimating equations. RESULTS: Exposure to SHS in infancy without prior exposure in utero was associated with an excess risk of food sensitization at age 4 years (OR 1.47, 95% CI 1.08-2.00), with comparable ORs at ages 8 and 16 years. In longitudinal analyses, an overall association was indicated between SHS in infancy and food sensitization up to age 16 years (OR 1.24, 95% CI 0.98-1.56). Maternal smoking during pregnancy was unrelated to sensitization up to 16 years of age. When sensitization was combined with concurrent symptoms of allergic disease, SHS in infancy was associated with an overall elevated risk of eczema with sensitization (OR 1.62, 95% CI 1.20-2.18). CONCLUSIONS: SHS exposure in infancy appears to increase the risk of sensitization to food allergens up to age 16 years, as well as eczema in combination with sensitization.

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1.

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.

Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma.

Cross-reactivity to fish and chicken meat - a new clinical syndrome.

BACKGROUND: Fish is one of the most allergenic foods. While clinical cross-reactivity among different fishes is a widely accepted feature of fish allergy, associations with other food allergies are not well understood. This study aims at analyzing the relevance of clinical cross-reactivity between fish and chicken meat in patients with allergy to chicken meat without sensitization to hen's eggs. METHODS: Patients with food allergy to fish and chicken meat (n = 29) or chicken meat only (n = 7) were recruited. IgE-reactive chicken proteins were identified (Edman, MS analysis) and quantified (ELISA). Allergens were used in IgE ELISA and skin testing. RESULTS: Chicken parvalbumin and two new allergens, aldolase and enolase, were identified at 12, 40, and 50 kDa, respectively. They were recognized by sIgE of 61%, 75%, and 83% of all patient sera which were in the majority of the cases positive for the fish homologues as well. Fish and chicken meat allergens were highly cross-reactive while high inhibition rates with fish or chicken allergens correlated with the patients' primary sensitization to fish or chicken. In cooked or roasted foods, enolase and aldolase were detectable in chicken breast while parvalbumin was detectable in chicken legs and wings. CONCLUSIONS: Fish and chicken meat are cross-reactive foods; both fish-allergic and chicken meat-allergic patients might be at risk of developing a food allergy to chicken meat or to fish, respectively. This clinical phenomenon is proposed to be termed 'fish-chicken syndrome' with cross-reactive allergens involved being parvalbumins, enolases, and aldolases.

Red meat allergic patients have a selective IgE response to the α-Gal glycan.

Galactose-α-1,3-galactose (α-Gal) is a mammalian carbohydrate with significance in a novel type of food allergy. Patients with IgE against α-Gal report severe allergic symptoms 3-6 h after consumption of red meat. We investigated whether IgE from red meat allergic patients recognizes other mammalian glycans than α-Gal or glycans from the plant kingdom and insects of importance in allergy. We found that none of the 24 red meat allergic patients investigated had an IgE antibody response against the other abundant mammalian glycan N-glycolylneuraminic acid or against cross-reactive carbohydrate determinants from plant or venom sources (nCup a 1, nArt v 1, and MUXF3). Deglycosylation of an α-Gal-containing protein, bovine thyroglobulin, significantly reduced the IgE response. In conclusion, we show that red meat allergic patients have a selective IgE response to the α-Gal glycan found in red meat. Other common glycans reactive in allergic disease are not targets of red meat allergic patients' IgE.

Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgE.

BACKGROUND: In Africa, peanuts are frequently consumed, but severe allergic reactions are rare. We investigated immunological patterns of clinical tolerance to peanut in peanut-sensitized but asymptomatic patients from central Africa compared to peanut-allergic and peanut-sensitized but asymptomatic patients from Sweden. METHODS: Sera from allergic patients (n = 54) from Zimbabwe sensitized to peanut but without allergic symptoms to peanut, and sera from peanut-allergic (n = 25) and peanut-sensitized but asymptomatic (n = 25) patients from Sweden were analyzed toward peanut allergen components (Ara h 1-3, 6, 8-9) and other allergen molecules from important allergen sources using microarray. IgE to Ara h 2 peptide epitopes was analyzed, and allergenic activity was assessed by basophil activation assay. RESULTS: Forty-six percent of the African and all peanut-allergic Swedish patients showed IgE toward one of the highly allergenic peanut allergens (Ara h 1-3, 6, 9). However, 48% of the African patients had IgE to cross-reactive carbohydrate determinants (CCDs) with low allergenic activity and 60% of the Swedish asymptomatic patients had IgE against the PR protein Ara h 8. IgG and IgG4 specificities and levels could not discriminate between the African asymptomatic and Swedish peanut-allergic patients. Asymptomatic patients almost lacked IgE to Ara h 2 peptides, which were recognized by peanut-allergic patients. Peanut IgE from peanut asymptomatic patients showed poor allergenic activity compared with IgE from peanut-allergic patients. CONCLUSIONS: Natural clinical tolerance to peanut in the African patients can be caused by IgE to low allergenic peanut components and by poor allergenic activity of peanut-specific IgE.

Are allergic multimorbidities and IgE polysensitization associated with the persistence or re-occurrence of foetal type 2 signalling? The MeDALL hypothesis.

Allergic diseases [asthma, rhinitis and atopic dermatitis (AD)] are complex. They are associated with allergen-specific IgE and nonallergic mechanisms that may coexist in the same patient. In addition, these diseases tend to cluster and patients present concomitant or consecutive diseases (multimorbidity). IgE sensitization should be considered as a quantitative trait. Important clinical and immunological differences exist between mono- and polysensitized subjects. Multimorbidities of allergic diseases share common causal mechanisms that are only partly IgE-mediated. Persistence of allergic diseases over time is associated with multimorbidity and/or IgE polysensitization. The importance of the family history of allergy may decrease with age. This review puts forward the hypothesis that allergic multimorbidities and IgE polysensitization are associated and related to the persistence or re-occurrence of foetal type 2 signalling. Asthma, rhinitis and AD are manifestations of a common systemic immune imbalance (mesodermal origin) with specific patterns of remodelling (ectodermal or endodermal origin). This study proposes a new classification of IgE-mediated allergic diseases that allows the definition of novel phenotypes to (i) better understand genetic and epigenetic mechanisms, (ii) better stratify allergic preschool children for prognosis and (iii) propose novel strategies of treatment and prevention.

Immunoproteomics of processed beef proteins reveal novel galactose-α-1,3-galactose-containing allergens.

BACKGROUND: Red meat allergy presents a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate α-Gal epitope. Food preparation and processing can influence the allergenicity of proteins. The aim of this study was to characterize the proteomic profile of different beef preparations and to investigate their α-Gal reactivity and potential allergenicity. METHODS: Extracts from raw, boiled, fried, and medium rare prepared beef were assessed by 2D PAGE for the comparison of protein profiles. IgE-binding proteins were identified using immunoblot-coupled proteomic analysis using sera from red meat-allergic patients. Presence of the α-Gal epitope was verified using anti-α-Gal antibody and IgE inhibition immunoblot with α-Gal. RESULTS: Multiple IgE-binding proteins were detected in the different beef preparations, many of which were also recognized by the anti-α-Gal antibody. Protein spots reacting with IgE in patient sera were analyzed by MS/MS, resulting in identification of 18 proteins with high identification scores. Seven of the 18 beef allergens identified using meat-allergic patient sera were also recognized by the anti-α-Gal monoclonal antibody, and four of them were stabile to thermal treatment. Furthermore, a dose-dependent inhibition of red meat-allergic patients' IgE to beef by α-Gal was demonstrated. CONCLUSIONS: We show that the α-Gal epitope is commonly present in IgE-reactive beef proteins recognized by meat-allergic patients. Seven novel α-Gal-containing IgE-binding proteins were identified, of which four were stable to heat treatment. Thus, the allergenicity of red meat proteins is preserved even upon different thermal cooking.

Identification of galactose-α-1,3-galactose in the gastrointestinal tract of the tick Ixodes ricinus; possible relationship with red meat allergy.

Patients with IgE antibodies against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to α-Gal and tick bites. We provide the first direct evidence that α-Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α-Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose-dependent inhibition of IgE responses to α-Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat-cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α-Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α-Gal. These results confirm that the α-Gal epitope is present in I. ricinus and imply host exposure to α-Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α-Gal and red meat allergy.

Dog saliva - an important source of dog allergens.

BACKGROUND: Allergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog. METHODS: IgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot. RESULTS: Greater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander-positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation. CONCLUSIONS: Dog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.

Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches.

Immunoglobulin E-mediated allergies affect more than 25% of the population. Allergen exposure induces a variety of symptoms in allergic patients, which include rhinitis, conjunctivitis, asthma, dermatitis, food allergy and life-threatening systemic anaphylaxis. At present, allergen-specific immunotherapy (SIT), which is based on the administration of the disease-causing allergens, is the only disease-modifying treatment for allergy. Current therapeutic allergy vaccines are still prepared from relatively poorly defined allergen extracts. However, with the availability of the structures of the most common allergen molecules, it has become possible to produce well-defined recombinant and synthetic allergy vaccines that allow specific targeting of the mechanisms of allergic disease. Here we provide a summary of the development and mechanisms of SIT, and then review new forms of therapeutic vaccines that are based on recombinant and synthetic molecules. Finally, we discuss possible allergen-specific strategies for prevention of allergic disease.

Altered immunoregulatory profile during anti-tumour necrosis factor treatment of patients with inflammatory bowel disease.

Inflammatory 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)-ß 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 accompanied by an increased percentage of CD69(+) cells among these cells after 6 weeks of treatment compared to before treatment (P < 0·01). There was also an increase of mucosal T helper type1 cells from weeks 2 to 6 (P < 0·05). In addition, CD25(+)TNF-RII(+) cells in the mucosa were decreased after 6 weeks of treatment compared to before treatment (P < 0·05). Before treatment, peripheral blood mononuclear cell baseline proliferation was increased when IL-10 was blocked (P < 0·01), but not after. In CD25(+) cell-depleted cultures proliferation increased after treatment (P < 0·05). Our data indicate that anti-TNF treatment leads to an induction of effector T cells. Anti-TNF therapy has no significant impact on regulatory T cells in IBD, although the composition of regulatory T cell subsets may change during treatment.

Conjunctival provocation with airborne allergen in patients with atopic keratoconjunctivitis.

BACKGROUND: Atopic 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. OBJECTIVE: To investigate the allergic reaction, to conjunctival provocation with airborne allergens, in patients with AKC. METHODS: Eleven 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. RESULTS: A significant change in clinical symptoms and signs, (redness and chemosis) was evident 10 min after provocation compared with baseline (P = 0.005) and compared with the unprovoked eye (P = 0.005) in AKC subjects. These parameters were normalized after 8 and 48 h. A significant increase for IFN-γ (P = 0.021) and IL-6 (P = 0.015), and a near significant increase for IL-10 (P = 0.066) were seen in the tear fluid of the challenged eye at 48 h after provocation vs. baseline and vs. the control eye for IFN-γ (P = 0.005), IL-6 (P = 0.028) and IL-10 (P = 0.008) in AKC subjects. CONCLUSION AND CLINICAL RELEVANCE: In this single dose allergen provocation study, AKC patients responded with a typical IgE-mediated allergic reaction. An increase in cytokines at 48 h after the challenge was demonstrated and might, with further studies, give us a better understanding of the nature of inflammation in AKC.