Hypoallergenic derivatives of Scylla paramamosain heat-stable allergens alleviated food allergy symptoms in Balb/c mice.

The design of hypoallergenic derivatives is a new strategy for allergen-specific immunotherapy. Although hypoallergenic derivatives of Scylla paramamosain (mud crab) heat-stable tropomyosin (TM) and myosin light chain (MLC) have been preliminarily explored, their allergenicity in vivo needs to be further studied. In this work, recombinant allergens (wtTM, wtMLC) and hypoallergenic derivatives (mtTM, mtMLC) were purified. IgE-binding frequencies of wtTM and wtMLC in 177 crab-sensitised patients were 32.8% and 11.9%, respectively. In the Balb/c mouse model, mtTM and mtMLC caused mild intestinal inflammation, did not activate T-helper (Th) 2 immune response (interleukin-4, anaphylactic mediator, IgE, and IgG1 antibodies were not significantly increased) but could significantly promote the production of interleukin-10, which equilibrated Th1/Th2 cells, thus alleviating allergic symptoms. Moreover, mtTM and mtMLC-induced rabbit/mice anti-IgG antibodies could effectively block wtTM and wtMLC binding to patients' sera IgE in vitro. These results indicate that hypoallergenic derivatives offer the promise for an immunotherapeutic regimen for crab allergy.

Identification of the Immunoglobulin E Epitope of Arginine Kinase, an Important Allergen from Crassostrea angulata.

Arginine kinase (AK) was identified as an allergen in Crassostrea angulata. However, little information is available about its epitopes. In this study, AK from C. angulata was registered to the World Health Organization/International Union of Immunological Societies allergen nomenclature committee to be named as Cra a 2. The immunoglobulin G/immunoglobulin E-binding capacity of Cra a 2 was significantly reduced after chemical denaturation treatment. Further, eight linear mimotopes and five conformational mimotopes of Cra a 2 were obtained using phage panning. In addition to six linear epitopes that have been identified, two linear epitopes were verified by a synthetic peptide, of which L-Cra a 2-2 was conserved in shellfish. Four conformational epitopes were verified by site-directed mutation, among which mutation of C-Cra a 2-1 affected the structure and reduced the immunoreactivity of Cra a 2 most significantly. Overall, the identified epitopes may lay a foundation for the development of hypoallergenic oyster products through food processing.

IgE Epitope Analysis for Scy p 1 and Scy p 3, the Heat-Stable Myofibrillar Allergens in Mud Crab.

Tropomyosin (Scy p 1) and myosin light chain (Scy p 3) are investigated to be important heat-stable allergens in Scylla paramamosain. However, the epitopes of Scy p 1 and Scy p 3 are limited. In this study, recombinant Scy p 1 and Scy p 3 had similar IgE-binding capacity to natural proteins. Mimotopes of Scy p 1 and Scy p 3 were analyzed by bioinformatics, phage display, and one-bead-one-compound technology. Ten linear epitopes of Scy p 1 and seven linear epitopes of Scy p 3 were identified by synthetic peptides and inhibition dot blot. Meanwhile, three conformational epitopes of Scy p 1 and seven conformational epitopes of Scy p 3 were verified by site-directed mutagenesis and the serological test. Furthermore, strong IgE-binding epitopes of Scy p 1 and Scy p 3 were conserved in multiple crustaceans. Overall, these epitopes could enhance our understanding of crab allergens, which lay the foundation for a cross-reaction.

Characterization, Epitope Identification, and Cross-reactivity Analysis of Tropomyosin: An Important Allergen of Crassostrea angulata.

Oyster is a common shellfish product in China, which is associated with food allergy. However, there is still lack of research on allergens in oysters. In this study, tropomyosin (TM), an important allergen of Crassostrea angulata, was purified and identified by mass spectrometry. Subsequently, TM was cloned and expressed, with a sequence of size 852 bp, encoding 284 amino acid residues. The results of circular dichroism, digestion assay, inhibition enzyme-linked immunosorbent assay, and basophil activation test showed that recombinant TM had similar physicochemical properties and immunological properties to native TM. Furthermore, two conformational mimotopes were obtained and 10 IgE linear epitopes were verified. Meanwhile, different degrees of cross-reactivity were observed between C. angulata TM and the other 8 shellfish TMs using antibodies and serological analysis, which may relate to the 3 conserved epitope regions. These findings are expected to provide a theoretical basis for the molecular diagnosis of oyster allergy and cross-reactivity among shellfish.

Identification and characterization of Crassostrea angulata arginine kinase, a novel allergen that causes cross-reactivity among shellfish.

Oyster is a common food that causes allergy. However, little information is available about its allergens and cross-reactivity. In this study, arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata. The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs. Furthermore, nine epitopes in oyster AK were verified using inhibition dot blots and inhibition enzyme linked immunosorbent assay, six of which were similar to the epitopes of shrimp/crab AK. The most conserved epitopes were P5 (121-133) and P6 (133-146), which may be responsible for the cross-reactivity caused by AK. These findings will provide a deeper understanding of oyster allergens and cross-reactivity among shellfish.

Linear Epitopes Play an Important Role in the Immunoglobulin G (IgG)/Immunoglobulin E (IgE)-Binding Capacity of Scy p 4.

Sarcoplasmic calcium-binding protein is a stable allergen in Scylla paramamosain and named Scy p 4. To explore the importance of linear epitopes in the immunoglobulin G (IgG)/immunoglobulin E (IgE)-binding capacity of Scy p 4, chemical denaturants were used to destroy the structure. Scy p 4 was reduced with dithiothreitol and subsequently alkylated with iodoacetamide (IAA). Furthermore, the structural analysis indicated that IAA-Scy p 4 was an unstructured protein. The inhibition enzyme-linked immunosorbent assay showed that IAA-Scy p 4 could inhibit the binding of Scy p 4 to sensitize serum, with inhibition rates reached 55%. Moreover, the linear mimotopes of Scy p 4 were predicted in silico. Three linear epitopes were verified by serological tests and named L-Scy p 4-1 (AA76-91), L-Scy p 4-2 (AA111-125), and L-Scy p 4-3 (AA137-146). Overall, these data provide an understanding of the relationship between the structure and allergenicity about Scy p 4, and the identified linear epitopes can be used for diagnosis and food processing of shellfish allergy.

IgE epitope analysis of sarcoplasmic-calcium-binding protein, a heat-resistant allergen in Crassostrea angulata.

Sarcoplasmic-calcium-binding protein (SCP) has been investigated as a novel allergen in Crassostrea angulata. Nevertheless, knowledge of its effector-cell-based allergic relevance and epitopes is limited. In this study, the heat-resistant allergen SCP was able to induce significant upregulation of CD63 and CD203c (p < 0.05), which showed obvious allergenicity in a basophil activation test. Furthermore, immunoinformatic tools, a one-bead-one-compound peptide library, and phage display technology were combined to analyze the allergenic epitopes of SCP. Five linear epitopes named L-SCP-1 (AA22-33), L-SCP-2 (AA64-75), L-SCP-3 (AA80-90), L-SCP-4 (AA107-116), and L-SCP-5 (AA144-159) were verified using serological tests. Additionally, two conformational epitopes (C-SCP-1 and C-SCP-2) were determined, and C-SCP-1 was located at one of the calcium-binding sites (AA106-117). Moreover, SCP showed weaker typical α-helical features and higher hydrophobicity after Ca2+ depletion, which reduced its IgE-binding capacity. Overall, these epitope data could enhance our understanding of oyster allergens, which could be used to develop hypoallergenic shellfish products.

The Association between Changes in Low Parathyroid Hormone Levels and Cardiac Function Decline in Maintenance Hemodialysis Patients: A Prospective Observational Study.

OBJECTIVES: The aim of this study was to investigate a possible association between changes in low parathyroid hormone (PTH) levels and cardiac function decline in maintenance hemodialysis (MHD) patients. METHODS: A total of 150 MHD patients were included and followed for 24 months. The enrolled patients were divided into 3 groups based on their PTH status at baseline and 24 months. Factors potentially involved in changes in the PTH level and cardiac function were compared using multivariate logistic regression analysis. RESULTS: At 24 months, the presence of low PTH levels increased by 26.7%. The main independent factors for low PTH levels were a low BMI, hemoglobin, and serum albumin and high serum calcium (p < 0.05). A persistently low PTH level at 24 months was associated with a decrease in the left ventricular ejection fraction (LVEF) and increase in valvular calcification (p < 0.05). Additionally, a decrease in PTH levels from normal or high to low values was associated with a decrease in LVEF and cardiac output (CO) and an increase in valvular calcification (p < 0.05). Furthermore, compared with those of the persistently low PTH level group, LVEF values were lower at 24 months in the group with a decrease from high/normal to low PTH level (p < 0.05). CONCLUSION: Persistently low PTH levels and changes in the PTH level from high/normal to low were associated with cardiac function decline in MHD patients. Moreover, a PTH level change from high/normal to low showed a stronger correlation with cardiac function decline.

Effects of the Maillard reaction on the epitopes and immunoreactivity of tropomyosin, a major allergen in Chlamys nobilis.

Scallop (Chlamys nobilis) causes an IgE-mediated food allergy; however, studies of the allergens in its musculus are not sufficiently comprehensive. In this context, the target protein was purified from scallops and confirmed to be the major allergen tropomyosin (TM) using proteomic technology and serological testing. Subsequently, seven potential IgE epitopes of TM were obtained using phage display technology with IgE enrichment from the serum of scallop-sensitized patients and identified via inhibition enzyme-linked immunosorbent assays. A method for the Maillard reaction of TM and xylose was established, and Maillard-reacted TM (MR-TM) showed significantly decreased immunobinding activity and CD63 and CD203c expression in basophils compared with TM. Furthermore, shotgun proteomics analysis showed that eleven specific amino acids (K12, R15, K28, K76, R125, R127, K128, R133, R140, K146, and K189) of the six IgE epitopes of TM were modified after the Maillard reaction. Overall, the immunoactivity of MR-TM was reduced, which provides a theoretical reference for the development of hypoallergenic foods.

Combination Processing Method Reduced IgE-Binding Activity of Litopenaeus vannamei by Modifying Lysine, Arginine, and Cysteine on Multiple Allergen Epitopes.

Allergic reactions occur after the whole food is ingested, rather than the purified allergen. The present study explores the low-allergenic food processing for Litopenaeus vannamei by analysis of macrostructure, digestibility, and immunoreactivity. Furthermore, the presence of modified amino acids on the reported IgE epitopes was analyzed by mass spectrometry. Results showed that the combination processing of Maillard reaction (shrimp meat with galactose) with high temperature-pressure at 115 °C obviously changed the macrostructure and increased the digestibility for the shrimp meat. Meanwhile, the processing significantly reduced the IgG/IgE-binding activity of the shrimp meat. The hypo-IgE-binding activity in processed shrimp may be due to the modification of lysine, arginine, and cysteine residues in antigen epitopes. This is a comprehensive assessment of the specific amino acid residues modified by glycation of multiple allergens in processed L. vannamei, which provides a new research method to explore the hypo-IgE-binding activity in food.

Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.

Site-Directed Mutations of Calcium-Binding Sites Contribute to Reducing the Immunoreactivity of the EF-Hand Sarcoplasmic Calcium-Binding Protein in Scylla paramamosain.

In order to reduce the immunoreactivity of sarcoplasmic calcium-binding protein (SCP), site-directed mutations were used to replace key amino acids in the conformational epitopes and calcium-binding sites. The mutant SCPs (mSCPs) were expressed in Escherichia coli, and their immunoreactivities were analyzed using iELISA and basophil activation assays. Furthermore, the structural changes of mSCPs were determined from the circular dichroism spectra. The iELISA results showed that mSCPs could effectively inhibit the binding of wild-type SCP (wtSCP) to sensitive serum, with inhibition rates that reached 90%. Moreover, mSCPs could downregulate the expression levels of CD63 and CD203c on the basophil surface. Compared with wtSCP, the peak values were significantly changed, and the calcium binding ability was impaired, which explained the decline in immunoreactivities of the mSCPs. All of the data confirmed that this approach was effective in reducing the immunoreactivity of SCP and could be applied to other shellfish allergens.

Identification and Cross-reactivity Analysis of Sarcoplasmic-Calcium-Binding Protein: A Novel Allergen in Crassostrea angulata.

Oysters are an important shellfish group known to cause food allergy; however, knowledge of their sensitization components and cross-reactivity is limited. This study aimed to identify a novel allergen in Crassostrea angulata and investigate its cross-reactivity. To this end, a 20 kDa protein was purified from oyster and confirmed to be a sarcoplasmic-calcium-binding protein (SCP) by LC-MS/MS. A 537 bp open reading frame was obtained from oyster SCP total RNA, which encoded 179 amino acids, and was expressed in Escherichia coli. According to the circular dichroism results, digestion assay, and inhibition ELISA, the recombinant SCP (rSCP) exhibited similar physicochemical properties and IgG-binding activity to native SCP. rSCP displayed stronger IgE-binding activity by immunological method. Moreover, a different intensity of cross-reactivity and sequence homology were demonstrated between shellfish species. Collectively, these findings provide novel insight into shellfish allergens, which can be used to aid in the in vitro diagnosis of oyster-sensitized patients.

Expression and epitope identification of myosin light chain isoform 1, an allergen in Procambarus clarkii.

Myosin light chain isoform 1 (MLC1) is reported to be a novel allergen in crayfish (Procambarus clarkii). However, little information is available about its allergic epitopes. In this study, recombinant crayfish MLC1 (rMLC1) was expressed and confirmed by mass spectrometry. Circular dichroic analysis and serological test were performed for the measuring of structural and immunological properties of rMLC1. Specific-protein-A-enriched IgG raised in rabbits against purified rMLC1 was used to screen a phage display random peptide library. Nine MLC1 mimotope clones were identified among 16 random clones after biopanning. Five conformational epitopes were identified with the program LocaPep, and mapped into 3 epitope regions at the antibody-binding interface of MLC1. MLC1 of crayfish showed high primary and secondary structure identity to MLC of other allergenic species, its epitopes were located in the structure conserved regions, and its cross-reactivity among related species was indicated by immunological assays.

Crystal Structure Analysis and Conformational Epitope Mutation of Triosephosphate Isomerase, a Mud Crab Allergen.

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab.

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.

Effects of thermal processing on digestion stability and immunoreactivity of the Litopenaeus vannamei matrix.

Many types of shellfish, including shrimp, are sometimes cooked before ingestion. Hence, it is necessary to investigate how cooking (boiling, pressure treatment or none (raw)) affects the structure, digestibility and immunoreactivity of multi-component shrimp muscle. Protein extraction, simulated gastrointestinal digestion, immunoreactivity, immunoglobulin E (IgE)-mediated human mast cell degranulation, morphology, particle size and UV absorbance scanning were used to investigate changes in the shrimp muscle upon treatment. The extractability of proteins and allergens was highest with 0.5 mol L-1 NaCl. Pressure treatment increased the digestibility and reduced the immunoreactivity of shrimp edible portions. Thermal processing induced the production of regular fiber bundles, blue shifts of absorbance peaks and reduction of particle size in the complex food matrix. These changes in macro- and micro-structure can further affect gastrointestinal digestibility and immunoreactivity due to the interactions between multiple components in the whole food. In conclusion, the digestibility, immunoreactivity and structure were altered by thermal processing of the complex food matrix.

Reducing Allergenicity to Arginine Kinase from Mud Crab Using Site-Directed Mutagenesis and Peptide Aptamers.

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.

Analysis of the Allergenic Epitopes of Tropomyosin from Mud Crab Using Phage Display and Site-Directed Mutagenesis.

Mud crab ( Scylla serrata), which is widely consumed, can cause severe allergic symptoms. Eight linear epitopes and seven conformational epitopes of tropomyosin (TM) from S. serrata were identified using phage display. The conformational epitopes were formed based on the coiled-coil structure of TM. Most of the epitopes were located in the regions where primary structures were conserved among crustacean TM. Twelve synthetic peptides were designed according to the epitopes and trypsin-cutting sites of TM, among them, three synthetic peptides (including one linear epitope and two conformational epitopes) were recognized by all of the patient sera using inhibitory dot blotting. A triple-variant (R90A-E164A-Y267A) was constructed based on the critical amino acids of the TM epitope. The IgE-binding activity of the triple-variant was significantly reduced compared with that of native TM. The results of phage display and site-directed mutagenesis offered new information regarding conformational epitopes of TM.

Effect of fish oil supplement in maintenance hemodialysis patients: a systematic review and meta-analysis of published randomized controlled trials.

PURPOSE: The purpose of this study was to systematically assess the efficacy of fish oil therapy on maintenance hemodialysis patients (MHD). METHODS: Electronic databases, including PubMed, Cochrane library, EMBase, and Web of Science, were searched for randomized controlled trials (RCTs) of fish oil versus placebo or no treatment in MHD patients. The study selection and data extraction were conducted independently by two reviewers, and statistical analysis was performed using RevMan software, version 5.2. RESULTS: A total of thirteen eligible RCTs involving 916 subjects (461 in the experimental group and 455 in the control group) were included. The meta-analysis showed that fish oil significantly reduced arteriovenous graft (AV-graft) events [risk ratio (RR) 0.71, 95 % confidence interval (CI) were (0.52, 0.97)] and cardiovascular events [RR (95 %CI) were 0.41 (0.26, 0.66)] in the fish oil group. In addition, compared with the control group, fish oil significantly decreased the Beck Depression Inventory (BDI) score [weighted mean difference (WMD) (95 %CI) were -11.91 (-15.88, -7.95)], serum intact parathyroidism (iPTH), C-reactive protein (CRP), and triglycerides (TG) [standard mean difference (SMD) (95 %CI) were -0.56 (-0.89, -0.23); -0.36 (-0.63, -0.09), and -0.41 (-0.68, -0.14), respectively]. However, the fish oil group did not differ significantly from the control group in albumin (ALB), hemoglobin (Hb), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and mortality. CONCLUSIONS: Fish oil reduced the risk of AV-graft events and cardiovascular events, and alleviated depression symptoms in MHD patients. It can also improve secondary hyperparathyroidism, micro-inflammation, and hypertriglyceridemia. But there is no evidence that fish oil can improve nutritional status and renal anemia.