The α-Gal epitope - the cause of a global allergic disease.

The galactose-α-1,3-galactose (α-Gal) epitope is the cause of a global allergic disease, the α-Gal syndrome (AGS). It is a severe form of allergy to food and products of mammalian origin where IgE against the mammalian carbohydrate, α-Gal, is the cause of the allergic reactions. Allergic reactions triggered by parenterally administered α-Gal sources appear immediately, but those triggered via the oral route appear with a latency of several hours. The α-Gal epitope is highly immunogenic to humans, apes and old-world monkeys, all of which produce anti-α-Gal antibodies of the IgM, IgA and IgG subclasses. Strong evidence suggests that in susceptible individuals, class switch to IgE occurs after several tick bites. In this review, we discuss the strong immunogenic role of the α-Gal epitope and its structural resemblance to the blood type B antigen. We emphasize the broad abundance of α-Gal in different foods and pharmaceuticals and the allergenicity of various α-Gal containing molecules. We give an overview of the association of tick bites with the development of AGS and describe innate and adaptive immune response to tick saliva that possibly leads to sensitization to α-Gal. We further discuss a currently favored hypothesis explaining the mechanisms of the delayed effector phase of the allergic reaction to α-Gal. We highlight AGS from a clinical point of view. We review the different clinical manifestations of the disease and the prevalence of sensitization to α-Gal and AGS. The usefulness of various diagnostic tests is discussed. Finally, we provide different aspects of the management of AGS. With climate change and global warming, the tick density is increasing, and their geographic range is expanding. Thus, more people will be affected by AGS which requires more knowledge of the disease.

Th2-skewed T cells correlate with B cell response to α-Gal and tick antigens in α-Gal syndrome.

Tick bites have been shown to transmit a novel form of severe food allergy, the galactose-α-1,3-galactose (α-Gal) syndrome (AGS). Cellular responses to α-Gal in patients with AGS have, to date, not been thoroughly scrutinized. Therefore, we investigated T and B cell proliferation, activation, and cytokine profiles in response to tick protein extract (TE) and α-Gal-free TE in patients with AGS and in healthy controls. T and B cells from both patients and controls proliferated in response to TE, but significantly more in patients with AGS. B cell proliferation, but not T cell proliferation, in patients with AGS was reduced by removing α-Gal from the TE. In addition, TE induced a clear Th2 cytokine profile in patients with AGS. Expression of CD23 by B cells correlated only to T cell proliferation. However, both B cell proliferation and CD23 expression were reduced when CD40L and IL-4 were blocked. A large portion of the IgG1 and IgE antibodies binding TE in patients with AGS were directed against the α-Gal epitope. We have, for what we believe to be the first time, investigated T and B cell responses to α-Gal carrying tick proteins in patients with AGS, which will be essential for the understanding of the immune response against an allergenic carbohydrate transmitted by ticks.

Cross-reactivity between tick and wasp venom can contribute to frequent wasp sensitization in patients with the α-Gal syndrome.

BACKGROUND: α-Gal syndrome (AGS) is a food allergy with severe delayed allergic reactions, mediated by IgE-reactivity to galactose-α1,3-galactose (α-Gal). AGS is strongly associated with tick bites. An increased incidence of venom sensitization has been found in AGS patients. Here, we evaluated the frequency of wasp sensitization in Swedish AGS patients and the possible cross-reactivity between wasp venom and tick proteins. METHODS: Sera from 136 Swedish AGS patients and 29 wasp-positive non-AGS control sera were analyzed for IgE-reactivity against wasp venom (Vespula spp.), the European tick Ixodes ricinus (Streptavidin ImmunoCAP), α-Gal and total IgE by ImmunoCAP. The presence of α-Gal on wasp venom proteins (Vespula vulgaris) was investigated by western blot (WB), and possible cross-reactivity between wasp venom and tick proteins by enzyme-linked immunosorbent assay and WB. Involvement of cross-reactive carbohydrate domains (CCDs) was also assessed. RESULTS: Wasp sensitization was present in 54% of AGS patients, although the IgE levels were low. Wasp sensitized patients had higher IgE levels to α-Gal and total IgE levels compared to non-wasp sensitized AGS patients. α-Gal was not detected in wasp venom, but cross-reactivity between wasp and tick proteins was demonstrated which was not dependent on CCDs. The same cross-reactivity was also observed in the control sera. Furthermore, 17 putative cross-reactive peptides were identified using an in silico approach. CONCLUSIONS: For the first time, cross-reactivity between wasp venom and tick proteins has been described. This may be a reason why the majority of Swedish AGS patients, who have all been tick bitten, are also sensitized against wasp.

Bovine γ-globulin, lactoferrin, and lactoperoxidase are relevant bovine milk allergens in patients with α-Gal syndrome.

BACKGROUND: Mammalian meat is the most common trigger of the allergic reactions in patients with α-Gal syndrome (AGS). Milk and dairy, although less often, also cause a significant number of allergic manifestations. The aim of this study was to identify α-Gal-containing bovine milk proteins with allergenic properties among AGS patients. METHODS: Thirty-eight AGS patients with IgE to milk were included in the study. Milk proteins were analyzed for the presence of α-Gal and for binding by patients' IgE using immunoblot, ImmunoCAP, and inhibition ELISA. Allergenicity of milk and milk proteins was assessed by basophil activation test. RESULTS: More than half of the AGS patients reported allergic reactions to milk or dairy products. Bovine γ-globulin (BGG), lactoferrin (LF), and lactoperoxidase (LPO) were identified as α-Gal carrying proteins which were recognized by AGS patients' IgE. Whey mirrored the anti-α-Gal and IgE reactivity of BGG, LF, and LPO. Eighty-nine percent of the patients displayed IgE to BGG, 91% to LF, and 57% to LPO. Inhibition of α-Gal-specific IgE binding was achieved by BGG, LF, LPO, and whey. These proteins also activated AGS patients' basophils. Interestingly, at lower concentrations, LF was the most potent inhibitor of IgE binding, and the most potent activator of basophils. CONCLUSION: BGG, LF, and LPO were all found to be relevant milk α-Gal-containing glycoproteins that bound AGS patients' IgE antibodies and activated their basophils. These proteins are probably involved in the allergic reactions to milk in AGS patients. LPO was for the first time shown to be an allergen.

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-α-1,3-galactose (α-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of α-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by α-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that α-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the α-Gal epitope remained for up to 2 h of digestion. Furthermore, α-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. α-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of α-Gal glycosylated BSA (bovine serum albumin) (BSA-α-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of α-Gal glycosylated proteins.

Clinical and Serological Characterization of the α-Gal Syndrome-Importance of Atopy for Symptom Severity in a European Cohort.

BACKGROUND: The galactose-α1,3-galactose (α-Gal) syndrome (AGS) is a novel form of food allergy. Patients experience delayed severe allergic reactions after mammalian meat consumption due to IgE antibodies directed against the carbohydrate α-Gal present in mammalian meat. The onset of the disease is associated with tick bites. OBJECTIVE: To characterize a cohort of patients with AGS from Sweden on a clinical and serological level, and identify risk factors for disease severity. METHODS: A total of 128 patients with symptoms after mammalian meat intake and IgE to α-Gal were included. Medical examination and diagnosis were made by an allergologist and questionnaires were filled in regarding onset of symptoms, tick exposure, and airborne allergies. Serum IgE reactivity against multiple food and airborne allergens, as well as protein extract from the tick Ixodes ricinus, was measured using ImmunoCAP. RESULTS: The majority of patients were middle aged, with equal gender distribution. Nearly all reported symptoms more than 2 hours after meat consumption. Urticaria (90%) and gastrointestinal symptoms (74%) were most common. Almost half of the patients suffered from anaphylaxis, and α-Gal IgE levels were significantly higher among these patients compared with those without anaphylaxis. Nearly all patients had been tick bitten and 75% had IgE against I. ricinus. More than half of the patients with AGS were atopic, and atopy increased the risk of anaphylaxis with pulmonary manifestations. Only 2 patients belonged to blood group B/AB. CONCLUSION: AGS is an upcoming food allergy where patients report severe symptoms and tick bites. Atopy was found to affect the manifestation of the disease in Swedish patients.

Selective COX-2 Inhibition Exerts No Negative Effects on Peripheral Blood Lymphocytes in Allergic Asthmatics.

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 stimulated expression of FOXP3 mRNA in CD4+ T cells. Proliferation of PBMCs to the major cat allergen Fel d 1 was slightly reduced by etoricoxib treatment in cat-allergic patients. CONCLUSIONS: Short-term treatment with the COX-2 inhibitor etoricoxib had a minor impact on T-cell responses, supporting its safe use also in subjects exposed to triggers of lymphocyte activation.

Development of a mouse model for chronic cat allergen-induced asthma.

BACKGROUND: Allergic asthma is a chronic inflammatory airway disease caused by exposure to airborne allergens. In order to develop novel therapies for allergic asthma, models that are relevant to human disease are needed. METHODS: Female BALB/c mice were presensitised subcutaneously with alum-adsorbed recombinant cat allergen Fel d 1, followed by intranasal challenges with cat dander extract spiked with recombinant Fel d 1 for 7 weeks. For reference, mice were presensitised and challenged with ovalbumin following the same protocol. Airway hyperresponsiveness, serum antibodies, airway inflammation and cell infiltration, and cytokines in lung tissue and bronchoalveolar lavage were measured. RESULTS: Mice presensitised with recombinant Fel d 1 and challenged with cat dander extract or presensitised and challenged with ovalbumin showed airway hyperresponsiveness in response to metacholine. Mice of the cat allergen model showed influx of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage, combined with increased levels of IL-17a and increased IL-4 mRNA expression in lung tissue. In contrast, mice sensitised and challenged with ovalbumin showed a predominant influx of eosinophils in bronchoalveolar lavage and had an increased expression of IL-5 in lung tissue. Both protocols induced features of lung tissue remodelling and allergen-specific antibody responses. CONCLUSIONS: The presented mouse model for cat allergen-induced asthma exhibits hallmarks of chronic allergic asthma, like airway hyperresponsiveness, a mixed neutrophilic/eosinophilic infiltration in bronchoalveolar lavage, expression of IL-17a and signs of remodelling in lung tissue. The model will provide a relevant platform for the development of novel treatment strategies.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BACKGROUND: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. OBJECTIVE: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. METHODS: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. RESULTS: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. CONCLUSION: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

HLA-DR(hi) and CCR9 Define a Pro-Inflammatory Monocyte Subset in IBD.

OBJECTIVES: It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inflammation, but the phenotype and inflammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inflammatory bowel disease (IBD). METHODS: Fifty-one patients with mild to severe ulcerative colitis (UC; n=31; UC-DAI 3-12) or Crohn's disease (CD; n=20; Harvey-Bradshaw indices (HBI) 2-16) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14(+)HLA-DR(hi) monocytes was monitored weekly in peripheral blood, using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients. RESULTS: The frequency of circulating CD14(+)HLA-DR(hi) monocytes was significantly higher in IBD patients with moderate to severe disease compared with healthy controls (P<0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14(+)HLA-DR(hi) monocytes was significantly reduced. CD14(+)HLA-DR(hi) monocytes produced high levels of inflammatory mediators, such as tumor necrosis factor (TNF)-α, and expressed the gut-homing receptor CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease. CONCLUSIONS: CD14(+)HLA-DR(hi) blood monocytes were increased in patients with active IBD. These monocytes exhibit a pro-inflammatory, gut-homing phenotype with regard to their TNF-α production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.

Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins.

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. ß-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.

Covalent coupling of vitamin D3 to the major cat allergen Fel d 1 improves the effects of allergen-specific immunotherapy in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (SIT) is currently the only curative treatment for allergy but the treatment needs to be improved. We hypothesize that covalent coupling of immunomodulating vitamin D3 to the major cat allergen Fel d 1 can enhance the beneficial effects of SIT to cat allergy. METHODS: We treated mice sensitized to Fel d 1 with subcutaneous injections of two doses of recombinant Fel d 1 coupled to 1α,25-dihydroxyvitamin D3 (rFel d 1:VD3) and compared to treatment with the same doses of rFel d 1 in a mouse model for cat allergy. Airway hyperresponsiveness (AHR), cytokines and cells in bronchoalveolar lavage (BAL), in vitro activation of splenocytes to rFel d 1, and Fel d 1-specific immunoglobulins were evaluated. RESULTS: Treatment with both doses of rFel d 1:VD3 decreased AHR, cellular influx and Th2 cytokines in BAL compared to untreated mice. High- and low-dose rFel d 1 treatment also decreased AHR and BAL Th2 cytokines, with less decrease for the low-dose treatment. Importantly, the total number of cells and eosinophils in BAL was markedly reduced at both high- and low-dose rFel d 1:VD3 compared to treatment with rFel d 1 alone. Finally, treatment with both rFel d 1 and rFel d 1:VD3 induced Fel d 1-specific serum IgG. CONCLUSION: Our results indicate a beneficial therapeutic effect of rFel d 1:VD3 on airway inflammation, AHR and rFel d 1-specific immune responses and thus suggest that this novel immunomodulatory candidate may improve both the efficacy and safety of SIT.

In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity.

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.