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The Milan criteria are widely accepted for indicating liver transplantation in patients with hepatocellular carcinoma (HCC). However, a 7% to 20% possibility of HCC recurrence remains, even among patients who fulfill the Milan criteria.We retrospectively reviewed 88 patients with HCC who underwent liver transplantation at Pusan National University Yangsan Hospital between May 2010 and December 2014. The risk factors for HCC recurrence were analyzed, and the overall survival and disease-free survival rates were calculated based on each risk factor.Seventeen patients (19.3%) experienced HCC recurrence. Multivariate analyses revealed that the independent risk factors for HCC recurrence were protein induced by vitamin K absence or antagonist II (PIVKA-II) levels of200 mAU/mL, levels of200 for alpha-fetoprotein (ng/mL) or PIVKA-II (mAU/mL), and microvascular invasion. Therefore, we defined the A-P 200 criteria as simultaneously exhibiting alpha-fetoprotein levels of ≤200 ng/mL and PIVKA-II levels of ≤200 mAU/mL. The 3-year overall survival rates among patients who fulfilled or exceeded the A-P 200 criteria were 89.2% and 80.0%, respectively (P = .79). The 3-year disease-free survival rates among patients who fulfilled or exceeded the A-P 200 criteria were 89.9% and 43.1%, respectively (P .001). We also applied the A-P 200 criteria to patient data from another major center and observed similar results.These findings confirm that the A-P 200 criteria can be used to predict recurrence after liver transplantation among patients with HCC.

We investigated the effect of a specific protein kinase C (PKC) inhibitor, bisindolylmaleimide I [BIM (I)], on L-type Ca2+ channels in rat ventricular myocytes. BIM (I) alone inhibited the L-type Ca2+ current in a concentration-dependent manner, with a Kd value of 3.31 ± 0.25 μM, and a Hill coefficient of 2.34 ± 0.23. Inhibition was immediate after applying BIM (I) in the bath solution and then it partially washed out. The steady-state activation curve was not altered by applying 3 μM BIM (I), but the steady-state inactivation curve shifted to a more negative potential with a change in the slope factor. Other PKC inhibitors, PKC-IP and chelerythrine, showed no significant effects either on the L-type Ca2+ current or on the inhibitory effect of BIM (I) on the L-type Ca2+ current. The results suggest that the inhibitory effect of BIM (I) on the L-type Ca2+ current is independent of the PKC pathway. Thus, our results should be considered in studies using BIM (I) to inhibit PKC activity and ion channel modulation.

Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane), belonging to a family of natural compounds that are abundant in broccoli, has received significant therapeutic interest in recent years. However, the molecular basis of its effects remains to be elucidated. In this study, we attempt to determine whether sulforaphane regulates the inflammatory response in an ovalbumin (OVA)-induced murine asthma model. Mice were sensitized with OVA, treated with sulforaphane, and then challenged with OVA. Sulforaphane administration significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Additionally, sulforaphane suppressed the increase in the levels of SOCS-3 and GATA-3 and IL-4 expression in the OVA-challenged mice. Collectively, our results demonstrate that sulforaphane regulates Th2 immune responses. This sutdy provides novel insights into the regulatory role of sulforaphane in allergen-induced Th2 inflammation and airway responses, which indicates its therapeutic potential for asthma and other allergic diseases. [BMB Reports 2012; 45(5): 311-316]

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.

Tuberculosis (TB) caused by Mycobacterium tuberculosis constitutes an ongoing threat to global health. An antigen that can induce dendritic cell (DC) maturation and lead to enhanced cellular immunity is crucial to the development of an effective TB vaccine. Here, we investigated the functional roles and the related signaling mechanism of the Rv0577 protein, a M. tuberculosis complex-restricted secreted protein involved in the methylglyoxal detoxification pathway. Rv0577 recognizes Toll-like receptor 2 (TLR2) and functionally induces DC maturation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF-α, IL-1β, IL-6, and IL-12p70) in DCs on MyD88-dependent signaling, mitogen-activated protein kinases, and nuclear factor κB signaling pathways. In addition, Rv0577-treated DCs activated naive T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, and induced T-cell proliferation, indicating that this protein possibly contributes to Th1-polarization of the immune response. More important, unlike LPS, Rv0577-treated DCs specifically induced the proliferation of memory CD4(+)/CD8(+)CD44(high)CD62L(low) T cells in the spleen of M. tuberculosis-infected mice in a TLR2-dependent manner. Taken together, these findings suggest that Rv0577 may regulate innate and adaptive immunity by interacting with TLR2, a finding that could be helpful in the design of new TB vaccines.

To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell-cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a down-regulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and β2-microglobulin (β2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8(+) T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8(+) T cells. In contrast, HSV-treated DCs that had been co-cultured with MCF-7 cells induced activation of CD8(+) T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-α and IFN-γ.

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.

Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis , a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis -derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4 + CD25 + Foxp3 + regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical relationship between the allergic immune response and helminth infection.

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA (AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4(+) T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8(+) T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4(+) and E7-specific CD8(+) T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8(+) effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.

DNA vaccines contribute to a promising new approach for the generation of cytotoxic T lymphocytes (CTL). DNA vaccines do have several disadvantages, including poor immunogenicity and oncogene expression. We used the natural killer T-cell (NKT) ligand α-galactosylceramide (α-GalCer) as an adjuvant to prime initial DNA vaccination; and used the potent immune-stimulatory tumor antigen-expressing dendritic cells (DCs) as a booster vaccination. A DNA vaccine expressing human papillomavirus (HPV) type 16 E7 (pcDNA3-CRT/E7) was combined with α-GalCer at the prime phase, and generated a higher number of E7-specific CD8(+) T-cells in vaccinated mice than vaccine used at boost phase. Therefore, priming with a DNA vaccine in the presence of α-GalCer and boosting with E7-pulsed DC-1 led to a significant enhancement of E7-specific CD8(+) effector and memory T-cells as well as significantly improved therapeutic and preventive effects against an E7-expressing tumor model (TC-1) in vaccinated mice. Our findings suggested that the potency of a DNA vaccine combined with α-GalCer could be further enhanced by boosting with an antigen-expressing DC-based vaccine to generate anti-tumor immunity.

Dendritic cell (DC)-based vaccines are recognized as a promising immunotherapeutic strategy against cancer. Various adjuvants are often incorporated to enhance the modest immunogenicity of DC vaccines. More specifically, many of the commonly used adjuvants are derived from bacteria. In the current study, we evaluate the use of apoptosis inhibitor 5 (API5), a damage-associated molecular pattern expressed by many human cancer cells, as a novel DC vaccine adjuvant. We showed that API5 can prompt activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. We also demonstrated that vaccination with API5-treated DCs pulsed with OVA, E7, or AH1-A5 peptides led to the generation of OVA, E7, or AH1-A5-specific CD8 + T cells and memory T cells, which is associated with long term tumor protection and antitumor effects in mice, against EG.7, TC-1, and CT26 tumors. Additionally, we determined that API5-mediated DC activation and immune stimulation are dependent on TLR4. Lastly, we showed that the API5 protein sequence fragment that is proximal to its leucine zipper motif is responsible for the adjuvant effects exerted by API5. Our data provide evidence that support the use of API5 as a promising adjuvant for DC-based therapies, which can be applied in combination with other cancer therapies. Most notably, our results further support the continued investigation of human-based adjuvants.

In this study, diamond thin films were deposited on tungsten carbide tools using surface-wave plasma-enhanced chemical vapor deposition (SWP-CVD). To eliminate the adverse effects of cobalt on the diamond deposition process, the cobalt was removed from the surface of the tools by etching with Murakami’s reagent for various times (30, 60, and 120 min). The cutting performance of the untreated and the diamond-coated WC tools was examined by performing cutting test on carbon fiber-reinforced plastic (CFRP). The results showed that all the diamond-coated tools exhibited great improvement on the durability and wear resistance compared to the uncoated one. In addition, the diamond-coated tool lift time is found to be proportional to the etching time. An increase more than twofold has been achieved when the etch time was increased from 30 min to 120 min.

Fretting occurs at the contact area between two materials under load and in the presence of minute relative surface motion by vibration or external force. Bearings, clutches, riveted and bolted lap joints are subjected to fretting damage. Friction coefficient, materials of the specimen, contact surface pressure, relative slip amplitudes, temperatures and environment have an effect on the fretting. In this study, fretting wear test is conducted with SCM415 (Cr–Mo alloy steel) which are much used for making gears and shafts because of its excellent machinability, good mechanical properties and low cost, compared with those of the existing machine structural steels. In order to determine the fretting wear type, fretting wear fixture which can be attached to the servo hydraulic fatigue testing machine is made. And then, specimens and fretting pad with a constant curvature are made of SCM415 materials. Different normal forces and displacement amplitudes are applied to the fretting pad and diamond-like carbon (DLC) is coated on the fretting pad for fretting wear test.

Objective: To overcome limitation of ex vivo manipulation against dendritic cells (DC) vaccines, we developed mannose-labeled poly(d,l-lactide-co-glycolide) nanoparticles (MN-PLGA-NPs). The MN-PLGA-NPs enabled selective delivery of tumor-specific antigen to mannose receptor overexpressed DCs without ex vivo manipulation. Methods: Conjugation of mannose (MN) and polyvinyl alcohol (PVA) was analyzed by H-NMR. Size and zeta potential of the MN-PLGA-NPs were examined by dynamic light scattering using an electrophoretic light scattering photometer. Selective delivery efficiency of MN-PLGA-NPs to DCs was examined by confocal microscope and flow cytometry. Migration of DCs containing MN-PLGA-NPs was assessed flow cytometry. Therapeutic efficacy of MN-PLGA(OVA+poly I:C)-NPs was examined in EG7 tumor-bearing mouse models. Biological effect and activated CD8+ T cells in tumor tissue were confirmed by immunohistochemistry assay. Results: Conjugation of MN and PVA was confirmed that the peaks for the CH2 of methylene group in PVA was observed at 1.48 ppm and 1.59 ppm, and CH of tetrahydropyran group in MN was observed at 3.40 ppm, 3.49 ppm, 3.76 ppm, and 5.41 ppm. Size and zeta potential of MN-PLGA-NPs were 200 nm formed spherical shape and -10 mV, respectively. Loading efficiency of OVA or poly I:C into MN-PLGA-NPs was 90 % or 45 %, individually. Intracellular uptake of MN-PLGA-NPs in DCs was increased through selective binding between MN and MN-receptor compared to PLGA-NPs. Additionally, DCs containing MN-PLGA-NPs exhibited efficient migration to popliteal lymph node. Therapeutic efficacy of MN-PLGA-NPs showed significant inhibition of tumor growth in EG7 tumor-bearing mice compared to the control (p < 0.001) and PLGA-NPs (p < 0.05), and showed 100% of survival up to 60 days. Additionally, MN-PLGA-NPs treatment group showed a greater number of activated CD8+ T cells in tumor tissue compared to the other groups (p < 0.001), and prevention effect of tumor growth resulted in effectively suppressed compared to the control (p < 0.001) or PLGA-NPs (p < 0.001). Conclusion: MN-PLGA-NPs is an attractive system that can directly activate DC in vivo without ex vivo manipulation, which overcome the limitation of existing DC vaccine. Keyword: dendritic cells, selective delivery, PLGA nanoparticle, cancer immunotherapy. Citation Format: Yeongseon Byeon, Ji Eun Won, Ga Hee Kim, Min Gi Kim, Yeong Min Park, Hee Dong HAN. Selective delivery of tumor specific antigen to dendritic cells by mannose-labeled PLGA nanoparticles to induce immune response for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4673.

Introduction: After cancer treatment using chemotherapy and radiotherapy, tumor microenvironment continually occurs inflammation, which depend on damage-associated molecular pattern (DAMPs) released from apoptotic tumor cells. In this case, tumor microenvironment is regarded as inflammation zone and induce various immune cells infiltration, pro- and anti-inflammatory cytokines production and interaction between immune cells and cancer cells. These, a dynamic and complex microenvironment induce a favorable state for tumor growth. Thus, recent studies assert that it is important to regulate excessively increased inflammatory response in the tumor microenvironment accordingly. Annexin V is a Ca2+-dependent phospholipid binding pro[[Unsupported Character - Codename ­]]tein and known to bind to exposed phosphatidylserine on the apoptotic cells. Several groups and our study have recently demonstrated that annexin V has anti-inflammatory effects by inhibiting bone-marrow derived dendritic cell (BMDCs) activation and pro-inflammtory cytokine production. So, we suggest that Annexin V as a therapeutic approach for controlling inflammatory response in cancer patients having increased inflammation in tumor microenvironment. Methods: The size and purity of recombinant proteins were assessed using SDS-PAGE. IVIS imaging system used to characterize the accumulation of Annexin V containing protein into tumor loci in tumor-bearing mice. Therapeutic efficacy was confirmed tumor-specific CD8+ T cells in PBMCs and splenocytes by Flow cytometry analysis. pro-, anti- inflammtory cytokines and various immune cells infiltration in tumor microenvironment were examined by ELISA and FACS. Cytotoxicity by tumor-specific CD8+ T cells of Luciferase-expressing tumor cells was observed by representative luminescence imaging system. Results: The Targeting efficiency of Annexin V protein into tumor microenvironment was increased compared to control. Therapeutic efficacy was demostrated activation of tumor-specific CD8+ T cells (P < 0.001), inhibition of tumor growth (P < 0.05) and increase of survival (P < 0.05) in tumor bearing mouse compared to control. AnnexinV protein administration was decreased significantly TGF-beta 1 cytokine production (P < 0.001) and immunosuppressive cells infiltration (P < 0.001) in tumor microenviroment compared to non-treated group. Conclusion: We identified multiple role of Annexin V protein, which has capacity of apoptotic tumor cell target, as regulator of excessive inflammation in tumor microenvironment. This results could primarily pave the road for the development of anti-tumor immunotherapy. Citation Format: Jung Hwa Park, Young Seob Kim, Hyun Jin Park, Gun Young Jang, T.c. Wu, Yeong-Min Park, Tae Heung Kang. AnnexinV: Multifunctional anticancer immunotherapeutic protein in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4066.

We investigated the effect of the phenylalkylamine Ca(2+) channel inhibitor verapamil on voltage-dependent K(+) (Kv) channels in rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Verapamil reduced the Kv current amplitude in a concentration-dependent manner. The apparent K(d) value for Kv channel inhibition was 0.82 microM. Although verapamil had no effect on the activation kinetics, it accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation by verapamil were 2.20+/-0.02 microM(-1) s(-1), and 1.79+/-0.26 s(-1), respectively. The steady-state activation and inactivation curves were unaffected by verapamil. The application of train pulses increased the verapamil-induced Kv channel inhibition. Furthermore, verapamil increased the recovery time constant, suggesting that the inhibitory effect of this agent was use-dependent. The inhibitory effect of verapamil was not affected by intracellular and extracellular Ca(2+)-free conditions. Another Ca(2+) channel inhibitor, nifedipine (10 microM) did not affect the Kv current, and did not alter the inhibitory effect of verapamil. Based on these results, we concluded that verapamil inhibited Kv current in a state-, time-, and use-dependent manner, independent of Ca(2+) channel inhibition.

Mucosal epithelia sense external stress signals and transmit them to the intracellular cascade responses. Ribotoxic stress-producing chemicals such as deoxynivalenol (DON) or other trichothecene mycotoxins have been linked with gastrointestinal inflammatory diseases by Fusarium-contamination. The purpose of this study was to test the hypothesis that DON evokes the epithelial sentinel signals of RNA-dependent protein kinase (PKR) and early growth response gene 1 (EGR-1), which together contribute to the pro-inflammatory cytokine interleukin 8 (IL-8) in human intestinal epithelial cells. PKR suppression by the dominant negative PKR expression attenuated DON-stimulated interleukin-8 production. Moreover, 1L-8 transcriptional activation by DON was also reduced by PKR inhibition in the human intestinal epithelial cells. Treatment with the PKR inhibitor also suppressed EGR-1 promoter activity, mRNA and protein induction, although mitogen-activated protein (MAP) kinases such as extracellular signal-regulated protein kinases (ERK) 1/2, p38, c-Jun N-terminal Kinase (INK) were little affected or even enhanced in presence of a PKR inhibitor. These patterns were also compared in the EGR-1-suppressed cells, which showed much more suppressed production of 1L-8. All things taken into consideration, DON-activated sentinel signals of EGR-1 via PKR mediated interleukin-8 production in human intestinal epithelial cells, which provide insight into the possible general mechanism associated with mucosal inflammation as an intestinal toxic insult by ribotoxic trichothecene mycotoxins.

The common word flavonoids is often used to classify a family of natural compounds, highly abundant in all higher plants, that have received significant therapeutic interest in recent years. Naringin is associated with a reduced risk of heart disease, neurodegenerative disease, cancer and other chronic diseases; however the molecular basis of this effect remains to be elucidated. Thus we attempted to elucidate the anti-allergic effect of Naringin in ovalbumin (OVA)-induced asthma model mice. The OVA-induced mice showed allergic reactions in the airways. These included an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid, an increase in inflammatory cell infiltration into the lung around blood vessels and airways, airway luminal narrowing, and the development of airway hyper-responsiveness (AHR). The administration of Naringin before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that Naringin plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of Naringin in terms of its effects on asthma in mice.

Suppression of an excessive systemic inflammatory response is a promising and potent strategy for treating endotoxic sepsis. Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan catabolism, may play a critical role in various inflammatory disorders. In this study, we report a critical role for IDO in the dysregulated immune response associated with endotoxin shock. We found that IDO knockout (IDO−/−) mice and 1-methyl-d-tryptophan-treated, endotoxin-shocked mice had decreased levels of the cytokines, TNF-α, IL-6, and IL-12, and enhanced levels of IL-10. Blockade of IDO is thought to promote host survival in LPS-induced endotoxin shock, yet little is known about the molecular mechanisms that regulate IDO expression during endotoxin shock. In vitro and in vivo, IDO expression was increased by exogenous IL-12, but decreased by exogenous IL-10 in dendritic cells and splenic dendritic cells. Interestingly, whereas LPS-induced IL-12 levels in serum were higher than those of IL-10, the balance between serum IL-12 and IL-10 following challenge became reversed in IDO−/−- or 1-methyl-d-tryptophan-treated mice. Our findings demonstrate that the detrimental immune response to endotoxin shock may occur via IDO modulation. Restoring the IL-12 and IL-10 balance by blocking IDO represents a potential strategy for sepsis treatment.

A potent anti-inflammatory activity was detected from methanol extracts prepared from roots of Rhodiola sacra. In the preliminary test, methanol extracts from the roots of R. sacra could potently attenuate histological change in the lung tissue after lipopolysaccharide (LPS)-treated inflammatory stimulation. The active compound has been identified as 3-(3,4-dihydroxy-phenyl)-acrylic acid phenethyl ester (caffeic acid phenethyl ester, CAPE). In vivo study on protective effect of R. sacra CAPE against inflammatory response by endotoxin shock in the LPS-induced inflammatory mice showed significantly attenuated the intravenous LPS-induced increase in plasma TNF-α and IL-1β concentration. R. sacra CAPE significantly inhibited LPS-induced nuclear transcription factor-κB (NF-κB) activation. It also down-regulates enhanced matrix metalloproteinase-9 (MMP-9) activity triggered by LPS in mouse lung. In the present study, the results indicate that the intake of R. sacra CAPE could provide health benefits, reducing the magnitude of the inflammatory process triggered by endotoxin shock and the production of inflammatory mediators.

Dentritic cell (DC)-based cancer immunotherapy faces challenges in both efficacy and practicality. However, DC-based vaccination requires multiple injections and elaborates ex vivo manipulation, which substantially limits their use. Therefore, we sought to develop a chitosan nanoparticle (CH-NP)-based platform for the next generation of vaccines to bypass the ex vivo manipulation and induce immune responses via active delivery of polyinosinic-polycytidylic acid sodium salt (poly I:C) to target Toll-like receptor 3 (TLR3) in endosomes. We developed CH-NPs encapsulating ovalbumin (OVA) as a model antigen and poly I:C as the adjuvant in an ionic complex. These CH-NPs showed increased in vivo intracellular delivery to the DCs in comparison with controls after injection into tumor-bearing mice, and promoted DC maturation, leading to emergence of antigen-specific cytotoxic CD8+ T cells. Finally, the CH-NPs showed significantly greater antitumor efficacy in EG.7 and TC-1 tumor-bearing mice compared to the control (p ex vivo manipulation, thus resulting in increased anticancer efficacy.

The migration of dendritic cells (DCs) to secondary lymphoid organs plays a crucial role in the initiation of adaptive immune responses. Although lipopolysaccharide enhances chemokine receptor 7 (CCR7) expression on DCs, the second signal for the migration of DCs toward the chemokine CCL19 remains unknown. In this study, we show that sphingosine kinase inhibitor (SKI) inhibits the migration of DCs toward CCL19 through the down-regulation of CCR7. Inhibition of p38 mitogen-activated protein kinase (MAPK) activation by SKI may be responsible for the SKI-mediated effects on the regulation of chemokine receptor expression. Impairment of DC migration by the inhibition of p38 MAPK and down-regulation of CCR7 expression may contribute to the protective effects of SKI in DC-related disorders. These results suggest that sphingosine kinase-mediated signalling plays a role in the innate and adaptive immune responses by altering DC migration.

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-gamma from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4(+) T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-kappaB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.

d-pinitol has been demonstrated to exert insulin-like and anti-inflammatory effects. However, the effects of the maturation and immunostimulatory functions of dendritic cells (DC) remain to be clearly elucidated. In this study, we have attempted to determine whether d-pinitol regulates surface molecule expression, cytokine production, endocytosis capacity, and underlying signaling pathways in murine bone marrow-derived DC. We also attempted to ascertain whether d-pinitol could influence Th1/Th2 immune response in vivo. The DC used in this study were derived from murine bone marrow cells, and were used as immature or LPS-stimulated mature DC. The DC were then assessed with regard to surface molecule expression, dextran-FITC uptake, cytokine production, capacity to induce T-cell differentiation, and underlying signaling pathways. d-pinitol was shown to significantly inhibit CD80, CD86, MHC class I, and MHC class II expression in the LPS-stimulated mature DC. The DC also evidenced impaired IL-12 expression and IFN-gamma production. The d-pinitol-treated DC were found to be highly efficient in regards to Ag capture via mannose receptor-mediated endocytosis. d-pinitol was also demonstrated to inhibit LPS-induced MAPKs activation and NF-kappaB nuclear translocation. Moreover, the d-pinitol-treated DC manifested impaired induction of Th1 responses, and normal cell-mediated immune responses. These novel findings provide new insight into the immunopharmacological role of d-pinitol in terms of its effects on DC. These findings also broaden current perspectives concerning our understanding of the immunopharmacological functions of d-pinitol, and have ramifications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (100 mum) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K(ATP) channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A(3) receptor inhibitor); these increases were inhibited by the inward rectifier K(+) (Kir) channel inhibitor, Ba(2+). These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (100 mum) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.

Infection with Shiga-like toxin (SLT)-producing Escherichia coli causes a spectrum of illnesses with high morbidity and mortality. Host mediators play an important role in the pathogenesis of SLT-I toxicity. We here investigated the effect of SLT-I on tumor necrosis factor-α (TNF-α) production, effect of TNF-α on glycolipid globotriaosyleramide (Gb3) expression, and relationship between Gb3 level and differential susceptibility of cells to SLT-I. In this study, we observed that detectable levels of TNF-α are produced 6 hrs after induction and continued to increase during 48 hrs by SLT-I. It was also found that Vero cells and dendritic cells expressed high levels of Gb3, 83% and 68%, respectively, and that macrophages had a low level of Gb3 (29%) and showed refractory to cytotoxicity against SLT-I. Vero cells and dendritic cells expressing high levels of Gb3 were highly susceptible to SLT-I. Furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. These results suggest that the expression of Gb3 is necessary, but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its receptor, Gb3, in the pathogenesis of E. coli O157 infection.

beta-Lapachone (LAPA) is a chemotherapeutic agent that can inhibit the expression of nitric oxide (NO) and inducible NO synthase (iNOS) in alveolar macrophages. No other information on the agent's anti-inflammatory activity has been reported. In the present study, we investigated the molecular mechanism of LAPA on lipopolysaccharide (LPS)-induced responses in BV2 microglia. Treatment of LAPA significantly inhibited NO and PGE(2) release in LPS-stimulated BV2 microglia. The inhibition of iNOS and COX-2 was also observed, suggesting the blockage of transcriptional levels. In addition, LAPA attenuated the expression of mRNA and proteins of proinflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in a dose-dependent manner. Moreover, LAPA exhibits anti-inflammatory properties by suppressing the NF-kappaB activation by blocking IkappaBalpha degradation and downregulating the ERK, p38 mitogen-activated protein kinase (MAPK) and Akt pathway. The results show that LAPA may be useful as a potential anti-inflammatory agent for attenuating inflammatory diseases.

Sphingosine kinase (Sphk) has been shown to be activated by growth factor and survival factors, and one of its products, sphingosine-1-phosphate, plays an important role in the regulation of various cellular responses. However, the effect of Sphk on the maturation and immunostimulatory function of dendritic cells (DCs) still remains largely unknown. In this study, we examined whether sphingosine kinase inhibitor (SKI) can influence co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) and cytokine production (IL-12 and IL-10) in murine bone marrow-derived DCs. SKI significantly inhibited co-stimulatory molecules in DCs. SKI suppressed IL-12 production by DCs and IFN-gamma production by T cells. In addition, SKI-inhibited LPS induced the translocation of nuclear factor-kappaB, whereas it did not affect the degradation of IL-1 receptor-associated kinase-1 by LPS. These novel findings provide new insight into the immunopharmacological role of SKI in terms of its effects on DCs. These findings open a possibility for further understanding of the immunopharmacological functions of SKI, as well as therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Upon activation, microglia release proinflammatory mediators that play important roles in eliciting neuroinflammatory responses associated with neurodegenerative diseases. The anti-inflammatory properties of eicosapentaenoic acid (EPA) have been known, however, the effects responsible for lipopolysaccharide (LPS)-induced activation remain poorly understood in microglia. In the present study, we investigated the effects of EPA on the expression of proinflammatory mediators in LPS-stimulated BV2 microglia. EPA significantly inhibited the release of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in a dose-dependent manner. EPA also attenuated the production of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and proinflammatory cytokines at mRNA and/or protein levels. Moreover, EPA suppressed NF-kappaB activation by blocking IkappaB degradation, and also blocked the mitogen-activated protein kinases (MAPKs) such as ERK, p38 and JNK, and the Akt pathway. The anti-inflammatory properties of EPA may be useful for ameliorating neurodegenerative diseases as well as suppressing LPS-induced shock.

The objective of the present study was to investigate the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the cell growth and apoptosis and its effect on the telomerase activity in human leukemic cell line U937. Exposure of U937 cells to TSA resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the up-regulation in proapoptotic Bax expression and down-regulation of antiapoptotic Bcl-2 and Bcl-X L . TSA treatment inhibited the levels of cIAP family members and induced the proteolytic activation of caspase-3, which was associated with concomitant degradation of poly(ADP-ribose)-polymerase and β-catenin protein. TSA treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by TSA treatment. We therefore conclude that TSA demonstrated antiproliferative and apoptosis-inducing effects on U937 cells in vitro, and that changes in Bcl-2 family protein levels as well as telomerase activity may play an important role in its mechanism of action.

Dendritic cells (DCs) are potent antigen presenting cells that are uniquely effective in generating primary immune responses. DCs that are manipulated to present tumor antigens induce antitumor immunity in animal models and preclinical human studies. A myriad of strategies have been developed to load tumor antigen effectively onto DCs. DC-tumor fusion presents a spectrum of tumor-associated antigens to helper T- and cytotoxic T-cell populations in the context of DC-mediated costimulatory signals. In this study, fusion cells (FCs) were generated with MCA-102 fibrosarcoma cells and murine bone marrow-derived myeloid DCs. The FCs coexpressed the DC-derived MHC class II and costimulatory molecules. The FCs also retained the functional properties of DCs and stimulated syngeneic T cell proliferation and interferon-gamma (IFN-gamma) production. Significantly, the results show that syngeneic T cells are primed by FCs to induce MHC class I-dependent lysis of MCA-102 fibrosarcoma. These findings indicate that fusions of tumor cells and DCs activate T-cell responses against syngeneic tumors.

It is well known that the quality of organs retrieved from brain-dead donors (DBDs) is better than those retrieved from circulatory death donors. However, in situations of organ retrieval from marginal DBDs, who are unstable despite intensive care, transplantation outcomes are not good. Organ ischemia is the most important determining factor in decreased organ quality in circulatory death donors and in some DBDs. Extracorporeal membrane oxygenation (ECMO) for management of DBDs can be an emergency countermeasure. The purpose of this report is to relay our experience with four cases of ECMO for DBD management. In all four cases, the donors were unstable and showed clear signs of ischemia despite intensive care, including ventilator use and administration of inotropic agents. Two donors had acute respiratory distress syndrome, and two exhibited dysfunctional oxygen delivery. ECMO was used to improve organ perfusion. ECMO resulted in an increased partial pressure of arterial oxygen increased and decreased lactic acid levels. Vital signs were stabilized, especially in the donors who were bleeding. The organ was successfully retrieved from each donor. Two livers (one of them was split), eight kidneys, and one pancreas were retrieved from four DBDs. All other organs were transplanted successfully, and there were no cases of primary nonfunction or delayed graft function. The ECMO machine is the most powerful supportive device for management of unstable DBDs. The use of ECMO in unstable DBDs can be beneficial in expanding the donor pool as well as improving the quality of retrieved organs.

Enteric drainage has been the main trend in solitary pancreas transplantation. Compared with bladder drainage, it does not cause metabolic or urologic complications, but there is no way to perform immunologic monitoring, except by graft pancreas biopsy. Additionally, although portal drainage of the graft vein is considered physiological drainage, it has more of a risk for surgical complications. To overcome these disadvantages, we successfully performed inferior vena cava (IVC)–duodenal drainage in pancreas alone transplantation. A 44-year-old man underwent pancreas alone transplantation. He had insulin-dependent diabetes because of chronic pancreatitis, thus he had taken a pancreatic enzyme. After right-sided medial visceral rotation, the IVC was dissected for anastomosis with a graft portal vein. The right common iliac artery was anastomosed with a Y-graft in the pancreas graft. The graft duodenum was anastomosed with recipient duodenum using the side-to-side manner. Postoperatively, he underwent protocol biopsies of the graft duodenum through endoscopy two times. There was no evidence of graft thrombosis or rejection. He had a normal glucose level without any diabetic drugs, and he required no pancreatic enzyme for digestion. The IVC-duodenum drainage procedure was a feasible method for preventing thrombosis and providing an opportunity for direct graft monitoring through endoscopy.

Introduction Although repeated liver transplantation (RLT) for irreversible graft failure is relatively rare, RLT is the only life-saving option available for a patient with failure of a previous liver transplant (LT). In cases in which failure of a previous LT is combined with TLS and exsanguination, waiting for organ allocation is not feasible and 2-stage liver transplantation (TSLT) is required. The aim of our case report was to describe the clinical management, including the criteria informing clinical decisions, for a patient who required 3 RLTs combined with TSLT. Case A 55-year-old man was admitted with liver cirrhosis associated with hepatitis B and multiple hepatocellular carcinomas. LT was performed using an emergent deceased donor graft of marginal quality. The graft was unsuccessful, with the patient showing hemodynamic deterioration and primary nonfunction of the graft. A total hepatectomy, with temporary portocaval shunt, was performed, with a second transplantation performed 3 days later. The second graft was from a 71-year-old, non–heart-beating donor, which resulted in a second episode of primary nonfunction. A third transplantation was performed 4 days later. The patient progressively recovered with extensive rehabilitation. Conclusion We report the successful outcome for a patient requiring 3 RLTs, with TSLT used as a bridge between transplants to reduce the duration of the anhepatic state. In selected cases, the combination of RLT and TSLT can provide a chance of survival from life-threatening liver failure.

Dendritic cell (DC) based cancer vaccines represent a promising immunotherapeutic strategy against cancer. To enhance the modest immunogenicity of DC vaccines, various adjuvants are often incorporated. Particularly, most of the common adjuvants are derived from bacteria. In the current study, we evaluate the use of a human pancreatic cancer derived protein, pancreatic adenocarcinoma upregulated factor (PAUF), as a novel DC vaccine adjuvant. We show that PAUF can induce activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. Furthermore, vaccination with PAUF treated DCs pulsed with E7 or OVA peptides leads to generation of E7 or OVA-specific CD8+ T cells and memory T cells, which correlate with long term tumor protection and antitumor effects against TC-1 and EG.7 tumors in mice. Finally, we demonstrated that PAUF mediated DC activation and immune stimulation are dependent on TLR4. Our data provides evidence supporting PAUF as a promising adjuvant for DC based therapies, which can be applied in conjunction with other cancer therapies. Most importantly, our results serve as a reference for future investigation of human based adjuvants.

Subsets of B cells inhibit various immune responses through their production of the cytokine interleukin-10 (IL-10). We found that IL-10–producing CD5 + B cells suppressed the immunoglobulin E (IgE)– and antigen-mediated activation of mast cells in vitro as well as allergic responses in mice in an IL-10–dependent manner. Furthermore, the suppressive effect of these B cells on mast cells in vitro and in vivo depended on direct cell-to-cell contact through the costimulatory receptor CD40 on CD5 + B cells and the CD40 ligand on mast cells. This contact enhanced the production of IL-10 by the CD5 + B cells. Through activation of the Janus-activated kinase–signal transducer and activator of transcription 3 pathway, IL-10 decreased the abundance of the kinases Fyn and Fgr and inhibited the activation of the downstream kinase Syk in mast cells. Together, these findings suggest that an important function of IL-10–producing CD5 + B cells is inhibiting mast cells and IgE-mediated allergic responses.

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including A549 lung adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN) sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment with SFN and TRAIL induced chromatin condensation, DNA fragmentation, annexin V staining and sub-G(1) phase DNA content. These indicators of apoptosis correlate with the induction of caspase-3 activity that results in the cleavage of poly(ADP-ribose) polymerase and the release of lactate dehydrogenase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. Inhibitors of ERK (PD98059) or Akt (LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although the activation of JNK was increased in response to combined treatment, inhibition of the JNK pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined SFN and TRAIL in human lung adenocarcinoma A549 cells through downregulation of ERK and Akt.

Apigenin, one of the most common flavonoids, has been shown to possess anti-inflammatory, anticarcinogenic, and free radical-scavenging properties. However, the influence of apigenin on the immunostimulatory effects and maturation of dendritic cells (DC) remains, for the most part, unknown. In this study, we have attempted to ascertain whether apigenin influences the expression of surface molecules, dextran uptake, cytokine production, and T-cell differentiation as well as the signaling pathways underlying these phenomena in murine bone marrow-derived DC. In the presence of apigenin, CD80, CD86, and major histocompatibility complex class I and II molecules, expressions on DC were significantly suppressed, and lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression was impaired. The DC proved highly efficient at antigen capture, as evidenced by the observation of mannose receptor-mediated endocytosis in the presence of apigenin. The LPS-induced activation of mitogen-activated protein kinase, the nuclear translocation of its nuclear factor-kappaB p65 subunit, and the induction of the T-helper 1 response were all impaired in the presence of apigenin, whereas the cell-mediated immune response remained normal. These findings provide new insight into the immunopharmacological functions of apigenin and its effects on DC, and they may also prove useful in the development of adjuvant therapies for individuals suffering from acute or chronic DC-associated diseases.

Epigallocatechin-3-gallate (EGCG), a major form of tea catechin, has anti-allergic properties. To elucidate the anti-allergic mechanisms of EGCG, we investigated its regulation of matrix metalloproteinase (MMP-9) expression in toluene diisocyanate (TDI)-inhalation lung tissues as well as TNF-α and Th2 cytokine (IL-5) production in BAL fluid. Compared with untreated asthmatic mice those administrated with EGCG had significantly reduced asthmatic reaction. Also, increased reactive oxygen species (ROS) generation by TDI inhalation was diminished by administration of EGCG in BAL fluid. These results suggest that EGCG regulates inflammatory cell migration possibly by suppressing MMP-9 production and ROS generation, and indicate that EGCG may be useful as an adjuvant therapy for bronchial asthma.

Silibinin is the primary active compound in silymarin. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, the effects of silibinin on the maturation and immunostimulatory activities exhibited by dendritic cells (DCs) remain, for the most part, unknown. In this study, we have attempted to determine whether silibinin can influence surface molecule expression, dextran uptake, cytokine production, capacity to induce T-cell differentiation, and the signaling pathways underlying these phenomena in murine bone marrow-derived DCs. Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with impairments of LPS-induced IL-12 expression in the DCs. Silibinin-treated DCs proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin also inhibited the LPS-induced activation of MAPKs and the nuclear translocation of the NF-kappaB p65 subunit. Additionally, silibinin-treated DCs evidenced an impaired induction of Th1 response, and a normal cell-mediated immune response. These findings provide new insight into the immunopharmacological functions of silibinin, especially with regard to their impact on the DCs. These findings expand our current understanding of the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated diseases.

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) which are responsible for protection from tumor challenge and regression of established metastatic tumor. It has been hypothesized that tumor lysate contains factors that may modulate DC maturation. In this study, we examined whether the uptake of tumor lysate (MCA-102 fibrosarcoma) could modulate DC phenotypes in vitro and whether the administration in vivo of tumor lysate-pulsed DC (TP-DC) could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens. It was investigated the uptake of tumor lysate by DC by means of flow cytometry and fluorescent microscope. Murine bone marrow-derived DC efficiently phagocytosed tumor lysate and after the uptake, the phenotype of TP-DC was surprisingly comparable to unpulsed-DC (UP-DC), exhibiting lower levels of CD80 (51%), CD86 (43%), and MHC class II (59%). Also, TP-DC did not enhance secretion of IL-12p70 (UP- vs. TP; 54.5+/-6.4 vs. 50.5+/-4.8 pg/ml, respectively), contrary to those activated with LPS (113.6+/-16.8 pg/ml). However, TP-DC vaccination in vivo increased the IFN-gamma production from splenocytes higher than that of UP-DC (TP- vs. UP-DC; 41029+/-1523 vs. 4752+/-590 pg/ml, respectively). Furthermore, the administration of TP-DC enhanced specific T cell responses against MCA-102 fibrosarcoma. These results demonstrate that augmentation of DC phenotype and function in vitro is not necessarily a prerequisite for TP-DC vaccination to successfully promote anti-tumor immunity in vivo.

Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.

Water-soluble proteoglycan (WSPG) of Agaricus blazei Murill has been known to stimulate the non-specific complements and humoral immune functions to act as polyclonal activators of B cells and to inhibit tumor growth and metastasis. However, little is known about its immunomodulating effects on murine bone marrow (BM)-derived dendritic cells (DC). In the present study, we examined the maturation process of murine BM-DC. BM cells were cultured in the presence of IL-4 and GM-CSF and the generated immature DC were stimulated with WSPG or LPS (WSPG-DC and LPS-DC, respectively) for 24 h. WSPG significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) II, as did LPS. IL-12p70 production in WSPG-DC was also identical to that in LPS-DC. The antigen-uptake capacity of WSPG-DC was determined by FITC-labeled dextran uptake. WSPG-DC lost dextran uptake capacity comparable to LPS-DC. The antigen-presenting capacity of WSPG-DC as analyzed by allogeneic T cell proliferation was significantly increased in comparison with immature DC, was identical to LPS-DC, and induced higher levels of IL-2 in responding T cells. These results indicate the immunomodulatory properties of WSPG, which might be therapeutically useful in the control of cancers and immunodeficient diseases through the up-regulation of DC maturation.

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the effect of curcumin on the maturation and immunostimulatory function of dendritic cells (DC) largely remains unknown. In this study, we examined whether curcumin can influence surface molecule expression, cytokine production, and their underlying signaling pathways in murine bone marrow-derived DC. DC were derived from murine bone marrow cells and used as immature or LPS-stimulated mature cells. The DC were tested for surface molecule expression, cytokine production, dextran uptake, the capacity to induce T cell differentiation, and their underlying signaling pathways. Curcumin significantly suppressed CD80, CD86, and MHC class II expression, but not MHC class I expression, in the DC. The DC also exhibited impaired IL-12 expression and proinflammatory cytokine production (IL-1β, IL-6, and TNF-α). The curcumin-treated DC were highly efficient at Ag capture, via mannose receptor-mediated endocytosis. Curcumin inhibited LPS-induced MAPK activation and the translocation of NF-κB p65. In addition, the curcumin-treated DC showed an impaired induction of Th1 responses and a normal cell-mediated immune response. These novel findings provide new insight into the immunopharmacological role of curcumin in impacting on the DC. These novel findings open perspectives for the understanding of the immunopharmacological role of curcumin and therapeutic adjuvants for DC-related acute and chronic diseases.

The aim of this study was to assess whether the use of antiplatelets and anticoagulants increased haemoptysis in patients with bronchiectasis. Cases (n = 242) with a history of haemoptysis were compared with controls (n = 242) without a history of haemoptysis. Of the 242 case patients, 16.5% took antiplatelets compared with 19.8% of controls (P = 0.346). The proportion of warfarin users did not differ between cases and controls (3.3% vs. 2.5%, P = 0.588). The use of these agents might not be associated with increased risk of haemoptysis in patients with bronchiectasis.