Casein, the major allergen in cow\'s milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.

BACKGROUND: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found.
METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen\'s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods.
RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients\' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients\' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree.
CONCLUSIONS: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.

UNASSIGNED: Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa. However, few studies focus on the distributional characteristics of allergens in AR patients in Southern Fujian Province, China.
UNASSIGNED: A skin prick test (SPT) was performed and eight common allergens including Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dpt), weeds, animal dander, molds, cockroaches, and mangoes were chosen.
UNASSIGNED: The positive reactions rate to the allergens was 65.79% in 6689 patients in Southern Fujian Province. Positive reactions to Df and Dpt had a negative association with age, whereas positive reactions to cockroach and weed had a positive association with age. A linear trend analysis demonstrated a significant positive relationship between positive reactions to various allergens from 2016 to 2019. Positive reactions to Df and Dpt were both correlated with the season. Positive reactions to Df, Dpt, cockroach and weed were related to disease duration and positive reactions to cockroach were correlated with city residence. Multivariate analysis revealed that male positive reactions gradually decreased with age (≤ 60), in contrast to female (≤ 60) positive reactions. Statistical difference was observed between the genders with regard to AR incidence from 2016 to 2019. The positive rate of skin tests was highest in summer in men, whereas in women it was lowest in summer. The gender composition ratios of positive cases in Xiamen, Zhangzhou, and Quanzhou cities differed significantly. The proportion of patients with positive reactions to the allergens in the three cities decreased with age. The highest proportions of patients with positive reactions all occurred during summer in the three cities. Furthermore, there were statistically significant differences in the age composition ratios across the seasons.
UNASSIGNED: This study analyzed the distributional characteristics of AR allergens in Southern Fujian Province, China. These findings will inform specific immunotherapy for AR patients.

Crustacean shellfish are major allergens in East Asia. In the present study, a major allergic protein in crustaceans, tropomyosin, was detected accurately using multiple reaction monitoring mode-based mass spectrometry, with shared signature peptides identified through proteomic analysis. The peptides were deliberately screened through thermal stability and enzymatic digestion efficiency to improve the suitability and accuracy of the developed method. Finally, the proposed method demonstrated a linear range of 0.15 to 30 mgTM/kgfood (R2 > 0.99), with a limit of detection of 0.15 mgTM/kg food and a limit of quantification of 0.5mgTM/kgfood and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.

Hemocyanin, an oxygen-transport protein, is widely distributed in the hemolymph of marine arthropods and mollusks, playing an important role in their physiological processes. Recently, hemocyanin has been recognized as a multifunctional glycoprotein involved in the immunological responses of aquatic invertebrates. Consequently, the link between hemocyanin functions and their potential applications has garnered increased attention. This review offers an integrated overview of hemocyanin\'s structure, physicochemical characteristics, and bioactivities to further promote the utilization of hemocyanin derived from marine products. Specifically, we review its implication in two aspects of food and aquaculture industries: quality and health. Hemocyanin\'s inducible phenoloxidase activity is thought to be an inducer of melanosis in crustaceans. New anti-melanosis agents targeted to hemocyanin need to be explored. The red-color change observed in shrimp shells is related to hemocyanin, affecting consumer preferences. Hemocyanin\'s adaptive modification in response to the aquatic environment is available as a biomarker. Additionally, hemocyanin is endowed with bioactivities encompassing anti-microbial, antiviral, and therapeutic activities. Hemocyanin is also a novel allergen and its allergenic features remain incompletely characterized.

BACKGROUND: The dosage of ovalbumin (OVA) during the sensitization stage is considered a crucial factor in the development of airway hyperresponsiveness (AHR). However, the inconsistent dosages of sensitizing OVA used in current studies and the lack of research on their impact on AHR are notable limitations.
METHODS: We examined the impact of increasing sensitizing doses of OVA in a murine asthma model, which entailed initial sensitization with OVA followed by repeated exposure to OVA aerosols. BALB/c mice were primed with doses of OVA (0, 10, 20, 50, and 100 μg) plus 1 mg Alum on Days 0 and 7, and were challenged with OVA aerosols (10 mg/mL for 30 min) between Days 14 and 17. Antigen-induced AHR to methacholine (MCh), as well as histological changes, eosinophilic infiltration, and epithelial injury were assessed.
RESULTS: The result indicated that there are striking OVA dose-related differences in antigen-induced AHR to MCh. The most intense antigen-induced AHR to MCh was observed with sensitization at 50 μg, while weaker responses were seen at 10, 20, and 100 μg. Meanwhile, there was a significant increase in eosinophil count with sensitization at 50 μg. The changes of AHR were correlated with total cells count, lymphocytes count, eosinophils count, and basophils count in bronchoalveolar lavage fluid; however, it did not correlate with histological changes such as cellular infiltration into bronchovascular bundles and goblet cell hyperplasia of the bronchial epithelium.
CONCLUSIONS: Overall, this study demonstrated that sensitization with 50 μg of OVA resulted in the most significant AHR compared to other dosages. These findings may offer valuable insights for future research on mouse asthma modeling protocols.

BACKGROUND: Cat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy.
METHODS: We first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA).
RESULTS: Among several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1.
CONCLUSIONS: We firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy.

Exposure to indoor allergens is a principal risk factor for allergic diseases. However, most of the previous studies on indoor allergens focused on very limited kinds of allergens in China. Knowledge of the simultaneous exposure to multiple allergens is still lacking. In this study, the residual profiles of 8 allergens were investigated in 166 dust samples from 11 cities in China. The house dust mite allergens including Der p 1, Der f 1, and Der 2 were detected in the range of <0.02-283.83 μg/g dust. The concentrations of dog allergen Can f 1 and cat allergen Fel d 1 varied widely, from <0.84-22,896.46 μg/g dust for Can f 1 and from <0.02-6298.96 μg/g dust for Fel d 1. Cockroach allergen Bla g 2 was detected in 68% of the samples but at a low level with a maximum of 9.44 μg/g dust. Comparatively low detection frequencies were found for mouse allergen Mus m 1 as 37% and for fungi allergen Asp f 2 as 24%. The frequency of cleaning sheets/bedding was negatively correlated to the levels of house dust mite allergens. The presence of pets indoors was associated with higher levels of pet allergens and lower levels of house dust mite allergens and cockroach allergen. Risk evaluation reveals that at least 4 allergens were found in more than 80% of the rooms and more than 2 allergens with median/high risk were detected in 42% of the rooms, indicating that simultaneous exposure to multiple allergens is prevalent in China.

Chronic allergen exposure can significantly induce p38 mitogen-activated protein kinase (MAPK) activation in asthma. p38 MAPK is involved in steroid resistance through phosphorylation of glucocorticoid receptors (GR) at S226. This study aims to investigate whether chronic allergen exposure can induce steroid resistance and whether it is associated with p38 MAPK activation in asthma. A mouse model of asthma was prepared by sensitizing and challenging mice with chronic ovalbumin (OVA) exposure. Key features of allergic asthma, encompassing bronchial hyperresponsiveness, pathology of lung tissues, cytokine profiles of inflammation in bronchoalveolar lavage fluid (BALF), and serum immunoglobulin (Ig)E concentration were evaluated. Furthermore, suppressive effects of corticosteroid on the splenocytes under stimulation of lipopolysaccharides, glucocorticoid receptor (GR) DNA binding ability of splenocytes, expression of GRα and phosphorylation of GR s226 in splenocytes, and p38 MAPK phosphorylation in splenocytes and lung tissues were determined. Chronic OVA exposure substantially induced airway hypersensitivity, leading to increased inflammatory infiltration in lung tissues. Additionally, it resulted in elevated levels of interleukin (IL)-4, IL-5, and IL-6 in BALF, as well as heightened levels of IgE in serum. Furthermore, OVA exposure substantially enhanced p38 MAPK phosphorylation in lung tissues. It also weakened the suppressive impacts of corticosteroids on splenocytes, impaired the GR DNA binding ability, and led to an enhanced phosphorylated state of GR S226 and p38 MAPK in splenocytes. Taken together, chronic allergen exposure contributes to steroid resistance in asthma, which is linked to an increased phosphorylated state of GR S226 and p38 MAPK.

Shrimp allergy is a serious public health problem. Epidemiological studies indicated the main cause of food allergy is the ingestion of shrimp and crab. Consequently, reducing the risk of shrimp and crab allergy is a major research focus. Few studies have demonstrated the effect of enzyme hydrolysis on reduction of shrimp allergens in food. In this study, we used papain, a commonly used enzyme in the food industry, as an ingredient to decrease levels of allergen tropomyosin in shrimp. 0-50U of papain was used to treat the shrimp meat, and then heated to measure the levels of tropomyosin, and determined the change of the protein secondary structure. The results showed that 20U of papain decrease the tropomyosin levels up to 80 %, which was further enhanced when combined with 3 min of heating. Furthermore, the result of Fourier-transform infrared spectroscopy (FTIR) showed alteration of secondary protein structure after the processing. The processing developed in this study may be an effective method used to change the levels and structure of tropomyosin.