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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.
Calvatia gigantea is a traditional Chinese medicinal fungus known for its diverse pharmacological effects, with polysaccharides being one of its main bioactive components. In this study, a novel homogeneous water-soluble polysaccharide (CGPA1) was successfully isolated and purified from the fruiting body of Calvatia gigantea. The molecular weight of CGPA1 was determined to be 11.69 kDa. Structural characterization was performed using a combination of high-performance gel permeation chromatography (HPGPC), Fourier-transform infrared spectroscopy (FTIR), methylation analysis, and nuclear magnetic resonance (NMR) spectroscopy. The analysis data revealed that CGPA1 is primarily composed of galactose, methyl-galactose, mannose, xylose, and glucose in a molar ratio of 60.01:27.16:10.34:1.83:0.67. The backbone structure predominantly consists of α-1,6-Gal-(1 → and α-1,6 (3-MeO)-Gal-(1→, with side chains of α-Gal and α-Man. Morphological analysis using SEM, XRD, and AFM, combined with Congo red experiments, suggesting that CGPA1 adopts an amorphous granular chain conformation. Both in vitro and in vivo tests confirmed that CGPA1 can promote the migration of MSF cells and accelerate the healing of acute injured wounds. It was found to facilitate skin re-epithelialization, and collagen deposition over the healing process.
Kepley CL, Wang Y, Yelton A, Siebert ER, Iweala OI
Current allergy and asthma reports •
Alpha-gal syndrome (AGS) is a novel allergic disease characterized by hypersensitivity responses following exposure to the glycan galactose-alpha-1,3-galactose (alpha-gal or α-Gal) attached to mammalian proteins and fats in food, supplements, and medications. Bites from hard-bodied ticks, including Amblyomma americanum (Aa or lone star tick) in the United States, have been identified as drivers of allergic sensitization to alpha-gal. Here, we review clinical presentation, epidemiology, diagnosis, and current understanding of the mechanistic drivers of AGS, with particular focus on the roles of allergic effector cells - mast cells (MCs) and basophils. We explore unique clinical characteristics of AGS through the lens of alpha-gal-specific IgE and MC activation in AGS. We propose potential explanations for delayed symptom onset, inconsistent symptom development, and persistence of allergic symptoms in some AGS patients despite removing all mammal products from the diet. Current evidence implicate bites from hard-bodied ticks as the primary sensitizing agent in AGS. However, there is emerging evidence that other ecto- and endoparasites may also induce alpha-gal-specific IgE. Multiparameter flow and mass cytometry and RNA sequencing have demonstrated an enrichment of unique populations of T, B, invariant natural killer T (iNKT), natural killer B (NKB) and MC progenitor cells in human volunteers with AGS. Recently developed mouse models of AGS will support future studies to identify which cells are critical for the development of AGS. In vitro models of the allergic effector phase of AGS using human sera and novel human alpha-gal-specific IgE monoclonal antibodies in humanized rat allergic effector cell lines, human basophils, human MC lines, and primary human MC cultures, confirm alpha-gal-induced allergic effector cell activation. They also provide a system to study potential alpha-gal-antigen-independent drivers of MC activation in AGS. Efforts are ongoing to understand the epidemiology and immune mechanisms of AGS. Novel reagents (e.g. alpha-gal-specific monoclonal antibodies) and murine AGS models will facilitate deeper investigation of tick-driven, alpha-gal-specific IgE and allergic effector cell-induced pathology in AGS.
International journal of biological macromolecules •
This study aimed to investigate the structural characterization, conformational properties, and hepatoprotectiveactivity of acidic polysaccharide (DNLPS-3) from Dendrobium nobile flower. DNLPS-3 with molecular weights of 15.65 KDa was composed of Fuc: Rha: Ara: Gal: Glc: Xyl: GalA: GlcA in a molar ratio of 0.26%: 2.38%: 4.47%: 3.55%: 0.43%: 17.84%: 69.80%: 1.26%. Structural analysis with methylation indicated that DNLPS-3 mainly contained T-Ara(f), T-Xyl(p), T-Gal(p)A, 3-Gal(p)A, 4-Gal(p)A, 3,4-Gal(p)A, 3,4-Glc(p), 2,4-Gal(p)A, 2,3,4-Gal(p), 4,6-Gal(p), 3,4,6-Gal(p). NMR results show that DNLPS-3 may be a pectin polysaccharide with →4)-α-Gal(p)A-(→main chain. In vitro experiments showed that DNLPS-3 exhibited significant hepatoprotective effects. The effect was achieved by reducing the activities of ALT and AST, suppressing the production of LDH and MDA, and enhancing the anti-oxidant activities of SOD and GSH, and the specific mechanism of its hepatoprotective effect is that DNLPS-3 up-regulates Nrf2/HO-1 pathway protein expression.
Wang Y, Hils M, Fischer A, Wölbing F, Biedermann T, et al.
Frontiers in immunology •
The prevalence of food allergy is rising and is estimated to approach 10%. Red meat allergy is the first known food allergy elicited by immunoglobulin E (IgE) antibodies recognizing a carbohydrate. Due to the loss of function of the alpha-1,3-galactosyltransferase ( gene in humans, the disaccharide galactose-α-1,3-galactose (α-Gal) cannot be synthesized and therefore became immunogenic. IgE sensitization is elicited through the skin by repetitive tick bites transmitting α-Gal. The underlying mechanisms regarding innate and adaptive immune cell activation, including the B-cell isotype switch to IgE, are poorly understood, requiring further research and physiologically relevant animal models. Here, we describe a new animal model of red meat allergy using percutaneous α-Gal sensitization of gene-edited -deficient pigs. Total and α-Gal-specific IgG, IgG1, IgG2, IgG4, and IgE levels were tracked. Further key factors associated with allergic skin inflammation, type 2 immunity, and allergy development were measured in PBMCs and skin samples. Significant increases in α-Gal-specific IgG1 and IgE levels indicated successful sensitization to the allergen α-Gal. Intracutaneous sensitizations with α-Gal recruited lymphocytes to the skin, including elevated numbers of T helper 2 (Th2) cells. Finally, α-Gal-sensitized pigs not only recognized α-Gal as non-self-antigen following α-Gal exposure through the skin but also developed anaphylaxis upon antigen challenge. Based on the similarities between the porcine and human skin, this new large animal model for α-Gal allergy should help to unveil the consecutive steps of cutaneous sensitization and aid the development of prophylactic and treatment interventions.
Glycosylation modification serves as a pivotal quality attribute in glycoprotein-based therapeutics, emphasizing its role in drug safety and efficacy. Prior studies have underscored the potential immunogenic risks posed by the presence of galactose-α-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (NeuGc) in glycoprotein formulations. This accentuates the imperative for developing robust qualitative and quantitative analytical methods to monitor these immunogenic epitopes, thereby ensuring drug safety. In the present investigation, α-Gal and NeuGc were accurately quantified using UPLC-FLR-MS/MS at the oligosaccharide level. Remarkably, α-Gal could be identified when the ion intensity ratio or the mass-to-charge ratio (m/z) of 528.19 to 366.14 exceeded 1. Concurrently, NeuGc and N-acetylneuraminic acid (NeuAc) could be unambiguously identified through their respective fragment ions at m/z 673.23 and m/z 657.23. Furthermore, relative quantification of α-Gal and NeuGc was achieved using fluorescence signals. This study introduces a sensitive and reliable analytical approach for the qualitative and quantitative assessment of α-Gal and NeuGc in glycoprotein pharmaceuticals. The methodology offers significant potential for application in process control and optimization of glycoprotein production, aimed at minimizing immunogenicity and enhancing product quality.