Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Effect of eicosapentaenoic acid ethyl ester on hypothyroid function.

Thyroid hormones affect reactions in almost all pathways of lipid metabolism. It has been reported that plasma free fatty acid (FFA) concentration in hypothyroidism is generally within the normal range. In this study, however, we show that plasma FFA concentration in some hypothyroid patients is higher than the normal range. Symptoms of thyroid dysfunction in these individuals were less severe than those of patients with lower plasma FFA concentrations. From these findings we hypothesized that the change in FFA concentration must correlate with thyroid function. Using an animal model, we then examined the effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E), a n-3 polyunsaturated fatty acid derived from fish oil, on thyroid function in 1-methyl-2-imidazolethiol (MMI)-induced hypothyroid rats. Oral administration of EPA-E inhibited reduction of thyroid hormone levels and the change of thyroid follicles in MMI-induced hypothyroid rats. These findings suggest that FFA may affect thyroid functions and EPA-E may prevent MMI-induced hypothyroidism.

Suppressive effect of the herbal medicine Oren-gedoku-to on cyclooxygenase-2 activity and azoxymethane-induced aberrant crypt foci development in rats.

The present study is part of a program to obtain effective chemopreventive agents with low toxicity from medicinal herbs and traditional herbal medicines. We previously reported that Oren (Coptidis rhizoma) and Ogon (Scutellariae radix) inhibit azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation. In the present investigation, we found Sanshishi (Gardeniae fructus) and the traditional herbal medicine Oren-gedoku-to (OGT), composed of Ogon, Oren, Sanshishi and Obaku, also have preventive potential. Sanshishi and OGT decreased the numbers of ACF to 25.2 and 59.4% of the control value at 2% in the diet, respectively. Adverse effects, evidenced by body weight loss, were weaker with OGT than component herbs. To investigate their mechanisms of action, the influence on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activities was studied. Both OGT and Sanshishi inhibited COX-2 but not COX-1, this presumably contributing to their suppressive effects on ACF development. The results suggest that OGT may be useful for colon cancer chemoprevention in terms of efficacy and toxicity.

Genipin, a metabolite derived from the herbal medicine Inchin-ko-to, and suppression of Fas-induced lethal liver apoptosis in mice.

BACKGROUND & AIMS: We showed previously that a Kampo (Chinese/Japanese herbal) medicine, Inchin-ko-to (ICKT), inhibits hepatocyte apoptosis induced by transforming growth factor beta1 in vitro. The present study investigated whether ICKT or its ingredients inhibit Fas-mediated liver apoptosis in vivo. METHODS: Acute liver injury was induced by an intravenous injection of anti-Fas antibody, Jo2. The effects of ICKT and its ingredients on lethality, histology, apoptotic index, serum transaminase levels, caspase activation, mitochondrial membrane potential (Deltapsi(m)), and mitochondrial permeability transition (MPT) were analyzed. Apoptosis in mouse hepatocytes in vitro was also evaluated. RESULTS: Pretreatment with ICKT rescued 75% of Jo2-treated mice and markedly suppressed liver apoptosis/injury. Genipin, an intestinal bacterial metabolite of geniposide that is a major ingredient of ICKT, was found to be an active principle of ICKT. Genipin also suppressed in vitro Fas-mediated apoptosis in primary-cultured murine hepatocytes. Activation of caspase 3 and 8 in the liver homogenate and rapid reduction of triangle uppsi(m) of hepatocytes isolated from Jo2-treated mice were inhibited by genipin preadministration. The resistance to Ca(2+)-induced MPT was enhanced in liver mitochondria of genipin-treated mice. CONCLUSIONS: These results suggest that the antiapoptotic activity of genipin via the interference with MPT is a possible mechanism for therapeutic effects of ICKT.

Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae.

Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver. The ATP7B gene, which encodes a copper transporting P-type ATPase, is defective in patients with Wilson disease. To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (delta ccc2), the yeast homologue of ATP7B. Wild type and the H1069Q mutant could rescue delta ccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease. In addition, the mutant containing only the sixth copper binding domain could rescue delta ccc2, indicating its functional importance.

Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA.

Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver. WND (ATP7B) gene, which encodes a putative copper transporting P-type ATPase, is defective in the patients. To investigate the in vivo function of WND protein as well as its intracellular localization, WND cDNA was introduced to the Long-Evans Cinnamon rat, known as a rodent model for Wilson's disease, by recombinant adenovirus-mediated gene delivery. An immunofluorescent study and a subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the Long-Evans Cinnamon rat, the plasma level of holoceruloplasmin, oxidase-active and copper-bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. These findings indicate that introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin and that the Golgi apparatus is the likely site for WND protein to manifest its function.

Inhibition of thymocyte apoptosis by berberine.

To find anti-apoptotic substances in plant resources, a microassay method for estimating DNA fragmentation was established using fluorochrome 3,5-diaminobenzoic acid dihydrochloride. Examination was made of various herbal medicines for inhibitory effects on glucocorticoid-induced apoptosis in thymocytes. Several Kampo medicines, e.g. Oren-gedoku-to and San'o-shashin-to, were found to inhibit dexamethasone-induced apoptosis in murine thymocytes. Some of these medicines contain Coptidis rhizoma (CR) as the major constituent, and the CR extract showed the most potent inhibitory activity on thymocyte apoptosis of more than 200 species of herbal extracts. The inhibition of apoptosis by CR extract was confirmed by the trypan blue exclusion test, lactate dehydrogenase release measurement, and morphological evaluation by electron microscopy. The benzodioxolo-benzoquinolizine alkaloid, berberine, and five berberine-type alkaloids, isolated from CR extract, had an inhibitory effect, whereas no effect was noted for the aporphin-type alkaloid magnoflorine. The inhibitory action of berberine was also demonstrated on etoposide- and camptothecin-induced apoptosis.

Temperature-sensitive phenotype of a mutant Sendai virus strain is caused by its insufficient accumulation of the M protein.

We investigated the process interrupting the production of a temperature-sensitive mutant strain of Sendai virus, Cl.151, at the nonpermissive temperature (38 degrees C). The amount of virus M protein increased up to 6-fold when the cells persistently infected with Cl.151 strain at 38 degrees C are transferred to 32 degrees C, while the amount of nucleocapsid proteins did not alter. Cl.151 strain could restore virus production at 38 degrees C not only by the supplementation of M protein of wild type (Z) strain but also by the supplementation of M protein of Cl.151 strain. Neither the amount of M mRNA nor the rate of synthesis of M protein was altered by temperature in cells infected with the Cl.151 strain. However, we found that M protein of Cl.151 virus, which has 3-amino acid alterations from the wild type, was highly unstable at 38 degrees C when expressed under the control of an actin promoter. These results clearly show that Sendai virus M protein has a critical role in the production of virus particles without affecting virus gene expression.

Ramie (Boehmeria nivea) pollen-induced bronchial asthma and allergenic cross-reactivity of ramie and Parietaria.

Ramie (Boehmeria nivea), a plant of the Urticaceae family, is widely distributed in the Nagasaki area, and has been established to be a cause of asthma. The rate of positive reactions to ramie in intradermal tests was 11.7% among adult asthmatic patients in the Nagasaki area. In this study, 10 patients were positive in provocation tests using ramie pollen. Ramie pollen-specific IgE antibodies were measured by ELISA, with the positive provocation test group showing higher O.D. values than the positive intradermal test group (p < 0.05). Ramie is of the same family as Parietaria, an important allergen in Europe. The cross-reactivity of ramie and Parietaria was examined by an ELISA inhibition test using P. officinalis and P. judaica (Pj10), but no cross-reactivity was found, suggesting that ramie may be a new independent allergen. As ramie is widely distributed throughout Japan and South-east Asia, further study is needed to determine whether it is an important allergen of the Urticaceae family in this region, as is Parietaria in Europe, and Urtica in America.

Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain.

Xeroderma pigmentosum (XP) is an autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer. Cells from people with this condition are hypersensitive to ultraviolet because of a defect in DNA repair. There are nine genetic complementation groups of XP, groups A-H and a variant. We have cloned the mouse DNA repair gene that complements the defect of group A, the XPAC gene. Here we report molecular cloning of human and mouse XPAC complementary DNAs. Expression of XPAC cDNA confers ultraviolet-resistance on several group A cell lines, but not on lines of other XP groups. Almost all group A lines tested showed abnormality or absence of XPAC messenger RNAs. These results indicate that a defective XPAC gene causes group A XP. The human and mouse XPAC genes are located on chromosome 9q34.1 and chromosome 4C2, respectively. Human XPAC cDNA encodes a protein of 273 amino acids with a zinc-finger motif.