Mixed Medicinal Mushroom Mycelia Attenuates Alzheimer's Disease Pathologies In Vitro and In Vivo.

Alzheimer's disease (AD) is characterized by memory impairment and existence of amyloid-ß (Aß) plaques and neuroinflammation. Due to the pivotal role of oxidative damage in AD, natural antioxidative agents, such as polyphenol-rich fungi, have garnered scientific scrutiny. Here, the aqueous extract of mixed medicinal mushroom mycelia (MMMM)-Phellinus linteus, Ganoderma lucidum, and Inonotus obliquus-cultivated on a barley medium was assessed for its anti-AD effects. Neuron-like PC12 cells, which were subjected to Zn2+, an Aß aggregator, were employed as an in vitro AD model. The cells pretreated with or without MMMM were assayed for Aß immunofluorescence, cell viability, reactive oxygen species (ROS), apoptosis, and antioxidant enzyme activity. Then, 5XFAD mice were administered with 30 mg/kg/day MMMM for 8 weeks and underwent memory function tests and histologic analyses. In vitro results demonstrated that the cells pretreated with MMMM exhibited attenuation in Aß immunofluorescence, ROS accumulation, and apoptosis, and incrementation in cell viability and antioxidant enzyme activity. In vivo results revealed that 5XFAD mice administered with MMMM showed attenuation in memory impairment and histologic deterioration such as Aß plaque accumulation and neuroinflammation. MMMM might mitigate AD-associated memory impairment and cerebral pathologies, including Aß plaque accumulation and neuroinflammation, by impeding Aß-induced neurotoxicity.

Neuroprotective Effects of Water Extract from Brown Algae Petalonia binghamiae in an Experimental Model of Focal Cerebral Ischemia In Vitro and In Vivo.

Focal cerebral ischemia (fCI) can result in brain injury and sensorimotor deficits. Brown algae are currently garnering scientific attention as potential therapeutic candidates for fCI. This study investigated the therapeutic effects of the hot water extract of Petalonia binghamiae (wPB), a brown alga, in in vitro and in vivo models of fCI. The neuroprotective efficacy of wPB was evaluated in an in vitro excitotoxicity model established using HT-22 cells challenged with glutamate. Afterward, C57/BL6 mice were administered wPB for 7 days (10 or 100 mg/kg, intragastric) and subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) operation, which was used as an in vivo fCI model. wPB co-incubation significantly inhibited cell death, oxidative stress, and apoptosis, as well as stimulated the expression of heme oxygenase-1 (HO-1), an antioxidant enzyme, and the nuclear translocation of its upstream regulator, nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-22 cells challenged with glutamate-induced excitotoxicity. Pretreatment with either dose of wPB significantly attenuated infarction volume, neuronal death, and sensorimotor deficits in an in vivo fCI model. Furthermore, the attenuation of oxidative stress and apoptosis in the ischemic lesion accompanied the wPB-associated protection. This study suggests that wPB can counteract fCI via an antioxidative effect, upregulating the Nrf2/HO-1 pathway.

Water Extract of Mixed Mushroom Mycelia Grown on a Solid Barley Medium Is Protective against Experimental Focal Cerebral Ischemia.

Although the individual consumption of medicinal mushrooms, including Phellinus linteus (PL), Ganoderma lucidum (GL), and Inonotus obliquus (IO), is known to be neuroprotective, the associated mechanisms underlying their therapeutic synergism on focal cerebral ischemia (fCI) have yet to be elucidated. This study aimed to demonstrate the neuroprotective effects of mixed mushroom mycelia (MMM) against experimental fCI. The water-fractions, ethanolic-fractions, and ethyl acetate-fractions of the MMM (PL, GL, and IO) grown in a barley medium using solid-state fermentation techniques were prepared and their protective effects against glutamate-induced excitotoxicity were compared in PC-12 cells. After the identification of the water extracts of MMM (wMMM) as the most suitable form, which possessed the lowest toxicity and highest efficacy, further analyses for evaluating the anti-apoptotic effects of wMMM, including Hoechst 33258-based nuclear staining, fluorescence-activated cell sorting, and reactive oxygen species (ROS) detection assays, were performed. Rats were subjected to a 90 min middle cerebral artery occlusion and reperfusion, after which a wMMM treatment resulted in significant dose-dependent improvements across a number of parameters. Furthermore, measurements of intracellular ROS and levels of antioxidant enzymes revealed a wMMM-mediated ROS attenuation and antioxidant enzyme upregulation. We suggest that wMMM is neuroprotective against fCI through its anti-apoptotic and anti-oxidative effects.

Ginseng Berry Prevents Alcohol-Induced Liver Damage by Improving the Anti-Inflammatory System Damage in Mice and Quality Control of Active Compounds.

The ginseng berry contains a variety of biologically active compounds and has a higher ginsenoside content than its roots. This study focused on the hepatoprotective activity of ginseng berry extract prepared by enzyme treatment (EGB) compared to the non-enzyme-treated ginseng berry extract (GB) and quality control of EGB. The feeding effect of EGB on alcohol-induced liver damage (AILD) was investigated by measuring the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared with those of EtOH-fed mice. Furthermore, cytokine levels in the culture supernatants of EGB- or GB-treated RAW 264.7 cells were determined by enzyme-linked immunosorbent assay. The developed method was applied to the simultaneous quantification of four major ginsenosides in EGB using UPLC-QTOF/MS. Treatment with EGB at a dose of 0.5 or 1 mg/mouse significantly suppressed the AST and ALT levels in mice with AILD. Enzyme-treated ginseng berry was also found to suppress the production of inflammatory mediators like nitric oxide (NO), tumor-necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, showing higher activity than that of GB. The amount of ginsenoside Re, F5, F3, and Rd in the EGB obtained using UPLC-QTOF/MS was 45.9, 3.3, 4.0, and 6.2 mg/g, respectively. These results suggest that EGB has a potential effect on AILD, and its hepatoprotective effect provides beneficial insights into developing new candidates for the prevention and cure of AILD. Also, this study demonstrated the utility of UPLC-QTOF/MS-based major compounds for quality control (QC) of EGB.

High sensitivity detection of active botulinum neurotoxin by glyco-quantitative polymerase chain-reaction.

The sensitive detection of highly toxic botulinum neurotoxin (BoNT) from Clostridium botulinum is of critical importance because it causes human illnesses if foodborne or introduced in wounds and as an iatrogenic substance. Moreover, it has been recently considered a possible biological warfare agent. Over the past decade, significant progress has been made in BoNT detection technologies, including mouse lethality assays, enzyme-linked immunosorbent assays, and endopeptidase assays and by mass spectrometry. Critical assay requirements, including rapid assay, active toxin detection, sensitive and accurate detection, still remain challenging. Here, we present a novel method to detect active BoNTs using a Glyco-quantitative polymerase chain-reaction (qPCR) approach. Sialyllactose, which interacts with the binding-domain of BoNTs, is incorporated into a sialyllactose-DNA conjugate as a binding-probe for active BoNT and recovered through BoNT-immunoprecipitation. Glyco-qPCR analysis of the bound sialyllactose-DNA is then used to detect low attomolar concentrations of BoNT and attomolar to femtomolar concentrations of BoNT in honey, the most common foodborne source of infant botulism.

Lactobacillus plantarum NCHBL-004 modulates high-fat diet-induced weight gain and enhances GLP-1 production for blood glucose regulation.

OBJECTIVES: This study investigated the therapeutic potential of Lactobacillus plantarum NCHBL-004 (NCHBL-004) in the treatment of obesity and associated metabolic disorders. METHODS: Mice were fed either a normal diet (ND) or a high-fat diet (HFD) with oral administration of NCHBL-004. After euthanasia, blood, liver and adipose tissue were collected. Furthermore, the microbiome and short-chain fatty acids (SCFAs) were analyzed from feces. RESULTS: Oral administration of live NCHBL-004 to mice fed a HFD resulted in notable reductions in weight gain, improvements in glucose metabolism, and maintenance of balanced lipid levels. A comparative analysis with other Lactobacillus strains highlighted the superior efficacy of NCHBL-004. Moreover, heat-killed NCHBL-004 demonstrated beneficial effects similar to those of live NCHBL-004. Additionally, administration of live NCHBL-004 induced glucagon-like peptide 1 (GLP-1) production and increased the levels of short-chain fatty acids (SCFAs), including acetate and propionate, in feces, positively influencing liver lipid metabolism and mitigating inflammation. Consistent with this, analysis of the gut microbiome following NCHBL-004 administration showed increases in SCFA-producing microbes with increased proportions of Lactobacillus spp. and a significant increase in the proportion of microbes capable of promoting GLP-1 secretion. CONCLUSIONS: These findings underscore the potential of both live and inactivated NCHBL-004 as potential therapeutic approaches to managing obesity and metabolic disorders, suggesting avenues for further investigation and clinical applications.

APRIL stimulates NF-κB-mediated HoxC4 induction for AID expression in mouse B cells.

Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cells. APRIL substantially increased both HoxC4 and AID expression, whereas BAFF induced the expression of AID but not HoxC4. To elucidate the underlying mechanisms, we constructed a HoxC4 gene promoter reporter vector and analyzed the promoter induction after APRIL stimulation. APRIL enhanced the HoxC4 promoter activity by 2.3-fold, and this increase disappeared when the second putative NF-κB-binding promoter element (NBE2) was mutated. Based on ChIP assays, we found that NF-κB bound to the HoxC4 promoter NBE2 region. Furthermore, the overexpression of NF-κB augmented the APRIL-induced HoxC4 promoter activity, while the expression of dominant negative-IκBα suppressed it. Taken together, our findings suggest that NF-κB mediates APRIL-induced HoxC4 transcription.

All-trans-retinoic acid rescues neurons after global ischemia by attenuating neuroinflammatory reactions.

Retinoic acid (RA) plays an important role in the developing mammalian nervous system. Based on this concept, some studies have demonstrated the beneficial effects of RA administration on neurogenesis in neuropathological diseases. Some investigations have revealed the anti-inflammatory effects of RA treatment in multiple systems, in addition to its role in neurogenesis. To date, however, the neuroprotective efficacy of RA after cerebral ischemia, especially in the context of its anti-inflammatory effects, has been poorly demonstrated. Additionally, to the best of our knowledge, experiments of the therapeutic efficacy of RA treatment in a transient global ischemic model in the Mongolian gerbil have been lacking worldwide. Here, we studied the neuroprotective effects and neurobehavioral outcomes of intraperitoneally administered all-trans-RA (ATRA; a synthetic form of RA) on brains with transient global ischemia that was induced with the bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model in the gerbil. In order to identify whether these neuroprotective mechanisms were due to the anti-inflammatory effects of ATRA, in vivo hippocampal expression of proinflammatory cytokines including tissue necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) after ATRA injection and in vitro levels of release of nitric oxide, TNF-α and IL-6 from lipopolysaccharide (LPS)-stimulated BV2 microglial cells after ATRA treatment were evaluated. The results showed that ATRA can protect pyramidal neurons in the hippocampal CA1 region against BCCAO-induced neuronal apoptosis and significantly reduce the extent of astrocytosis and microglial activation. In addition, the ischemia-induced neurobehavioral changes were normalized by ATRA injection. Consistent with these phenotypic data, we observed the diminishing effects of ATRA treatment on the production of proinflammatory mediators (e.g., TNF-α and IL-6) in hippocampal homogenates and LPS-stimulated BV2 cells, and these effects were dose-dependent. These results suggest a beneficial role of ATRA in the attenuation of global cerebral ischemia due to its anti-inflammatory properties, resulting in, at least partly, the inhibition of microglial secretion of variable proinflammatory cytokines.

Nicotine alleviates alcohol-induced memory and long-term potentiation impairment.

Alcohol and nicotine are routinely abused together. There is one plausible explanation that comorbidity can alleviate alcohol-induced cognitive impairment. However, the mechanism involved is not known. The aim of this report was to evaluate the interactive effects of alcohol coadministration with nicotine on hippocampal memory and long-term potentiation (LTP). C57BL/6 mice were distributed into 4 treatment groups: control, alcohol, nicotine, and alcohol plus nicotine. All mice received tap water or alcohol solution and saline or nicotine. In water maze test, the alcohol group showed significant decreases in hippocampus function and acquisition training than control, nicotine, and combined treatment groups. The alcohol group also showed a significantly shorter latency in entering the foot-shock compartment than control, nicotine, and combined treatment groups in a passive avoidance test. Theta burst stimulation was adopted to induce concrete LTP in CA1 field recording using hippocampal slice. Alcohol alone administration failed to maintain LTP. Nicotine alone administration did not alter hippocampal LTP. There were no negative effects of alcohol on hippocampal LTP in mice administrated with nicotine. The current study successfully demonstrated beneficial effects of nicotine on alcohol induced memory impairment accompanied by hippocampal LTP impairment after one week of co-administration and one-day withdrawal.

Bovine Lactoferricin Induces Intestinal Epithelial Cell Activation through Phosphorylation of FAK and Paxillin and Prevents Rotavirus Infection.

We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.

NMR-Based Metabolomics Approach to Investigate the Effects of Fruits of Acanthopanax sessiliflorus in a High-Fat Diet Induced Mouse Model.

The prevalence of obesity is rapidly increasing and is recognized as a serious health problem. To investigate metabolic changes in an obese model after administration of Acanthopanax sessiliflorus, mice were divided into four groups: normal diet, high-fat diet (HFD), HFD with treatment fenofibrate, and A. sessiliflorus fruit extract. The liver tissue of mice was analyzed using nuclear magnetic resonance (NMR) spectrometry-based metabolomics. In multivariate statistical analyses, the HFD group was discriminated from the normal diet group, and the group fed A. sessiliflorus fruit was discriminated from the HFD group. In biomarker analysis between the HFD group and the group fed A. sessiliflorus fruit, alanine, inosine, formate, pyroglutamate, taurine, and tyrosine, with AUC values of 0.7 or more, were found. The levels of these metabolites were distinguished from the HFD mouse model. Changes in these metabolites were confirmed to act on metabolic pathways related to antioxidant activity.

Improved isolation methods for mucosal leukocytes from small and large intestines in rats.

We optimized the isolation protocol for intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from the rat small intestine, and LPLs from even the rat large intestine. The major population of IELs in the small intestine was considered to be from the villus epithelia. The cytotoxicity of mucosal leukocytes was comparable among isolated fractions from both the small and large intestines, regardless of the population differences. Further analyses of the cells collected from other lymphoid tissues demonstrated that CD161(+) cells selectively accumulated in the intestinal lamina propria and did not recirculate through the lymph ducts. Our modified isolation protocol enables the collection of mucosal immune cells from the rat intestines without any deterioration of cell function and could contribute to a better understanding of dietary influences on the mucosal immune system.

Korean mistletoe lectin (KML-IIU) and its subchains induce nitric oxide (NO) production in murine macrophage cells.

Synthesis of nitric oxide (NO) is one of the important effector functions of innate immune cells. Although several reports have indicated mistletoe lectins induce immune cells to produce cytokines, studies regarding the activities of the lectins in the production of NO have been very limited. Here, we report on the induction of NO synthesis in a murine macrophage cell line, RAW264.7, by Korean mistletoe lectin (KML-IIU). When the macrophage cells were treated with KML-IIU in the presence of a suboptimal concentration of IFN-gamma, NO production was induced in a concentration-dependent manner. Significantly higher levels of NO were induced by subchains of the KML-IIU (A and B), which have lower toxicities, as compared to the hololectin. Furthermore, expression of the inducible nitric oxide synthase (iNOS) gene was elevated in accordance with the level of NO production. When the synthase was inhibited by iNOS inhibitors (L-NIL and L-NAME), NO production was specifically reduced in a concentration-dependent manner. Our studies demonstrate that the KML-IIU and its subchains induce NO production in murine macrophage cells via activation of the iNOS gene expression, suggesting that the KML-IIU subchains may be used as an immunomodulator to enhance the effector functions of innate immune cells.

Effect of gamma irradiation on spleen cell function and cytotoxicity of doxorubicin.

The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-gamma and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated doxorubicin (NIRD) was used as a control. The mice injected with NIRD (2mg/kg body weight for 5 days, 24h interval) showed a considerable decrease (P<0.05) in the body, spleen weight, proliferation and cytokine release (IL-2 and IFN-gamma) as compared to control. However, a non-significant variation was observed in IRD treated mice compared with normal. Tumor bearing mice treated with NIRD and IRD (2mg/kg body weight, five doses at 48 h interval) showed diverse results on spleen cell cytokine release, proliferation and metastasis. HPLC results revealed the formation of several trace level degradation (P<0.05) products of IRD. IRD displayed a non-significant variation of cytotoxicity on B16BL6 cells, and low percentage (P<0.01) of cardiotoxicity on H9c2 cells as compared to NIRD. Altogether, this present study emphasis that gamma irradiation altered the property of doxorubicin.

Ginsenoside-Rb2 and 20(S)-Ginsenoside-Rg3 from Korean Red Ginseng Prevent Rotavirus Infection in Newborn Mice.

It is well known that Korean red ginseng has various biological activities. However, there is little knowledge about the antiviral activity of Korean red ginseng and its ginsenosides. In this study, we addressed whether oral administration of ginsenoside-Rb2 and -Rg3 is able to protect against rotavirus (RV) infection. The protective effect of ginsenosides against RV infection was examined using an in vivo experiment model in which newborn mice (10-day-old) were inoculated perorally (p.o.) with 1.5 × 106 plaque-forming units/mouse of RV strain SA11. When various dosages of ginsenoside-Rb2 (25-250 mg/kg) were administered 3days, 2 days, or 1 day before virus challenge, treatment with this ginsenoside at the dosage of 75 mg/kg 3days before virus infection most effectively reduced RV-induced diarrhea. In addition, consecutive administration of ginsenoside-Rb2 (75 mg/kg) at 3 days, 2 days, and 1 day before virus infection was more effective than single administration on day -3. The consecutive administration of ginsenoside-Rb2 also reduced virus titers in the bowels of RV-infected mice. In an experiment to compare the protective activity between ginsenoside-Rb2 and its two hydrolytic products (20(S)- and 20(R)-ginsenoside-Rg3), 20(S)-ginsenoside-Rg3, but not 20(R)-ginsenoside-Rg3, prevented RV infection. These results suggest that ginsenoside-Rb2 and its hydrolytic product, 20(S)-ginsenoside-Rg3, are promising candidates as an antiviral agent to protect against RV infection.

Ovalbumin modified by gamma irradiation alters its immunological functions and allergic responses.

It is well known that gamma (gamma)-ray irradiation results in the alteration of biological function of bioactive materials such as proteins, saccharides and lipids. In this study the effect of gamma-irradiation on the chemical and immunological property of an allergen, ovalbumin (OVA), was investigated. Irradiation of more than 10 kGy resulted in the alteration of the structure of OVA. However, OVA treated with 10 kGy irradiation (10 kGy-OVA), but not 100 kGy-OVA, fully maintained immunological reactivity to a monoclonal antibody specific to the intact allergen (clone 14). Mice immunized with 10 kGy- as well as 100 kGy-OVA showed significantly lower antibody response to the allergen than those with intact OVA in a gamma-ray dosage-dependent manner. Especially immunization of both 10 kGy- and 100 kGy-OVA induced a significant decrease of OVA-specific IgE. Splenocytes of mice immunized with irradiated OVA showed a significant reduction in OVA-specific T cell proliferation and the secretion of Th1-type (IFN-gamma and IL-2) and Th2-type cytokines (IL-4 and IL-6). The expression of T cell activation markers such as CD25 and CD44 was also down-regulated in T cells of mice immunized with irradiated OVAs. These results suggest that gamma-ray irradiation of OVA suppress humoral and cellular immune responses specific to the allergen OVA, and the modification method with gamma-irradiation may be available for the control of allergy.

Isolation and characterization of two Korean mistletoe lectins.

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.

Isolation of fatty acids with anticancer activity from Protaetia brevitarsis larva.

In this study, biologically active compounds were isolated from Protaetia brevitarsis larva (PBL) by dichloromethane extraction. The dichloromethane extract from PBL was highly cytotoxic to various cancer cells. From a silica gel column chromatograpy of this extract, we obtained four fractions (F-2, F-4, F-5 and F-7) having apoptosis-inducing activity. These fractions induced DNA ladder and caspase-3 activation during apoptosis in colon 26 tumor cells. In 1H and 13C NMR and mass spectral analysis of the fraction F-2 showing the highest apoptosis-inducing activity, we found that the fraction was composed of three free fatty acids such as palmitic acid, (Z)-9-octadecenoic acid and octadecenoic acid. These results indicate that the dichloromethane extract of PBL includes anticancer components composed of at least three fatty acids, and apoptosis-inducing activity of the extract was mediated by caspase-3 activation in tumor cells.

Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells.

Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall ß-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1.

Induction of Oral Tolerance by Gamma-Irradiated Ovalbumin Administration.

Oral administration of soluble antigen can induce peripheral tolerance to the antigen. This study was conducted to evaluate whether gamma-irradiated ovalbumin (OVA) can induce oral tolerance. To investigate this, we administrated intact or irradiated OVA to mice, induced allergic response using intact OVA and alum, then compared humoral and cellular immune responses. Mice treated with gammairradiated OVA had less OVA-specific IgE compared with those who were administered intact OVA. There was no difference in levels of OVA-specific IgG+A+M, IgG1, and IgG2a. Splenocytes of mice administered irradiated OVA showed similar OVA-specific T cell proliferation and secretion of IFN-γ and IL-4. However, there was an increase in IL-2 and a decrease of IL-6 secretion in mice treated with irradiated OVA. These results indicate that gamma-irradiated OVA have similar effects to intact OVA on antigen tolerance.