Oral Administration of Alginic Acid Oligosaccharide Suppresses IgE Production and Inhibits the Induction of Oral Tolerance

We have found that alginic acid oligosaccharide (ALGO) enhanced Th1 by promoting IL-12 production, suggesting that ALGO can be applied as an anti-allergic food. In this study we examined both positive and negative functions of ALGO. First we investigated the anti-allergic activity of ALGO, as a positive function, when orally administered. IgE production was significantly inhibited in mice fed ALGO as compared to control mice. This result indicates that ALGO had anti-allergic activity even when orally administered. On the other hand, we also found a negative function of ALGO. Oral co-administration of a protein antigen and ALGO inhibited the induction of oral tolerance to the protein. These data indicate the potential of ALGO as an anti-allergic food material and the necessity of further examination to determine a safe method application.     

Lactobacillus helveticus SBT2171 alleviates allergic symptoms in a murine model for pollen allergy

ABSTRACT

Lactic acid bacteria are known to have various health-promoting effects and are highly expected to find applications in anti-allergic food materials. In this study, we focused on Lactobacillus helveticus SBT2171 (LH2171), which reportedly modifies some unique immune responses and ameliorated symptoms of patients allergic to mites and house dust in the previous studies. We examined the effect of LH2171 on cytokine production by antigen-stimulated murine naïve splenocytes in vitro and demonstrated that it inhibited IL-4 and IL-13 production while enhancing IFN-γ and IL-10 production. Then, we examined the anti-allergic effect of LH2171 in vivo using a murine model of pollen allergy and found that LH2171 reduced the sneezing frequency when orally administered to mice. We successfully confirmed the immune modulatory activity of LH2171 and its anti-allergic activity against inhaled antigens. These evidences would contribute to identifying the anti-allergic mechanism of LH2171.

Abbreviations: ALDH: aldehyde dehydrogenase; EGCG: epigallocatechin gallate; LAB: lactic acid bacteria; LH2171: Lactobacillus helveticus SBT2171; NALT: nasal-associated lymphoid tissue; OVA: ovalbumin     

Inhibitory effect of porphyran, prepared from dried "Nori", on contact hypersensitivity in mice

Porphyran is a major component of the red algae, Porphyra tenera and P. yezoensis, which are processed into a sheet type of dried food, "Nori". Porphyran has been reported to activate murine macrophages by in vitro and i.p. injection studies. The contact hypersensitivity (CHS) reaction in mice is commonly used as a model to evaluate the anti-allergic activity of food and food components. We therefore studied the effect of porphyran on the CHS reaction in Balb/c mice to evaluate anti-allergic activity of porphyran. We found that an oral administration of porphyran (2% in drinking water) suppressed the CHS reaction (ear edema) induced by 2,4,6-trinitrochlorobenzene. We also found that porphyran suppressed the serum level of IgE and the production of interferon-gamma (IFN-gamma) in the challenged ear lobe. We conclude from these results that the CHS reaction was suppressed by oral porphyran due to the decreased serum level of IgE and the production of IFN-gamma in the challenged ear lobe.     

An in vitro effect of coffee on the antigen-specific immune responses of naïve splenocytes

Coffee is a globally consumed beverage with potential health benefits. However, there are few reports about the effects of coffee on immunological functions. We previously reported that in an allergic mouse model, coffee intake prevented allergy development through augmentation of interleukin (IL)-12p40. In order to investigate the anti-allergic activity of coffee, we examined the effect of coffee on antigen (Ag)-specific responses of immune cells in vitro. Coffee treatment suppressed proliferation and IL-2 secretion of mouse splenocytes in the same way as splenocytes from mice administered coffee orally. However, IL-12p40 secretion decreased significantly as a result of in vitro coffee treatment, which was contrary to the results obtained from experiments of mice administered coffee orally. Therefore, modification associated with oral administration might influence the anti-allergic activity of coffee.     

Identification of DK419, a potent inhibitor of Wnt/β-catenin signaling and colorectal cancer growth

The Wnt signaling pathway is critical for normal tissue development and is an underlying mechanism of disease when dysregulated. Previously, we reported that the drug Niclosamide inhibits Wnt/β-catenin signaling by decreasing the cytosolic levels of Dishevelled and β-catenin, and inhibits the growth of colon cancers both in vitro and in vivo. Since the discovery of Niclosamide’s anthelmintic activity, a growing body of literature indicates that Niclosamide is a multifunctional drug. In an effort to identify derivatives of Niclosamide with improved pharmacokinetic properties that maintain the multifunctional drug activity of Niclosamide for clinical evaluation, we designed DK419, a derivative containing a 1H-benzo[d]imidazole-4-carboxamide substructure, using the structure-activity relationships (SAR) of the Niclosamide salicylanilide chemotype. Similar to Niclosamide, we found DK419 inhibited Wnt/β-catenin signaling, altered cellular oxygen consumption rate and induced production of pAMPK. Moreover, we found DK419 inhibited the growth of CRC tumor cells in vitro, had good plasma exposure when dosed orally, and inhibited the growth of patient derived CRC240 tumor explants in mice dosed orally. DK419, a derivative of Niclosamide with multifunctional activity and improved pharmacokinetic properties, is a promising agent to treat colorectal cancer, Wnt-related diseases and other diseases in which Niclosamide has demonstrated functional activity.     

Inhibitory activity of a ceramide library on interleukin-4 production from activated T cells

Allergic diseases are hypersensitivity disorders associated with the production of specific immunoglobulin E (IgE) to environmental allergens. Interleukin (IL)-4, produced primarily by CD4(+) T cells, is an important stimulus for the switch of the antibody isotype to IgE in both mice and humans. In this study we investigated the inhibitory activity of IL-4 production in activated T cells by screening ceramide derivatives prepared by solid phase combinatorial chemistry. Many ceramide derivatives significantly inhibited IL-4 production in T cells. In particular, ceramide derivatives with a lauroyl group showed strong inhibitory activities on IL-4 production in both phorbol 12-myristate 13-acetate (PMA)-activated EL4 T cells and antigen-primed cells, suggesting that they can be used as compounds for the development of anti-allergic agents.     

Murine B7-H3 is a negative regulator of T cells

T cell activation is regulated by the innate immune system through positive and negative costimulatory molecules. B7-H3 is a novel B7-like molecule with a putative receptor on activated T cells. Human B7-H3 was first described as a positive costimulator, most potently inducing IFN-gamma production and cellular immunity. In this study we examined the expression and function of mouse B7-H3. B7-H3 is mostly expressed on professional APCs; its expression on dendritic cells appears to be up-regulated by LPS. In contrast to human B7-H3, we found that mouse B7-H3 protein inhibited T cell activation and effector cytokine production. An antagonistic mAb to B7-H3 enhanced T cell proliferation in vitro and led to exacerbated experimental autoimmune encephalomyelitis in vivo. Therefore, mouse B7-H3 serves as a negative regulator of T cell activation and function.     

The inhibitory effect of anti-allergic agent suplatast tosilate (IPD-1151T) on methacholine- and allergen-induced bronchoconstriction in sensitized mice. asakazu@med.showa-u.dc.jp

The influence of an anti-allergic agent, suplatast tosilate (IPD-1151T; (+/-)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenyl-carbamoyl]-ethyl] dimethylsulfonium p-toluenesulfonate) on allergic bronchoconstriction induced by allergen and methacholine (MCh) were examined in mice. BALB/c mice were sensitized by intraperitoneal injection of dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) mixed with A1(OH)3 (DNP-KLH). IPD-1151T was administered orally once a day for either 5 or 14 days in doses of 10, 30 or 100 mg/kg. Bronchoconstriction was measured 24h after the final drug administration. IPD-1151T inhibited both antigen- and MCh-mediated bronchoconstriction in actively sensitized mice. The inhibition induced was closely related to the dose and frequency of oral administration of the agent. We also examined the effect of IPD-1151T on IgE production in response to DNP-KLH immunization. IPD-1151T inhibited dose-dependently both total and specific IgE concentrations in serum prepared from mice 15 days after immunization. These results strongly indicate that IPD-1151T inhibits IgE production in vivo and results in attenuating effect on bronchoconstriction.     

Grifola frondosa extract and ergosterol reduce allergic reactions in an allergy mouse model by suppressing the degranulation of mast cells

ABSTRACT

The increasing number of patients suffering from allergic diseases is a global health problem. Grifola frondosa is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that G. frondosa extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow–derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells in vivo. Our findings suggest that G. frondosa, including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.     

Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown guinea pigs

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.     

Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice

Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-alpha production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with beta-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-alpha is added or overexpressed. Thus, (c) the enhanced TGF-alpha production when cells are not in three- dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-alpha. The data suggest that ECM upregulates WAP by downregulating TGF-alpha production. We also propose that changes in TGF-alpha activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.     

PACAP inhibits tumor growth and interferes with clusterin in cervical carcinomas

Secretory clusterin (sCLU), an anti-apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo-resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt–Raf–ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.     

Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var. saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated mast cells

The present study was performed to investigate the anti-allergy potentials of glycoprotein (90 kDa) isolated from Opuntia ficus-indica var. saboten MAKINO (OFI glycoprotein) in vivo (ICR mice) and in vitro (RBL-2H3 cells). At first, to know whether the OFI glycoprotein has an inhibitory ability for allergy in vivo, we evaluated the activities of allergy-related factors such as histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in compound 48/80 (8 ml/kg BW)-treated ICR mice. After that, we studied to found the effect for anti-allergy in vitro such as nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK) 1/2, arachidonic acid, and cyclooxygenase-2 (COX-2) in compound 48/80 (5 μg/ml)-treated RBL-2H3 cells. Our results showed that the OFI glycoprotein (5 mg/kg) inhibited histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in mice serum. Also OFI glycoprotein (25 μg/ml) has suppressive effects on the expression of MAPK (ERK1/2), and on protein expression of anti-allergic proteins (iNOS and COX-2). Thus, we speculate that the OFI glycoprotein is an example of natural compound that blocks anti-allergic signal transduction pathways.     

Severe growth retardation and short life span of double-mutant mice lacking Xpa and exon 15 of Xpg

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XP-G/CS) with characteristics, such as growth retardation and a short life span. In the previous studies, we generated four Xpg mutant mice with two different C-terminal truncations, null, or a base substitution mutation to identify the protein region that causes the onset of CS, and found that the CS-causing mutations, null or a deletion of the last 360 amino acids, completely inhibited the NER activity of mouse XPG (Xpg), but the non-CS-causing mutations, XpgD811A (base substitution that eliminates the nuclease activity of Xpg) or XpgDeltaex15 (deletion of the exon 15 corresponding to the last 183 amino acids), resulted in the retention of residual NER activity. To understand why mutations that completely eliminate the NER activity of Xpg cause CS but those that abolish the nuclease activity without totally eliminating the NER activity of Xpg do not result in CS, we made a series of Xpg mutant mice with Xpa-null mutant allele and found that mice with the non-CS-causing deletion mutation (XpgDeltaex15) exhibited the CS phenotype when XPA was also absent but the base substitution mutation (XpgD811A) that eliminated the Xpg nuclease activity did not. These results indicate that Xpg has a second function, beside NER, and that the disruption of this second function (deletion of the last 183 amino acids) when combined with an NER defect causes CS. When we compared amino acid sequences corresponding to the exon 15 of Xpg, a significant homology was conserved among vertebrates, but not in Drosophila and Saccharomyces cerevisiae. These observations suggest that the second function of XPG may be conserved only in vertebrates and CS symptoms may occur in its absence.     

Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10

Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.     

Ivermectin suppresses tumour growth and metastasis through degradation of PAK1 in oesophageal squamous cell carcinoma

Oesophageal squamous cell carcinoma (ESCC), the most common form of oesophageal malignancies in the Asia‐Pacific region, remains a major clinical challenge. In this study, we found that ivermectin, an effective antiparasitic drug that has been approved for patients to orally treat onchocerciasis for over 30 years, displayed potent antitumour activity against ESCC cells in vitro and in nude mice. We demonstrated that ivermectin significantly inhibited cell viability and colony formation, and induced apoptosis through a mitochondrial‐dependent manner in ESCC cells. Ivermectin also abrogated ESCC cell migration, invasion, as well as the protein levels of MMP‐2 and MMP‐9. Mechanistically, ivermectin strongly inhibited the expression of PAK1; by further gain‐ and loss‐of‐function experiments, we confirmed that PAK1 played a crucial role in ivermectin‐mediated inhibitory effects on ESCC cells. In addition, the data indicated that ivermectin promoted PAK1 degradation through the proteasome‐dependent pathway. Additionally, ivermectin synergized with chemotherapeutic drugs including cisplatin and 5‐fluorouracil to induce apoptosis of ESCC cells. Interestingly, the in vivo experiments also confirmed that ivermectin effectively suppressed tumour growth and lung metastasis of ESCC. Collectively, these results indicate that ivermectin exerts a potent antitumour activity against ESCC and is a promising therapeutic candidate drug for ESCC patients, even those carrying metastasis.