International Immunopharmacology

In this study, we synthesized (±)-tylophorine malate (NK-007), an analog of tylophorine (DCB3503), and analyzed its anti-inflammatory effect in vivo using a dextran sulfate sodium (DSS)-induced colitis model and an acetic acid-induced colitis model. As indicated by disease activity index (DAI) and degree of macroscopic colonic damage, NK-007 can significantly suppress colitis. To delineate the underlying mechanism, we have explored the influence of NK-007 on the production of TNF-α by murine primary bone marrow-derived dendritic cells (BMDCs) as well as monocyte/macrophage cell line Raw 264.7 triggered by lipopolysaccharide (LPS). For both types of innate immune cells, NK-007 showed a potent TNF-α inhibitory effect, and has in addition reduced the expression of IL-12 in BMDCs. Moreover, Raw cells treated with NK-007 also showed decreased phosphorylation of NF-κB, which may explain the protective immune-regulatory effect of NK-007 for experimental colitis.

The efficacy of a potential therapeutic vaccine against chronic hepatitis B virus (HBV) infection depends on the development of strong and multi-specific T cell responses. The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward HBV core antigen (HBcAg) has been shown to be critical for the outcomes of HBV chronic infection. In this study we have identified a previously undescribed HLA-A*0201-restricted HBcAg-specific CTL epitope (HBcAg₆₄₋₇₂, C₆₄₋₇₂, ELMTLATWV). T2 binding assay showed that C₆₄₋₇₂ had high affinity to HLA-A*0201 molecule. Functionally, the peptide C₆₄₋₇₂ could induce peptide-specific CTLs both in vivo (HLA-A2.1/K(b) transgenic mice) and in vitro (PBLs of healthy HLA-A2.1+ donors), as demonstrated by interferon-γ (IFN-γ) secretion upon stimulation with C₆₄₋₇₂-pulsed T2 cells or autologous human dendritic cells (DCs) respectively. HLA-A*0201-C₆₄₋₇₂ tetramer staining revealed the presence of a significant population of C₆₄₋₇₂-specific CTLs in C₆₄₋₇₂-stimulated CD8+ T cells. Furthermore, the peptide-specific cytotoxic reactivity and the production of perforin and granzyme B of CTLs also increased after stimulation with C₆₄₋₇₂-pulsed autologous DCs. These results indicate that the newly identified epitope C₆₄₋₇₂ has potential to be used in the development of immunotherapeutic approaches to HBV infection.

The TNF-α expression-inhibitory effect of lactic acid bacteria (LAB) isolated from kimchi were measured in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Among the LAB evaluated, Lactobacillus plantarum CLP-0611 inhibited the IL-1β and IL-6 expression, as well as the NF-κB and AP1 activation in LPS-stimulated peritoneal macrophages. Therefore, we investigated its inhibitory effect on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. TNBS significantly induced colon shortening, as well as myeloperoxidase activity and macroscopic score. Oral administration of CLP-0611 significantly reduced TNBS-induced body weight loss, colon shortening, myeloperoxidase activity, IRAK-1 phosphorylation, NF-κB and MAP kinase (p38, ERK, JNK) activation, and iNOS and COX-2 expression. CLP-0611 also inhibited TNBS-induced expression of TNF-α, IL-1β, and IL-6. However, IL-10 expression was induced. CLP-0611 also induced the production of M2 macrophage markers (IL-10, arginase I and CD206). Based on these findings, CLP-0611 inhibits TLR-4-linked NF-κB and MAPK signaling pathways and polarizes M1 to M2-like macrophages, thus ameliorating colitis.

4b,11b-Dihydroxy-4b,11b-dihydro-12H-indeno[1,2-b]naphtho[2,3-d]furan-12-one (DHFO), an easily synthesisable, orally bioavailable and relatively non-toxic small molecule synthesised in our lab, was previously reported to possess anti-oxidant, 5-lipoxygenase(LOX) inhibitory, anti-inflammatory and peripheral analgesic activities. The present work deals with exploration of DHFO's efficacy in immunopathogenic chronic inflammatory conditions - arthritis and allergy. In carrageenan-induced inflammatory air pouch, which resembles the arthritic synovium, DHFO effectively reduced inflammatory redness and swelling and neutrophil infiltration. In complete Freund's adjuvant-induced arthritis, DHFO significantly decreased paw oedema and nitrite levels with efficacy comparable to diclofenac. DHFO inhibited neutrophil activation (observed as decreased MPO levels), in both the in vivo models of inflammation. Interestingly, DHFO did not ulcerate the gastrointestinal tract, while diclofenac was observed to be extremely ulcerogenic. In antigen-induced active and passive anaphylaxis (allergy) models, DHFO dose-dependently prevented mesenteric mast cell (MC) degranulation with efficacy comparable to ketotifen. DHFO also inhibited compound 48/80-induced paw oedema and peritoneal MC degranulation. DHFO stabilised p815 murine MCs stimulated by compound 48/80 and calcium ionophore-A23187, indicating an action downstream of calcium mobilisation. DHFO's anti-allergic mechanism could be two-pronged involving (1) inhibition of IgE production and/or (2) MC stabilisation. DHFO inhibited lipopolysaccharide (LPS)-induced pro-inflammatory mediator release (ROS, NO, IL-6 levels) and COX2 expression in RAW264.7 murine macrophages. Protein expression studies confirmed DHFO's ability to reduce nuclear levels of NF-κB in LPS-stimulated macrophages. Thus, DHFO is a promising non-ulcerogenic synthetic small molecule lead for immunopathogenic chronic inflammatory conditions.

CKD712, (S)-1-(α-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, was considered as a new effective drug candidate to sepsis, based on its anti-inflammatory activity. It was reported that CKD712 inhibited various signal pathways which play a key role in production of proinflammatory cytokines. Here, we examined the effect of CKD712 on the secretion of high mobility group box 1 (HMGB1), which is one of the proinflammatory cytokines. CKD712 can reduce Gram-negative lipopolysaccharide (LPS)- and Gram-positive lipoteichoic acid (LTA)-stimulated HMGB1 secretion in RAW264.7 and human peripheral blood monocytes (PBMo), and also reduce LPS-induced nucleocytoplasmic translocation of HMGB1 1h before or after LPS treatment. CKD712 could dose-dependently inhibit the activation of PI3K and PI3K-dependent kinase 1 (PDK1), which are involved in HMGB1 secretion signaling pathway. In addition, CKD712 inhibited classical protein kinase C (cPKC), the effective kinase for phosphorylation of HMGB1 for secretion, however, had no effect on histone acetyl-transferase activity, which is another mechanism known for HMGB1 secretion. Thus, we suggest that CKD712 could inhibit LPS- and LTA-stimulated HMGB1 secretion through the inhibition of HMGB1 phosphorylation by inhibiting PI3K-PKC signaling pathway.

The aim of this study is to investigate the anti-inflammatory and immunosuppressive effects of ZL-5015 (1,3-dicyclopentyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylic acid diethyl ester) in order to determine its potential as a lead compound to develop novel drugs with both anti-inflammatory and immunosuppressive activities. Inflammatory in vivo models (specifically, acetic acid-induced mouse writhing, xylene-induced mouse ear swelling and carrageenan-induced rat paw edema) and in vitro models (specifically, lipopolysaccharide (LPS)-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) by mouse peritoneal macrophages and RAW264.7 cells) were used to evaluate the anti-inflammatory activities. Immunological in vivo models (specifically, rabbit red blood cells (RRBC)-induced mouse hemolysin production, 2,4-dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) and adjuvant-induced rat arthritis) and in vitro models (specifically, concanavalin A (Con A) and LPS-stimulated mouse splenocyte proliferation) were applied to estimate the immunosuppressive effects. It was found that ZL-5015 significantly decreased acetic acid-induced mouse writhing, xylene-induced mouse ear swelling, and carrageenan-induced rat paw edema at the doses from 25 to 100mg/kg, and inhibited mouse hemolysin production, DTH response, and adjuvant-induced rat arthritis at the doses from 50 to 200mg/kg. The compound appeared to be more potent in inhibition of inflammation than in suppression of immune function, as judged by the minimal statistically effective dose. The in vitro studies revealed that ZL-5015 greatly inhibited the production of NO, PGE2 and TNF-α, slightly promoted IL-10 production and suppressed the splenocyte proliferation stimulated by Con A or LPS at the concentrations from 10 to 40μM. In conclusion, our study demonstrates that the tetrahydropyrimidine derivative, ZL-5015, has both anti-inflammatory and immunosuppressive activities, although its potency is not satisfactory. Therefore ZL-5015 should be considered as a lead compound for further structural modification in the continuing search for novel and effective drugs in this area.

In Crohn's disease (CD), epidemiological data and animal studies suggest that vitamin D (vitD) has protective immune-modulating properties. 1,25-dihydroxyvitamin D3 and dexamethasone (DEX) induce interleukin (IL)-10 productions in healthy controls (HC) T cells. We studied if 1,25-dihydroxyvitamin D3 with and without DEX could induce IL-10 production, downregulate pro-inflammatory Interferon (IFN)-gamma and Tumor Necrosis Factor (TNF)-alpha production, and influence cell kinetics in peripheral CD4+ T cells from CD patients. CD4+ T cells were separated from peripheral blood from CD patients and HC. Cells were activated by anti-CD3 and anti-CD28 in the presence of 1,25-dihydroxyvitamin D3 and/or DEX. Cytokine levels, proliferation, and apoptosis were measured following 7 days of culture. In T cells from CD patients, 1,25-dihydroxyvitamin D3 and DEX increased IL-10 production from a median of 0.08 ng/ml to 0.2 ng/ml (p<0.01) and downregulated IFN-gamma production from 8.3 ng/ml to 3.1 ng/ml (p<0.01). The induced IL-10 increase in cultures from HC (0.2 ng/ml to 1.0 ng/ml, p<0.01) was significantly higher than in CD patients (p<0.05). In CD cultures, the IL-4 production increased from 0.3 ng/ml to 0.5 ng/ml (p<0.01) and IL-6 production from 2.5 ng/ml to 6.1 ng/ml (p<0.05). Similar changes in cytokine levels were observed with 1,25-dihydroxyvitamin D3 independently of DEX. In addition, 1,25-dihydroxyvitamin D3 and DEX decreased proliferation and reduced viability of T cells. We found that 1,25-dihydroxyvitamin D3 with and without DEX stimulation increased IL-10 and reduced IFN-gamma production. These findings suggest that vitD may play a therapeutic role in CD.

We recently designed a series of N-[4-(t-amino-yl)-but-2-yn-1-yl] isoindoline-1,3-diones as anti-inflammatory compounds, called ZM compounds. These ZM compounds were categorized according to the nature of the cyclic amino groups into ZM2, ZM3, ZM4, and ZM5 and were shown to reduce carrageenan-induced inflammation, inhibit cyclooxygenase (1 and 2) and have less adverse effects than the common non-steroidal anti-inflammatory drugs. In the present study, we are examining the potential effects of ZM compounds in modulating cytokines production in vivo and in vitro from stimulated spleen cells, CD4+ CD25+ve T regulatory cells and CD4+CD25-ve T helper cells. Six hours following oral administration of 20mg/kg of ZM4 and ZM5 compounds reversed LPS-induced TGF-β suppression whereas ZM2, ZM3, ZM4, and ZM5 reversed LPS-induced TNF-α and IL-12 increase in mice spleen. In addition, increasing concentrations of ZM2, ZM4 and ZM5 increased significantly TGF-β1 production, whereas ZM3, ZM4 and ZM5 suppressed only TNF-α production in LPS and LPS+PMA stimulated spleen cells. Furthermore, only ZM5, enhanced significantly TGF-β1 production from LPS and LPS+PMA stimulated CD4+CD25+ve cells (p<0.001), whereas none of the ZM compounds modulated TNF-α from CD4+CD25-ve T helper cells. These results indicate that ZM5 (N-{4-(2-Azepan-1-yl)-but-2-yn-1-yl}isoindoline-1,3-dione) enhances TGF-β production from CD4+CD25+ve cells independent of protein kinase C activation and suggest that all ZM compounds suppress TNF-α from monocytes/macrophage cells. In conclusion, these ZM compounds have potential to be used use as anti-inflammatory agents and further studies to show the possibility of utilizing these basic aminoacetylenic isoindolines in autoimmune mediated inflammatory diseases are warranted.

There have been a number of reports showing that the crude beta-glucan fraction prepared from various kinds of Basidiomycetes mushrooms acts as anti-cancer and anti-allergic reagent through stimulation of IFN-gamma production. It has been reported, however, that the exposure of the airway to beta-(1,3) d-glucan, contained in house dust, indoor moulds and some bacteria, potentiates the airway allergic response. It seems likely that the discrepant effects on immune function may be related to such factors as differences of the administration route, average molecular weight and water solubility. We isolated a new low-molecular-weight (about 100 kDa) beta-glucan from Aureobasidium pullulans 1A1 strain of black yeast, and found that it had low viscosity and was water-soluble. In this study, we examined the effects of water-soluble low-molecular-weight beta-(1-->3) and 50-80% branched beta-(1-->6) glucan (LMW-beta-Glucan) isolated from A. pullulans on the ova-albumin (OVA)-treated allergic reaction in mice. Feeding standard laboratory diets containing 0.5 and 1% LMW-beta-Glucan significantly inhibited the OVA-specific IgE elevation compared to that in OVA-sensitized mice fed standard laboratory diet alone (control). Furthermore, feeding standard laboratory diets containing 0.5 and 1% LMW-beta-Glucan inhibited the reduction of IL-12 and IFN-gamma production from splenocytes and the reduction of CD8- and IFN-gamma-positive cell number in the small intestine of the OVA-sensitized mice. These findings suggest that anti-food allergic action of LMW-beta-Glucan may be due to the inducing IFN-gamma production in the small intestine and splenocytes.

We recently elucidated the structure of a highly branched 1,3-beta-D-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of beta-D-glucans are known to depend on their structures, the effects of a highly branched 1,3-beta-D-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-gamma and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-alpha in DBA/2 mouse-derived splenocytes in vitro.

Glucans have a long history as nonspecific biological modulators. A novel glucan-Phycarine-was isolated from sporophytes of Laminaria digitata. Phycarine showed significant stimulation of phagocytic activity as well as potentiation of synthesis and release of IL-1, IL-6 and TNF-alpha. In addition, Phycarine increased NK cell-mediated killing of tumor cells both in vitro and in vivo while acting via complement receptor type 3 (CR3) receptors.

Soluble beta-1,3-glucan has been demonstrated to protect against infection and shock in rats and mice, and clinical studies suggest that administration of soluble glucans to trauma/surgical patients decreases septic complications and improves survival. However, little is known about the precise mechanisms by which glucans influence the state of activation of blood cells, which are responsible for the fulminant cytokine production and the activation of the coagulation system observed in serious gram-negative infection. We studied therefore the effect of an underivatized, soluble yeast beta-1,3-glucan and lipopolysaccharide (LPS), either alone or in combination, on tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 secretion and monocyte tissue factor (TF) expression in human whole blood. As expected, LPS induced the secretion of substantial amounts of all measured parameters, whereas only minor amounts of TNFalpha, IL-6, and IL-10 were induced by beta-glucan itself. However, beta-glucan itself induced the production of significant amounts of IL-8 and TF. Soluble beta-1,3-glucan had a strong synergistic effect on the LPS-induced secretion of IL-8, IL-10, and on monocyte TF activity, but not on TNFalpha and 1L-6 production. On the other hand, soluble beta-glucan strongly primed LPS stimulation of all parameters, including TNFalpha and IL-6. beta-Glucan also induced detectable neutrophil degranulation within 15 min, whereas a response to LPS was first detected after 90 min. In conclusion, soluble beta-1,3-glucan upregulated leukocyte activity, both on its own and in concert with LPS.

In this study we demonstrated that the oral administration of β-1,3-glucan (Imunoglucan®) protects mice from a lethal dose of Listeria monocytogenes (LM) when administered prophylactically for 10 days at the doses of 150 and 300 mg/kg, with survival rates up to 40%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with LM, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors revealed an increased colony-stimulating activity (CSA) in the serum of infected mice pre-treated with Imunoglucan®. The treatment also restored the reduced ability of stromal cells to display myeloid progenitors in long-term bone marrow cultures (LTBMC) and up-regulated IL-6 and IL-1α production by these cells in the infected mice, which was consistent with higher number of non-adherent cells. Additional studies to investigate the levels of interferon-gamma (INF-γ) in the supernatant of splenocyte cultures demonstrated a further increase in the level of this cytokine in infected-treated mice, compared to infected controls. In all cases, no differences were observed between the responses of the two optimal biologically effective doses. In contrast, no significant changes were produced by the treatment with the 50mg/kg dose. In addition, no changes were observed in normal mice treated with the three doses used. All together our results suggest that orally given Imunoglucan® indirectly modulates immune activity and probably disengages Listeria induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1α, IL-6, and INF-γ).

The signaling mechanism of the novel (1,4)-alpha-D-glucan (RR1) isolated from the medicinal plant Tinospora cordifolia was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4 degrees C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-alpha synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-alpha synthesis. RR1 induced TNF-alpha synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-kappaB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-kappaB in a time- and dose-dependent manner and this modulation of nuclear NF-kappaB activity is associated with the degradation of I-kappaB alpha thus facilitating the translocation of NF-kappaB into the nucleus. RR1-induced NF-kappaB activity peaks at 8 h of RR1 stimulation while I-kappaB alpha degradation occurred within 1 h of stimulation. RR1-induced NF-kappaB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-alpha-D-glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-kappaB translocation and cytokine production.

The present study investigated the effects of the anthraquinone derivative (O,O'-bis-(3'-iodopropyl)-1,4-dihidroxyanthraquinone - DIPDHAQ), mitoxantrone analog, in an experimental autoimmune encephalomyelitis (EAE) model. The results showed that DIPDHAQ treatment improved the clinical signs of the disease (n=10; vehicle: 3.8 ± 0.3; DIPDHAQ: 1.4 ± 0.9). The improvement was associated with a decrease of inflammatory cells, demyelination, IL-17, IFN-γ, IL-12p40, IL-6, TGF-β, CCL5 and CCL20 levels in the spinal cord. DIPDHAQ presented a low cytotoxicity when in vitro assays were performed. Therefore, the findings suggest a major role for DIPDHAQ in multiple sclerosis, disease characterized as an autoimmune inflammatory disorder against myelin proteins of the brain and spinal cord. The attenuation of inflammation and consequently improvement of clinical signs, involving a decrease of pro-inflammatory cytokines and the low cytotoxicity of DIPDHAQ, suggest that this compound could be used as an alternative treatment for autoimmune diseases in the central nervous system.

Fructose-1,6-diphosphate (FDP), a high-energy glycolytic pathway intermediate, is reported to have a salutary effect in endotoxic shock and sepsis, but its underlying mechanism of action in inflammation is incompletely understood. In this study, our aim was to examine the function of FDP on acute lung injury (ALI) induced by lipopolysaccharide (LPS). We found that in vitro pretreatment with FDP remarkably repressed the production of TNF-alpha and IL-6 in murine alveolar macrophages MH-S exposed to LPS. In the mouse model of LPS-induced inflammatory lung injury, intravenous precondition of a single 400 mg/kg dose of FDP resulted in a significant reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage leucocyte content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Furthermore, histopathologic examination indicated that alveolitis with inflammatory cells infiltration and alveolar hemorrhage in the alveolar space was less severe in the FDP-treated mice than in the mice treated by LPS alone at 24 h. Additionally, pretreatment with FDP markedly decreased the transcription of TNF-alpha, IL-6 and inducible NO synthase (iNOS), and suppressed the nuclear translocation of NF-kappaB in lung tissues in response to LPS challenge. These results thus suggested that FDP plays an anti-inflammatory role in LPS-mediated acute lung injury, possibly through abrogation of NF-kappaB activation.

Sepsis remains an important and life-threatening problem, and is the most common cause of death in the intensive care unit. One promising therapeutic candidate for protection against injury in sepsis is fructose-1,6-bisphosphate (FBP), a high-energy glycolytic pathway intermediate. The objective of the study was to establish a role for FBP on the immune system, especially in lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy humans by gradient centrifugation. T-lymphocytes were stimulated for 96 h with phytohemagglutinin (PHA) and varying concentration of FBP. Fructose-1,6-bisphosphate at concentrations between 1.2 and 10 mM decreased proliferation of T-lymphocytes and reduced the viability only at concentrations 5.0 and 10 mM. The levels of soluble IL-2 receptor were reduced at FBP concentrations between 1.2 and 10 mM. In conclusion, this study demonstrates that FBP has important effect on immunomodulatory and this result can be correlated with the protection against injury in sepsis.

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKβ and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKβ. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-β (IFN-β) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation. Copyright © 2015. Published by Elsevier B.V.

Background: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent and caspase-independent cell apoptosis. It has been verified that Omi/HtrA2 is related to apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria. Ucf-101 is a specific inhibitor of Omi/HtrA2 and it has been demonstrated that Ucf-101 has organ protective effects in a variety of in vitro and in vivo studies. The aim of our study was to examine the neuroprotective effects of Ucf-101 on cerebral oxidative injury and cognitive impairment in septic rats. Methods: Male Sprague Dawley rats are subjected to cecal ligation and puncture (CLP) or sham-operated laparotomy. Rats were divided into 4 groups: (1) a sham group plus normal saline (10 mL/kg); (2) a sham group plus Ucf-101 (10 umol/kg); (3) CLP plus normal saline (10 mL/kg); and (4) CLP plus Ucf-101 (10 umol/kg). Brain tumor necrosis factor (TNF)-α level, caspase-3 and caspase-9 activities, malondialdehyde (MDA) content and catalase (CAT) activities were examined. TUNEL staining was utilized to evaluate the amount of apoptosis and the cognitive function was evaluated by the MWM test. The study also assessed the clinical scores of animals and the survival time for the 7-day period. Results: CLP resulted in a poor survival rate, evidence of hippocampal oxidative injury, cell apoptosis and cognitive dysfunction as well as elevated TNF-α level and caspases activities, increased weight loss and clinical scores. Ucf-101 pre-treatment could significantly inhibit caspases activities and cell apoptosis, reduce TNF-α and MDA levels, slightly reverse CAT activities in the brain and attenuate this CLP effect on cognitive dysfunction. In addition, the survival rate and survival time was significantly improved by pre-treatment with Ucf-101. Conclusions: The present results demonstrated that ucf-101 has the neuroprotective effects on cerebral oxidative injury and cognitive impairment in septic rats.

The role of lactic acid bacteria in gut mucosal immunity was investigated by comparing the enhanced effects in the Peyer's patches and spleen of BALB/c mice fed daily with Lactobacillus paracasei subsp. paracasei NTU 101 for 3 to 9 weeks. After feeding with Lactobacillus, the percentage of CD4+ T cells in both Peyer's patches and the spleen was significantly increased; however, expression of CD 154 molecules, which play a pivotal role in cell-to-cell communication, on CD4+ T cells and the percentage of B220+ B cells increased only in Peyer's patches. Compared with systemic serum IgA, Peyer's patch-derived immunomodulation induced higher levels of intestinal IgA+-producing cells in the lamina propria. Our data also showed that feeding with Lactobacillus induced stronger CD4+ T cell-dendritic cell interaction, enhanced CD4+ T cell and B cell proliferation, and increased IL-1beta, IL-10, IL-12, IFN-gamma, and TNF-alpha mRNA expression in Peyer's patches, but not in the spleen. Here, we demonstrate that following Lactobacillus treatment, Peyer's patches exhibited a more distinct capacity to induce CD4+ T cell-dendritic cell interactions, lymphocyte proliferation, and cytokine secretion than the spleen, and thereby promoted greater intestinal IgA production that could enhance immunosurveillance to prevent intestinal infections or other intestinal pathologies.

Dendritic cells (DCs) are the most potent antigen presenting cells of the immune system as they can act as initiators, stimulators and regulators of the immune response. Human DCs are most commonly generated for clinical use by in vitro differentiation of monocytes with exogenous cytokines. Here, we investigate the effect of LCOS 1013 on the production of mature Mo-DCs. LCOS 1013 is a new bacterial component from walls of gram(+)Klebsellia pneumoniae bacteria that contain some OmpA glycoproteins. Purified peripheral blood monocytes were cultured for 6 days with IL-4 and GM-CSF in order to obtain immature dendritic cells (Im-MoDCs). On day six, Im-MoDCs were matured with either LCOS 1013, TNF alpha, LPS or CD40-Ligand. LCOS 1013 matured Mo-DCs (LCO-DCs) showed a higher expression of DC-LAMP, CD80, CD83, CD54 and CD40 than TNF alpha, LPS and CD40L matured Mo-DCs. Interestingly, LCO-DCs exhibited high expression of full competent CCR7 and high secretion of IL-12 during their maturation. Functionally, LCO-DCs have equivalent potency to trigger mixed leukocyte reaction and antigen-specific reaction and polarize immune response towards Th1 way. Moreover, we found that LCOS 1013 activates DCs through TLR2. LCOS 1013 represents an attractive therapeutic maturation agent of DCs allowing the production of Mo-DCs with high capacity to migrate and to induced Th1 immune responses.

Beta-sitosterol (SITO) is a potential candidate for cancer chemotherapy, however, little is known about the cellular and molecular mechanisms in cancer cells. We herein identified how SITO induces anti-proliferation and cell death in MCA-102 fibrosarcoma cells. SITO exposure induced-apoptosis and the cell death resulted from a significant loss of the Bcl-2 and the inhibitor of apoptosis protein (IAP) family (XIAP, cIAP-1 and cIAP-2), and increased Bax with an alteration of p53 and p21. SITO-induced cell death significantly also increased caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage, and caspase-3 inhibitor z-DEVD-fmk significantly inhibited SITO-induced cell death. These data suggest that the activation of caspase-3 is associated with SITO-induced-apoptosis. Treatment with SITO also induced phosphorylation of extracellular-signal regulating kinase (ERK) and p38 mitogen-activated protein kinase (MARK), but not c-Jun N-terminal kinase (JNK). A specific ERK inhibitor PD98059 significantly blocks SITO-induced-apoptosis, whereas a JNK inhibitor SP600125 has no affect. A p38 MAPK inhibitor SB203580 very slightly suppressed cell death. The induction of apoptosis was also accompanied by an inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases SITO-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of SITO is mediated through the activation of ERK and the block of the PI3K/Akt signal pathway in MCA-102 cells. Therefore, SITO has a strong potential as a therapeutic agent for preventing cancers such as fibrosarcoma.

The aim of this study was to investigate whether the vitamin D analogue KH 1060 could exert a suppressive action on Tumor necrosis factor-alpha (TNF-alpha). The chimeric anti-TNF-alpha monoclonal antibody (anti-TNF), alone or in combination with KH 1060, was also used. KH 1060 (0.01, 0.1, 1 nM) significantly inhibited cell proliferation, determined after 5 days by [3H]thymidine incorporation, when peripheral blood mononuclear cells (PBMC), obtained from healthy subjects, were stimulated with phytohaemagglutinin (PHA) and incubated for 24 h in the absence and in the presence of lipopolysaccharide (LPS). In the same experimental conditions, anti-TNF exerted a significant inhibition on PBMC proliferation, at the lowest doses (0.001, 0.01 microg/ml) in the absence of LPS, and at 0.001, 1, 10 microg/ml in its presence. A synergistic inhibition was registered combining KH 1060 and anti-TNF, at well-defined concentrations. 0.1 nM KH 1060 produced a significant decrease in TNF-alpha levels, determined by ELISA, although less remarkable than in the presence of anti-TNF. This decrease was synergistic, associating 0.1 nM KH 1060 and 0.1 microg/ml anti-TNF. VDR protein levels were increased by 0.1 nM KH 1060, 0.1 microg/ml anti-TNF or their combination. The protein levels of two oncogenes, Bax and Bcl-2, remained unchanged, when PBMC were incubated with KH 1060, anti-TNF or their combination in the absence of LPS, while, in its presence, an increase was registered. The demonstrated anti-TNF-alpha effect of KH 1060 may suggest for this compound an immunosuppressive action and the possibility to synergistically act with other drugs.

The active form of vitamin D, 1,25(OH)(2)D(3), exerts important effects on proliferation and differentiation of many cell types, and immunoregulatory activities in particular on T cell-mediated immunity. The aim of this study was to investigate whether KH 1060, a vitamin D analogue, could decrease tumor necrosis factor-alpha (TNF-alpha) levels in patients with inflammatory bowel disease (IBD). PBMC proliferation was determined by [(3)H]thymidine incorporation. TNF-alpha levels were measured by ELISA kit; VDR, Bcl-2 and Bax protein levels with Western blot analysis. KH 1060 inhibited PBMC proliferation and decreased TNF-alpha levels in IBD patients and this effect was synergistic with anti-TNF-alpha. VDR protein levels were significantly increased by PBMC treatment with KH 1060 or anti-TNF-alpha or their combination in ulcerative colitis (UC) patients, and decreased in Crohn's disease (CD) patients, treating the cells with KH 1060. In UC patients an increase in Bcl-2 and Bax levels was observed incubating, PBMC with KH 1060 or anti-TNF-alpha or their combination. In CD patients a slight decrease in Bcl-2 levels was registered when anti-TNF alone or in association with KH 1060 was used. Bax protein levels were slightly increased in the presence of KH 1060 alone or in combination with anti-TNF. This study shows that KH 1060 acts as an immunomodulator on PBMC, acting as TNF-alpha inhibitor. This finding provides strong evidence that vitamin D status could be an important regulator of immunity IBD.

Recent evidence has suggested that miRNA is implicated in the immune response of allergic and inflammatory diseases. However, little is known about its role in the mechanism that underlies the establishment of pro-allergic DCs in allergic rhinitis is not fully understood. This study assessed whether and how microRNA (miR)-106b regulates the pro-allergic properties of DCs upon allergen stimulation in vitro. Bone marrow-derived dendritic cells (BMDCs) were generated and stimulated with ovalbumin (OVA) to identify the miRNA expression profile. After transfection with miR-106b mimics and inhibitors OVA-activated BMDCs were further evaluated for surface marker expression using flow cytometry, cytokine production using ELISA and subsequent effects on Th2 cell polarisation using flow cytometry. Moreover, the upstream controllers and potential target proteins of miR-106b were examined in a western blot analysis. Results showed that MiR-106b expression was significantly inhibited in activated BMDCs upon OVA stimulation (p<0.05). Surface marker expression (e.g., MHC class II, CD80 and CD86) was significantly upregulated after the transfection of an miR-106b inhibitor (p<0.05), and the proportion of GATA-3(+) T cells was significantly increased among CD4(+) T cells that were cocultured with miR-106b inhibitor-pretreated BMDCs (p<0.05). Conversely, IL-12 production from OVA-activated BMDCs and the proportion of T-bet(+) T cells increased significantly in a coculture of CD4(+) T cells and miR-106b mimics-transfected BMDCs (p<0.05). The early growth response (Egr)-2 was identified via luciferase reporter assays as a target gene of miR-106b, and significant Egr-2 upregulation was observed in OVA-activated BMDCs following transfection with a miR-106b inhibitor (p<0.05). In conclusion, our results suggest that miR-106b negatively regulates the pro-allergic properties of BMDCs and subsequent Th2 polarisation upon OVA stimulation and might represent a promising therapeutic target for allergic inflammation. Copyright © 2015. Published by Elsevier B.V.

The etiopathogenesis of Hashimoto's thyroiditis (HT) has not been clearly elucidated although the role of chronical inflammation and endothelial dysfunction has been established. The imbalance between pro- and anti-inflammatory cytokines may play a role in the etiology. The aim of the present study was to investigate whether cytokine gene polymorphisms are associated with HT, and to evaluate the relationship between genotypes and clinical/laboratory manifestation of HT. Tumor necrosis factor α (TNFα) G-308A (rs 1800629), interleukin-6 (IL-6) G-174C (rs 1800795) and IL-10 G-1082A (rs 1800896) single nucleotide polymorphisms (SNPs) in DNA from peripheral blood leukocytes of 190 patients with HT and 231 healthy controls were investigated by real-time PCR combined with melting curve analysis using fluorescence-labeled hybridization probes. There was no notable risk for HT afflicted by TNFα -308, IL-6 -174 and IL-10 -1082 polymorphisms alone. However, carriers of variant alleles of both IL-10 -1082 and TNFα -308 polymorphisms had four-fold times higher risk for HT in comparison with non-carriers. Additionally, concomitant presence of both mutant IL-10 -1082 A and IL-6 -174 C alleles raised three-fold the HT risk. Our results suggest that the combined effects of TNFα -308, IL-6 -174 and IL-10 -1082 variant alleles may be more decisive to induce functional differences and modify the risk for HT.

Interleukin 22 (IL-22) is a cytokine induced during both innate and adaptive immune responses. It can effect an acute phase response, implicating a role for IL-22 in mechanisms of inflammation. IL-22 requires the presence of the IL-22 receptor (IL-22R) and IL-10 receptor 2 (IL-10R2) chains, two members of the class II cytokine receptor family (CRF2), to effect signal transduction within a cell. We studied the interaction between human IL-22 and the extracellular domains (ECD) of its receptor chains in an enzyme-linked immunoabsorbant assay (ELISA)-based format, using biotinylated IL-22 (bio-IL-22) and receptor-fusions containing the ECD of a receptor fused to the Fc of hIgG1 (IL-22R-Fc and IL-10R2-Fc). IL-22 has measurable affinity for IL-22R-Fc homodimer and undetectable affinity for IL-10R2. IL-22 has substantially greater affinity for IL-22R/IL-10R2-Fc heterodimers. Further analyses involving sequential additions of receptor homodimers and cytokine indicates that the IL-10R2(ECD) binds to a surface created by the interaction between IL-22 and the IL-22R(ECD), and thereby further stabilizes the association of IL-22 within this cytokine-receptor-Fc complex. Both a neutralizing rat monoclonal antibody, specific for human IL-22, and human IL-22BP-Fc, an Fc-fusion of the secreted IL-22 binding-protein and proposed natural antagonist for IL-22, bind to similar cytokine epitopes that may overlap the binding site for IL-22R(ECD). Another rat monoclonal antibody, specific for IL-22, binds to an epitope that may overlap a separate binding site for IL-10R2(ECD). We propose, based on this data, a temporal model for the development of a functional IL-22 cytokine-receptor complex.

The aim of this study was to elucidate the molecular mechanisms involved in the therapeutic effects of proanthocyanidins from grape seeds (GSPE) on recurrent ulcerative colitis (UC) in rats. GSPE in doses of 100, 200, and 400mg/kg were intragastrically administered per day for 7 days after recurrent colitis was twice-induced by TNBS. The levels of GSH, as well as the activity of GSH-Px and SOD in colon tissues were measured by biochemical methods. The expression levels of tumor necrosis factor-α (TNF-α) and the nuclear translocation levels of nuclear factor-kappa B (NF-κB) in the colon tissues were measured by enzyme-linked immunosorbent assay methods. Western blotting analysis was used to determine the protein expression levels of inhibitory kappa B-alpha (IκBα), inhibitor kappa B kinase (IKKα/β), phosphorylated IκBα and phosphorylated IKKα/β. GSPE treatment was associated with a remarkable increased the activity of GSH-Px and SOD with GSH levels in TNBS-induced recurrent colitis rats as compared to the model group. GSPE also significantly reduced the expression levels of TNF-α, p-IKKα/β, p-IκBα and the translocation of NF-κB in the colon mucosa. GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response and promoting damaged tissue repair to improve colonic oxidative stress. Moreover, GSPE inhibited the TNBS-induced inflammatory of recurrent colitis though blocking NF-κB signaling pathways.

As an important immune mediator, PGE2 plays an important role in the immune tolerance, autoimmune diseases, immune regulation and tumor immunotolerance. PGE2 is considered to be a promising candidate for the control of the immune diseases. To further understand the immuno-modulating effects of PGE2 on CD4+ T cells, in vitro investigation was conducted in the present study. The results showed that PGE2 inhibited the proliferation of T cells in vitro in a dose-dependent manner. Gene expression profiling showed that 1716 genes were down regulated and 73 genes were up regulated with a change of 1.5 fold. Several signal transduction pathways were involved, such as TNF-α and NF-kB signaling pathway, T cell receptor signaling pathway, IL-2 signaling pathway, and MAPK pathway. The results showed that PGE2 inhibited IFN-γ, TNF-α and IL-4 production by CD4+ T cells 24h after cell culture. A comparison between IFN-γ and IL-4 production showed that PGE2 enhanced the relative ratio of IL-4 to IFN-γ in CD4+ T cells culture, and regulated CD4+ T cells toward Th2 cell development. The results of the present study indicated that PGE2 has the potential to treat Th1-mediated inflammatory diseases by regulating CD4+ T cells toward Th2 cell immune response.

Sunitinib is a receptor tyrosine kinase inhibitor (TKI) that is front-line therapy for metastatic renal cell carcinoma (mRCC). Its antitumor activity is related to its ability to block tumor cell and tumor vasculature cell signaling via several TKI receptors (i.e. vascular endothelial growth factor receptors VEGFRs, platelet-derived growth factors (PDGFs), and stem cell factors). Sunitinib also targets myeloid derived suppressor cells (MDSCs) significantly reducing their accumulation in the peripheral blood and reversing T cell (IFNγ) suppression in both mRCC patients and in murine tumor models. This reduction in immune suppression provides a rationale for combining sunitinib with immunotherapy for the treatment of certain tumor types. Despite these encouraging findings, however, we have observed that sunitinib has variable impact at reducing MDSCs and restoring T cell function within the tumor microenvironment. Given the immunosuppressive and proangiogenic activities of MDSC, it seems plausible that their persistence may contribute to the resistance that develops in sunitinib-treated patients. While sunitinib reduced tumor infiltrating MDSCs in Renca and CT26-bearing mice, coinciding with strong to modest decreases in tumor size respectively, it was ineffective at reducing MDSCs (<35% reduction in Gr1+CD11b+) or tumor burden in 4T1-bearing mice. Persistence of intratumor MDSCs was paralleled by depressed intratumor T cell IFNγ response and increased GM-CSF expression. Additionally, in vitro and in vivo experiments showed that GM-CSF prolongs survival of MDSCs, thus protecting them from the effects of sunitinib via a pSTAT5-dependent pathway. Although preliminary, there is evidence of intratumor MDSC resistance in some mRCC patients following sunitinib treatment. Intratumor MDSC persistence and T cell IFNγ response post nephrectomy in patients receiving sunitinib in a neoadjuvant setting are being compared to RCC patients undergoing nephrectomy without prior sunitinib treatment. Tumors from untreated patients showed suppressed T cell IFNγ response along with substantial expression of MDSCs (5% of total digested cells). Thus far, tumors from 5/8 neoadjuvant patients showed persistence of intratumor MDSCs and low T cell IFNγ production post sunitinib treatment, findings that parallel results from untreated tumors. In the remaining 3 neoadjuvant patients, intratumor MDSCs were detected at low levels which coincided with a T cell IFNγ response similar to that observed with normal donor peripheral T cells. GM-CSF's role in promoting MDSC survival in patient tumors is supported by the observation that GM-CSF is produced in short-term RCC cultures at levels capable of protecting MDSCs from sunitinib-induced cell death. Additionally, persistence of MDSC also may be associated with increased expression of proangiogenic proteins, such as MMP9, MMP8, and IL-8 produced by tumor stromal cells or infiltrating MDSCs. Indeed our findings suggest that the most dominate MDSC subset in RCC patients is the neutrophilic population that produces proangiogenic proteins. We propose that the development of sunitinib resistance is partly mediated by the survival of MDSCs intratumorally, thereby providing sustained immune suppression and angiogenesis.

To detect the expression of cadherin-11 and its correlation with synovitis in osteoarthritis (OA), to explore the mechanism of over expression of cadherin-11 and its role in migratory or invasive capacity of fibroblast-like synoviocytes (FLS). Synovitis severity was recorded according to Krenn's scoring system in 25 osteoarthritis patients undergoing total knee arthroplasty. Cadherin-11 expression in OA synoviums and its correlation with synovitis score and systemic inflammation markers were explored. After induction with Interleukin-1 beta (IL-1β) or tumor necrosis factor-alpha (TNF-α),cadherin-11 expression on OA FLS was assessed by qPCR and western blot,the capacity of migration and invasion of OA FLS was tested by transwell assay, and matrix metalloproteinase-2 production was assessed with ELISA as cadherin-11 expression was up regulated after infection with cadherin-11 cDNA-containing lentivirus, also when cadherin-11 expression was knocked down after infection with cadherin 11 shRNA containing lentivirus. Cadherin-11 expression in OA synovium showed significant differences among different grades of synovitis. Cadherin-11 in the lining layer was positively correlated with hyperplasia of the lining layer, density of the resident cells, inflammatory infiltrate, total synovitis score and D-dimer. Cadherin-11 in the sublining layer was positively correlated with the density of the resident cells, inflammatory infiltrate, total synovitis score and erythrocyte sedimentation rate. IL-1β or TNF-α could up-regulate cadherin-11 expression on OA FLS at transcriptional and protein level. Over expression of cadherin-11 increased migratory or invasive capacity of OA FLS, while cadherin-11 knock down reduced migratory or invasive capacity and MMP-2 production in OA FLS. The over expression of cadherin-11 in osteoarthritis is positively correlated with synovitis severity, and can be driven by proinflammatory cytokines on OA FLS; cadherin-11 increases migratory or invasive capacity and MMP-2 production of fibroblast-like synoviocytes of osteoarthritis. Copyright © 2015. Published by Elsevier B.V.

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-α and IFN-γ released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF-α and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-α and IFN-γ. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-α whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

Objective: To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells. Methods: γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells. Results: After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35μg/ml to 18.75μg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group. Conclusion: Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.

We have previously reported that nasally administered Lactobacillus fermentum CJL-112 (CJL-112) efficiently improves resistance against lethal influenza infection in both mice and chicken. The aim of the present study was to understand the underlying mechanisms of the significant anti-influenza activity of this lactobacilli strain. In vitro, co-culturing of the chicken macrophage cell line HD-11 with CJL-112 significantly increased nitric oxide (NO) production. In vivo, CJL-112 was nasally administered to BALB/c mice for 21days prior to influenza A/NWS/33 (H1N1) virus (IFV) infection. Significant up-regulation of T-helper 1 (Th1) cytokines (IL-2, IFN-γ) was observed, while the levels of T-helper 2 (Th2) cytokines (IL-4, IL-5, IL-10) was either reduced or unchanged than that in control mice were. Furthermore, IgA and specific anti-influenza IgA levels increased significantly in the treated mice than those in untreated mice. Therefore, CJL-112 likely protects the mice against lethal IFV infection via stimulation of macrophages, activation of Th1 and augmentation of IgA production, when directly delivered into the respiratory tract.

To describe how systemic disease is treated in a large cohort of Spanish patients with primary Sjögren syndrome (pSS) in daily practice, focusing on the adequacy of therapies for the level of systemic activity measured by ESSDAI score. By December 2014, our database included 1120 consecutive patients who fulfilled the 2002 classification criteria for SS. Therapeutic schedules were classified into 4 categories: no systemic therapies, hydroxychloroquine (HCQ) and/or low dose glucocorticoids (GCS) (<20mg/day), high dose GCS (>20mg/day) and use of second-line therapies (immunosuppressive agents, intravenous immunoglobulins [IVIG] and/or rituximab [RTX]). There were 1048 (94%) females and 72 (6%) males, with a mean age at diagnosis of 54 years. The main drug-based therapeutic approaches for systemic pSS during follow-up were HCQ in 282 (25%) patients, GCS in 475 (42%, at doses >20mg/day in 255-23%), immunosuppressive agents in 148 (13%), IVIG in 25 (2%) and RTX in 35 (3%) patients. HCQ was associated with a lower risk of death (adjusted HR of 0.57, 95% 0.34-0.95). We classified 16 (7%) of the 255 patients treated with >20mg GCS and 21/148 (14%) treated with immunosuppressive agents, patients as inadequately treated, mainly associated with articular involvement of low/moderate activity. The management of pSS should be organ-specific, using low dose GCS in patients with moderate systemic activity, limiting the use of high dose GCS and second-line therapies to refractory or potentially severe scenarios. The use of systemic therapies for dryness, chronic pain or fatigue is not warranted. Copyright © 2015 Elsevier B.V. All rights reserved.

OK-432 (Picibanil), a Streptococcal immunotherapeutic agent, has been used for immunotherapy of various cancers as a biological response modifier (BRM). However, OK-432 contains multiple components consisting of immunotherapeutic ones and contaminants which may weaken the effects or exert side-effects. In this study, we investigated extraction of contaminants from OK-432 using Triton X-114 (TX-114)-water phase partitioning and examined an antitumor effect of the resulting preparation. OK-432 was subjected to TX-114 partitioning to give residual precipitate designated as OK-TX-ppt. OK-TX-ppt exerted no TLR2-mediated activity, but induced interleukin (IL)-6 in human PBMC. OK-TX-ppt also induced tumor necrosis factor (TNF)-alpha, IL-10, IL-12, and interferon (IFN)-gamma in PBMC. Moreover, IFN-gamma-inducing activity of OK-TX-ppt was significantly higher and IL-10 production was lower than that of OK-432. In tumor-bearing mice model, administration of OK-TX-ppt i.p. extended the survival time of Meth-A-bearing mice compared to OK-432. OK-TX-ppt also increased the levels of IL-12 and IFN-gamma in mouse spleen cells in vitro. These results indicated that TX-114 partitioning removed some contaminants, which attenuates the antitumor effect, from OK-432 and increase the immunotherapeutic effects of OK-432.

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that affects about 1% of the population worldwide. RA is mainly manifested by persistent synovitis and progressive joint destruction. The aim of the present study was to examine the anti-arthritis effects of SND-117, a sinomenine bivalent that is obtained from the structure modification of a clinically available anti-RA drug, sinomenine. The arthritis model (CIA) was established by immunizing DBA/1 mice with type II collagen, and the arthritis scores including inflammation, joint destruction and bone erosion were assessed after booster immunization for 3weeks. The levels of cytokines such as IL-1β, IL-6 and TNF-α were analyzed by quantitative PCR and ELISA. The TNF-α induced NF-κB activation in fibroblast-like synovial cells (FLSCs) was analyzed by Western blot. SND-117 significantly relieved the inflammatory symptoms of collagen-induced arthritis, reduced bone erosion and joint destruction in CIA mice. The serum levels of IL-1β, IL-6 and TNF-α of CIA mice were markedly decreased by SND-117. SND-117 also strongly inhibited the phosphorylation and nuclear translocation of NF-κB p65 in FLSCs upon TNF-α stimulation. These data demonstrated that SND-117 could effectively block the pathogenesis of collagen-induced arthritis in CIA mice via inhibition of NF-κB signaling, and might provide potential clinic benefits in rheumatoid arthritis management. Copyright © 2015. Published by Elsevier B.V.

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) plays an important role in inflammation. However, the role of 11β-HSD1 in rheumatoid arthritis (RA) remains unknown. The purpose of this study was to evaluate the therapeutic effects of a selective 11β-HSD1 inhibitor BVT-2733 in collagen-induced arthritis (CIA) and its underlying mechanisms. CIA mice were treated with BVT-2733 (100mg/kg, orally) or vehicle twice daily for 2weeks. Arthritis score and joint histology were investigated. The levels of pro-inflammatory cytokines as well as anti-type II collagen antibody (anti-CII) were detected by ELISA. Western blot analysis was used to assess the activation of NF-κB and NLRP1 inflammasome in joint tissues and in human RA synovial cells. BVT-2733 treatment attenuated the arthritis severity and anti-CII level in CIA mice. BVT-2733 also decreased the levels of serum TNF-α, IL-1β, IL-6 and IL-17. BVT-2733 treatment also significantly reduced synovial inflammation and joint destruction. NF-κB activation and NLRP1 inflammasome assembly were also inhibited in arthritic joints and human RA synovial cells. In conclusion, BVT-2733 exhibits an anti-inflammatory effect on CIA. This protective effect is, at least partly, mediated by inhibition of the NF-κB and NLRP1 inflammasome signaling pathways. 11β-HSD1 inhibition may represent a potential therapeutic target for RA patients.

15-Deoxy-delta(12,14)-prostaglandin J2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma). Because PPARgamma is highly expressed in macrophages obtained from stimulant-elicited peritonitis, but not in resident peritoneal macrophages, the effect of dPGJ2 was tested on innate functions of macrophages. dPGJ2 inhibited adhesion and phagocytosis of Escherichia coli. Inhibition of these functions by dPGJ2 was not mediated via the adhesion molecule Mac-1. In addition, dPGJ2 inhibited chemotaxis toward zymosan-activated serum and it also inhibited the production of superoxide anion when macrophages were stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OPZ), but not lipopolysaccharide. Similarly, dPGJ2 inhibited the production of hydrogen peroxide when macrophages were stimulated with either PMA or OPZ. These studies suggest that dPGJ2 may be a negative regulator of macrophage functions.

Analogous to reported immunomodulatory effects of probiotics, this study was performed to analyse the immunomodulatory properties of prebiotic oligosaccharides that share chemical characteristics with human milk oligosaccharides. A mixture containing galacto- and fructo-oligosaccharides (GOS/FOS; ratio 9:1) was tested at dietary doses between 1% and 10% (w/w of total diet) in an influenza vaccination model, using 10 C56BL/6JolaHsd mice per group. The modulation of vaccine specific delayed-type hypersensitivity (DTH) responses was studied as a marker of T-helper 1 (Th1) immunity, as well as other immune parameters. GOS/FOS enhanced DTH responses dose-dependently (optimum at 5% w/w of total diet; 41.4+/-14.1% increased compared to controls, p<0.05). No significant changes were detected on splenocyte proliferation or vaccine-specific antibody concentrations. Simultaneously, GOS/FOS dose-dependently increased the proportion of faecal bifidobacteria and lactobacilli (maximal effect at 10% w/w of total diet; 16.8+/-2.4% and 5.8+/-1.3% increased compared to controls respectively, p<0.01 for both parameters). In a comparative experiment, GOS/FOS and FOS/inulin (both at 2% w/w of total diet) induced similar significant effects on the gut microbiota. In contrast to GOS/FOS, FOS/inulin did not enhance DTH responses, indicating that an increase in the proportions of bifidobacteria and lactobacilli is not sufficient for an immunomodulatory effect in this model. The use of GOS/FOS in dietary products might provide an opportunity to stimulate the adaptive immune response in a Th1-direction and subsequently inhibit infections and Th2-related immune disorders in humans, for instance allergies. Clinical studies are being performed to confirm this.

Licochalcone, a constituent of licorice, has antitumor, antimicrobial, and anti-inflammatory effects. Recently, licochalcone E was isolated from the roots of Glycyrrhiza inflata and its biological functions are not fully examined. In this study, we investigated its ability to modulate production of IL-12p40, a common subunit of IL-12 and IL-23. Licochalcone E dose-dependently inhibited IL-12p40 production from lipopolysaccharide-stimulated RAW264.7 macrophage cells. The repressive effect was mapped to a region in the IL-12 gene promoter containing a binding site for NF-kappaB. Furthermore, licochalcone E decreased binding to the NF-kappaB site in RAW264.7 macrophage cells. Using a chronic allergic contact dermatitis model induced by repeated application of oxazolone, we showed that licochalcone E inhibited the increased IL-12p40 expression and ear thickness induced by oxazolone. Taken together, licochalcone E inhibits IL-12p40 production and has therapeutic potential to reduce skin inflammation.

Heat-killed Lactobacillus plantarum strain L-137 (HK L-137) is a more potent inducer of interleukin (IL)-12 than other heat-killed Lactobacillus strains. To elucidate the mechanism involved in this IL-12p40 induction, we compared HK L-137 with heat-killed L. plantarum strain JCM1149 (HK JCM1149) by nuclear magnetic resonance and mass spectrometry. Results showed that HK L-137 contained lipoteichoic acid (LTA) with a chemical structure similar to that of JCM1149, except for a lower degree of glucosyl substitution in the poly(glycerol phosphate) backbone. Lysozyme sensitivity and electrophoretic moiety analysis revealed that HK L-137 exposed more LTA on its cell surface than HK JCM1149. Phagocytosis of HK L-137 by splenic adherent cells was significantly greater than that of HK JCM1149. Anti-LTA antibody and anti-scavenger receptor-A (SR-A) antibody selectively inhibited phagocytosis of HK L-137, as well as IL-12p40 production, by splenic adherent cells. Thus, a higher efficiency of phagocytosis of HK L-137 via SR-A for LTA is responsible for the potent IL-12p40 induction. Copyright © 2015. Published by Elsevier B.V.

Inflammatory bowel disease (IBD), typified by Crohn's disease and ulcerative colitis, is a common disorder characterized by recurrent and serious inflammation of the gastrointestinal tract. Recent immunologic advances have established that T cells and inflammatory cytokines play a pivotal role in the gastrointestinal inflammation of IBD. However, many cytokines not only elicit inflammation but also protect host against microbial invasion. Hence, suppression of these dual-purpose cytokines often exposes the patients to significant risk of infection. Recent research on Interleukin (IL)-23, IL-17, and IL-17 producing T cells has become the vanguard of further understanding the contribution of cytokines to autoimmune and inflammatory diseases. IL-23 is a newly discovered member of the IL-12-related cytokine family, and is primarily involved in the differentiation of pathogenic T cells characterized by their production of IL-17. IL-17 is a potent inflammatory mediator implicated in a number of autoimmune diseases. The discovery of this IL-23/IL-17-mediated inflammatory axis is having a profound impact on the elucidation of T cell-mediated pathogenesis as well as development of novel therapeutic targets. In this review, we discuss the current literature and present our recent studies on the role of IL-23 and IL-17 in the pathogenesis of IBD. Controlling the expression/production of IL-23 and IL-17 is an approach that would allow the development of a novel treatment strategy with more anti-inflammatory efficacy and potentially with less suppressive effects on host defenses.

Inhalation of adenosine-5'-monophosphate (AMP) causes bronchoconstriction in asthma but not in healthy subjects. Bronchoconstriction upon AMP inhalation is thought to occur by histamine release and subsequent binding to receptors on airway smooth muscle cells. To explain enhanced sensitivity to AMP in asthma, mast cell expression of the adenosine A2A and A2B receptors and histamine release were measured after incubation of human mast cell line 1 (HMC-1) cells with AMP and the non-specific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) for 1.5 and 6 h. To establish a Thelper-2 environment resembling the asthma phenotype, HMC-1 cells were additionally cultured with IL-4 and IL-13 alone or stimulated with the combination of both cytokines and AMP and NECA. To study effects of prolonged presence of the inflammatory environment, the cells were pre-incubated overnight (18 h) with IL-4 and IL-13 and additionally stimulated with AMP and NECA for 1.5 or 6 h. AMP and NECA hardly affected adenosine receptor expression but increased IL-8 secretion. Incubation with IL-4 and IL-13 for 6 h increased adenosine A2A receptor expression and histamine secretion, but decreased IL-8 secretion. The combination of IL-4, IL-13, and AMP/NECA for 6 h increased A2B receptor expression and IL-8 secretion. Overnight stimulation with IL-4, IL-13 and subsequent stimulation with AMP/NECA for 1.5 h decreased A2AR expression which was accompanied by increased histamine secretion. These results suggest a role for decreased A(2A)R expression in enhanced adenosine responsiveness as observed in asthma.

This study investigated whether aldose reductase (AR) inhibition affects interleukin (IL)-13-induced mucus production in the human bronchial epithelial cell line-16 (HBE16) cells. The HBE16 cells were cultured with AR inhibitors (zopolrestat) or were transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with 10 ng/ml IL-13 for 2h. The levels of mucin (MUC)5AC mRNA and protein were measured by using RT-PCR or ELISA. Intracellular reactive oxygen species (ROS) were measured fluorimetrically with the CM-H2DCFDA probe. Western blotting was performed to determine the levels of AR, phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and phosphorylated Janus kinase 2 (p-JAK2). The results show that treatment with zopolrestat or transfection with AR siRNA significantly suppressed IL-13-stimulated MUC5AC mRNA and protein in the HBE16 cells (P<0.05). AR inhibition could suppress IL-13-induced ROS generation, the phosphorylation of JAK2/STAT6 pathway and the activation of nuclear factor (NF)-kappa B, thereby decreasing mucus production in vitro (all P<0.05). Therefore, the inhibition of AR could be a therapeutic target for mucus hypersecretion in chronic inflammation lung disease.

To examine the mechanisms of diabetes-enhanced inflammation, ear inflammation was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in streptozotocin (STZ)-injected diabetic and control mice. The inflammatory response was determined from ear thickness and histology. The mRNA expression of several inflammation-related genes 8, 24 and 32h after TPA treatment was determined by quantitative real-time RT-PCR. Ear thickness did not differ between the two groups at 8h, but was greater in the diabetic mice than control mice at 24 and 32h (late phase). STZ-diabetic conditions variously affected TPA-induced gene expression. The changes 8h after TPA treatment probably reflected transcriptional regulation, and the genes were divided into three groups, up-regulated (IL-6, MCP-1, HO-1 and SOCS3), unregulated (IL-1beta, TNF-alpha and IL-10) and down-regulated (RANTES) genes. TPA-induced gene expression of cytokines, except for RANTES, peaked at 8h and significantly declined in the late phase in control mice, while the expression of IL-1beta and TNF-alpha did not decline in the late phase in the diabetic mice. This result indicated the destabilization process for these mRNA, a type of post-transcriptional regulation, to be impaired under STZ-induced diabetic conditions; however, TPA-induced gene and protein expression of TTP, an RNA-binding protein involved in mRNA decay, were adversely enhanced in the diabetic mice. These findings suggested that STZ-induced diabetes affected the transcriptional and post-transcriptional control of TPA-induced inflammation, and greater mRNA levels of IL-1beta and TNF-alpha in the late phase were probably responsible for the diabetes-enhanced inflammation.