Comparison of adjuvant mechanisms of medium-chain triacylglycerol in a mouse FITC-induced contact hypersensitivity model.

The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA.

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

The role of Piper chaba Hunt. and its pure compound, piperine, on TRPV1 activation and adjuvant effect.

BACKGROUND: Piper chaba Hunt. is used as an ingredient in Thai traditional preparation for arthritis. Its isolated compound is piperine which shows anti-inflammatory activity. Piperine produces a burning sensation because it activates TRPV1 receptor. The TRPV1 activation involved with the analgesic and adjuvant effect. P. chaba Hunt. has not been reported about TRPV1 activation and adjuvant effect. The aim of this study was to investigate the effect of P. chaba extract and piperine on TRPV1 receptor, which is considered as a target for analgesic and their adjuvant effects to support the development of an analgesic drug from herbal medicine. METHODS: The effect of P. chaba extract and piperine on HEK cells expressing TRPV1 channel was examined by calcium imaging assay. Adjuvant effects of P. chaba extract and piperine were investigated by a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) model in mice. RESULTS: P. chaba extract induced calcium influx with EC50 value of 0.67 µg/ml. Piperine induced calcium influx with EC50 value of 0.31 µg/ml or 1.08 µM. For mouse CHS model, we found that 1% piperine, 5% piperine, 1% P. chaba extract and 5% P. chaba extract significantly enhanced sensitization to FITC as revealed by ear swelling responses. CONCLUSION: P. chaba extract and piperine activated TRPV1 channel and enhanced contact sensitization to FITC.

Lettuce-derived secretory IgA specifically neutralizes the Shiga toxin 1 activity.

MAIN CONCLUSION: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

Enhancement of mouse contact hypersensitivity appears with a short chain triacylglycerol but not with a long chain one.

The prevalence of skin allergies could be partly due to the increased exposure to chemicals from consumer products. Chemicals that can enhance hypersensitivity caused by other chemicals are the focus of this study. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. We have also found that tributyrin, a triacylglycerol (TAG) with three butyric acids, enhances sensitization to FITC. To elucidate such an enhanced skin sensitization might be based on a general feature of TAG, we compared tributyrin and triolein, a natural TAG, as to an adjuvant effect on FITC-CHS. Triolein is the dominant TAG in olive oil and contains long chain mono-unsaturated fatty acids. Unlike tributyrin and dibutyl phthalate (DBP), triolein did not exhibit an adjuvant effect. With triolein, enhancement of FITC-presenting CD11c+ dendritic cell trafficking to draining lymph nodes was weak, and the activation status of DC, as revealed as CD86 expression, was low. We found a difference in the pattern of skin cytokine production, i.e., that thymic stromal lymphopoietin was produced with DBP and interleukin-1ß with tributyrin. Triolein did not induce either of these cytokines. This illustrates that the adjuvant effect of tributyrin on FITC-CHS is not a general phenomenon for TAGs. Although beneficial effects may be expected through oral administration of tributyrin, the effect on skin immune systems should be considered.

Plant-derived secretory component forms secretory IgA with shiga toxin 1-specific dimeric IgA produced by mouse cells and whole plants.

KEY MESSAGE: A key module, secretory component (SC), was efficiently expressed in Arabidopsis thaliana. The plant-based SC and immunoglobulin A of animal or plant origin formed secretory IgA that maintains antigen-binding activity. Plant expression systems are suitable for scalable and cost-effective production of biologics. Secretory immunoglobulin A (SIgA) will be useful as a therapeutic antibody against mucosal pathogens. SIgA is equipped with a secretory component (SC), which assists the performance of SIgA on the mucosal surface. Here we produced SC using a plant expression system and formed SIgA with dimeric IgAs produced by mouse cells as well as by whole plants. To increase the expression level, an endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), was added to mouse SC (SC-KDEL). The SC-KDEL cDNA was inserted into a binary vector with a translational enhancer and an efficient terminator. The SC-KDEL transgenic Arabidopsis thaliana produced SC-KDEL at the level of 2.7% of total leaf proteins. In vitro reaction of the plant-derived SC-KDEL with mouse dimeric monoclonal IgAs resulted in the formation of SIgA. When reacted with Shiga toxin 1 (Stx1)-specific ones, the antigen-binding activity was maintained. When an A. thaliana plant expressing SC-KDEL was crossed with one expressing dimeric IgA specific for Stx1, the plant-based SIgA exhibited antigen-binding activity. Leaf extracts of the crossbred transgenic plants neutralized Stx1 cytotoxicity against Stx1-sensitive cells. These results suggest that transgenic plants expressing SC-KDEL will provide a versatile means of SIgA production.

Skin Sensitization to Fluorescein Isothiocyanate Is Enhanced by Butyl Paraben in a Mouse Model.

Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c+ dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.

Adjuvant effect of short chain triacylglycerol tributyrin on a mouse contact hypersensitivity model.

Little attention has been paid to chemicals that can enhance hypersensitivity caused by other chemicals. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. Furthermore, phthalate esters with such an enhancing effect were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels, which are expressed on a part of sensory neurons, using a TRPA1-expressing cell line. In this study, we examined these activities of esters comprising glycerol and a short chain fatty acid, i.e. dibutyrin and tributyrin. We carried out chemical synthesis of dibutyrin isomers. Each dibutyrin isomer weakly activated TRPA1 and slightly enhanced skin sensitization to FITC. Unexpectedly, TRPA1 activation and enhancement of FITC-CHS were much more evident in the presence of tributyrin. Mechanistically, tributyrin induced increased dendritic cell trafficking from the skin to draining lymph nodes. Tributyrin enhanced interferon-γ (IFN-γ) production by draining lymph nodes, while its effect on interleukin-4 (IL-4) production was relatively less prominent. These results suggested that tributyrin concomitantly caused TRPA1 activation and an adjuvant effect on FITC-CHS.

An Aliphatic Ester Diisopropyl Sebacate Exhibited an Adjuvant Effect on Fluorescein Isothiocyanate-Induced Contact Hypersensitivity Mouse Models.

Alternative plasticizers have become more popular due to health concerns about phthalate esters. We demonstrated that phthalate esters enhanced skin sensitization to fluorescein isothiocyanate (FITC) in mouse contact hypersensitivity models. Alternative plasticizers have not been well studied as to their effect on the immune system. We previously found that diisopropyl adipate (DIPA), an aliphatic dicarboxylic acid ester, enhanced skin sensitization to FITC. Sebacate esters are also widely used as alternative plasticizers. Here we tested diisopropyl sebacate (DIPS), which has the same alcohol with an aliphatic dicarboxylic acid of longer chain, using BALB/c mice. The results showed that DIPS facilitated skin sensitization to FITC and increased FITC-presenting dendritic cell trafficking from the skin to draining lymph nodes. Furthermore, DIPS activated transient receptor potential ankyrin 1 (TRPA1). The latter feature has been commonly observed for phthalate esters and DIPA, which have adjuvant effects. In summary, the adjuvant effect of a sebacate ester was demonstrated in a mouse model.

Dibutyl Phthalate Rather than Monobutyl Phthalate Facilitates Contact Hypersensitivity to Fluorescein Isothiocyanate in a Mouse Model.

Dibutyl phthalate (DBP) is a plasticizer used for many consumer products including cosmetics. Potential health concerns regarding DBP include reproductive and developmental toxicity, endocrine disruption and neurotoxicity. DBP is a high priority chemical as to reduction of exposure of children to it. Through reproductive toxicity studies, monobutyl phthalate (MBP) has been proposed to be the active metabolite derived from DBP. We previously demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We have also shown that DBP enhanced skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model. Through MBP formation by esterase in the skin, it is possible that MBP exerts a major effect on the biological activity we observed. To test this possibility, we directly compared DBP and MBP. A more than 40-fold higher concentration of MBP as compared with DBP was required for activation of TRPA1 in vitro. Unlike DBP, MBP did not enhance skin sensitization to FITC. These results demonstrated that DBP directly, i.e., not through its metabolite MBP, activates TRPA1 and enhances FITC-CHS. It is noteworthy that butyl benzoate, a related compound, activated TRPA1 and enhanced FITC-CHS.

Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA.

Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

Dibutyl Maleate and Dibutyl Fumarate Enhance Contact Sensitization to Fluorescein Isothiocyanate in Mice.

Di-n-butyl phthalate (DBP), a phthalate ester, has been shown to have an adjuvant effect on fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse models. Di-n-butyl maleate (DBM), widely used as a plasticizer for industrial application, has been reported to cause dermatitis in humans. DBM is a butyl alcohol ester of di-carboxylic acid that represents a part of the DBP structure, while di-n-butyl fumarate (DBF) is a trans isomer of DBM. We examined whether DBM or DBF exhibits an adjuvant effect like DBP does. When BALB/c mice were epicutaneously sensitized with FITC in the presence of DBM or DBF, the FITC-specific CHS response was enhanced, as we have observed for DBP. As to underlying mechanisms, DBM and DBF facilitated the trafficking of FITC-presenting CD11c(+) dendritic cells (DCs) from skin to draining lymph nodes and increased the cytokine production by draining lymph nodes. In conclusion, DBM and DBF may have an effect that aggravates contact dermatitis through a skin sensitization process.

Lack of Impact of High Dietary Vitamin A on T Helper 2-Dependent Contact Hypersensitivity to Fluorescein Isothiocyanate in Mice.

Overuse of vitamin A as a dietary supplement is a concern in industrialized countries. High-level dietary vitamin A is thought to shift immunity to a T helper 2 (Th2)-dominant one, resulting in the promotion of allergies. We have been studying a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model that involves Th2-type immunity. We fed a diet with a high retinyl palmitate content (250 international units (IU)/g diet) or a control diet (4 IU/g diet) to BALB/c mice for three weeks. No augmentation of FITC-induced CHS was found in mice fed the diet with a high vitamin A content, although accumulation of the vitamin was confirmed in the livers of these animals. The results indicated that relatively short-term feeding of the high-level vitamin A diet did not influence the Th2-driven response at a stage with significant retinol accumulation in the liver. The results were in contrast to the high-dose pyridoxine diets that produced a reduced response in FITC-induced CHS.

Adjuvant Effect of an Alternative Plasticizer, Diisopropyl Adipate, on a Contact Hypersensitivity Mouse Model: Link with Sensory Ion Channel TRPA1 Activation.

Due to health concerns about phthalate esters, the use of alternative plasticizers is being considered. Phthalate esters enhance skin sensitization to fluorescein isothiocyanate (FITC) in mouse models. We have demonstrated that phthalate esters stimulate transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We also found a correlation between TRPA1 activation and the enhancing effect on FITC-induced contact hypersensitivity (CHS) when testing various types of phthalate esters. Here we investigated the effects of an alternative plasticizer, diisopropyl adipate (DIA). Activation of TRPA1 by DIA was demonstrated by calcium mobilization using Chinese hamster ovary cells expressing TRPA1 in vitro. The effect of DIA was inhibited by a TRPA1-specific antagonist, HC-030031. The presence of DIA or dibutyl phthalate (DBP; positive control) during skin sensitization of BALB/c mice to FITC augmented the CHS response, as revealed by the level of ear-swelling. The enhancing effect of DIA was inhibited by in vivo pretreatment with HC-030031. FITC-presenting CD11c(+) dendritic cell (DC)-trafficking to draining lymph nodes was facilitated both by DIA and by DBP. DBP and DIA were similarly active in the enhancement of interferon-γ production by draining lymph nodes, but the effect on interleukin-4 production was weaker with DIA. Overall, DIA activated TRPA1 and enhanced FITC-induced CHS, as DBP did. The adjuvant effects of adipate esters may need to be considered because they are used as ingredients in cosmetics and drug formulations topically applied to the skin.

Recombinant immunoglobulin A specific for influenza A virus hemagglutinin: production, functional analysis, and formation of secretory immunoglobulin A.

Secretory immunoglobulin (Ig) A (SIgA), comprised of dimeric IgA and secretory component (SC), is believed to provide a defense mechanism on the mucosal surface. Influenza A virus (IAV) hemagglutinin (HA)-specific SIgA is thought to play an important role in the prevention of IAV infection. However, the topical application of preformed IAV-specific SIgA has not been shown to prevent IAV infection. This is due to the difficulty in the production of antigen-specific IgA monoclonal antibodies (mAbs) and monoclonal SIgA. Here, a recombinant hybrid IgA (HIgA) was established that utilizes variable regions of an HA-specific mouse IgG mAb and the heavy chain constant region of a mouse IgA mAb. We expressed the dimeric HIgA in Chinese hamster ovary-K1 (CHO-K1) cells. When in vitro IAV infection of Madin-Darby canine kidney (MDCK) cells was tested, 10 times lower concentrations of HIgA were able to inhibit it as compared with an HA-specific IgG with the same variable regions. A functional hybrid secretory IgA (HSIgA) was also produced through incubation of the dimeric HIgA with recombinant mouse SC in vitro. It was demonstrated that HSIgA could be separated from the dimeric HIgA on size exclusion chromatography. This study provides a basic strategy for investigating the role of SIgA upon IAV infection on the mucosal surface.

Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies.

Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by size-exclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.

Stable expression and characterization of monomeric and dimeric recombinant hybrid-IgG/IgA immunoglobulins specific for Shiga toxin.

Antigen-specific immunoglobulin A (IgA) may be useful for preventing infectious diseases through passive immunization on the mucosal surface. We previously established mouse IgA and IgG monoclonal antibodies (mAbs) specific for the binding subunit of Shiga toxin 1 (Stx1B). We also developed a recombinant hybrid-IgG/IgA, in which variable regions from the IgG mAb were present. The binding activity of recombinant hybrid-IgG/IgA was verified by transient expression. Aiming at a constant supply, we established Chinese hamster ovary cells stably expressing monomeric or dimeric hybrid-IgG/IgA. The cDNAs encoding heavy and light chains were co-expressed for the monomeric hybrid-IgG/IgA, while those encoding heavy, light, and joining chains were co-expressed for the dimeric one. Serum-free culture supernatants of the cloned transfectants were subjected to size-exclusion chromatography. The elution patterns showed that the binding to immobilized Stx1B and the immunoblot signals of assembled immunoglobulins were correlated. In the transfectant for the dimeric hybrid-IgG/IgA, both monomers and dimers were observed. Size-exclusion chromatography enabled us to prepare a sample of the dimeric hybrid-IgG/IgA devoid of the monomeric one. The monomeric and dimeric forms of hybrid-IgG/IgA were prepared from the respective transfectants to examine the neutralization of Stx1. After pretreatment with monomeric or dimeric hybrid-IgG/IgA, the cytotoxicity of Stx1 toward Vero cells was abolished. Furthermore, the dimeric form was more than 10-fold more effective than the monomeric one in terms of toxin neutralization. These results suggest that the tetravalent feature of the binding sites of the dimeric hybrid-IgG/IgA contributes to the efficacy of toxin neutralization.

Production of hybrid-IgG/IgA plantibodies with neutralizing activity against Shiga toxin 1.

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.

Lack of transient receptor potential melastatin 8 activation by phthalate esters that enhance contact hypersensitivity in mice.

We studied the involvement of sensory neurons in skin sensitization to allergens using a mouse model in which the T-helper type 2 response is essential. Skin sensitization to fluorescein isothiocyanate (FITC) has been shown to be enhanced by several phthalate esters, including dibutyl phthalate (DBP). For different types of phthalate esters, we found a correlation between the ability of transient receptor potential (TRP) A1 activation and that of enhancing skin sensitization. A TRPA1-specific antagonist, HC-030031, was shown to suppress skin sensitization in the presence of DBP. However, since phthalate esters also activate TRPV1, phthalate esters could activate other types of TRP channels non-selectively. Furthermore, sensitization to FITC is also enhanced by menthol, which activates TRPA1 and TRPM8. Here we established an in vitro system for measuring TRPM8 activation. The selectivity for TRPM8 was established by the fact that two TRPM8 agonists (menthol and icilin) induced calcium mobilization, whereas agonists of TRPA1 and TRPV1 did not. We demonstrated that phthalate esters do not activate TRPM8. TRPA1-antagonist HC-030031 did not inhibit TRPM8 activation induced by menthol or icilin. These results show that phthalate esters activate TRPA1 and TRPV1 with selectivity. TRPM8 activation is not likely to be involved in the sensitization to FITC.