Comparative analysis of asian carps parvalbumin reveals the divergence pattern of major fish allergen.

BACKGROUND: Asian carps, a popular freshwater fish globally, are valued for their flavor and serve as a crucial protein source, especially for infants. However, grass carp parvalbumin is highly allergenic, surpassing the allergenicity of fish like salmon and cod. The allergenic potential of parvalbumin in other Asian carps remains unknown, underscoring the need for allergen identification to improve the precision of fish allergy diagnosis and treatment. OBJECTIVE: To identify all parvalbumin homologs in Asian carps and investigate the role of gene divergence in allergenic homolog formation. METHODS: Three annotated genomes of Asian carp, including grass carp, black carp and bighead carp, were constructed using a hybrid assembly approach. Through sequence homology at the genomic level, all the homologs of major fish allergens were identified. Bioinformatics tools were then employed to reveal the gene structures, expression levels, and protein conformations of parvalbumin. RESULTS: Grass carp genome analysis showed nine parvalbumin homologs, with Cid_PV2 most similar to Cten i 1. Bighead and black carp genomes had ten homologs, including potentially allergenic Mpi_PV7 and Hno_PV7. Tissue-specific expression patterns revealed alternative usage of parvalbumin homologs. Gene duplication events expanded parvalbumin copies in bony fish, with two gene clusters identified in Asian carp genomes. CONCLUSION: All the homologs of Asian carps' parvalbumin were accurately identified and gene divergence contributed to the formation of allergenic homologs. Together with a comprehensive gene sequence profile of carps' parvalbumin, those could be applied to achieve a more precise clinical diagnostic test.

Shrimp Extract Exacerbates Allergic Immune Responses in Mice: Implications on Clinical Diagnosis of Shellfish Allergy.

Tropomyosin has been identified as the major cross-reactive shellfish allergen, but recent studies showed the presence of other clinically relevant allergens. This study aims at determining the allergic immune responses of mice sensitized with raw and boiled shrimp extracts in comparison to recombinant tropomyosin (rTM). Female Balb/c mice were intragastrically sensitized and challenged with raw, boiled shrimp or rTM. Systemic, cellular and humoral allergic responses were compared, while allergenicity of the extracts was also compared by skin prick test (SPT) and immunoblot on shrimp allergic subjects. We showed that rTM and shrimp extracts induced IgE- and Th2-mediated allergic responses in mice, distinguished by remarkable intestinal inflammation in small intestine across all regimens. Notably, boiled shrimp extract exhibited the highest sensitization rate (73.7% of mice developed positive TM-specific IgE response) when compared with raw extract (47.8%) and rTM (34.8%). Mice sensitized with boiled extract manifested the highest allergen-specific IgE and Th2 cytokine responses than the others. Immunoblot results indicated that tropomyosin remained the major allergen in extract-based sensitization and had stronger allergenicity in a heat-treated form comparing to untreated TM, which was in line with the SPT results that boiled extract induced larger wheal size in patients. Hemocyanin and glycogen phosphorylase were also identified as minor allergens associated with manifestation of shrimp allergy. This study shows that boiled extract enhanced sensitization and Th2 responses in agreement with the higher allergenicity of heat-treated TM. This study thus presents three shrimp allergy murine models suitable for mechanistic and intervention studies, and in vivo evidence implies higher effectiveness of boiled extract for the clinical diagnosis of shellfish allergy.

Real-World Sensitization and Tolerance Pattern to Seafood in Fish-Allergic Individuals.

BACKGROUND: Seafood is a common cause of food allergy and anaphylaxis, but there are limited published real-world data describing the clinical presentation of fish and shellfish allergies. OBJECTIVE: This study aimed to examine the clinical characteristics, immunological profile, and tolerance pattern to fish, crustaceans, and mollusks in fish-allergic individuals. METHODS: Patients presenting with IgE-mediated fish allergy between 2016 and 2021 were recruited. A comprehensive sensitization profile including specific IgE and skin prick test to various fish and shellfish species and a detailed clinical history including individuals' recent seafood consumption were evaluated. RESULTS: A total of 249 fish-allergic individuals (aged 4.2 ± 5.8 years) were recruited from 6 allergy clinics in Hong Kong, and they had experienced their fish-allergic reaction 2.2 ± 3.4 years before enrollment. Seventy-five subjects (30%) reacted to either grass carp, salmon, grouper, or cod in oral food challenges. We identified an IgE sensitization gradient that corresponded to the level of ß-parvalbumin in fish. In total, 40% of fish-allergic individuals reported tolerance to 1 or more types of fish, more commonly to fish with a lower ß-parvalbumin level such as tuna and salmon, compared with ß-parvalbumin-rich fish such as catfish and grass carp. Despite fish and shellfish cosensitization, 41% of individuals reported tolerance to crustaceans, mollusks, or both, whereas shellfish avoidance occurred in half of the fish-allergic individuals, of whom 33% lacked shellfish sensitization. CONCLUSIONS: Fish allergy commonly presents in early childhood. A considerable proportion of fish-allergic patients are selectively tolerant to certain fish, typically those with lower levels of ß-parvalbumin. There is an unmet need to promote precision medicine for seafood allergies.

Genomics of Shrimp Allergens and Beyond.

Allergy to shellfishes, including mollusks and crustaceans, is a growing health concern worldwide. Crustacean shellfish is one of the "Big Eight" allergens designated by the U.S. Food and Drug Administration and is the major cause of food-induced anaphylaxis. Shrimp is one of the most consumed crustaceans triggering immunoglobulin E (IgE)-mediated allergic reactions. Over the past decades, the allergen repertoire of shrimp has been unveiled based on conventional immunodetection methods. With the availability of genomic data for penaeid shrimp and other technological advancements like transcriptomic approaches, new shrimp allergens have been identified and directed new insights into their expression levels, cross-reactivity, and functional impact. In this review paper, we summarize the current knowledge on shrimp allergens, as well as allergens from other crustaceans and mollusks. Specific emphasis is put on the genomic information of the shrimp allergens, their protein characteristics, and cross-reactivity among shrimp and other organisms.

Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions.

In an attempt to address the significant morbidity, mortality, and economic cost associated with tunneled dialysis catheter (TDC) dysfunction, we report the development of nitric oxide-releasing dialysis catheter lock solutions. Catheter lock solutions with a range of NO payloads and release kinetics were prepared using low-molecular-weight N-diazeniumdiolate nitric oxide donors. Nitric oxide released through the catheter surface as a dissolved gas was maintained at therapeutically relevant levels for at least 72 h, supporting clinical translatability (interdialytic period). Slow, sustained NO release from the catheter surface prevented bacterial adhesion in vitro by 88.9 and 99.7% for Pseudomonas aeruginosa and Staphylococcus epidermidis, respectively, outperforming a burst NO-release profile. Furthermore, bacteria adhered to the catheter surface in vitro prior to lock solution use was reduced by 98.7 and 99.2% for P. aeruginosa and S. epidermidis, respectively, when using a slow releasing NO donor, demonstrating both preventative and treatment potential. The adhesion of proteins to the catheter surface, a process often preceding biofilm formation and thrombosis, was also lessened by 60-65% by sustained NO release. In vitro cytotoxicity of catheter extract solutions to mammalian cells was minimal, supporting the non-toxic nature of the NO-releasing lock solutions. The use of the NO-releasing lock solution in an in vivo TDC porcine model demonstrated decreased infection and thrombosis, enhanced catheter functionality, and improved outcome (i.e., likelihood of survival) as a result of catheter use.

Differential patterns of fish sensitization in Asian populations: Implication for precision diagnosis.

BACKGROUND: The current diagnostics of fish allergy lack sufficient accuracy such that more reliable tests such as component-resolved diagnosis (CRD) are urgently needed. This study aimed at identifying fish allergens of salmon and grass carp and evaluating the sensitization pattern in fish allergic subjects from two distinct populations in Asia. METHODS: One hundred and three fish allergic subjects were recruited from Hong Kong (67 subjects) and Japan (46 subjects). Western blot and mass spectrometry were used to identify allergens from salmon and grass carp. Fish allergens were purified and tested against 96 sera on ELISA to analyze patients' sensitization pattern. The protein profiles of salmon meat prepared under different cooking methods until core temperature reached 80 °C were evaluated by SDS-PAGE and mass spectrometry. RESULTS: Three common allergens between salmon and grass carp, namely enolase, glycerldehyde-3-phosphate dehydrogenase (GAPDH) and parvalbumin, and two salmon-specific allergens collagen and aldolase were identified. Parvalbumin was the major allergen for both fishes showing an overall sensitization rate of 74.7%, followed by collagen (38.9%), aldolase (38.5%) and enolase (17.8%). Japanese subjects showed more diverse allergen sensitization pattern and more frequent IgE-binding to heat-labile salmon allergens. Compared with steaming and boiling, cooking by baking and frying retained more fish proteins inclusive of heat-labile allergens. CONCLUSIONS: Fish allergic patients from different Asian populations show varying fish allergen sensitization profiles. The relevant extracts and components for diagnosis are population-dependent but parvalbumin and collagen are important biomarkers. Cooking methods modify allergen composition of salmon and appear to influence patients' allergic manifestations.

Emerging approaches in the diagnosis and therapy in shellfish allergy.

PURPOSE OF REVIEW: Despite the high prevalence of shellfish allergy, the clinical management of seafood allergy has remained unchanged over decades. Here, we examined the current status in the diagnosis and clinical management of shellfish allergy and highlighted the imminent need for more specific diagnostic methods, as well as effective and safe therapeutic approaches for shellfish allergy. RECENT FINDINGS: With the advancement in the molecular identifications and definition of reactive epitopes of shellfish allergens, new diagnostic designs such as component-resolved diagnosis, basophil activation test (BAT) and the emerging IgE-crosslinking-induced luciferase expression are emerging. Furthermore, various allergen-specific immunotherapy strategies (such as shellfish extracts and allergens, hypoallergens, hypoallergen DNA vaccines, mimotopes and peptide-based therapies) are being explored at preclinical stages whereas limited nonallergen specific immunotherapy approaches are under clinical trials. SUMMARY: With an increasing understanding of the underlying immunological mechanisms and molecular features of shellfish allergy, the future for developing precise diagnostic and therapeutic strategies to better manage shellfish allergy is promising.

Comprehending the allergen repertoire of shrimp for precision molecular diagnosis of shrimp allergy.

BACKGROUND: Clinical management of shrimp allergy is hampered by the lack of accurate tests. Molecular diagnosis has been shown to more accurately reflect the clinical reactivity but the full spectrum of shrimp allergens and their clinical relevance are yet to be established. We therefore sought to comprehend the allergen repertoire of shrimp, investigate and compare the sensitization pattern and diagnostic value of the allergens in allergic subjects of two distinct populations. METHODS: Sera were collected from 85 subjects with challenge-proven or doctor-diagnosed shrimp allergy in Hong Kong and Thailand. The IgE-binding proteins of Penaeus monodon were probed by Western blotting and identified by mass spectrometry. Recombinant shrimp allergens were synthesized and analyzed for IgE sensitization by ELISA. RESULTS: Ten IgE-binding proteins were identified, and a comprehensive panel of 11 recombinant shrimp allergens was generated. The major shrimp allergens among Hong Kong subjects were troponin C (Pen m 6) and glycogen phosphorylase (Pen m 14, 47.1%), tropomyosin (Pen m 1, 41.2%) and sarcoplasmic-calcium binding protein (Pen m 4, 35.3%), while those among Thai subjects were Pen m 1 (68.8%), Pen m 6 (50.0%) and fatty acid-binding protein (Pen m 13, 37.5%). Component-based tests yielded significantly higher area under curve values (0.77-0.96) than shrimp extract-IgE test (0.70-0.75). Yet the best component test differed between populations; Pen m 1-IgE test added diagnostic value only in the Thai cohort, whereas sensitizations to other components were better predictors of shrimp allergy in Hong Kong patients. CONCLUSION: Pen m 14 was identified as a novel shrimp allergen predictive of challenge outcome. Molecular diagnosis better predicts shrimp allergy than conventional tests, but the relevant component is population dependent.

Cell-Based Functional IgE Assays Are Superior to Conventional Allergy Tests for Shrimp Allergy Diagnosis.

BACKGROUND: The diagnosis of shellfish allergy currently relies on patient history, skin prick test (SPT), and serum specific IgE (sIgE) quantification. These methods lack sufficient diagnostic accuracy, whereas the gold standard of oral food challenges is risky and burdensome. Markers of reactivity and severity of allergic reactions to shellfish will improve clinical care of these patients. OBJECTIVES: This study compared the diagnostic performance of SPT, sIgE, basophil activation test (BAT), and IgE crosslinking-induced luciferase expression (EXiLE) test for shrimp allergy. METHODS: Thirty-five subjects with documented history of shrimp allergic reactions were recruited and grouped according to results of double-blind, placebo-controlled food challenge (DBPCFC). In addition to routine diagnostics, BAT (Flow CAST) and EXiLE test with shrimp extract and tropomyosin were performed. RESULTS: Of 35 subjects, 15 were shrimp allergic with pruritus, urticaria, and itchy mouth on DBPCFC, whereas 20 were tolerant to shrimp. Tropomyosin only accounted for 53.3% of sensitization among subjects with challenge-proven shrimp allergy. BAT using shrimp extract as stimulant showed the highest area under curve value (0.88), Youden Index (0.81), likelihood ratio (14.73), odds ratio (104), and variable importance (4.27) when compared with other assays and tropomyosin diagnosis. Results of BAT significantly correlated with those of EXiLE (r = 0.664, P < .0001). CONCLUSIONS: BAT is a more accurate diagnostic marker for shrimp allergy than SPT and shrimp sIgE, whereas the EXiLE test based on an IgE crosslinking assay is a good alternative to BAT. Tropomyosin may not be the most important shrimp allergen in Chinese, which warrants further investigation to search for other major allergens and diagnostic markers.

Seafood Allergy in Asia: Geographical Specificity and Beyond.

Asian countries have unique ways of food processing and dietary habits that may explain the observed differences in the prevalence, natural history, epidemiology and sensitization pattern of food allergic diseases when compared to western countries. Per capita consumption of seafood, including fish and shellfish, is well above the global average for many Asian countries because of their coastal geographical location and rich seafood supply. The wide availability and high abundance of seafood in Asian countries have shaped a diverse way of processing and eating this major food group. Such unique features have significant impact on the sensitization profile and allergenicity of Asians to fish and shellfish. For example, fish and shellfish are eaten raw in some countries that may promote sensitization to heat-labile allergens not otherwise seen in other regions. Fermented fish sauce is commonly used as a condiment in some countries which may promote fish sensitization. Shrimp head and shrimp roe are regarded as delicacies in some countries, but their allergen profiles are yet to be characterized. Freshwater fish and shellfish are a common food source in many Asian countries but the allergenicity of many such species remains unknown. In this review, we discuss factors that may contribute to differences in molecular profile and sensitization pattern for fish and shellfish that are observed in Asian populations and revisit the current status of seafood allergy in this part of the world. Acknowledging the similarities and differences of seafood allergy patterns between Asian and western populations can help us refine a better strategy for diagnosing and managing seafood allergy.

T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy.

Food allergy has been rising in prevalence over the last two decades, affecting more than 10% of the world population. Current management of IgE-mediated food allergy relies on avoidance and rescue medications; research into treatments that are safer and providing guaranteed and durable curative effects is, therefore, essential. T-cell epitope-based immunotherapy holds the potential for modulating food allergic responses without IgE cross-linking. In this chapter, we describe the methods in evaluating the therapeutic capacities of immunodominant T-cell epitopes in animal models of food allergy. Moreover, we explain in detail the methods to measure the allergen-specific antibody levels, prepare single-cell suspension from spleen, and prepare small intestine for immunohistochemical analysis of eosinophils and Foxp3+ cells.

Molecular and immunological characterization of grass carp (Ctenopharyngodon idella) parvalbumin Cten i 1: A major fish allergen in Hong Kong.

BACKGROUND: Grass carp is the most commonly consumed fish species in Hong Kong. The allergenicity of grass carp and its allergen content are yet to be reported. This study characterized the major allergen in grass carp and investigated its allergenicity. METHODS: Sixty-nine subjects with history of IgE-mediated allergic reaction to grass carp were recruited. The protein content in steamed grass carp extract was resolved by SDS-PAGE, and the major allergen was identified by immunoblotting with serum from subjects allergic to grass carp. The identity of allergen was elucidated by mass spectrometry and amino acid sequence obtained by amplifying the specific gene from cDNA library of grass carp. The cross-reactivity between parvalbumins from grass carp and other phylogenetically close (common carp) or commercially important (cod and salmon) species was investigated by competitive inhibition ELISA. RESULTS: A major IgE-binding protein was found at approximately 9 kDa and identified as parvalbumin by immunoblotting and mass spectrometry. Grass carp parvalbumin was more allergenic than common carp, salmon, and cod parvalbumins despite sharing high sequence homology. This newly identified major allergenic parvalbumin isoform from grass carp was registered as Cten i 1 in the World Health Organization and International Union of Immunological Societies allergen database. CONCLUSIONS: Grass carp parvalbumin is identified as the major fish allergen in Hong Kong. The strong allergenicity of Cten i 1 contributes to the high IgE reactivity of grass carp. Grass carp, among other fish species, should be considered when managing fish-allergic patients.

Overcoming Shellfish Allergy: How Far Have We Come?

Shellfish allergy caused by undesirable immunological responses upon ingestion of crustaceans and mollusks is a common cause of food allergy, especially in the Asia-Pacific region. While the prevalence of shellfish allergy is increasing, the mainstay of clinical diagnosis for these patients includes extract-based skin prick test and specific IgE measurement while clinical management consists of food avoidance and as-needed use of adrenaline autoinjector should they develop severe allergic reactions. Such a standard of care is unsatisfactory to both patients and healthcare practitioners. There is a pressing need to introduce more specific diagnostic methods, as well as effective and safe therapies for patients with shellfish allergy. Knowledge gained on the identifications and defining the immuno-molecular features of different shellfish allergens over the past two decades have gradually translated into the design of new diagnostic and treatment options for shellfish allergy. In this review, we will discuss the epidemiology, the molecular identification of shellfish allergens, recent progress in various diagnostic methods, as well as current development in immunotherapeutic approaches including the use of unmodified allergens, hypoallergens, immunoregulatory peptides and DNA vaccines for the prevention and treatment of shellfish allergy. The prospect of a "cure "for shellfish allergy is within reach.

Modulating Shrimp Tropomyosin-Mediated Allergy: Hypoallergen DNA Vaccines Induce Regulatory T Cells to Reduce Hypersensitivity in Mouse Model.

Shellfish allergy is one of the most common food allergies, with tropomyosin as the major cross-reactive allergen. However, no allergen-specific immunotherapy is clinically available. Recently, we designed two shrimp hypoallergens MEM49 and MED171. This study aimed to examine and compare the efficacy of the MEM49- and MED171-based DNA vaccines (pMEM49 and pMED171) in modulating shrimp allergy in a murine model of shrimp tropomyosin sensitivity. Intradermal immunization of BALB/c mice with pMEM49 or pMED171 effectively down-modulated allergic symptoms, tropomyosin-specific IgE levels, intestinal Th2 cytokines expression, and inflammatory cell infiltration. Both pMEM49 and pMED171 increased the frequency of regulatory T cells, but to a greater extent by pMED171 with upregulation of gut-homing molecules integrin-α4ß7. The functionality of the pMED171-induced Treg cells was further illustrated by anti-CD25-mediated depletion of Treg cells and the adoptive transfer of CD4+CD25+Foxp3+Treg cells. Collectively, the data demonstrate that intradermal administration of pMED171 leads to the priming, activation, and migration of dermal dendritic cells which subsequently induce Treg cells, both locally and systemically, to downregulate the allergic responses to tropomyosin. This study is the first to demonstrate the potency of hypoallergen-encoding DNA vaccines as a therapeutic strategy for human shellfish allergy via the vigorous induction of functional Treg cells.

IGFBP-2 stimulates calcium/calmodulin-dependent protein kinase kinase 2 activation leading to AMP-activated protein kinase induction which is required for osteoblast differentiation.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2-/- mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.

Inhibition of Aberrant IGF-I Signaling in Diabetic Male Rat Retina Prevents and Reverses Changes of Diabetic Retinopathy.

Hyperglycemia results in inhibition of cleavage of integrin-associated protein (IAP) thereby allowing it to bind to SHPS-1 which results in pathophysiologic changes in endothelial function. This study determined if an anti-rat IAP antibody directed against the SHPS-1 binding site which disrupts IAP/SHPS-1 association could inhibit these pathophysiologic changes. The anti-IAP antibody inhibited IGF-I-stimulated SHPS-1, p52Shc, MAP kinase phosphorylation, and proliferation in endothelial cells. To determine if it could reverse established pathophysiologic changes in vivo, this antibody or normal rat IgG F(ab)2 was injected intraperitoneally for 6 weeks into rats that had diabetes for 4 weeks. Optical coherence tomography (OCT) showed that retinal thickness increased at 4 weeks and this increase was maintained in rats treated with the control antibody for an additional 6 weeks. The increase was reversed by anti-IAP antibody treatment (84.6 ± 2.0 compared to 92.3 ± 2.5 µm, p < 0.01). This value was similar to nondiabetic animals (82.2 ± 1.6 µm, p, NS). The anti-IAP antibody also decreased retinal vascular permeability (0.62 ± 0.12 vs. 0.96 ± 0.25%/g/h, p < 0.001). To determine if it was effective after local injection, this antibody or control was administered via intravitreal injection. After 3 weeks, retinal thickness increased to 6.4 ± 2.8% in diabetic rats, and IAP antibody treatment prevented this increase (0.8 ± 2.5%, p < 0.01). It also prevented the increase of retinal vascular permeability (0.92 ± 0.62 vs. 1.63 ± 0.99%/g/h, p < 0.001). Biochemical analyses of retinal extracts showed that the anti-IAP antibody inhibited IAP/SHPS-1 association and SHPS-1 phosphorylation. This resulted in inhibition of AKT activation and VEGF synthesis in the retina: changes associated with increased vascular permeability. We conclude the anti-rat IAP antibody disrupts IAP/SHPS-1 association and attenuates aberrant IGF-I signaling thereby preventing or reversing the progression of retinal pathophysiological changes.

Immunotherapy of Food Allergy: a Comprehensive Review.

Food allergy imposes a severe global health burden, and thus, there is a dire need for safe and effective treatments. Allergen-specific immunotherapy (AIT) is currently the only approach to restore immune tolerance through administrating increasing doses of allergen extracts. Unfortunately, the development of AIT for food allergies has been impeded by the frequent anaphylactic side effects during the course of treatment. The emergence of component-resolved diagnosis has greatly improved our ability to identify causative allergens and revolutionized the design of AIT. Molecular features such as IgE-binding epitopes and T cell epitopes have been elucidated in most major food allergens, inspiring the use of multiple strategies to manipulate the allergens and design safer alternatives to AIT. Although these allergen-modifying approaches are currently restricted to preclinical characterization and animal studies, the employment of these strategies has certainly paved the way for improving the safety of existing AIT. A safe and effective AIT for food allergy is not far beyond reach.