Tomoharu Yokooji

Common food allergens and their IgE-binding epitopes

Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cows milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cows milk, wheat, shrimp, and peanut.

Role of intestinal efflux transporters in the intestinal absorption of methotrexate in rats

The role of intestinal efflux transporters such as P‐glycoprotein (P‐gp), breast cancer resistance protein (BCRP) and multidrug resistance‐associated proteins (MRPs) in intestinal absorption of methotrexate was examined in rats. In everted intestine, the mucosal efflux of methotrexate after application to serosal side was higher in jejunum than ileum, and the efflux in jejunum was suppressed by pantoprazole, a BCRP inhibitor, and probenecid, an MRP inhibitor, but not by verapamil, a P‐gp inhibitor. The mucosal methotrexate efflux in ileum was suppressed by pantoprazole, but not by other inhibitors. On the other hand, the serosal efflux of methotrexate after application to mucosal side was greater in ileum than jejunum, and was suppressed by probenecid. In in‐vivo rat studies, the intestinal absorption of methotrexate was significantly higher when methotrexate was administered to ileum than jejunum. Pantoprazole increased methotrexate absorption from jejunum and ileum. Probenecid increased the absorption of methotrexate from jejunum but decreased the absorption from ileum, as evaluated by peak plasma methotrexate levels. In conclusion, BCRP and MRPs are involved in the regional difference in absorption of methotrexate along the intestine, depending on their expression sites.

Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

BACKGROUND Non-steroidal anti-inflammatory drugs (NSAIDs), especially aspirin, and food additives (FAs) may exacerbate allergic symptoms in patients with chronic idiopathic urticaria and food-dependent exercise-induced anaphylaxis (FDEIA). Augmentation of histamine release from human mast cells and basophils by those substances is speculated to be the cause of exacerbated allergic symptoms. We sought to investigate the mechanism of action of aspirin on IgE-mediated histamine release. METHODS The effects of NSAIDs, FAs or cyclooxygenase (COX) inhibitors on histamine release from human basophils concentrated by gravity separation were evaluated. RESULTS Benzoate and tartrazine, which have no COX inhibitory activity, augmented histamine release from basophils similar to aspirin. In contrast, ibuprofen, meloxicam, FR122047 and NS-398, which have COX inhibitory activity, did not affect histamine release. These results indicate that the augmentation of histamine release by aspirin is not due to COX inhibition. It was observed that aspirin augmented histamine release from human basophils only when specifically activated by anti-IgE antibodies, but not by A23187 or formyl-methionyl-leucyl-phenylalanine. When the IgE receptor signaling pathway was activated, aspirin increased the phosphorylation of Syk. Moreover, patients with chronic urticaria and FDEIA tended to be more sensitive to aspirin as regards the augmentation of histamine release, compared with healthy controls. CONCLUSIONS Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

Characterization of intestinal absorption of mizoribine mediated by concentrative nucleoside transporters in rats.

Mizoribine, an imidazole nucleoside, is an inhibitor of purine synthesis and has been used as an orally available immunosuppressive agent in human renal transplantation. In the present study, the intestinal absorption of mizoribine was characterized by examining the contribution of concentrative nucleoside transporters (CNT1, CNT2) in rats. When mizoribine was administered orally in conscious rats, the bioavailability of mizoribine estimated by urinary excretion percentage of unchanged mizoribine was a dose dependent: 53.1+/-6.0% at 5 mg/kg and 24.0+/-5.1% at 20 mg/kg. In in-situ loop studies, the disappearance rate, or absorption rate, of mizoribine from the intestinal lumen was comparable between 1 and 5 mg/kg, but significantly lower at 25 mg/kg. Coadministration of adenosine (a substrate of both CNT1 and CNT2), thymidine (a CNT1 substrate) and inosine (a CNT2 substrate) significantly suppressed the intestinal mizoribine absorption, depending on the nucleoside concentrations coadministered. Gemcitabine (a pyrimidine nucleoside analogue, a CNT1 substrate) and ribavirin (a purine nucleoside analog, a CNT2 substrate) also significantly suppressed the mizoribine intestinal absorption. Bile salts such as sodium cholate and sodium glycocholate (10 mM) also significantly suppressed the intestinal mizoribine absorption, but not ribavirin absorption. Mizoribine is an amphoteric compound, however, the suppression of intestinal absorption by bile salts was not ascribed to the electrostatic interaction or micellar formation between mizoribine and bile salts. In conclusion, the intestinal absorption of mizoribine is mediated by CNT1 and CNT2, and nucleoside-derived drugs such as gemcitabine and ribavirin can suppress the intestinal absorption of mizoribine. Bile salts such as sodium glycocholate were also found to cause interaction with mizoribine.

Site‐specific bidirectional efflux of 2,4‐dinitrophenyl‐S‐glutathione, a substrate of multidrug resistance‐associated proteins, in rat intestine and Caco‐2 cells

The site‐specific function of multidrug‐resistance‐associated proteins (MRPs), especially MRP2 and MRP3, was examined in rat intestine and human colon adenocarcinoma (Caco‐2) cells. The MRP function was evaluated pharmacokinetically by measuring the efflux transport of 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), an MRP substrate, after application of 1‐chloro‐2,4‐dinitrobenzene (CDNB), a precursor of DNP‐SG. The expression of rat and human MRP2 and MRP3 was analysed by Western blotting. The rat jejunum exhibited a higher apical MRP2 and a lower basolateral MRP3 expression than ileum. In accordance with the expression level, DNP‐SG efflux to the mucosal surface was significantly greater in jejunum, while serosal efflux was greater in ileum. Site‐specific bidirectional efflux of DNP‐SG was also observed in in‐vivo studies, in which portal and femoral plasma levels and biliary excretion rate of DNP‐SG were significantly higher when CDNB was administered to ileum. Caco‐2 cells also showed a bidirectional efflux of DNP‐SG. Probenecid, an MRP inhibitor, significantly suppressed the mucosal efflux in jejunum and serosal efflux in ileum. In contrast, probenecid significantly suppressed both apical and basolateral efflux of DNP‐SG in Caco‐2 cells, though the inhibition was of small magnitude. In conclusion, the efflux of DNP‐SG from enterocytes mediated by MRPs exhibited a significant regional difference in rat intestine, indicating possible variability in intestinal bioavailabilities of MRP substrates, depending on their absorption sites along the intestine.

Modulation of intestinal transport of 2,4-dinitrophenyl-S-glutathione, a multidrug resistance-associated protein 2 substrate, by bilirubin treatment in rats

The effect of bilirubin treatment on intestinal transport of 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), a substrate of multidrug resistance‐associated protein 2 (MRP2), after application of 1‐chloro‐2, 4‐dinitrobenzene (CDNB), a precursor of DNP‐SG, was examined in rat intestine by the in‐vitro everted sac, in‐situ re‐circulating perfusion, and in‐situ loop methods. CDNB was taken up rapidly by jejunum and ileum, and the consequent intestinal efflux of DNP‐SG, a glutathione conjugated metabolite of CDNB, was significantly higher in jejunum than in ileum in the in‐situ and in‐vitro studies. Co‐administration of bilirubin (100 μM), as well as probenecid (1 mM) or ciclosporin (100 μM), with CDNB decreased the DNP‐SG efflux in jejunum significantly, but not in ileum. The suppression of DNP‐SG efflux in jejunum was also observed after intravenous administration of bilirubin (85.5 μmol kg−1), in which plasma bilirubin glucuronide levels were approximately 100 μM. In the in‐vitro metabolism study, bilirubin exerted no significant effect on CDNB metabolism in the intestinal S9 fraction (supernatant of 9000 g). These results suggested that the diseased states accompanied with hyperbilirubinaemia might have increased the intestinal absorption, or oral bioavailability, of MRP2 substrates by suppressing MRP2 function at the proximal intestinal region.

Site-specific contribution of proton-coupled folate transporter/haem carrier protein 1 in the intestinal absorption of methotrexate in rats.

Objectives Methotrexate is reportedly a substrate for proton‐coupled folate transporter/haem carrier protein 1 (PCFT/HCP1) and reduced folate carrier 1 (RFC1). In this study, we examined the contribution of PCFT/HCP1 and RFC1 in the intestinal absorption of methotrexate in rats.

Intestinal absorption of lysozyme, an egg-white allergen, in rats: kinetics and effect of NSAIDs.

The absorption pathway(s) of a representative food allergen, lysozyme, and the mechanisms of lysozyme absorption facilitated by non-steroidal anti-inflammatory drugs were examined by intestinal closed-loop and re-circulating perfusion methods in rats. The absorption rate of fluorescein isothiocyanate (FITC)-labeled lysozyme in the proximal intestine was higher than that for a marker of non-specific absorption, FD-10, and was suppressed by colchicine (endocytosis inhibitor). Aspirin increased the absorption of FITC-lysozyme in the proximal intestine with no effects on tissue accumulation. Diclofenac facilitated FITC-lysozyme absorption, but meloxicam and loxoprofen exerted no effects on absorption. Co-administration of misoprostol (synthetic prostaglandin-E1 analog) with aspirin significantly ameliorated the aspirin-facilitated absorption of FITC-lysozyme to the same level as that seen with controls. Thus, lysozyme absorption was mediated by endocytic and paracellular pathways in the proximal intestine, and was facilitated by aspirin and diclofenac after impairment of the paracellular pathway. Misoprostol may suppress the allergen absorption facilitated by aspirin.

Effect of genistein, a natural soy isoflavone, on the pharmacokinetics and intestinal toxicity of irinotecan hydrochloride in rats.

The effect of genistein, a natural soy isoflavone, on pharmacokinetics and intestinal toxicity, or late‐onset diarrhoea, of irinotecan hydrochloride (CPT‐11) was examined in rats.

Modulated function of tissue efflux transporters under hyperbilirubinemia in rats.

The effect of hyperbilirubinemia on the function of tissue efflux transporters such as multidrug resistance-associated proteins (Mrps) and organic anion transporting polypeptides (Oatps) was examined by measuring tissue accumulation of 2,4-dinitrophenyl-S-glutathione (DNP-SG) after intravenous administration of 1-chloro-2,4-dinitrobenzene (CDNB), a precursor of DNP-SG, in rats. DNP-SG is known as a substrate of both Mrps and Oatps. Hyperbilirubinemia was induced by a bolus intravenous administration of bilirubin. Treatment with probenecid, an inhibitor for both Mrps and Oatps, significantly increased DNP-SG concentrations in the brain, heart, liver, kidney, jejunum, spleen and skeletal muscle as compared with those in control rats, suggesting the expression of some probenecid-sensitive efflux transporters in these tissues. Rats with more than 70 microM of unconjugated/conjugated bilirubin in plasma exhibited significantly higher DNP-SG concentrations in the brain, liver, jejunum, and skeletal muscle. These results suggested that probenecid-sensitive efflux transporters in tissues were suppressed functionally under hyperbilirubinemia. In conclusion, hyperbilirubinemia accompanied by obstructive jaundice is caused by various disease states, which may increase harmful toxicities of exogenously administered Mrps and/or Oatps substrate drugs at various tissues, by suppressing the efflux transporters function systemically.