Analysis of the allergenicity and B cell epitopes in tropomyosin of shrimp (Litopenaeus vannamei) and correlation to cross-reactivity based on epitopes with fish (Larimichthys crocea) and clam (Ruditapes philippinarum).

Tropomyosin (TM) is a highly conserved protein that considered as the major allergen of crustacean and mollusk species, while, fish-TM also shares high homology with low allergenicity. In this study, the amino acid sequence, B cell epitopes and allergenicity of shrimp (Litopenaeus vannamei), which is widely consumed, were evaluated by using immunoinformatic tools, dot-blot, enzyme-linked immunosorbent assay (ELISA) and mediator release assay. Meanwhile, cross-reactivity of allergic epitopes of fish-TM, shrimp-TM and clam-TM were assessed. Results showed that three IgE-binding epitopes (X1: 47-61, QKRMQQLENDLDQVQ; X2: 97-108, EDLERSEERLNT and X3: 244-257, RSVQKLQKEVDRLE) of shrimp-TM also exhibited degranulation ability. In comparison with epitopes from shrimp-TM, those from clam-TM showed high cross-reactivity (>80%) and degranulation ability, while those from fish-TM showed low cross-reactivity (<20%). These findings would apply a new understanding of the cross-reactivity of TM from fish, shrimp and clam in terms of allergenic epitopes.

Influence of nonthermal extraction technique and allergenicity characteristics of tropomyosin from fish (Larimichthys crocea) in comparison with shrimp (Litopenaeus vannamei) and clam (Ruditapes philippinarum).

Recent reports showed that patients could be sensitized to fish tropomyosin (TM), who exhibited clinical symptoms. However, little information is available on differences in TM immune cross-reactivity among fish, shrimp and clam. Moreover, allergenicity might change during the food processing owing to the change of protein structure. In this study, we developed a nonthermal extraction technique to purified TM, IgG/IgE binding, cross-reactivity and structures were compared. Results showed that raw and boiled fish-TM were not cross reactive and had weak recognition of shrimp, while, shrimp-TM and clam-TM were cross reactive. The ELISA further confirmed that fish-TM was not able to trigger allergic immune response in shrimp sensitive subjects, while, surface hydrophobicity of fish-TM was higher. The study demonstrated that fish-TM, being with high sequence similarity, did not have cross-reactivity with shrimp and clam-TM. They could have a variable degree of IgE binding depending on subject sensitivity and allergenicity.

Banana lectin (BanLec) induces non-specific activation of basophils and mast cells in atopic subjects.

Summary: Dietary lectins play a major role in the activation of mast cells / basophils by bridging cell surface IgE glycans to release histamine and other mediators. In the present study, the effect of mannose / glucose-specific banana lectin (BanLec) on the activation of mast cells / basophils from non-atopic and atopic subjects has been investigated. BanLec was purified from banana pulp in a yield of 7 mg/kg. Leukocytes isolated from heparinized blood of non-atopic / atopic subjects were used for quantitation of the released histamine. Approximately 28.2% of the atopics (n = 117) was positive by skin prick test (SPT) to purified BanLec (100 µg/mL concentration), and all the non-atopics (n = 20) were negative. Maximal release of histamine was seen at 2 µg of BanLec. In percent histamine release, an increase of 35-40% is observed in case of atopics (n = 7) compared to non-atopics (n = 5), and the histamine release from atopic and non-atopic subjects correlates fairly well with the total serum IgE levels (R2 = 0.817). BanLec also induces release of histamine (26.7%) from mast cells present in rat peritoneal exudate cells. BanLec can significantly activate and degranulate mast cells and basophils by cross-linking the trimannosidic core mannose of IgE glycans in atopic population as compared to non-atopic population; the activation is marginal in the case of non-atopics.

Immunomodulatory glc/man-directed Dolichos lablab lectin (DLL) evokes anti-tumour response in vivo by counteracting angiogenic gene expressions.

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti-cancer agents. The present investigation sought to demonstrate the anti-proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non-specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)-2 production. The DLL-conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in-vivo anti-tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL-treated mice showed an up-regulated immunoregulatory cytokine IL-2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF-κB), hypoxia inducible factor 1α (HIF-1 α), matrix metalloproteinase (MMP)-2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti-angiogenic molecule in cancer therapeutics.

Microwave assisted rapid synthesis and biological evaluation of stable copper nanoparticles using T. arjuna bark extract.

Terminalia arjuna (T. arjuna) bark extract is used to reduce Cu(2+)→Cu(0) under microwave irradiation. The formation of copper nanoparticles (CuNPs) is monitored by recording the UV-Vis absorption spectra for surface plasmon resonance (SPR) peak, ~535 nm. The intensity of SPR increased linearly with increasing temperature of the reaction mixture. The formation mechanism of CuNPs is supported by the observed marginal decrease in pH and an increase in solution potential (E) of the reaction mixture. X-ray diffraction (XRD) pattern of the CuNPs agrees with the reported data for Cu metal and the crystallite size is ~23 nm. Fourier transform infrared spectroscopy (FT-IR) and solid-state (13)C NMR shows the presence of plant residues on the CuNPs, i.e., in situ bio-capping is possible by this method. Thermo gravimetric (TG) analysis shows the thermal degradation of plant residue and the conversion of Cu to CuO. Field emission electron microscopic (FESEM) image shows uniform spherical particles obtained here. Elemental analysis by energy dispersive X-ray (EDX) analysis confirms the presence of Cu alone, as expected. The in vitro antimicrobial activity is found to be effective for CuNPs dried at RT when compared to CuNPs dried at 70 °C. In addition, CuNPs shows very good antioxidant property.

Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety.

Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15-fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop.

Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods.

RATIONALE: Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity. METHODS: Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS-PAGE. Residual stained protein bands were measured by image analysis. RESULTS: The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins. CONCLUSIONS: Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.

Allergy to eggplant (Solanum melongena) caused by a putative secondary metabolite.

We describe a case of allergy caused by ingestion of eggplant in an atopic subject. Symptoms included urticaria, itching of the throat, and hoarseness. Skin prick test (SPT) was positive with 4 varieties of eggplant; however, allergen-specific immunoglobulin E was not detected. SPT with fractions of green long eggplant extract obtained by dialysis and ultrafiltration suggested the allergen to be less than 10 kd. SPT following acetone precipitation of eggplant extract revealed that the allergen was present in the supernatant portion. Further analysis by size-exclusion chromatography of the 10 kd filtrate of eggplant extract on Sephadex G-25 followed by SPT of fractions revealed that the causative allergen was a low molecular weight nonprotein secondary metabolite of less than 1 kd. To our knowledge, this is the first report of allergy to the ingestion of eggplant in which a nonprotein secondary metabolite has been detected as an allergen.

Potato lectin activates basophils and mast cells of atopic subjects by its interaction with core chitobiose of cell-bound non-specific immunoglobulin E.

A major factor in non-allergic food hypersensitivity could be the interaction of dietary lectins with mast cells and basophils. Because immunoglobulin E (IgE) contains 10-12% carbohydrates, lectins can activate and degranulate these cells by cross-linking the glycans of cell-bound IgE. The present objective focuses on the effect of potato lectin (Solanum tuberosum agglutinin; STA) for its ability to release histamine from basophils in vitro and mast cells in vivo from non-atopic and atopic subjects. In this study, subjects were selected randomly based on case history and skin prick test responses with food, pollen and house dust mite extracts. Skin prick test (SPT) was performed with STA at 100 microg/ml concentration. Histamine release was performed using leucocytes from non-atopic and atopic subjects and rat peritoneal exudate cells. SPT on 110 atopic subjects using STA showed 39 subjects positive (35%); however, none showed STA-specific IgE; among 20 non-atopic subjects, none were positive by SPT. Maximal histamine release was found to be 65% in atopic subjects (n = 7) compared to 28% in non-atopic subjects (n = 5); the release was inhibited specifically by oligomers of N-acetylglucosamine and correlates well with serum total IgE levels (R(2) = 0.923). Binding of STA to N-linked glycoproteins (horseradish peroxidase, avidin and IgG) was positive by dot blot and binding assay. As potato lectin activates and degranulates both mast cells and basophils by interacting with the chitobiose core of IgE glycans, higher intake of potato may increase the clinical symptoms as a result of non-allergic food hypersensitivity in atopic subjects.