IgE and anaphylaxis specific to the carbohydrate alpha-gal depend on IL-4.

BACKGROUND: Alpha-gal (Galα1-3Galß1-4GlcNAc) is a carbohydrate with the potential to elicit fatal allergic reactions to mammalian meat and drugs of mammalian origin. This type of allergy is induced by tick bites, and therapeutic options for this skin-driven food allergy are limited to the avoidance of the allergen and treatment of symptoms. Thus, a better understanding of the immune mechanisms resulting in sensitization through the skin is crucial, especially in the case of a carbohydrate allergen for which underlying immune responses are poorly understood. OBJECTIVE: We aimed to establish a mouse model of alpha-gal allergy for in-depth immunologic analyses. METHODS: Alpha-galactosyltransferase 1-deficient mice devoid of alpha-gal glycosylations were sensitized with the alpha-gal-carrying self-protein mouse serum albumin by repetitive intracutaneous injections in combination with the adjuvant aluminum hydroxide. The role of basophils and IL-4 in sensitization was investigated by antibody-mediated depletion. RESULTS: Alpha-gal-sensitized mice displayed increased levels of alpha-gal-specific IgE and IgG1 and developed systemic anaphylaxis on challenge with both alpha-gal-containing glycoproteins and glycolipids. In accordance with alpha-gal-allergic patients, we detected elevated numbers of basophils at the site of sensitization as well as increased numbers of alpha-gal-specific B cells, germinal center B cells, and B cells of IgE and IgG1 isotypes in skin-draining lymph nodes. By depleting IL-4 during sensitization, we demonstrated for the first time that sensitization and elicitation of allergy to alpha-gal and correspondingly to a carbohydrate allergen is dependent on IL-4. CONCLUSION: These findings establish IL-4 as a potential target to interfere with alpha-gal allergy elicited by tick bites.

α-Gal present on both glycolipids and glycoproteins contributes to immune response in meat-allergic patients.

BACKGROUND: The α-Gal syndrome is associated with the presence of IgE directed to the carbohydrate galactose-α-1,3-galactose (α-Gal) and is characterized by a delayed allergic reaction occurring 2 to 6 hours after ingestion of mammalian meat. On the basis of their slow digestion and processing kinetics, α-Gal-carrying glycolipids have been proposed as the main trigger of the delayed reaction. OBJECTIVE: We analyzed and compared the in vitro allergenicity of α-Gal-carrying glycoproteins and glycolipids from natural food sources. METHODS: Proteins and lipids were extracted from pork kidney (PK), beef, and chicken. Glycolipids were purified from rabbit erythrocytes. The presence of α-Gal and IgE binding of α-Gal-allergic patient sera (n = 39) was assessed by thin-layer chromatography as well as by direct and inhibition enzyme-linked immunosorbent assay. The in vitro allergenicity of glycoproteins and glycolipids from different meat extracts was determined by basophil activation test. Glycoprotein stability was evaluated by simulated gastric and intestinal digestion assays. RESULTS: α-Gal was detected on glycolipids of PK and beef. Patient IgE antibodies recognized α-Gal bound to glycoproteins and glycolipids, although binding to glycoproteins was more potent. Rabbit glycolipids were able to strongly activate patient basophils, whereas lipid extracts from PK and beef were also found to trigger basophil activation, but at a lower capacity compared to the respective protein extracts. Simulated gastric digestion assays of PK showed a high stability of α-Gal-carrying proteins in PK. CONCLUSION: Both α-Gal-carrying glycoproteins and glycolipids are able to strongly activate patient basophils. In PK and beef, α-Gal epitopes seem to be less abundant on glycolipids than on glycoproteins, suggesting a major role of glycoproteins in delayed anaphylaxis upon consumption of these food sources.

Component-resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals.

BACKGROUND: Furry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea-pigs still relies on animal dander extracts which do not allow to define the primary sensitization source. OBJECTIVE: To identify major guinea-pig allergens and to evaluate their potential as marker allergens for in vitro IgE-diagnosis in comparison with dander extracts. METHODS: A group of patients allergic to guinea-pig (n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea-pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA. RESULTS: The combination of 4 guinea-pig lipocalin allergens, including 2 new lipocalins, Cav p 1.0201 and Cav p 6.0101, and the previously characterized lipocalins Cav p 2 and Cav p 3, enabled the identification of 90% of all patients allergic to guinea-pig. The vast majority had specific IgE to Cav p 1 (83%). Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE-cross-reactive with these allergens. In the group of cat- and dog-allergic patients, 73% had also specific IgE to guinea-pig dander. However, only 27% of the cat /dog-allergic patients had specific IgE to any of the non-cross-reactive guinea-pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea-pig dander could be explained by IgE-cross-reactivity among serum albumins and certain lipocalins. CONCLUSIONS AND CLINICAL RELEVANCE: The availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea-pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component-resolved IgE-diagnosis of allergy to small furry pets.

The basophil activation test differentiates between patients with alpha-gal syndrome and asymptomatic alpha-gal sensitization.

BACKGROUND: Galactose-alpha-1,3-galactose (alpha-gal) syndrome is characterized by the presence of serum specific IgE antibodies to alpha-gal and delayed type I allergic reactions to the carbohydrate alpha-gal after consumption of mammalian (red) meat products and drugs of mammalian origin. Diagnostics currently rely on patient history, skin tests, determination of serum specific IgE antibodies, and oral food or drug challenges. OBJECTIVE: We sought to assess the utility of different basophil parameters (basophil reactivity and sensitivity, the ratio of the percentage of CD63+ basophils induced by the alpha-gal-containing allergen to the percentage of CD63+ basophils after stimulation with anti-FcεRI antibody [%CD63+/anti-FcεRI], and area under the dose-response curve [AUC]) as biomarkers for the clinical outcome of patients with alpha-gal syndrome compared with subjects with asymptomatic alpha-gal sensitization. METHODS: In addition to routine diagnostics, a basophil activation test (Flow CAST) with different concentrations of alpha-gal-containing allergens (eg, commercially available alpha-gal-carrying proteins and pork kidney extracts) was performed in 21 patients with alpha-gal syndrome, 12 alpha-gal-sensitized subjects, and 18 control subjects. RESULTS: Alpha-gal-containing allergens induced strong basophil activation in a dose-dependent manner in patients. Basophil reactivity at distinct allergen concentrations, the %CD63+/anti-FcεRI ratio across most allergen concentrations, the AUC of dose-response curves, and basophil allergen threshold sensitivity (CD-sens) with pork kidney extract were significantly higher in patients with alpha-gal syndrome compared with those in sensitized subjects. All parameters were negative in control subjects. CONCLUSION: The basophil activation test should be considered as an additional diagnostic test before performing time-consuming and potentially risky oral provocation tests. The %CD63+/anti-FcεRI ratio for all allergens and AUCs for pork kidney were the best parameters for distinguishing patients with alpha-gal syndrome from subjects with asymptomatic alpha-gal sensitization.

Hypoallergenic animals: A promise of hope for allergic patients?

Furry pets are beloved companion animals; horse riding is a popular leisure activity. So-called hypoallergenic animals have gained high interest as sensitization to animal dander and allergy to furry animals are widespread. Allergen immunotherapy to furry animals is still limited, and allergen avoidance in addition to symptomatic pharmaceutical treatment is often the only available option. Patients with an existing allergy to furry animals or with an atopic background are seeking for a hypoallergenic alternative. This review summarizes current knowledge and discusses future strategies.

Mammalian derived lipocalin and secretoglobin respiratory allergens strongly bind ligands with potentially immune modulating properties.

Allergens from furry animals frequently cause sensitization and respiratory allergic diseases. Most relevant mammalian respiratory allergens belong either to the protein family of lipocalins or secretoglobins. Their mechanism of sensitization remains largely unresolved. Mammalian lipocalin and secretoglobin allergens are associated with a function in chemical communication that involves abundant secretion into the environment, high stability and the ability to transport small volatile compounds. These properties are likely to contribute concomitantly to their allergenic potential. In this study, we aim to further elucidate the physiological function of lipocalin and secretoglobin allergens and link it to their sensitizing capacity, by analyzing their ligand-binding characteristics. We produced eight major mammalian respiratory allergens from four pet species in E.coli and compared their ligand-binding affinities to forty-nine ligands of different chemical classes by using a fluorescence-quenching assay. Furthermore, we solved the crystal-structure of the major guinea pig allergen Cav p 1, a typical lipocalin. Recombinant lipocalin and secretoglobin allergens are of high thermal stability with melting temperatures ranging from 65 to 90°C and strongly bind ligands with dissociation constants in the low micromolar range, particularly fatty acids, fatty alcohols and the terpene alcohol farnesol, that are associated with potential semiochemical and/or immune-modulating functions. Through the systematic screening of respiratory mammalian lipocalin and secretoglobin allergens with a large panel of potential ligands, we observed that total amino acid composition, as well as cavity shape and volume direct affinities to ligands of different chemical classes. Therefore, we were able to categorize lipocalin allergens over their ligand-binding profile into three sub-groups of a lipocalin clade that is associated with functions in chemical communication, thus strengthening the function of major mammalian respiratory allergens as semiochemical carriers. The promiscuous binding capability of hydrophobic ligands from environmental sources warrants further investigation regarding their impact on a molecule's allergenicity.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.