Food Allergy and Anaphylaxis Research (28)HIV Research and Treatment (18)Allergic Rhinitis and Sensitization (18)HIV/AIDS drug development and treatment (11)Contact Dermatitis and Allergies (10)
Alpha-gal Syndrome (AGS) is a potentially life-threatening allergy caused by an IgE-mediated immune response to galactose-α-1,3-galactose (alpha-gal), a carbohydrate epitope present in most mammalian meats. Currently, strict avoidance of mammalian meat remains the primary management strategy for affected individuals, and alpha-gal-free beef is not commercially available. Here, we leverage cultivated meat as a biotechnology platform to address this unmet clinical need by engineering alpha-gal-free bovine muscle cells. Using CRISPR/Cas9 genome editing, we disrupted GGTA1, the gene encoding α1,3-galactosyltransferase, in immortalized bovine satellite cells (iBSCs). High-efficiency editing produced clonal GGTA1 knockout iBSCs harboring a homozygous frameshift mutation. Flow cytometry and immunofluorescence confirmed loss of the alpha-gal epitope, while bulk RNA-seq indicated minimal disruption of global gene expression and preserved myogenic differentiation capacity. Importantly, lysates from GGTA1 knockout iBSCs elicited substantially reduced basophil activation in assays using plasma from a patient with AGS, indicating reduced basophil activation consistent with reduced allergenic potential. Together, these findings establish a proof of concept for engineering AGS-compatible cultivated meat and demonstrate the potential of cultivated meat technologies to address human health challenges.
Wang Y, Zhang Y, Dudley C, Fitzgerald K, Carson AS, et al.
The Journal of allergy and clinical immunology •
In alpha-gal syndrome (AGS), IgE antibodies form against the glycan galactose-α-1,3-galactose (alpha-gal) in mammal products rather than food proteins. Alpha-gal glycolipids and glycoproteins activate human basophils sensitized with AGS plasma in an IgE-dependent fashion. However, it is unknown whether alpha-gal specific IgE (sIgE), independent of other blood proteins, is sufficient for mediating basophil and mast cell activation. We sought to determine whether alpha-gal antigens could activate passively sensitized rat basophilic leukemia (RBL) SX-38 cells, which express human IgE receptors and are commonly used to model allergen/IgE-mediated mast cell activation in food protein allergy. Using the clustered regularly interspaced short palindromic repeats technology, we created a novel, alpha-gal-deficient RBL cell line, alpha-gal knockout RBL SX-38, passively sensitizing cells with sera from AGS donors or with novel alpha-gal sIgE clones, and then stimulated with alpha-gal glycoproteins. To assess effector cell activation, we measured cell surface expression of activation marker CD63 by flow cytometry and mediator release through β-hexosaminidase release assays. After alpha-gal antigen stimulation, the percentage of CD63 alpha-gal knockout RBL SX-38 cells sensitized with AGS sera increased 3-fold compared with cells sensitized with control serum. Select human AGS IgE clones facilitated alpha-gal antigen-dependent and antigen-independent CD63 upregulation. Cells sensitized with pooled AGS sera released β-hexosaminidase in an alpha-gal-independent fashion. We saw no β-hexosaminidase release above background in cells sensitized with alpha-gal sIgE clones. Certain alpha-gal-specific human IgE clones may partially activate allergic effector cells independent of antigen, potentially lowering thresholds for subsequent alpha-gal-induced or antigen-independent allergic effector cell degranulation. This may affect duration and severity of allergic symptoms in patients with AGS.
Jones VM, Nakahara H, Choudhary SK, Dunbar NM, Szczepiorkowski ZM, et al.
Transfusion •
The bites of certain tick species can induce galactose-α-1,3-galactose (alpha-gal) IgE formation. Individuals with alpha-gal IgE can become allergic to meat, a condition termed alpha-gal syndrome. Alpha-gal is structurally related to B antigen. Recent case reports (Gilstad et al., 2023 and Miller et al., 2024) suggest that transfusing group B plasma or platelets to group O individuals with alpha-gal IgE can trigger severe allergic transfusion reactions. However, mechanistic evidence is lacking. A 76-year-old group O man undergoing heart surgery became profoundly hypotensive after receiving 50 mL of a group B platelet unit. He recovered with diphenhydramine treatment. We investigated potential causes of anaphylaxis (alpha-gal syndrome; IgA deficiency; protamine, and rocuronium allergies). The platelet unit and patient samples were analyzed by flow cytometry. Indirect basophil activation tests (iBATs) were performed on the patient's plasma. The patient's serum tryptase level spiked, consistent with anaphylaxis. His serum tested positive for alpha-gal IgE (1.49 kU/L). Flow cytometric analyses demonstrated that: (1) the transfused platelets expressed substantial B antigen, and (2) the patient's plasma contained IgE-recognizing alpha-gal and B antigen > A antigen. In iBATs, resting allogeneic basophils incubated with the patient's plasma showed equivalent (albeit weak) activation when stimulated with either alpha-gal or B antigen. We report a case of anaphylactic shock in an O patient transfused with a B platelet unit. The in vitro data suggest, but do not prove, that the reaction was mediated by recipient alpha-gal IgE. Further studies are needed to establish whether transfusion-related alpha-gal syndrome ("TRAGS") is a true clinical entity.
Alpha-gal syndrome (AGS) is an IgE-mediated allergy to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Alpha-gal is found in the tissues of non-catarrhine mammals, and the characteristic delayed reactions are caused by the consumption of red meat, visceral organs, dairy, gelatin, and other products, including medications sourced from non-primate mammals. Although the syndrome is not nationally notifiable, it is estimated that 450,000 cases exist, making AGS the tenth-most common food allergy in the US. The syndrome is profoundly influenced by geographic locale, reflecting the important role of tick bites in sensitization. However, the specific immune cells, their interactions, and the downstream signaling cascades triggered by tick bites are not well understood. To address this gap, we conducted a comprehensive study to analyze the immune cells in the blood of AGS subjects compared to those of healthy controls. Peripheral blood mononuclear cell preparations were enriched for B cells from the same patient and sequentially labeled with sample tags and BD AbSeq oligo-conjugated antibodies. Sequencing libraries were prepared to targeted mRNA, antibody-oligonucleotides, and sample tags from AGS and control subjects. Multimodal analysis of both transcriptomes and surface marker expression of immune cells at the single-cell level was used to profile the immune response in AGS. Several clusters of cells were unique to AGS subjects, including natural killer B (NKB) cells, natural killer T (NKT) cells and most notably, a circulating mast cell progenitor population. In addition, subjects with AGS had increased expression of several genes involved in immunomodulation and type 2 immunity. Although rare, we identified and characterized alpha-gal-specific memory B cells and alpha-gal-specific IgE-secreting cells. Our findings also revealed the presence of IgE-secreting transcripts, along with other classes of immunoglobulins (Ig), in a single cell, suggesting a unique pattern of Ig gene arrangements and class switching in B cells. Tick bites appear to induce a population of circulating mast cell progenitors and innate-like cells, such as NKT and NKB cells, that may play a critical role in sensitization to alpha-gal. Furthermore, alpha-gal-specific IgE is secreted by a heterogeneous population of B cells, including CCR6-proficient B cells and CCR6-deficient plasmablast/plasma cells.
Sharma SR, Hussain S, Choudhary SK, Commins SP, Karim S
Ticks and tick-borne diseases •
Alpha-Gal Syndrome (AGS) is a delayed allergic reaction triggered by IgE antibodies targeting galactose-α-1,3-galactose (α-gal), prevalent in red meat. Its global significance has increased, with over 450,000 estimated cases in the United States alone. AGS is linked to tick bites, causing sensitization and elevated α-gal specific IgE levels. However, the precise mechanisms and tick intrinsic factors contributing to AGS development post-tick bites remain unclear. This study aims to characterize the alpha-gal conjugated lipid antigens in Amblyomma americanum (Am. americanum) salivary glands and saliva. Nanospray ionization mass spectrometry (NSI-MS) analysis revealed the identification of α-gal bound lipid antigens in Am. americanum saliva. Additionally, the activation of basophils by extracted alpha-gal bound lipids and proteins provides evidence of their antigenic capabilities.
Taylor ML, Kersh GJ, Salzer JS, Jones ES, Binder AM, et al.
Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology •
Alpha-gal syndrome (AGS) is an allergy to galactose-α-1,3-galactose (alpha-gal), a carbohydrate found in most mammals. Evidence indicates that AGS develops after a tick bite, and in the United States, AGS is most associated with bites from Amblyomma americanum (lone star tick); however, not all persons bitten by ticks develop clinical AGS. To investigate intrinsic risk factors associated with the development of AGS. We performed a case-control study among adults presenting for diagnosis or management of AGS at an allergy clinic in North Carolina during 2019 to 2020 and compared them with controls enrolled from 2 nearby internal medicine clinics. A questionnaire gathered epidemiologic and tick exposure data, and blood was obtained for alpha-gal-specific IgE and other testing. The 82 enrolled case patients and 191 controls did not differ significantly by age or sex. Case patients were more likely than controls to have A or O blood types (non B-antigen), have experienced childhood allergies, and have a family history of AGS and other food allergies. Case patients were also more likely to report experiencing long healing times for insect bites or stings and a family history of allergy to stinging or biting insects. This study suggested that intrinsic factors contribute to risk of developing AGS. Some traits are genetic, but common behaviors among households and family units likely also contribute. Identification of these risk factors can inform personal risk, aid health care providers in understanding susceptible populations, and contribute to ongoing understanding of AGS epidemiology.
Tick-borne diseases (TBDs) other than Lyme disease, such as spotted fever group rickettsiosis, ehrlichiosis, and galactose-α-1,3-galactose (α-gal) syndrome, are an emerging public health issue. Long-term sequelae secondary to Ehrlichia or Rickettsia infection are uncommon; however, musculoskeletal symptoms are often attributed to prior tick exposure. To evaluate the potential associations between prior exposure to TBDs and musculoskeletal symptoms, including radiographic osteoarthritis. This cross-sectional study analyzed serum samples from the fourth visit (2017-2018) of the Johnston County Osteoarthritis (JoCo OA) project, an ongoing longitudinal, population-based study in Johnston County, North Carolina. Biospecimen testing and analysis were performed between May 2022 and November 2023. Participants in the JoCo OA project are noninstitutionalized White and Black Johnston County residents 45 years or older. The primary outcome was seropositivity with Ehrlichia IgG, Rickettsia IgG, and/or α-gal IgE and musculoskeletal symptoms. Secondary outcomes included risk factors associated with elevated α-gal IgE and weighted population point prevalence rates. Participants completed questionnaires, underwent physical assessments, and provided biospecimens for serological testing. Multivariable models were used to estimate associations of interest. Of the 605 participants who completed the fourth visit of the JoCo OA project, 488 (80.7%) had serum samples available for testing. The 488 participants had a median (IQR) age of 72 (68-78) years and included 336 females (68.9%) and 161 Black (33.0%) and 327 White (67.0%) individuals. The overall weighted point prevalence was 8.6% (95% CI, 5.9%-11.3%) for Ehrlichia IgG, 17.1% (95% CI, 12.6%-21.5%) for Rickettsia IgG, and 19.6% (95% CI, 15.3%-23.8%) for α-gal IgE level greater than 0.1 IU/mL. Only α-gal IgE was associated with knee pain, aching or stiffness (mean ratio, 1.30; 95% CI, 1.09-1.56). Antibodies to Rickettsia, Ehrlichia, and α-gal were not associated with symptomatic radiographic knee osteoarthritis. Male sex (odds ratio [OR], 2.63; 95% CI, 1.55-4.47), current smoker status (OR, 3.55; 95% CI, 1.38-9.18), and an attached tick bite in the past 5 years (OR, 3.99; 95% CI, 2.22-7.15) were all risk factors that were associated with α-gal IgE level greater than 0.1 IU/mL. Despite only 84 individuals (17.2%) recalling a tick bite in the past 5 years, 178 (36.5%) had evidence of prior tick-borne exposure, suggesting frequent human-tick interactions. Results of this cross-sectional study indicate no association between Ehrlichia or Rickettsia seropositivity and chronic musculoskeletal symptoms or osteoarthritis. Further investigation is needed into the pathogenesis of α-gal syndrome and interventions to reduce human-tick interactions.
Li Z, Ma A, Miller I, Starnes R, Talkington A, et al.
Journal of controlled release : official journal of the Controlled Release Society •
Polyethylene glycol (PEG) is frequently used in various protein and nanomedicine therapeutics. However, various studies have shown that select PEGylated therapeutics can induce production of anti-PEG antibodies (APA), potentially culminating in rapid clearance from the systemic circulation, loss of efficacy and possibly increased risks of allergic reactions. Although IgE is a frequent cause of immediate hypersensitivity reactions (IHR), the role of IgE APA in PEG-related IHR is not well understood, due in part to a lack of standardized assays for measuring IgE APA. Here, we developed a rigorous competitive ELISA method to measure the concentrations of various APA isotypes, including IgE, with picomolar sensitivities. In a small number of serum samples from patients with known PEG allergy, the assay allowed us to detect a strong correlation between IgG and IgE APA in individuals with history of allergic reactions to PEG or PEGylated drugs, but not between IgM and IgE APA. We detected appreciable levels of IgG and IgM APA in individuals with history of alpha-gal allergy, however, they were not elevated relative to those detected in other healthy controls, and we found no pre-existing IgE APA. While preliminary and should be further investigated, these results suggest that differences in the route and mechanism of PEG exposure may drive variability in APA response.
Sharma SR, Choudhary SK, Vorobiov J, Commins SP, Karim S
bioRxiv : the preprint server for biology •
Alpha-Gal Syndrome (AGS) is a delayed allergic reaction due to specific IgE antibodies targeting galactose-α-1,3-galactose (α-gal), a carbohydrate found in red meat. This condition has gained significant attention globally due to its increasing prevalence, with more than 450,000 cases estimated in the United States alone. Previous research has established a connection between AGS and tick bites, which sensitize individuals to α-gal antigens and elevate the levels of α-gal specific IgE. However, the precise mechanism by which tick bites influence the hosťs immune system and contribute to the development of AGS remains poorly understood. This study investigates various factors related to ticks and the host associated with the development of AGS following a tick bite, using mice with a targeted disruption of alpha-1,3-galactosyltransferase (AGKO) as a model organism. Lone-star tick () and gulf-coast tick () nymphs were used to sensitize AGKO mice, followed by pork meat challenge. Tick bite site biopsies from sensitized and non-sensitized mice were subjected to mRNA gene expression analysis to assess the host immune response. Antibody responses in sensitized mice were also determined. Our results showed a significant increase in the titer of total IgE, IgG1, and α-gal IgG1 antibodies in the lone-star tick-sensitized AGKO mice compared to the gulf-coast tick-sensitized mice. Pork challenge in -sensitized mice led to a decline in body temperature after the meat challenge. Gene expression analysis revealed that bites direct mouse immunity toward Th2 and facilitate host sensitization to the α-gal antigen, while did not. This study supports the hypothesis that specific tick species may increase the risk of developing α-gal-specific IgE and hypersensitivity reactions or AGS, thereby providing opportunities for future research on the mechanistic role of tick and host-related factors in AGS development.
Kersh GJ, Salzer J, Jones ES, Binder AM, Armstrong PA, et al.
Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology •
The disaccharide galactose-α-1,3-galactose (alpha-gal) is expressed in mammals other than humans, apes, and old-world monkeys. In humans, elevated immunoglobulin E (IgE) antibodies specific for alpha-gal can result in allergic hypersensitivity known as alpha-gal syndrome (AGS). Case reports and series suggest that tick bites can induce alpha-gal-specific IgE (sIgE) antibodies. To evaluate tick exposure as a risk factor for AGS and elevated alpha-gal sIgE level. We conducted a case-control study comparing patients with AGS from a North Carolina allergy clinic with controls who were patients at a nearby internal medicine clinic. Cases and controls were administered a questionnaire to obtain information about demographics, home environment, outdoor activities, and recollection of tick bite. Serum samples taken at the time of enrollment were tested for total IgE, alpha-gal sIgE, and antibodies to other tick-borne pathogens. The patients with AGS were more likely to recall finding a tick on themselves (odds ratio [OR], 11.20; 95% confidence interval [CI], 4.97-25.15), live near wooded forest (OR, 2.27; 95% CI, 0.92-5.55), and spend 17 or more hours per week outdoors in wooded areas (OR, 5.58; 95% CI, 2.56-12.19). The patients with AGS were also more likely to report 4 or more tick bites (OR, 33.05; 95% CI, 9.92-155.12) and reactions at the site of tick bites (OR, 7.93; 95% CI, 3.74-16.80). Furthermore, elevated alpha-gal sIgE level was observed in 33% of the controls and was associated with tick exposure in the controls (OR, 4.25; 95% CI, 2.21-8.18). The results define tick bite as a risk factor for AGS and elevated alpha-gal sIgE level.
Sharma SR, Choudhary SK, Vorobiov J, Commins SP, Karim S
Frontiers in immunology •
Alpha-Gal Syndrome (AGS) is a delayed allergic reaction due to specific IgE antibodies targeting galactose-α-1,3-galactose (α-gal), a carbohydrate found in red meat. This condition has gained significant attention globally due to its increasing prevalence, with more than 450,000 cases estimated just in the United States alone. Previous research has established a connection between AGS and tick bites, which sensitize individuals to α-gal antigens and elevate the levels of specific IgE. However, the precise mechanism by which tick bites influence the host's immune system and contribute to the development of AGS remains poorly understood. This study investigates various factors related to ticks and the host associated with the development of AGS following a tick bite, using mice with a targeted disruption of alpha-1,3-galactosyltransferase (AGKO) as a model organism. Lone-star tick () and gulf-coast tick () nymphs were used to sensitize AGKO mice, followed by pork meat challenge. Tick bite site biopsies from sensitized and non-sensitized mice were subjected to mRNA gene expression analysis to assess the host immune response. Antibody responses in sensitized mice were also determined. Our results showed a significant increase in the total IgE, IgG1, and α-gal IgG1 antibodies titers in the lone-star tick-sensitized AGKO mice compared to the gulf-coast tick-sensitized mice. Pork challenge in -sensitized mice led to a decline in body temperature after the meat challenge. Gene expression analysis revealed that bites direct mouse immunity toward Th2 and facilitate host sensitization to the α-gal antigen. This study supports the hypothesis that specific tick species may increase the risk of developing α-gal-specific IgE and hypersensitivity reactions or AGS, thereby providing opportunities for future research on the mechanistic role of tick and host-related factors in AGS development.
The Journal of thoracic and cardiovascular surgery •
Galactose-α-1,3-galactose (alpha-gal) is a carbohydrate that is ubiquitously expressed in all mammals except for primates and humans. Patients can become sensitized to this antigen and develop alpha-gal syndrome (AGS), or a red meat allergy. Symptoms range from generalized gastroenteritis and malaise to anaphylaxis, and in endemic areas, the prevalence can be as high as 20%. Although AGS patients commonly avoid alpha-gal by avoiding meat, patients have also developed symptoms due to animal-derived medical products and devices. With the rise in transcatheter aortic valve replacement, we investigate the immunogenicity of common cardiac materials and valves. To assess the in vitro immunoglobulin E response toward common medical products, including cardiac patch materials and bioprosthetic valves in patients with AGS. Immunoblot and immunohistochemistry techniques were applied to assess immunoglobulin E reactivity to various mammalian derived tissues and medical products for patients with AGS. AGS serum showed strong reactivity to all of the commercially available, nonhuman products tested, including various decellularized cardiac patch materials and bioprosthetic aortic valves. AGS serum did not react to tissues prepared using alpha-gal knockout pigs. Despite commercial decellularization processes, alpha-gal continues to be present in animal-derived medical products, including bioprosthetic valves. Serum from patients with AGS demonstrates a strong affinity for these products in vitro. This may have serious potential implications for sensitized patients undergoing cardiac surgery, including early valve failure and accelerated coronary artery disease.
Alpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS. Eight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs. Compared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature. TSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.