Qing-Mei Liu

Anti-Food Allergic Activity of Sulfated Polysaccharide from Gracilaria lemaneiformis is Dependent on Immunosuppression and Inhibition of p38 MAPK

Polysaccharides from Gracilaria lemaneiformis in particular possess various bioactive functions, but their antiallergic activity remains incompletely defined. Sulfated polysaccharide from Gracilaria lemaneiformis (GLSP) was obtained by water extraction and ethanol precipitation followed by column chromatography. BALB/c mice, RBL-2H3, and KU812 cells were used for verifying the anti food allergic activity of GLSP. According to the results of mice experiment, GLSP was able to alleviate allergy symptoms, to reduce TM-specific IgE and IgG1, to suppress Th2 cell polarization, and to promote the function of regulatory T (Treg) cells. In addition, GLSP had the ability to inhibit the function of RBL-2H3 cells. Furthermore, GLSP inhibited the activation of KU812 via suppression of p38 mitogen-activated protein kinase (MAPK). In conclusion, immunosuppression as well as the reduction in the level of p38 MAPK may contribute to GLSPs putative activity against food allergy. GLSP may be used as a functional food component for allergic patients.

Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain

Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharide from Porphyra haitanensis

The immunoregulatory activity of sulfated polysaccharide from Porphyra haitanensis (PHPS) was investigated in a RAW264.7 macrophages cell model and a BALB/c murine model. The subpopulation of dendritic cells (DCs) and regulatory T cells (Tregs) from PHPS-treated mice splenocytes were also measured by flow cytometry. Consistent with previous reports, we showed that PHPS increased the phagocytosis of RAW264.7 macrophages, and enhanced the secretion of interleukin (IL)-6, IL-10 and tumor necrosis factor-α (TNF-α). Meanwhile, PHPS induced the production of nitric oxide via the Jun N-terminal kinase (JNK) and the Janus kinase (JAK2) signaling pathways in RAW264.7 macrophages. Furthermore, PHPS promoted the proliferation of mice lymphocytes, inducing the generation of TNF-α and IL-10 in vivo, as well as the subpopulation of CD4+ splenic T lymphocytes, DCs, and Tregs. These results indicated that PHPS plays key roles in immunoregulation and may be apply to develop new health foods.

Assessment of the sensitizing capacity and allergenicity of enzymatic cross‐linked arginine kinase, the crab allergen

SCOPE The enzymatic cross-linking of an allergen by food processing may alter its sensitization potential. In this study, the IgE-binding activity and allergenicity of cross-linked thermal polymerized arginine kinase (CL-pAK) were investigated. METHODS AND RESULTS The IgE-binding activity and stability of CL-pAK were analyzed by immunological and proteomics methods. The sensitization and potency to induce oral tolerance of CL-pAK were tested using in vivo assays and a cell model. According to the results of inhibition of ELISA, the half inhibitory concentration of AK after cross-linking changed from 1.13 to 228.36 μg/mL. The results of in vitro digestion demonstrated that CL-pAK showed more resistance to gastrointestinal digestion than native AK. Low allergenicity and capacity to induce oral tolerance in mice were shown by the sera levels of AK-specific antibodies and T-cell cytokine production. Exposure of RBL-2H3 cells to CL-pAK compared with AK, resulted in lower levels of mast degranulation and histamine. CONCLUSION Enzymatic cross-linking with thermal polymerization of AK by tyrosinase and caffeic acid had high potential in mitigating IgE-binding activity and allergenicity, which were influenced by altering the molecular and immunological features of the shellfish protein.

Allergenicity and Oral Tolerance of Enzymatic Cross-Linked Tropomyosin Evaluated Using Cell and Mouse Models

The enzymatic cross-linking of proteins to form high-molecular-weight compounds may alter their sensitization potential. The IgG-/IgE-binding activity, digestibility, allergenicity, and oral tolerance of cross-linked tropomyosin with tyrosinase (CTC) or horseradish peroxidase (CHP) were investigated. ELISA results demonstrated CTC or CHP reduced its IgE-binding activity by 34.5 ± 1.8 and 63.5 ± 0.6%, respectively. Compared with native tropomyosin or CTC, CHP was more easily digested into small fragments; CHP decreased the degranulation of RBL-2H3 cells and increased endocytosis by dendritic cells. CHP can induce oral tolerance and reduce allergenicity in mice by decreasing IgE and IgG1 levels in serum, the production of T-cell cytokines, and the percentage composition of dendritic cells. These findings demonstrate CHP has more potential of reducing the allergenicity than CTC via influencing the morphology of protein, changing the original method of antigen presentation, modulating the Th1/Th2 immunobalance, and inducing the oral tolerance of the allergen tropomyosin.

Suppression of Th2 immune responses by the sulfated polysaccharide from Porphyra haitanensis in tropomyosin-sensitized mice.

The sulfated polysaccharide from Porphyra was hypothesized to exhibit immunoregulatory, anti-tumor and anti-inflammatory activity, but its anti-allergic activity is not fully understood. Therefore, the aim of this study was to isolate sulfated polysaccharide from Porphyra haitanensis (PHPS) and investigate its anti-allergic potential using a tropomyosin (TM)-induced mouse allergy model. Intraperitoneal injection of PHPS suppressed the allergic reaction by modulating serum IgE, IgG1 and IgG2a levels in mice. In particular, when PHPS was injected prior to the first immunization with TM, the IgE level decreased by 34.2% compared with the control (PBS) group. Oral therapeutic administration of PHPS to TM-sensitized mice decreased histamine release and repaired the pathology in the jejunum of the small intestine. In vitro, the mRNA expressions of the TM-induced Th2 cytokines (interleukin-4 (IL-4), IL-5 and IL-13) in splenic lymphocytes were reduced by PHPS; however, the expression of Th1 and regulatory cytokines (interferon gamma (IFN-γ) and IL-10) were up-regulated in PHPS-treated splenic lymphocytes. In the splenic lymphocyte supernatant, the IL-4, IL-13 and IFN-γ levels were also regulated by PHPS. Moreover, PHPS induced IFN-γ secretion via the Jun N-terminal kinase (JNK) and Janus kinase 2 (JAK2) signaling pathways. Therefore, these results suggest that PHPS suppresses the TM-induced allergic reaction, possibly by modulating the imbalance of the Th1/Th2 immune response.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

Eucheuma cottonii Sulfated Oligosaccharides Decrease Food Allergic Responses in Animal Models by Up-regulating Regulatory T (Treg) Cells

In the present study, the anti-food allergy activity of Eucheuma cottonii sulfated oligosaccharide (ESO) was investigated. ESO was obtained by enzymatic degradation and purified by column chromatography. RBL-2H3 cells and BALB/c mouse model were used to test the anti-food allergy activity of ESO. The effects of ESO on the regulatory T (Treg) cells and bone marrow-derived mast cells (BMMCs) were investigated by flow cytometry. The results of in vivo assay showed that ESO decreased the levels of mast cell protease-1 and histamine and inhibited the levels of specific IgE by 77.7%. In addition, the production of interleukin (IL)-4 and IL-13 was diminished in the ESO groups compared to the non-ESO-treated group. Furthermore, ESO could up-regulate Treg cells by 22.2-97.1%. In conclusion, ESO decreased the allergy response in mice by reducing basophil degranulation, up-regulating Treg cells via Forkhead box protein 3 (Foxp3), and releasing IL-10. ESO may have preventive and therapeutic potential in allergic disease.

Inhibitory activities of compounds from the marine actinomycete Williamsia sp. MCCC 1A11233 variant on IgE-mediated mast cells and passive cutaneous anaphylaxis

The compounds of the deep-sea-derived marine Williamsia sp. MCCC 1A11233 (CDMW) were isolated, which are secondary metabolites of the actinomycetes. In this study, seven kinds of CDMW were found to decrease degranulation and histamine release in immunoglobulin E (IgE)-mediated rat basophilic leukemia (RBL)-2H3 cells. The production of cytokines (tumor necrosis factor-α, interleukin-4) was inhibited by these CDMW in RBL-2H3 cells, and their chemical structures were established mainly based on detailed analysis of their NMR spectra. CDMW-3, CDMW-5, and CDMW-15 were further demonstrated to block mast cell-dependent passive cutaneous anaphylaxis in IgE-sensitized mice. Bone marrow mononuclear cells (BMMCs) were established to clarify the effect of CDMW-3, CDMW-5, and CDMW-15 on mast cells. The seven kinds of CDMW decreased the degranulation and histamine release of BMMCs. Furthermore, flow cytometry results indicated that CDMW-3, CDMW-5, and CDMW-15 increased the annexin+ cell population of BMMCs. In conclusion, CDMW-3, CDMW-5, and CDMW-15 have obvious antiallergic activity due to induction of the apoptosis of mast cells.

Deep-sea-derived butyrolactone I suppresses ovalbumin-induced anaphylaxis by regulating mast cell function in a murine model

Deep-sea-derived butyrolactone I (BTL-I), which was identified as a type of butanolide, was isolated from Aspergillus sp. Ovalbumin (OVA)-induced BALB/c anaphylaxis was established to explore the antifood allergic activity of BTL-I. As a result, BTL-I was able to alleviate OVA-induced allergy symptoms, reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and decrease the population of mast cells in the spleen and mesenteric lymph nodes. BTL-I also significantly suppressed mast-dependent passive cutaneous anaphylaxis. Additionally, the maturation of bone marrow-derived mast cells (BMMCs) declined as BTL-I caused down-regulation of c-KIT receptors. Furthermore, molecular docking analyses revealed that BTL-I interacted with the inhibitory receptor, FcγRIIB. In conclusion, the reduction of mast cell function by deep-sea-derived BTL-I as well as its interactions with the inhibitory receptor, FcγRIIB, may contribute to BTL-I-related protection against food anaphylaxis.