Karolinska Institutet

Increasing prevalence of galactose-α-1,3-galactose sensitization in the Danish general adult population.

Alpha-gal syndrome is a novel food allergy to the oligosaccharide galactose-α-1,3-galactose (alpha-gal) present in mammalian meat. Tick bites are considered an important route of sensitization to alpha-gal. Data on alpha-gal sensitization in the general population is scant. We utilized a unique data source of repeated population-based health examination studies to assess prevalence, time trends, risk factors, and characteristics of alpha-gal sensitization. Alpha-gal sensitization was assessed in >11.000 adults from four health examination studies of randomly invited residents in the Copenhagen region conducted in 1990-1991, 2011-2012, 2012-2015, and 2016-2017. Alpha-gal sensitization was defined as serum specific IgE (sIgE) to alpha-gal ≥0.1 kU/L; ≥0.35 kU/L; ≥0.7 kU/L; ≥3.5 kU/L. The population was characterized according to genetically determined ABO blood group, aeroallergen sensitization, and pets at home. The prevalence of sIgE to alpha-gal ≥0.1 kUA/L was 1.3% in 1990-1991, 3.7% in 2012-2015 and 3.2% in 2016-2017. Of those sensitized to alpha-gal >97% reported to consume red meat at least once a week, even for sIgE to alpha-gal ≥3.5 kU/L. Male sex, older age, aeroallergen sensitization, cat at home, and blood group A were associated with increased odds of alpha-gal sensitization. The known protective effect of blood group B was confirmed. In this general adult population, the prevalence of alpha-gal sensitization had doubled from 1990-1991 to 2016-2017. This could potentially be due to increased tick exposure and an increased atopic predisposition.

Allergenic potency of various foods of mammalian origin in patients with α-Gal syndrome.

The α-Gal syndrome (AGS) is an emerging allergy to mammalian food caused by IgE-mediated reactions to the carbohydrate galactose-α-1,3-galactose (α-Gal). Mammalian food sources contain α-Gal, but the amount differs. The objective of this study was to investigate the allergenic potency of various foods of mammalian origin among AGS patients. Twenty-six AGS patients were included. Food extracts from innards, lean meats, processed meat products, milk, and whey were analyzed. Immunoblot, ELISA, immunofluorescence, and basophil activation test were used to determine the α-Gal content, characterize IgE binding, and assess foods' allergenicity. The determined amount of α-Gal, IgE reactivity to food extracts, and food extract potencies to activate patients' basophils correlated well with each other. Pork and beef kidney showed the highest allergenicity. Beef liver and bacon showed allergenicity comparable to that of lean meats. Game meat seemed to have a higher allergenic potency than meats from farm-raised animals. The processed meat products liver pâté and black pudding, despite lower α-Gal content, demonstrated moderate allergenicity. Milk showed the lowest allergenicity. IgE reactivity to food extracts was highly similar for all patients and strongly dominated by the α-Gal epitope. The allergenic potency of mammalian meat depends on the origin of the meat, the different cuts, and type of processing, with innards posing the greatest risk to AGS patients. Even processed mammalian meat constitutes a risk. Dairy products show the lowest risk. This study highlights the importance of analyzing even more foods to improve the management of AGS.

Living close to the forest is a risk factor for galactose-α-1,3-galactose sensitization.

The α-gal syndrome is a food allergy to galactose-α-1,3-galactose present in mammalian meat; it usually develops after tick bites. Despite being considered an emerging health problem, little is known about the geographic risk factors and patterns of α-gal sensitization. The aim of this study was to assess geographic clusters of α-gal sensitization and determine whether distance from home address to the forest and tick abundance are risk factors for α-gal sensitization in a Danish adult population. We utilized 3 population-based health examination studies that were conducted in 2011-2017 and covered a total of 8742 participants living in a suburban area. All participants were screened for a level of serum-specific IgE to α-gal of 0.1 kU/L or higher. Their home addresses were linked with CORINE Land Cover data and a tick prediction model. The associations of (1) distance to the forest and (2) tick abundance with α-gal sensitization were assessed by using multivariable logistic regression. There were 344 cases of galactose-α-1,3-galactose-specific IgE level being 0.1 kU/L; we identified 1 geographic cluster of cases. For every 1 km closer a participant lived to the forest, the odds ratio of α-gal sensitization was 1.09 (95% CI = 1.05-1.13). There were no associations between predicted tick nymph abundance and α-gal sensitization. In a general, individuals residing in a suburban area had 9% higher odds of being sensitized to α-gal the closer they lived to a forest, thus supporting exposure to ticks as a risk factor for sensitization to α-gal.

Stereoselective Synthesis of the Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C-Type Lectin Receptors.

In this work, we have discovered that the Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc trisaccharide, a fragment of the B antigen Type-1, is a new ligand of two C-type lectin receptors (CLRs) i. e. DCAR and Mincle which are key players in different types of autoimmune diseases. Accordingly, we report here on a straightforward methodology to access pure Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc trisaccharide. A spacer with a terminal primary amine group was included at the reducing end of the GlcNAc residue thus ensuring the further functionalization of the trisaccharide Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc.

A case of alpha-gal syndrome: Recall urticaria and 10 years of measurements of IgE to galactose-α-1,3-galactose.

Alpha-gal IgE level can change rapidly. Reassessment of a patient's alpha-gal IgE level may be helpful in the patient's clinical follow-up. Pruritus related to the site of a previous tick bite strengthens the diagnosis of alpha-gal syndrome.

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.

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. Sera from 136 Swedish AGS patients and 29 wasp-positive non-AGS control sera were analyzed for IgE-reactivity against wasp venom ( spp.), the European tick (Streptavidin ImmunoCAP), α-Gal and total IgE by ImmunoCAP. The presence of α-Gal on wasp venom proteins () 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. 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 approach. 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.

Alpha-gal sensitization among young adults is associated with male sex and polysensitization.

A pediatric case of tick-bite-Induced meat allergy and recall urticaria.

Alpha-gal syndrome should be suspected also in children in case of typical allergic symptoms in the evening or night during tick seasons and the event of recall urticaria. It is, however, still a challenge for both patients and clinicians.

Elucidating the α-Gal syndrome at the molecular allergen level.

Course of IgE to α-Gal in a Swedish population of α-Gal syndrome patients.

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

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. 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. 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. 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.

Carbohydrate epitopes currently recognized as targets for IgE antibodies.

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be "classical" cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.

Unusual and Unexpected Allergic Reactions Can Be Unraveled by Molecular Allergy Diagnostics.

The fifth class of immunoglobulin, immunoglobulin E (IgE) was discovered in 1967 and has had immense importance for the understanding, diagnosis, and treatment of allergic disease. More than 50 years have passed and efforts to characterize, standardize, and refine allergens with the aim to improve clinical diagnosis and allergen-specific immunotherapy are still ongoing. Another important breakthrough was made in 1999 with the introduction of component-resolved diagnostics (CRD), making it possible to quantify IgE antibodies against individual allergen proteins for diagnostic purposes at a molecular level. The progress and developments made in allergy diagnosis often originate from clinical observations and case studies. Observant physicians and health-care personnel have reported their findings in the medical literature, which in turn has inspired researchers to become involved in clinical research. Allergists continuously encounter new allergies and are often asked by their patients how to prevent new reactions. In the current article, we focus on recent clinical observations that can now be explained by CRD. The examples taken concern allergic reactions toward peanuts, tree nuts, lemon kernels, health drinks, meat, insects, dog dander, cannabis, and semen. We now have an improved understanding of why patients may react in a serious or unexpected way, as illustrated by these examples, yet many other clinical observations remain unexplained. The aim of this review is to highlight the importance of clinical observations among allergic patients, focusing on systemic, or unusual and unexpected allergic reactions, where component-testing has further refined the diagnosis of IgE-mediated allergy.

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

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. To characterize a cohort of patients with AGS from Sweden on a clinical and serological level, and identify risk factors for disease severity. 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. 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. 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.

Allergenomics of the tick Ixodes ricinus reveals important α-Gal-carrying IgE-binding proteins in red meat allergy.

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.

On the cause and consequences of IgE to galactose-α-1,3-galactose: A report from the National Institute of Allergy and Infectious Diseases Workshop on Understanding IgE-Mediated Mammalian Meat Allergy.

The mammalian meat allergy known as the "α-Gal syndrome" relates to IgE specific for galactose-α-1,3-galactose (α-Gal), an oligosaccharide that is present in cells and tissues of nonprimate mammals. The recognition of delayed reactions to food derived from mammals in patients with IgE to α-Gal and also the association with tick bites have been increasing worldwide. In 2018, the National Institute of Allergy and Infectious Diseases, Division of Allergy, Immunology and Transplantation, sponsored a workshop on this emerging tick-related disease. International experts from the fields of tick biology, allergy, immunology, infectious disease, and dermatology discussed the current state of our understanding of this emerging medical condition. The participants provided suggestions for specific research priorities and for the development of resources to advance our knowledge of the mechanisms, diagnosis, management, and prevention of this allergic disease. This publication is a summary of the workshop and the panel's recommendations are presented herein.

Definitive chemoradiotherapy plus cetuximab for cancer in the oesophagus or gastro-oesophageal junction.

Chemoradiotherapy is standard treatment for localized oesophageal cancer unsuitable for surgery. We aimed to evaluate the efficacy of cetuximab in combination with chemoradiotherapy. This non-randomised multicentre phase II trial recruited patients aged 18-75 with WHO performance status 0-2 having squamous cell carcinoma or adenocarcinoma in the oesophagus or gastro-oesophageal junction, T2-4, N0-3, M0 not suitable for surgery. Chemotherapy was three 21-day cycles of fluorouracil 750 mg/m2 D1-5 and oxaliplatin D1 (cycle 1:130mg/m 2, cycle 2-3:85 mg/m 2). Radiotherapy was 50Gy in 2Gy/fraction, 5 days a week, concurrent with cycle 2 and 3 and weekly cetuximab. The primary objective was loco-regional control at one year. 52 patients were included. 51 were eligible for toxicity and survival analysis and 46 for recurrence analysis. Full radiotherapy dose was delivered to 80%, 75% received all three cycles of chemotherapy and 75% received four or more doses of cetuximab. The most common related grade III-IV adverse events were gastro-intestinal(16), hypersensitivity(6) and infection(5). There were two drug-related deaths. Within six months from the end of treatment, six patients died from complications from fistulas. The loco-regional control rate at one year was 47.3%(95%CI 30.9%-62.1%). Overall survival at three years was 29.1%(95% CI 17.4-41.9%). Oxaliplatin and fluorouracil given concurrent with radiotherapy and cetuximab had an acceptable safety profile and showed a clinical response in patients with locoregionally advanced oesophageal cancer unsuitable for surgery. However, the primary end-point was not met, and the addition of cetuximab to definitive chemoradiotherapy cannot be recommended.

Galactose α-1,3-galactose phenotypes: Lessons from various patient populations.

To review published studies on galactose α-1,3-galactose (α-gal), a carbohydrate epitope found on proteins and lipids in nonprimate mammals and present in foods (particularly organ or fat-rich red meat) and medications, where it causes delayed-onset and immediate-onset anaphylaxis. A literature search for the terms galactose α-1,3-galactose and α-gal using PubMed and Embase was performed. Studies on α-gal were included in this review. Several species of ticks contain α-gal epitopes and possibly salivary adjuvants that promote high titer sensitization and clinical reactivity. Risk factors for α-gal syndrome include exposure to ticks of particular species. Age and sex differences seen in various cohorts possibly reflect the prevalence of these exposures that vary according to setting. The reason and mechanisms for delayed onset of food-related anaphylaxis and the preponderance of abdominal reactions are not clear but may involve the kinetics of allergen digestion and processing or immunologic presentation via a different mechanism from usual immediate-type food allergy.

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

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. 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/IgG (ELISA) responses were determined. Basophil activation tests were performed. 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 IgG and IgG 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. Red meat-allergic patients showed significantly higher α-Gal IgE, IgG , and IgG 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.

α-Gal on the protein surface affects uptake and degradation in immature monocyte derived dendritic cells.

Red meat allergy is characterized by an IgE response against the carbohydrate galactose-α-1,3-galactose (α-Gal), which is abundantly expressed on glycoproteins from non-primate mammals. The mechanisms of how α-Gal is processed and presented to the immune system to initiate an allergic reaction are still unknown. The aim of this study was to reveal whether the presence of α-Gal epitopes on the protein surface influence antigen uptake and processing in immature monocyte-derived dendritic cells (iMDDCs). Immature MDDCs were prepared from healthy blood donors and red meat allergic patients. We found an increased internalization of α-Gal carrying proteins over time in iMDDCs by flow cytometric analysis, which was independent of the donor allergic status. The uptake of α-Gal carrying proteins was significantly higher than the uptake of non-α-Gal carrying proteins. Confocal microscopy revealed α-Gal carrying proteins scattered around the cytoplasm in most iMDDCs while detection of proteins not carrying α-Gal was negligible. Fluorescent detection of protein on SDS-PAGE showed that degradation of α-Gal carrying proteins was slower than degradation of non-α-Gal carrying proteins. Thus, the presence of α-Gal on the protein surface affects both uptake and degradation of the protein, and the results add new knowledge of α-Gal as a clinically relevant food allergen.

Peptidomics of an in vitro digested α-Gal carrying protein revealed IgE-reactive peptides.

The mammalian carbohydrate galactose-α1,3-galactose (α-Gal) causes a novel form of food allergy, red meat allergy, where patients experience severe allergic reactions several hours after red meat consumption. Here we explored gastric digestion of α-Gal glycoproteins using an in vitro model. Bovine thyroglobulin (BTG), a typical α-Gal carrying glycoprotein, was digested with pepsin. The resulting peptides were characterised by SDS PAGE, immunoblot and ImmunoCAP using sera from 20 red meat allergic patients. During pepsinolysis of BTG, a wide range of peptide bands was observed of which 14 to 17 kDa peptides remained stable throughout the gastric phase. The presence of the α-Gal epitope on the obtained peptides was demonstrated by an anti-α-Gal antibody and IgE from red meat allergic patients. The α-Gal digests were able to inhibit up to 86% of IgE reactivity to BTG. Importantly, basophil activation test demonstrated that the allergenic activity of BTG was retained after digestion in all four tested patients. Mass spectrometry-based peptidomics revealed that these peptides represent mostly internal and C-terminal parts of the protein, where the most potent IgE-binding α-Gal residues were identified at Asn, Asn and Asn. Thus allergenic α-Gal epitopes are stable to pepsinolysis, reinforcing their role as clinically relevant food allergens.

Anaphylactic Reactions to Novel Foods: Case Report of a Child With Severe Crocodile Meat Allergy.

Availability of "exotic" foods is steadily increasing. In this report, we describe the first case of anaphylaxis to crocodile meat. The patient was a 13-year-old boy with severe immunoglobulin E-mediated allergy to chicken meat. When tasting crocodile meat for the first time, he developed an anaphylactic reaction. Cross-reactivity between chicken and crocodile meat was suspected to have triggered this reaction. Basophil activation and immunoglobulin E testing confirmed the boy's allergic reaction to crocodile meat proteins. Molecular analysis identified a crocodile α-parvalbumin, with extensive sequence homology to chicken α-parvalbumin, as the main cross-reactive allergen. We conclude that crocodile meat can be a potent food allergen and patients with allergy to chicken meat should be advised to avoid intake of meat from crocodile species. Both foods and people travel around the world and accessibility to exotic foods is steadily growing. As a result, novel allergic cross-reactivities are likely to become a challenge in the management of food allergy and, as our report illustrates, cross-reactivity has to be considered even between foods that might not intuitively be perceived as related.

IgE reactivity to α-Gal in relation to Lyme borreliosis.

An association between tick bites, the development of immunoglobulin E (IgE) antibodies to galactose-α-1, 3-galactose (α-Gal) and red meat allergy has recently been reported. Here we wanted to elucidate the relation between tick exposure, IgE antibodies to α-Gal and Lyme borreliosis (LB). In the highly LB endemic area of Kalmar County, Sweden, serum samples and health inquiries from 518 blood donors were included. All sera were investigated for multiple IgG anti-Borrelia antibodies using a multiplex assay (recomBead, Mikrogen). In addition, three serially collected sera over a six month period from 148 patients with clinically defined erythema migrans (EM) were included. IgE antibodies against α-Gal were determined using ImmunoCAP (Thermo Fisher Scientific). In blood donors reporting previous LB (n = 124) IgE to α-Gal was found in 16%, while in donors denying previous LB but with multiple anti-Borrelia antibodies (n = 94; interpreted as asymptomatic LB) 10% were IgE α-Gal-positive. Finally, in donors without Borrelia antibodies denying previous LB (n = 300) 14% showed IgE to α-Gal. No significant difference in proportions among the groups were found. In EM patients, IgE to α-Gal was found in 32/148 (22%) at diagnosis, 31/148 (21%) after two-three months and 23/148 (16%) after six months. A significant reduction of proportion and level of IgE to α-Gal was found between the second and third sample (p<0.01). A positive IgE anti α-Gal was more common among men compared with women both in blood donors and in EM patients (p≤0.01). IgE to α-Gal reactivity was common in a tick endemic area but showed no significant relation to previous LB. IgE anti-α-Gal reactivity in EM patients peaked within three months of diagnosis of EM, after which it waned indicating that recent tick exposure is of importance in α-Gal sensitization. Furthermore, IgE anti α-Gal was more common in men compared with women.

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

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. 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. 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. 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.

The red meat allergy syndrome in Sweden.

In the last decade, a novel type of food allergy presenting with severe allergic reactions several hours after consumption of red meat has been recognized. The allergic responses are due to IgE antibodies directed against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) found in mammalian meat. This review presents the red meat allergy syndrome in Sweden, discusses the features of the immune response to carbohydrates, and highlights the presence of heat stable α-Gal-containing proteins in meat. The number of diagnosed red meat allergy cases in Sweden has increased significantly over the past few years. All patients have been tick bitten. Our recent work has shown that α-Gal is present in the European tick (), thus potentially explaining the strong association between anti-α-Gal IgE and tick bites, with development of red meat allergy as a secondary phenomenon. Further studies using immunoproteomics have identified novel α-Gal-containing meat proteins that bound IgE from red meat allergic patients. Four of these proteins were stable to thermal processing pointing to the fact that the allergenicity of red meat proteins is preserved in cooked meat. In keeping with the fact that the α-Gal epitope is structurally related to the blood group B antigen, a positive association with the B-negative blood groups among our red meat allergic patients was noted. A selective IgE reactivity to the pure carbohydrate moiety was observed when investigating the specificity of the α-Gal immune response. IgE from red meat allergic patients does not recognize the other major mammalian carbohydrate, N-glycolylneuraminic acid (Neu5Gc), also present in high amounts in red meat. Furthermore, neither common cross-reactive carbohydrate determinants (CCDs) from plants nor venoms are targets of the IgE response in these patients. Taken together, the α-Gal carbohydrate has shown to be a potentially clinically relevant allergen that should be taken into account in the diagnosis of food allergy. Many new findings in the field of red meat allergy have been obtained during the past years, but further efforts to understand the process of digestion, absorption, and delivery of α-Gal-containing molecules to the circulation are needed.

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.

Allergy to furry animals: New insights, diagnostic approaches, and challenges.

The prevalence of allergy to furry animals has been increasing, and allergy to cats, dogs, or both is considered a major risk factor for the development of asthma and rhinitis. An important step forward in the diagnosis of allergy to furry animals has been made with the introduction of molecular-based allergy diagnostics. A workshop on furry animals was convened to provide an up-to-date assessment of our understanding of (1) the exposure and immune response to the major mammalian allergens, (2) the relationship of these responses (particularly those to specific proteins or components) to symptoms, and (3) the relevance of these specific antibody responses to current or future investigation of patients presenting with allergic diseases. In this review research results discussed at the workshop are presented, including the effect of concomitant exposures from other allergens or microorganisms, the significance of the community prevalence of furry animals, molecular-based allergy diagnostics, and a detailed discussion of cat and dog components.

Galactose-α-1,3-Galactose Allergy Is Not a Hitherto Unrecognized Cause of Chronic Spontaneous Urticaria.

Tick bite-induced galactose-α-1,3-galactose (α-Gal) IgE and subsequent ingestion of red meat may cause delayed severe allergic reactions including urticaria, gastrointestinal symptoms or anaphylaxis. We tested the hypothesis that increased levels of IgE to α-Gal due to tick bites and the subsequent ingestion of red meat or meat products may possibly be an un(der)recognized cause of chronic spontaneous urticaria (CSU). Levels of IgE to α-Gal and total IgE were measured (ImmunoCAP, Phadia AB/Thermo Fisher Scientific) in 83 patients (61 female and 22 male, median age 43 years, range 18-82) from the Department of Dermatology and Allergy, Charité - Universitätsmedizin, Berlin, Germany. All had been clinically diagnosed with moderate-to-severe CSU of a median duration of 2.9 years (range 0.1-50). Eighty of the 83 patients (96%) had undetectable (<0.1 kUA/l) serum levels of IgE against α-Gal. The levels in the remaining 3 were all low (0.25, 0.4 and 3.1 kUA/l). In no patient, including those with measurable serum levels of IgE against α-Gal, was eating red meat associated with the development of symptoms of urticaria. Our results indicate that an allergic response to α-Gal is highly unlikely to be a hitherto unrecognized common cause of CSU.

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

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. 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. 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. 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.

Red meat allergy in Sweden: association with tick sensitization and B-negative blood groups.

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.

Impaired allergy diagnostics among parasite-infected patients caused by IgE antibodies to the carbohydrate epitope galactose-α 1,3-galactose.

The carbohydrate epitope galactose-α 1,3-galactose (α-Gal) is abundantly expressed on nonprimate mammalian proteins. We have recently shown that α-Gal is responsible for the IgE binding to cat IgA, a newly identified cat allergen (Fel d 5). We sought to investigate the diagnostic relevance of IgE antibodies to Fel d 5 and α-Gal among parasite-infected patients from central Africa without cat allergy compared with patients with cat allergy from the same region. Sera from 47 parasite-infected patients and 31 patients with cat allergy were analyzed for total IgE and IgE antibodies against cat dander extract (CDE) by using the ImmunoCAP system. Inhibition assay was performed with α-Gal on solid phase-bound CDE. The presence of IgE specific for the major cat allergen Fel d 1, Fel d 5, and α-Gal was analyzed by means of ELISA. Among the 47 parasite-infected patients, 85% had IgE antibodies against α-Gal (OD; median, 0.175; range, 0.102-1.466) and 66% against Fel d 5 (OD; median, 0.13; range, 0.103-1.285). Twenty-four of the parasite-infected patients were sensitized to CDE, and 21 of them had IgE antibodies to Fel d 5 and α-Gal. There was no correlation between IgE levels to CDE and rFel d 1 among the parasite-infected patients but a strong correlation between CDE and Fel d 5 and α-Gal (P < .001). Among the group with cat allergy, only 5 patients had IgE to α-Gal, and nearly 75% (n = 23) had IgE to rFel d 1 (median, 7.07 kU(A)/L; range, 0.51-148.5 kU(A)/L). In contrast, among the patients with cat allergy, there was a correlation between IgE levels to CDE and rFel d 1 (P < .05) but no correlation between CDE and Fel d 5 and α-Gal. IgE to α-Gal causes impaired allergy diagnostics in parasite-infected patients. Screening for IgE to rFel d 1 and other allergens without carbohydrates might identify patients with true cat sensitization/allergy in parasite-infested areas.

The carbohydrate galactose-alpha-1,3-galactose is a major IgE-binding epitope on cat IgA.

Monoclonal antibodies to the epitope alpha-Gal-(1-4)-beta-Gal-(1- of Moraxella catarrhalis LPS react with a similar epitope in type IV pili of Neisseria meningitidis.

Murine monoclonal antibodies (MAbs) against the A, B and C LPS serotypes of M. catarrhalis were generated and their binding specificity was examined in an enzyme-linked immunosorbent assay (ELISA). Two broadly cross-reactive monoclonal antibodies (MCA1 and MCC2) against the outer core region of LPS were further characterized. A panel of synthetic glycoproteins and glycolipids was used to determine the binding specificity of the MAbs. MCA1 and MCC2 bound specifically to alpha-Gal-(1-4)-beta-Gal of galabiose and globotriose glycoconjugates. The reactivity of the MAbs with galabiose was higher than that with globotriose. The MAbs could recognize the alpha-Gal-(1-4)-beta-Gal epitope only when it was in a terminal position. MCA1 was further shown to react with a similar epitope in the glycosylated type IV pili of N. meningitidis, which has been shown to contain a 1-4 linked digalactose at the terminal part of the saccharide present in the pili. MCA1 could efficiently recognize this epitope indicating that it was exposed on the surface of the pili.

Extracorporeal ("ex vivo") connection of pig kidneys to humans. II. The anti-pig antibody response.

Pig kidneys were extracorporeally "ex vivo" connected to the circulation of two volunteer male dialysis patients (Breimer et al., this issue). The patients were pretreated by daily plasmapheresis for 3 consecutive days, which reduced the anti-pig lymphocytotoxic titer from 8 to 2 in the first patient and from 8 to 1 in the second patient. The anti-pig hemagglutinating titers were reduced from 32 to 4 in the first patient and from 2 to 1 in the second patient. No drugs, except heparin, were given. The perfusion lasted for 65 min in patient 1 and the experiment was terminated due to increased vascular resistance in the pig kidney. Ultrastructural investigation showed a picture similar to a hyperacute vascular rejection. Immunohistochemical studies showed a weak staining of IgM antibodies, but no IgG in the small arteries and glomeruli. The pig kidney of patient 2 was perfused for 15 min and the experiment terminated due to serious side effects of the patient. Light and electron microscopical investigation showed virtually no structural changes of the kidney tissue and immunostaining for human antibodies was negative. In both patients, serum samples collected 2-5 weeks postperfusion showed a strong anti-pig antibody titer rise (up to 512) which thereafter declined but stabilized on a higher level than before the experiment. The antibody response in the two patients was different. In patient 1, the major anti-pig antibodies directed to carbohydrate antigens were of IgG (IgG1 and IgG2 subclasses) type, while the IgM response was less prominent and virtually no IgA antibodies were produced. Despite the short duration of the perfusion in patient 2, a humoral immune response was seen that was mainly confined to the IgA immunoglobulin class (IgA1 subclass). Blood group glycospingolipid fractions, prepared from the contralateral kidney of the donor pigs, were used for immunostaining with patient serum samples. In both patients, the antibodies produced after the perfusion, mainly recognized the Galα1-3Gal epitope both as part of the "linear B" pentasaccharide but also on more complex carbohydrate structures. Patient 1 was HLA-immunized before the experiment due to a kidney allograft and had a panel reactivity of 85% before the perfusion. No change in the panel reactivity of HLA-antibodies was found after the perfusion experiments. Patient 2 had no HLA antibodies before and remained negative after the perfusion. Patient serum samples collected before and after the perfusion were tested for reactivity against human endothelial cell lines. No antibodies were generated.

Studies of the binding activity of phage G13 to synthetic trisaccharides analogous to binding structures in Salmonella typhimurium and Escherichia coli C core saccharide. Correlation between conformation and binding activity.

Phage G13 binds to the carbohydrate part of lipopolysaccharides from rough mutants of Salmonella and Escherichia coli as the first event of infection. Equilibrium dialysis inhibition studies with native and synthetic trisaccharides as inhibitors suggested that phage G13 recognizes branched oligosaccharides having 6-O-alpha- or 7-O-alpha-glycosyl groups with alpha-Man(1----3) [alpha-Man(1----6)]Man (Man[Man]Man) and alpha-Glc(1----3)-[alpha-Hep(1----7)] alpha-Hep(1----3) alpha-Hep(1----5)Kdo as the smallest saccharides with inhibitory activity (Wollin et al., 1989). Of four synthetic analogues to Man[Man]Man only Man(1----3)[alpha-Gal(1----6)]alpha-Man-OMe (Man[Gal]-Man) and alpha-Glc(1----3)[alpha-Hep(1----7)]alpha-Hep-OMe (Glc[Hep]Hep) inhibited the binding of labelled E. coli C core nonasaccharide ligand to G13 with activities which were 10- and 15-fold lower than Man[Man]Man. The trisaccharides alpha-Man(1----3)[alpha-Glc(1----6)[alpha-Man-OMe (Man[Glc]Mann) and alpha-Man(1---3)[alpha-Tal(1----6)]alpha-Man-OMe (Man[Tal]Man) showed no inhibition at concentrations 75-fold higher than Man[Man]Man. Minimum energy conformation calculations of the saccharides using the GESA method showed that the 6-O-alpha-Man group in Man[Man]Man and the 7-O-alpha-Hep group SL805 pentasaccharide expose their OH-2 and OH-3 groups in a similar way and these are postulated to be key structural features for binding activity. The importance of hydroxy groups at certain positions is implied from the fact that both manno- and galacto-isomers are active. We also conclude that the O6-C6-C5-O5-C1 region of the 3-O-alpha-glycosyl group in the Man[Man]Man trisaccharide, or part of it, is important for the G13 binding activity.

Pathogenesis of urinary tract infections--amoxicillin induces genital Escherichia coli colonization.

The aim of this study was to examine whether a vaginal Escherichia coli colonization, mimicking the one seen in UTI-prone females, could be induced by amoxicillin administration. In adult cynomolgus monkeys, who carry the alpha-Gal-1-4-beta-Gal receptor for P-fimbriae, we have investigated the ability of a clinical isolate of P-fimbriated E. coli to colonize the mucous membranes of the genital tract. When the vagina was washed with a suspension of the P-fimbriated strain, a persistent colonization was obtained in only 17% (4/24) of the experiments. When such colonization attempts were performed during vaginal amoxicillin administration, a persistent and heavy colonization of the vagina occurred in 5/5 attempts. The findings do not contradict our hypothesis that antibiotics given to UTI-prone females may facilitate the establishment of the abnormal colonization of the genital tract.