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Purpose: To investigate the potential anti-inflammatory effects of the seeds of Opuntina humifusa and its active constituents.Methods: The extract of O. humifusa seeds was tested for the inhibition of nitric oxide (NO) production in liposaccharide (LPS)-stimulated RAW 264.7 cells using Griess reagent. The active constituents were isolated using bioassay-guided isolation methods. The effects of the active constituent on NO, proinflammatory cytokines, nuclear factor-kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis.Results: The seed extract of O. humifusa significantly attenuated LPS-induced NO production in RAW 264.7 cells (p < 0.05). Bioassay-guided fractionation resulted in the isolation of isoamericanin A as an active constituent. Isoamericanin A reduced LPS-induced production of NO, iNOS, and proinflammatory cytokines (TNF-α and IL-6) in a concentration-dependent manner (p < 0.05). Furthermore, the effect was accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and the decreased phosphorylation of IκB in the cytosol induced by LPS (p < 0.05).Conclusion: The seed extract of O. humifusa and its active constituent, isoamericanin A, have antiinflammatory effects in LPS-stimulated RAW 264.7 cells, suggesting that they have potentials as antiinflammatory agents. Keywords: Opuntia humifusa seeds, Isoamericanin A, Nitric oxide, RAW 264.7 cells, NF-kappa B

We aimed to investigate possible anticancer effects of Amtak-ssamchae (a newly bred cabbage line containing more carotenoids) in an in vitro pancreatic-tumor cell model and an in vivo model (Panc02 cells). In an in vitro assay, Amtak-ssamchae significantly reduced the viability of Panc02 cells relative to normal ssamchae. Furthermore, an Amtak-ssamchae extract significantly reduced the tumor size in mice inoculated with Panc02 cells, and this effect was accompanied by changes in the cell population in/near the tumor and spleen. Treatment with the extract of the new cabbage line blocked regulatory-T-cell recruitment, which is known as the main pathological mechanism of action of pancreatic cancer to avoid host immunity. Additionally, Amtak-ssamchae (not the normal line) contained β-carotene (at 27.9 ± 2.1 μg/g). Thus, we suggest that the Amtak-ssamchae extract could be a resource for natural supplements and functional foods for combating pancreatic cancer.

Our study aimed to investigate the effects of the polysaccharide-rich extract of Phragmites rhizoma (PEP) against water immersion restraint (WIR) stress and forced swimming-induced fatigue. Exposure to WIR stress significantly increased the ulcer index, bleeding score, the weight of the adrenal gland, blood glucose concentrations, total cholesterol, cortisol, and creatine kinase (CK). The weight of the spleen decreased significantly. In addition, myeloperoxidase (MPO) and thiobarbituric acid-reactive substance (TBARS) were significantly upregulated by WIR stress. The antioxidative factors such as glutathione (GSH) and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the stomach were decreased by WIR stress. Alterations induced by WIR stress were effectively reversed by pretreatment with PEP. The swimming endurance capacity of mice was significantly prolonged by the oral administration of PEP. Swimming-induced fatigue significantly reduced the body weight; however, the injection of PEP inhibited the decrease of body weight. The PEP-treated group had significantly lower CK levels in plasma, an indicator of muscle damage. These results indicated that PEP has anti-stress and anti-fatigue effects, which are mediated by suppressing the hyperactivation of the hypothalamus-pituitary-adrenal axis, and antagonism of the oxidative damages induced by WIR stress and prolonged swimming times.

This study aimed to examine the effects of Lactobacillus plantarum, a lactic acid bacteria strain isolated from kimchi, on the development of low-grade inflammation and type 2 diabetes mellitus (T2DM) exacerbated by chronic stress. C57BL/6 mice were fed either a high-fat diet (HFD) and randomized into an HFD group or a group that was fed an HFD and subjected to chronic cold exposure-related stress (HFDS), or mice were fed a normal diet (ND) and randomized into an ND group or a group that was fed an ND and subjected to chronic cold exposure-related stress (NDS). Lactobacillus plantarum LRCC5310 (10(8), 10(10) CFU) and LRCC5314 (10(8), 10(10) CFU) as well as L. gasseri BNR17 (10(8) CFU), as a positive control, were administered orally twice every day to all the mice for 12 weeks. The expression of Glut4 and adiponectin, main glucose transporter-related genes, was upregulated in the LRCC5310- and LRCC5314-treated groups. Levels of serum proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6]) and of mRNAs of proinflammatory genes (Tnf-α, Il-6, Ccl2, leptin) were elevated in HFDS mice. The expression of proinflammatory genes was downregulated in LRCC5310- and LRCC5314-treated groups; this was not the case for Tnf-α expression in HFDS mice. Levels of serum corticosterone and mRNA levels of stress-related genes (Npy, Y2r) were decreased in lactic acid bacteria (LAB)-fed groups, with only LRCC5314 downregulating Npy expression in HFDS mice. These results suggest that the LAB strains can normalize the expression of metabolic genes, inhibit inflammatory responses, and suppress stress in HFDS mice.

Summary: Background: Exosomes are small extracellular vesicles secreted by various types of cells. Exosomes play an important role in intercellular communication by serving as vehicles for transferring proteins, lipids, mRNAs, microRNAs, and DNAs to recipient cells. This study investigated the role of exosomes in gastric cancer and the aging process by using a large number of human serum samples from young and aged individuals with or without gastric cancer. Methods: The age- and cancer-associated changes in exosome levels and exosomal cytokine content were measured. Exosomes in human serum were isolated by using a total exosome isolation reagent, followed by a quantification of isolated exosomes using a MicroBCA assay. Exosomal cytokines were measured by ELISA. Results: Serum exosome levels are increased by cancer and aging. Exosomal levels of TNF-α and TGF-β are increased, whereas IL-10 levels are reduced in gastric cancer. In addition, the cancer-associated changes in exosomal cytokines remain constant with age, even though aging affects every type of immune cells and immunomodulating factors. Conclusion: Gastric cancer and aging affect serum exosome and exosomal cytokines levels. Future studies would require the analysis of more diverse cytokines in exosomes derived from multiple types of cancer. Keywords: exosome, microvesicle, aging, cancer, cytokine

Commensal bacteria interact with mucosal immune or epithelial cells to modulate the immune system. Probiotics have been proposed as a novel preventive or therapeutic method for immunological diseases because of their ability to alter intestinal microflora. Herein, lactobacilli isolated from kimchi were tested for their capacity to modulate intestinal allergy in mice. Lactic acid bacteria suppressed IL-4, IL-5, IL-13, and IL-17A cytokine production in splenocytes from OVA-sensitized mice in vitro. In a mouse model of food allergy, lactobacilli significantly inhibited the expression of Th2-associated gene products such as GATA3, IL-4, IL-5, IL-9, and IL-13 in the small intestine. Moreover, pro-inflammatory cytokine levels were lower in the lactobacillus group. Lactic acid bacteria also modulated mast cell infiltration and activation. Serum IgE/IgG2a and IgG1/IgG2a ratios were altered to alleviate Th2 responses. In conclusion, lactobacilli from kimchi effectively regulated intestinal allergy by suppressing Th2 responses and modulating mast cell function and recruitment.

Alzheimer&rsquo, s disease (AD) is a progressive, neurodegenerative brain disorder associated with loss of memory and cognitive function. Beta-amyloid (A&beta, ) aggregates, in particular, are known to be highly neurotoxic and lead to neurodegeneration. Therefore, blockade or reduction of A&beta, aggregation is a promising therapeutic approach in AD. We have previously reported an inhibitory effect of the methanol extract of Perilla frutescens (L.) Britton (Lamiaceae) and its hexane fraction on A&beta, aggregation. Here, the hexane fraction of P. frutescens was subjected to diverse column chromatography based on activity-guided isolation methodology. This approach identified five asarone derivatives including 2,3-dimethoxy-5-(1E)-1-propen-1-yl-phenol (1), &beta, asarone (2), 3-(2,4,5-trimethoxyphenyl)-(2E)-2-propen-1-ol (3), asaronealdehyde (4), and &alpha, asarone (5). All five asarone derivatives efficiently reduced the aggregation of A&beta, and disaggregated preformed A&beta, aggregates in a dose-dependent manner as determined by a Thioflavin T (ThT) fluorescence assay. Furthermore, asarone derivatives protected PC12 cells from A&beta, aggregate-induced toxicity by reducing the aggregation of A&beta, and significantly reduced NO production from LPS-stimulated BV2 microglial cells. Taken together, these results suggest that asarone derivatives derived from P. frutescens are neuroprotective and have the prophylactic and therapeutic potential in AD.

Our study aimed to investigate the effects of the polysaccharide-rich extract of

Solanum melongena or eggplant is a species of the nightshade family. According to Korean folk medicine, S. melongena stalk possesses excellent therapeutic effects on warts, burns, and many inflammatory diseases, such as stomatitis, arthritis, and gastritis. In order to investigate the anti-inflammatory effects of S. melongena stalk, an ethanol extract of the stalk was prepared, and fractionated into hexane, dichloromethane, ethyl acetate, n-butanol, and water fractions. The results showed that the ethyl acetate and n-butanol fractions contained high levels of phenol and reduced artificial free radicals. In contrast, the hexane and dichloromethane fractions decreased the production of nitric oxide, pro-inflammatory cytokines, and prostaglandin E2, despite the presence of low levels of phenols implying that other compounds than phenols are involved in anti-inflammatory reactions. The data suggest that S. melongena stalk possesses pharmacological activity and might be useful for development of antiox...

Background T helper 17 (Th17) cells, which are differentiated from CD4+ T cells, drive inflammation, leading to autoimmune diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease. Therefore, inhibiting Th17 polarization could be a therapeutic target for inflammatory diseases. Purpose We investigated the inhibitory effect of Fraxinus rhynchophylla (Oleaceae) on Th17 differentiation and found its active component. Study design The activity of F. rhynchophylla and its active constituent was verified using CD4+ cells extracted from C57BL/6 mice. Methods Micro-environment for Th17 polarization was provided to CD4+ cells and the effect of treatment with samples was measured by enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blot. Results The extract of F. rhynchophylla Hance and its chemical constituent, α-amyrin acetate, which was isolated via bioassay-guided isolation, significantly inhibited Th17 polarization as revealed when interleukin (IL)-17, a characteristic cytokine produced by Th17 cells, was measured. Furthermore, the inhibitory effect of α-amyrin acetate was compared to the amyrin derivatives, α-amyrin and β-amyrin. All displayed a suppressive effect on Th17 polarization and all reduced the expression of single transducer and activator of transcription 3 (STAT3) and retinoic acid receptor-related orphan receptor γt (RORγt), which are crucial transcription factors regulating Th17 differentiation. α-Amyrin acetate, however, exhibited the most prominent effects, which indicates that the functional group, acetate, might strengthen the inhibitory effect on Th17 differentiation. Conclusion Taken together, these results suggest that the extract of F. rhynchophylla and its active constituent, α-amyrin acetate, could be applied as a potential therapeutic agent for autoimmune disorders such as rheumatoid arthritis.

The potential risks that electromagnetic fields (EMF) pose to human physiology have been debated for several decades, especially considering that EMF is almost omnipresent and some occupations involve regular exposure to particularly strong fields. In the present study, the effects of 60 Hz 0.3 mT EMF on CD4+ T cells were evaluated. Production of T cell related cytokines, IFN-γ and IL-2, was not altered in CD4+ T cells that were exposed to EMF, and cell proliferation was also unaffected. The expression of genes present in a subset of Th17 cells was upregulated following EMF exposure, and the production of effector cytokines of the IL-17A subset also increased. To determine signaling pathways that underlie these effects, phosphorylation of STAT3 and SMAD3, downstream molecules of cytokines critical for Th17 induction, was analyzed. Increased SMAD3 phosphorylation level in cells exposed to EMF, suggesting that SMAD3 may be at least in part causing the increased Th17 cell production. Differentiation of Treg, another CD4+ T cell subset induced by SMAD3 signaling, was also elevated following EMF exposure. These results suggest that 60 Hz 0.3 mT EMF exposure amplifies TGF-β signaling and increases the generation of specific T cell subsets.

Background Systemic lupus erythematosus (SLE) is associated with B cell hyperactivity, and lupus nephritis (LN), in particular, is promoted by the production of autoantibodies and immune complex deposition. Bruton’s tyrosine kinase (BTK) plays critical roles in B cell receptor-related and Fc receptor-related signaling. We aimed to investigate the impact of therapeutic intervention with HM71224 (LY3337641), a selective BTK inhibitor, on the development of murine SLE-like disease features. Methods We examined the therapeutic effects of HM71224 on SLE-like disease features in MRL/lpr and NZB/W F1 mice. The disease-related skin lesion was macroscopically observed in MRL/lpr mice, and the impact on splenomegaly and lymphadenopathy was determined by the weight of the spleen and cervical lymph node. The renal function was evaluated by measuring blood urea nitrogen, serum creatinine, and urine protein, and the renal damage was assessed by histopathological grading. Survival rate was observed during the administration period. The impact of B cell inhibition was investigated in splenocytes from both mice using flow cytometry. Autoantibody was measured in serum by ELISA. Results HM71224 effectively suppressed splenic B220+GL7+, B220+CD138+, and B220+CD69+ B cell counts, and anti-dsDNA IgG and reduced splenomegaly and lymph node enlargement. The compound also prevented skin lesions caused by lupus development, ameliorated renal inflammation and damage with increased blood urea nitrogen and creatinine, and decreased proteinuria. Furthermore, HM71224 also decreased mortality from lupus development in both mouse models. Conclusion Our results indicate that inhibition of BTK by HM71224 effectively reduced B cell hyperactivity and significantly attenuated the development of SLE and LN in rodent SLE models.

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-κB) translocation.

Purpose : To investigate the potential immune-enhancing effects of fourteen natural plant extracts on mouse macrophage RAW 264.7 cells. Methods : Fourteen plant extracts from 7 different plants were tested on RAW 264.7 cells to determine their immunostimulant activities. Methylthiazolydiphenyltetrazolium bromide (MTT) and Griess assays were performed to evaluate cell viability and nitric oxide (NO) production, respectively. Then, immune related proteins were measured by western blot analysis, while cytokines and phagocytic activity were determined by enzyme-linked immunosorbent assay (ELISA) method. Results : Among the 14 plant extracts, the hot water extract of Agastache rugose was selected based on the screening results on NO production. The hot water extract of A. rugose significantly increased NO production in a concentration-dependent manner without any cytotoxicity. In addition, the expression levels of proteins (iNOS and COX-2) and cytokines (TNF-α, IL-1β, IL-6 and IL-12) closely related to immune reaction were also significantly upregulated. Furthermore, phagocytic activity of RAW 264.7 cells significantly increased following treatment with A. rugosa . Conclusion : The hot water extract of A. rugosa exhibits significant immune-stimulant activity. Therefore, A. rugosa can be used as a natural resource for immune enhancement or dietary supplement. Keywords : Immune enhancing activity, Macrophage polarization, Natural plant extracts, Agastache rugosa , RAW 264.7

Activated macrophages have been simply de?ned as cells that secrete in?ammatory mediators and kill intracellular pathogens until few years ago. Recent studies have proposed a new classification system to separate activated macrophages based on their functional phenotypes: host defense, wound healing, and immune regulation. Regulatory macrophages can arise following innate or adaptive immune responses and hinder macrophage-mediated host defense and inflammatory functions by inhibiting the production of pro-inflammatory mediators. In this study, we investigated whether PD-1 and PD-L1 interaction between macrophages and T cells alters macrophage activities. Our data provide evidence for PD-1/PD-L1 engagement inducing a regulatory profile in macrophages. Regulatory macrophages derived from PD-L1 signaling lost their host defense activity, which consists of the production of pro-inflammatory cytokine IL-6 and the exhibition of increased IL-10, SPHK1 and LIGHT gene levels in early phases of LPS stimulation. This differentiation seems to occur through excessive activation of TLR4 downstream MAPK signaling pathways. Regulatory macrophages induced from PD-1/PD-L1 interaction decrease inflammatory mediators and produce anti-inflammatory cytokines, so this macrophage subset has been under considerable attention as a possible immune regulation mechanism. Understanding and modulating regulatory macrophages may lead to new approches to treat or prevent auto-immune diseases such as type I diabetes, rheumatic syndrome and hypersensitivity-related diseases, which are concerned with the overproduction of inflammatory cytokines in macroages.

Type 1 diabetes (T1D) is caused by dysregulation of the immune system in the pancreatic islets, which eventually leads to insulin-producing pancreatic β-cell death and destabilization of glucose homeostasis. One of the major characteristics of T1D pathogenesis is the production of inflammatory mediators by macrophages that result in destruction or damage of pancreatic β-cells. In this study the inflammatory microenvironment of T1D was simulated with RAW264.7 cells and MIN6 cells, acting as macrophages and pancreatic β-cells respectably. In this setting, peroxiredoxin-1, an anti-oxidant enzyme was knocked down to observe its functions in the pathogenesis of T1D. RAW264.7 cells were primed with lipopolysaccharide and co-cultured with MIN6 cells while PRX-1 was knocked down in one or both cell types. Our results suggest that hindrance of PRX-1 activity or the deficiency of this enzyme in inflammatory conditions negatively affects pancreatic β-cell survival. The observed decrease in viability of MIN6 cells seems to be caused by nitric oxide production. Additionally, it seems that PRX-1 affects previously reported protective activity of IL-6 in pancreatic β cells as well. These results signify new, undiscovered roles for PRX-1 in inflammatory conditions and may contribute toward our understanding of autoimmunity.

Objectives In the present study, we aimed to examine the anti-atopic properties of bile from the cat fish, Silurus asotus, to determine its possible use as a pharmaceutical product. Methods The anti-atopic activities of cat fish bile were examined in a non-cell antioxidant, in-vitro assay (splenocytes and mast cells) and a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like mouse model. Results The results of these experiments revealed that Silurus asotus bile (SAB) scavenges radicals and protects proteins from superoxide attacks, suggesting that SAB suppresses the T helper (Th) type 2-skewed immune response. Th1/Th2 mRNA cytokines (interleukin (IL)-2, interferon (IFN)-γ and IL-4) from mouse splenocytes were effectively inhibited, and the release of β-hexosaminidase in RBL-2H3 mast cells was significantly suppressed by SAB. These results were supported by screening the Th1/Th2 cytokine mRNAs (IL-2, IFN-γ and IL-4) from lymph nodes in DNCB-treated mice. More dramatic results were observed in the histological changes at higher SAB concentrations (5%) compared to the therapeutic control, visualized using hematoxylin–eosin (H&E) staining. Conclusions The results presented in this study suggest that SAB may provide functional advantages with regard to treating atopic dermatitis because of its antioxidant and immune-suppressive effects.

Beta-amyloid (Aβ) is considered as one of the major causes of Alzheimer`s disease. This study examined the neuroprotective effects of chlorogenic acid, a naturally occurring polyphenol which is distributed widely in plants, fruits and vegetables, against Aβ-induced toxicity. Aβ decreased significantly the viability of PC12 cells. This was accompanied by an increase in the intracellular calcium levels and cleaved caspase-3. In addition, Aβ induced an increase in Bax, and a decrease in Bcl-2 compared to the controls. However, a pre-treatment with chlorogenic acid rescued the PC12 cells from Aβ by attenuating the elevated intracellular calcium levels and reducing the levels of the apoptosis related proteins, including caspase-3, Bcl-2 and Bax. These results suggest that the protective effects of chlorogenic acid are, at least in parts, by attenuating the intracellular calcium influx and reducing apoptosis induced by Aβ.

Aims: Atopic dermatitis (AD) is marked by elevated levels of immunoglobulin E and skin lesions such as oedema and haemorrhage. Kimchi is a Korean fermented food that contains beneficial bacteria for human health. In this study, Lactobacillus plantarum CJLP55, CJLP56, CJLP133 and CJLP136 isolated from Kimchi were investigated for their capacity to inhibit AD. Methods and Results: The three strains, CJLP55, CJLP133 and CJLP136, suppressed AD-like skin lesions, high serum IgE levels and epidermal thickening. The three strains diminished the accumulation of eosinophils and mast cells into topical inflammatory sites and the enlargement of axillary lymph nodes, which are responsible for the dorsal dermatitis. CJLP55, CJLP133 and CJLP136 decreased production of type 2 cytokines such as IL-4 and IL-5 in lymph node cell culture. CJLP133 and CJLP136 increased IFN-γ secretion, while CJLP55 enhanced IL-10 production. Conclusions: The three strains isolated from Kimchi suppress house-dust mite-induced dermatitis in NC/Nga mouse, a representative animal model of human AD. Significance and Impact of the Study: These findings suggest that lactobacilli isolated from Kimchi inhibit AD, probably by altering the balance of Th1/Th2 ratio or inducing IL-10 production.

E-mail: soypark23@dankook.ac.krReceived January 17, 2014, Accepted March 10, 2014Key Words : Buddleja officinalis , Acetogenic glycoside, Hydrolysis, NMRBuddleja officinalis Maximowicz, which belongs to theBuddlejaceae, is a shrub tree and widely distributed in Asia,Africa, and America. It has been regarded as a traditionalherbal medicine for the treatment of inflammation, con-junctivitis, headache, and clustered nebulae.

－ T-cell activation depends on signals received by the T-cell receptor and CD28 co-stimulatory receptor. Since B7.1 and B7.2 molecules expressed on the surface of antigen presenting cells provide co-stimulatory signals through CD28 to T-cells, an inhibitor of CD28-B7.1/B7.2 binding has been proposed as a therapeutic agent for suppression of excessive T-cell activity. Although anti-B7.1/B7.2 antibodies are known to block B7.1 and B7.2 molecules, their effects on intracellular events in antigen presenting cells remain unclear. In this study, anti-B7.1/B7.2 antibodies decreased secretion of nitric oxide and pro-inflammatory cyto-kines such as TNF-α, IL-1β, and IL-12 in LPS-activated RAW264.7 macrophage-like cells and peritoneal macrophages. Moreover, anti-B7.1/B7.2 antibodies inhibited IκBα phosphorylation and down-regulated expression of co-stimulatory molecules including B7.1, B7.2, and PD-L1 in LPS-stimulated peritoneal macro-phages. These findings suggest that CTLA4-Ig and anti-B7.1/B7.2 antibodies may be candidates to treat chronic inflammatory diseases and autoimmune responses caused by excessive activation of both T-cells and macrophages.Keywords: Anti-B7.1/B7.2 antibodies, RAW264.7, Peritoneal macrophage, Co-stimulatory molecule, Pro- inflammatory cytokine

In pancreatic islets, free radical formation produced upon exposure to proinflammatory cytokines mediates β cell destruction, which ultimately leads to type 1 diabetes (T1D). In this study, we examined whether laccase, a family of the blue copper protein, can be successfully used to prevent β cells from cytokine-mediated apoptosis. Non-obese diabetic (NOD) mice were used for these experiments. In parallel, the RINm5f β cell line was employed as a model system for in vitro experiments. The results demonstrated that laccase effectively scavenged peroxinitrite, which can be formed by nitric oxide, and upregulated the expression of antioxidant enzymes, such as manganese superoxide dismutase (MnSOD) and catalase. Interestingly, laccase balanced pro- (Bax) and anti-apoptotic (Bcl-2) proteins in terms of both the mRNA and protein levels with a downregulation of cytochrome c protein in RINm5f cells. In addition, laccase maintained blood glucose concentrations at a normal level with a simultaneous increase in plasma insulin levels during the spontaneous induction of diabetes in NOD mice. In conclusion, the antioxidant potentials of laccase in scavenging free radicals and upregulation of antioxidant enzymes may exert its pro-survival effect by counteracting the increased intracellular oxidative stress, and, consequently, by inhibiting apoptosis induced by cytokine-mediated activation during the course of T1D.

The cytokines which is produced by allergen-specific T helper (Th) cells play a pivotal role in the pathogenesis of asthma. Asthma is caused by exaggerated T-helper 2 (Th2)-based immune responses. It is suggested that controlling such Th2-based response is necessary for asthma therapy. The current therapies for asthma focus primarily on control of symptoms and suppression of inflammation, without affecting the underlying cause. So, we examined that anti-asthmatic drugs might have play a certain role in Th2/Th1 balance. Splenocytes isolated from ovalbumin (OVA)-sensitized mice cultured with anti-asthmatic drugs. It is well known that Th2 and Th1 immune responses can balance one another, as Th2 mediators suppress Th1 responses and Th1 mediators similarly inhibit Th2 responses. But salmeterol inhibits both of Th1 and Th2 mediators, which salmeterol is a suppressor of immune responses not only a suppressor of Th2-based immune responses. Aminophylline is a weak suppressor of immune responses. But ipratropium and cromoglycate don't have any suppressor effect to Th2-driven responses. They only have suppressor effect to Th1 immune responses. Salmeterol, ipratropium, aminophylline, and cromoglycate augmented mRNA levels of CRTH2, EP2, and IP2 receptors in OVA-sensitized splenocytes. It is well known that the up-regulation of CRTH2 - PGD2 receptor - results in restraint of eosinophil recruitment and that the increment of IP and EP2 - PGI2 and PGE2 receptor, respectively - may induce the accumulation of cAMP that decrease the effector function of T cells. Moreover salmeterol and cromoglycate increase the mRNA expression of PGD2 synthase. These findings indicate that anti-asthma agents may alleviate the immunological responses that cause the asthmatic diseases.

Alzheimer’s disease (AD) is the most common form of dementia among the elderly, causing progressive cognitive dysfunction, particularly memory loss. Recently, modulation of Aβ toxicity, which is on...

We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p

−Immunosuppressive therapy after organ transplantation is routinely used to prevent rejection of theorgan, because this decreases the risk of adverse events, infection, and malignancies. Recently, ursodeoxy-cholic acid (UDCA), which is isolated from the dried bile of adult Chinese bears, has been shown to reducethe incidence and severity of acute rejection of liver allograft during early phase of liver transplantation. There-fore, in this study, we investigated the effect of UDCA on the proliferation of splenocytes exposed to PMA plusionomycin. Our results demonstrated that UDCA decreased the splenocytes’ proliferation in a dose-depen-dent manner. The decreased cell proliferation was accompanied with the decreased secretion of cytokinessuch as IL-2, IFN-γ and TNF-α. In addition, the pretreatment of UDCA on splenocytes stimulated with PMAplus ionomycin decreased the mRNA levels of cytokines (IL-2, IFN-γ and TNF-α) and costimulatory molecules(B7.2 and PD-L1). These results suggest the beneficial effect of UDCA on organ transplantation by decreas-ing lymphocyte proliferation.

Considering the importance of inflammation and apoptosis in neurodegenerative conditions, the potential suppressive effects of the Rg3, a by-product obtained during the steaming of red ginseng, may indicate that Rg3 could provide a beneficial therapeutic approach to treating or preventing neurodegenerative disease. We investigated the effect of Rg3 on Abeta42-mediated microglial activation and inflammation-mediated neurotoxicity in murine BV-2 microglial and Neuro-2a neuroblastoma cells, respectively. Rg3 effectively reduced inflammatory cytokine expression in Abeta42-treated BV-2, and inhibited the binding of NF-kappaB p65 to its DNA consensus sequences, and significantly reduced the expression of TNF-alpha in activated microglia. Pretreatment with Rg3 increased the survival rate of Neuro-2a exposed to TNF-alpha. These observations suggest that Rg3 reduced neurotoxicity by inhibiting chronic inflammation through the suppression of activated microglia. In addition, the expression of pro-inflammatory cytokines in BV-2 stimulated by Abeta42 was decreased but not eliminated by Rg3 when binding to the macrophage scavenger receptor type A (MSRA) was blocked with fucoidan. This implies that the inflammatory response may not be exclusively triggered via MSRA. More interestingly, iNOS was almost completely inhibited in the presence of Rg3 when MSRA binding was blocked with fucoidan. Moreover, Rg3 increased the expression of MSRA in BV-2 transfected with siRNA targeting MSRA mRNA, and this increased MSRA expression may play a role in the phagocytosis of Abeta42 peptides. Our results indicate that inhibition of the inflammatory repertoire of microglia, neuroprotection, and increased MSRA expression induced by Rg3 may at least partly explain its therapeutic effects in chronic neurodegenerative diseases.

Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFNγ in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.

In anti-cancer therapies, radiotherapy and chemotherapy show a superior inhibition effect on cancer cell growth, but those are very toxic to normal tissues and organs. Particularly, drugs for neutropenia, one of chemotherapy agents, which suppress the function of bone marrow, are needed to be controlled in terms of their dosage and therapy period because of their side effect. Phellinus linteus polysaccharide (PL) has been reported to increase the number of splenocytes and bone marrow cells. PL has been shown to decrease the side effects of cyclophosphamide (CYC) treatment to the cancer patients. PL showed no effects in semisolid clonogenic assay, suggesting that PL doesn`t contain substantial compounds to substitute for colony stimulating factors (CSFs). On the other hand, PL increased the expression of SCF, IL-3, GM-CSF, TPO genes. These results indicate that PL may promote the growth and proliferation of splenocytes and bone marrow cells through indirect or CSFs-dependent pathway, which may lead to a hematopoiesis.

Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

NF-kappaB is a key regulator of transcription after TCR and costimulatory receptor ligation. To determine the role of T cell-intrinsic NF-kappaB activation in acute allograft rejection, we used IkappaBalphaDeltaN-Tg mice (H-2b) that express an inhibitor of NF-kappaB restricted to the T cell compartment. We have previously shown that these mice permanently accept fully allogeneic (H-2d) cardiac grafts and secondary donor skin grafts, and that splenocytes from these tolerant mice have reduced alloreactivity when restimulated in vitro. These results were compatible with either deletion or suppression of allospecific T cells as possible mechanisms of tolerance. The aim of this study was to investigate the mechanism of transplant tolerance in these mice. IkappaBalphaDeltaN-Tg mice did not have increased numbers or function of CD4+ CD25+ regulatory T cells either before or after cardiac transplantation. In addition, tolerance could not be transferred to fresh NF-kappaB-competent T cells and was not permissive for linked suppression to skin grafts sharing donor and third-party alloantigens, suggesting that dominant suppression is not the mechanism by which IkappaBalphaDeltaN-Tg mice achieve tolerance. In contrast, overexpression of the antiapoptotic protein Bcl-xL in T cells from IkappaBalphaDeltaN-Tg mice resulted in effective rejection of cardiac allografts and correlated with an increased frequency of splenocytes producing IFN-gamma in response to alloantigen. Together, these results suggest that the death of alloreactive T cells may be partly responsible for the transplantation tolerance observed in mice with defective T cell-intrinsic NF-kappaB activation.

IL-2-deficient mice develop a lymphoproliferative and autoimmune disease characterized by autoimmune hemolytic anemia (AHA) and inflammatory bowel disease. We have previously reported that IL-2 is necessary for optimal up-regulation of CTLA-4, an inducible negative regulator of T cell activation. In this study, we have tested the hypothesis that reduced expression of CTLA-4 in IL-2-deficient T cells contributes to the pathogenesis of disease in IL-2-deficient mice. Expression of CTLA-4 as a transgene completely prevented lymphoaccumulation and AHA in IL-2-deficient mice. The normalization of T cell numbers was due to inhibition of expansion of conventional CD4+CD25− T cells rather than to rescue of the numbers or function of CD4+CD25+ regulatory T cells, suggesting that CTLA-4 expression on conventional T cells plays a role in maintaining normal T cell homeostasis. In addition, the inhibitory effect of the CTLA-4 transgene on T cell expansion was at least in part independent of CD28 expression. Our results suggest that deficient CTLA-4 expression on conventional T cells contributes to the pathophysiology of the lymphoproliferative disease and AHA in IL-2-deficient mice. Thus, restoring CTLA-4 expression in T cells may be an attractive strategy to control clinical autoimmune diseases in which CTLA-4 expression is reduced.

The role of secondary lymphoid organs in adaptive immune responses following transplantation is controversial. To examine the requirement for peripheral lymphoid organs in mounting immune responses to transplantation antigens, lymphotoxin alpha-deficient (LTalpha-/-) and LTbeta-receptor-deficient (LTbetaR-/-) mice that lack lymph nodes and Peyer's patches were used as recipients of fully allogeneic heart and skin grafts. Splenectomized LTalpha-/- and LTbetaR-/- mice effectively rejected skin and cardiac allografts, although with delayed kinetics when compared with wild-type controls. In addition, initial skin allograft challenge in splenectomized LTbetaR-/- mice resulted in accelerated rejection of subsequent donor cardiac allografts when compared with heart rejection in nonsensitized controls. Thus, although peripheral lymphoid organs play an important role in allowing allograft responses to occur, they do not appear to be absolutely required for either acute allograft rejection, or T-cell priming. These results suggest that immunologic events capable of leading to allograft rejection can successfully occur at sites other than classical secondary lymphoid organs.

Natural engagement of CTLA-4 on host B7 limits T cell activation. We hypothesized that therapeutic cross-linking of CTLA-4 in vivo may further inhibit T cell function and prevent allograft rejection. However, none of the currently available CTLA-4-binding reagents have ligating properties when injected in vivo. The observation that surface-immobilized anti-CTLA-4 mAb inhibits T cell activation in vitro prompted us to develop a membrane-bound single-chain anti-CTLA-4 Ab (7M). To model whether tissue expression of 7M could suppress allograft rejection, we examined the ability of H-2Ld-specific TCR-transgenic T cells to reject 7M-expressing allogeneic tumor cells injected s.c. Expression of 7M significantly inhibited allogeneic rejection in mice that received CTLA-4+/+ but not CTLA-4−/− T cells. Furthermore, CTLA-4+/+ T cells that had encountered 7M-expressing tumors in vivo acquired defects in cytokine production and cytotoxicity. Thus, deliberate ligation of CTLA-4 in vivo potently inhibits allogeneic T cell responses.