Kunihiko Hashimoto

A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice.

Background: Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions: the higher viscosity displayed the stronger potency. However, the high-viscous GM powders, ordinary konjac powder and highly purified GM were mostly unsuitable for the application to various food industries. Our aims are to develop new physiological functions of low-viscous GM powder, pulverized GM or re-granulated fine GM, using a murine model of atopic dermatitis. Methods: Male 4-week-old NC/Nga mice were fed for 8 weeks on diets containing 5% of two high-viscous and two low-viscous GM powders, respectively. Results: Striking suppression against the aggravation of dermatitis, the increase in scratching behaviors, and the rise in IgE levels was recognized only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. Eczema prevention in the fine GM-fed mice was accompanied by a significant decrease in their plasma IFN-γ levels, a positive regulatory cytokine for atopic skin inflammation. Conclusion: Only the pulverized GM possessed the ability to suppress the development of dermatitis in NC/Nga mice. This is the new immunomodulatory function of low-viscous GM with a small particle size.

Dietary Pulverized Konjac Glucomannan Prevents the Development of Allergic Rhinitis-Like Symptoms and IgE Response in Mice

Konjac is a traditional Japanese food with a peculiar texture, and it has been suggested that its main ingredient, konjac glucomannan (KGM), ameliorates metabolic disorders such as diabetes and hypercholesteremia. We have found that feeding with pulverized KGM (PKGM) prevents skin inflammation in a murine model of atopic dermatitis. Here, we show that dietary PKGM suppresses allergic rhinitis-like symptoms in mice upon immunization and nasal sensitization with ovalbumin (OVA). The PKGM-fed mice showed a much lower frequency of sneezing than in control animals. We also found that PKGM supplementation exclusively suppressed OVA-specific IgE response without affecting IgG1/IgG2a responses as well as systemic Th1/Th2 cytokine production. These results suggest that PKGM can be a beneficial foodstuff in preventing nasal allergy such as seasonal pollinosis.

Dietary Pulverized Konjac Glucomannan Suppresses Scratching Behavior and Skin Inflammatory Immune Responses in NC/Nga Mice

Background: Feeding with pulverized konjac glucomannan (PKGM) suppresses the development of eczema and hyper-IgE production in NC/Nga mice, a model of atopic dermatitis. This study aimed to examine the effects of PKGM on scratching behavior and skin inflammatory immune responses in NC/Nga mice. Methods: Four-week-old NC/Nga mice were maintained for 8 or 9 weeks on diet containing PKGM. Scratching behavior and clinical symptoms were evaluated every 2 weeks. Effects of PKGM on cutaneous inflammation were evaluated by histopathological analysis. Local expression levels of substance P and proinflammatory cytokines were measured by ELISA. Results: An increase in scratching behavior was evident from 6 weeks of age in control mice, but this symptom was dose-dependently inhibited in PKGM-fed mice. Continuous PKGM feeding then significantly inhibited eczematous skin lesions including hyperkeratosis, dermal mastocytosis and eosinophilia. Concomitantly, cutaneous overproductions of substance P, IL-10, IL-4, and TNF-α were all suppressed in PKGM-fed mice. Conclusions: PKGM feeding markedly suppressed development of scratching behavior, substance P expression with mastocytosis, and skin inflammatory immune responses in NC/Nga mice.

Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

Background:Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. Objectives:We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. Methods:We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. Results: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. Conclusions:We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.

Molecular Cloning and Immunochemical Characterization of a New Japanese Cedar Pollen Allergen Homologous to Plant Subtilisin-Like Serine Protease

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.