α-Linolenic Acid Screened by Molecular Docking Attenuates Inflammation by Regulating Th1/Th2 Imbalance in Ovalbumin-Induced Mice of Allergic Rhinitis

α-Linolenic acid (ALA) is a natural essential fatty acid widely found in plant seed oils and beans, which shows positive anti-inflammatory and antiallergic effects. In our previous study, ALA was proven to bind tightly to the seven protein targets closely associated with allergic rhinitis (AR) by molecular docking, which indicates that ALA may have a potential role in the treatment of AR. A mouse model of AR induced by ovalbumin (OVA) was adopted in this study to explore the therapeutical effect and potential mechanism of ALA in treating AR. Results demonstrated that ALA remarkably relieved the nasal symptoms, reduced the OVA-sIgE level in the serum, relieved the histopathological injuries, and downregulated the mRNA expression levels of IL-6 and IL-1β in the nasal mucosa. ALA also remarkably moderated the imbalance of Th1/Th2 cells, increased the mRNA expression levels of T-bet and STAT1, and reduced GATA3 and STAT6. ALA was proven to have a substantial therapeutic effect on mice with AR, and the underlying mechanism was likely to be the regulation of Th1/Th2 imbalance through the JAK/T-bet/STAT1 and JAK/GATA3/STAT6 pathways. This study provides a specific experimental basis for the clinical use and drug development of ALA in the treatment of AR.     

Mast cells play a key role in Th2 cytokine-dependent asthma model through production of adhesion molecules by liberation of TNF-α

Mast cells are well recognized as key cells in allergic reactions, such as asthma and allergic airway diseases. However, the effects of mast cells and TNF-α on T-helper type 2 (Th2) cytokine-dependent asthma are not clearly understood. Therefore, an aim of this study was to investigate the role of mast cells on Th2 cytokine-dependent airway hyperresponsiveness and inflammation. We used genetically mast cell-deficient WBB6F1/J-Kitw/Kitw-v (W/Wv), congenic normal WBB6F1/J-Kit+/Kit+ (+/+), and mast cell-reconstituted W/Wv mouse models of allergic asthma to investigate the role of mast cells in Th2 cytokine-dependent asthma induced by ovalbumin (OVA). And we investigated whether the intratracheal injection of TNF-α directly induce the expression of ICAM-1 and VCAM-1 in W/Wv mice. This study, with OVA-sensitized and OVA-challenged mice, revealed the following typical histopathologic features of allergic diseases: increased inflammatory cells of the airway, airway hyperresponsiveness, and increased levels of TNF-α, intercellular adhesion molecule (ICAM)-1, and vascular cellular adhesion molecule (VCAM)-1. However, the histopathologic features and levels of ICAM-1 and VCAM-1 proteins in W/Wv mice after OVA challenges were significantly inhibited. Moreover, mast cell-reconstituted W/Wv mice showed restoration of histopathologic features and recovery of ICAM-1 and VCAM-1 protein levels that were similar to those found in +/+ mice. Intratracheal administration of TNF-α resulted in increased ICAM-1 and VCAM-1 protein levels in W/Wv mice. These results suggest that mast cells play a key role in a Th2 cytokine-dependent asthma model through production of adhesion molecules, including ICAM-1 and VCAM-1, by liberation of TNF-α.     

Higher infiltration by Th17 cells compared with regulatory T cells is associated with severe acute T-cell-mediated graft rejection

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were 11.6 ± 12.2 cells/mm2 and 5.6 ± 8.0 cells/mm2, respectively. The average Treg/Th17 ratio was 5.6 ± 8.2. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury (P < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.     

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.     

Synthesis and biological activity of hydroxylated analogs of RCAI-80

RCAI-80 is one of the ester analogs of KRN7000 (α-galactosylceramide). This compound released mainly T helper 2 (Th2) cytokines, such as IL-4 rather than T helper 1 (Th1) cytokines, such as IFNγ from the invariant natural killer T (iNKT) cells. In addition, it has been known that some of the hydroxylated derivatives of KRN7000 make the cytokine secretion bias to Th2 by decreasing the IFNγ production to almost zero. This time, the three compounds having these two characteristic properties, namely an ester group and also some extra hydroxy groups existing on the ester side chain and/or on the 2-acyloxy-3,4-dihydroxyoctadecyl main chain of RCAI-80, were synthesized to examine the biological activities. As a result, it was found that these compounds made the cytokine secretion skew to Th2. Therefore, their effectiveness for experimental autoimmune encephalomyelitis (EAE) was examined. It was recognized that one of them showed moderate suppression of EAE symptom.     

In vitro induction of anterior gradient-2-specific cytotoxic T lymphocytes by dendritic cells transduced with recombinant adenoviruses as a potential therapy for colorectal cancer

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration, and cellular transformation, and is one of the specific mRNA markers for circulating tumor cells in patients with gastrointestinal cancer. We investigated the feasibility of AGR2 as a potent antigen for tumor immunotherapy against colorectal cancer (CRC) cells using dendritic cells (DCs) transduced with a recombinant adenovirus harboring the AGR2 gene (AdAGR2). DCs transduced with a recombinant adenovirus encoding the AGR2 gene (AdAGR2/DCs) were characterized. These genetically-modified DCs expressed AGR2 mRNA as well as AGR2 protein at a multiplicity of infection of 1,000 without any significant alterations in DC viability and cytokine secretion (IL-10 and IL-12p70) compared with unmodified DCs as a control. In addition, AdAGR2 transduction did not impair DC maturation, but enhanced expression of HLA-DR, CD80, and CD86. AdAGR2/DCs augmented the number of IFN-γ-secreting T-cells and elicited potent AGR2-specific cytotoxic T lymphocytes capable of lysing AGR2-expressing CRC cell lines. These results suggest that AGR2 act as a potentially important antigen for immunotherapy against CRC in clinical applications.     

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3⁺CCR4^-CD4⁺ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3⁺CCR4^-CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4⁺ or CXCR3⁺CCR4^-CD4⁺ T cells with DC (CD4⁺/DC or CXCR3⁺CD4⁺/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3⁺CCR4^-CD4⁺ and CD4⁺ T cells on DC phenotype expression, CXCR3⁺CD4⁺/DC in CTL culture were able to expand number of CD8⁺ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3⁺CCR4^-CD4⁺ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.     

TCP复合BMP-2诱导hMSCs成骨分化的协调机制研究

目的探讨TCP复合BMP-2诱导h MSCs成骨分化的协调机制。方法分别制备TCP支架及TCP复合BMP-2支架材料,自3名志愿者身上分离h MSCs进行传代扩增,取第3代细胞接种备好的支架材料,在复合后的第1、3、7天测量ALP的活性并进行比较。结果在复合后第1天,两组间ALP活性值比较无明显差异(P0.05);在复合后第3、7天两组间ALP活性值比较差异显著(P0.05);TCP与细胞培养组1、3、7天的ALP活性值差异不显著,无统计学意义(P0.05);TCP复合BMP-2与细胞培养组的1、3、7天的ALP活性值随着培养时间的延长,逐渐升高。结论 BMP-2能协调TCP促进h MSCs的分化效应,基因治疗可能为骨骼疾病提供一种新的治疗途径。     

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

Phosphorylation of ERK-Dependent NF-κB Triggers NLRP3 Inflammasome Mediated by Vimentin in EV71-Infected Glioblastoma Cells

Enterovirus 71 (EV71) is a dominant pathogenic agent that may cause severe central nervous system (CNS) diseases among infants and young children in the Asia-pacific. The inflammasome is closely implicated in EV71-induced CNS injuries through a series of signaling pathways. However, the activation pathway of NLRP3 inflammasome involved in EV71-mediated CNS injuries remains poorly defined. In the studies, EV71 infection, ERK1/2 phosphorylation, and activation of NLRP3 are abolished in glioblastoma cells with low vimentin expression by CRISPR/Cas9-mediated knockdown. PD098059, an inhibitor of p-ERK, remarkably blocks the vimentin-mediated ERK1/2 phosphorylation in EV71-infected cells. Nuclear translocation of NF-κB p65 is dependent on p-ERK in a time-dependent manner. Moreover, NLRP3 activation and caspase-1 production are limited in EV71-infected cells upon the caffeic acid phenethyl ester (CAPE) administration, an inhibitor of NF-κB, which contributes to the inflammasome regulation. In conclusion, these results suggest that EV71-mediated NLRP3 inflammasome could be activated via the VIM-ERK-NF-κB pathway, and the treatment of the dephosphorylation of ERK and NF-κB inhibitors is beneficial to host defense in EV71-infected CNS.     

Carnosic Acid Protects INS-1 β-Cells against Streptozotocin-Induced Damage by Inhibiting Apoptosis and Improving Insulin Secretion and Glucose Uptake

Carnosic acid (CA), a natural polyphenolic diterpene derived from Rosmarinus officinalis, has been proven to possess a broad spectrum of medicinal properties. Nevertheless, no studies on its impact on pancreatic β-cells have been conducted to date. Herein, clonal rat INS-1 (832/13) cells were pretreated with CA for 24 h and then incubated with streptozotocin (STZ) for 3 h. Several functional experiments were performed to determine the effect of CA on STZ-induced pancreatic β-cell damage, including cell viability assay, apoptosis analysis, and measurement of the level of insulin secretion, glucose uptake, malondialdehyde (MDA), reactive oxygen species (ROS), and proteins expression. STZ treatment decreased cell survival, insulin secretion, glucose uptake, and increased apoptosis, MDA, and ROS production in INS-1 cells. Furthermore, protein expression/phosphorylation analysis showed significant down-regulation in insulin, PDX-1, PI3K, AKT/p-AKT, and Bcl₂. On the other hand, expression of BAX and BAD and cleaved PARP were significantly increased. Interestingly, preincubation with CA reversed the adverse impact of STZ at the cellular and protein expression levels. In conclusion, the data indicate that CA protects β-cells against STZ-induced damage, presumably through its modulatory effect on the different pathways, including the Pi3K/AKT/PDX-1/insulin pathway and mitochondria-mediated apoptosis.     

Crocins from Crocus sativus L. in the Management of Hyperglycemia. In Vivo Evidence from Zebrafish

Diabetes mellitus is a disease characterized by persistent high blood glucose levels and accompanied by impaired metabolic pathways. In this study, we used zebrafish to investigate the effect of crocins isolated from Crocus sativus L., on the control of glucose levels and pancreatic β-cells. Embryos were exposed to an aqueous solution of crocins and whole embryo glucose levels were measured at 48 h post-treatment. We showed that the application of crocins reduces zebrafish embryo glucose levels and enhances insulin expression. We also examined whether crocins are implicated in the metabolic pathway of gluconeogenesis. We showed that following a single application of crocins and glucose level reduction, the expression of phosphoenolpyruvate carboxykinase 1 (pck1), a key gene involved in glucose metabolism, is increased. We propose a putative role for the crocins in glucose metabolism and insulin management.     

Immunomodulatory Effects by Photodynamic Treatment of Glioblastoma Cells In Vitro

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.     

喷涂法构筑二氧化硅纳米粒子涂层及其光学和润湿性质

采用Stöber方法,通过调节反应温度及乙醇和水的体积,合成了不同粒径的二氧化硅纳米粒子.以合成的粒径为20 nm的二氧化硅纳米粒子为原料,采用简单、方便的喷涂方法在玻璃片上构筑了纳米粒子涂层.在550 ℃煅烧二氧化硅纳米粒子涂层,增强了二氧化硅纳米粒子在玻璃片上的附着力.用1H, 1H, 2H, 2H-全氟辛基三乙氧基硅烷修饰之后,二氧化硅纳米粒子涂层的表面润湿性由亲水性转变为疏水性.通过喷涂法制备的二氧化硅纳米粒子涂层具有减反增透效果,当二氧化硅纳米粒子质量分数为0.48%、循环喷涂沉积数为3时,涂层在可见光范围内的最大透光率可达95.5%.用扫描电子显微镜观测涂层表面形貌发现,喷涂法制备的涂层是均匀的、可控的.喷涂技术构筑纳米粒子涂层具有简单快速、可大面积应用等优点.     

β-甲萘醌催化氧化合成技术研究进展

综述了不同原料合成K系维生素重要中间体β-甲萘醌的不同方法及相应催化氧化体系建立的研究进展。与传统工业生产工艺相比较,着重分析以β-甲基萘为原料所采用的各催化剂类别及其催化氧化合成路线的特点和不足;结合国内外催化材料使用和国内生产现状,提出环境友好绿色催化氧化合成β-甲萘醌的未来发展趋势及研究方向。     

基质固相分散-高效液相色谱法测定袋泡茶中黄曲霉毒素

提出了基质固相分散-高效液相色谱法测定袋泡茶中黄曲霉毒素B1、B2、G1和G2含量的方法。优化的试验条件为:1样品与分散剂(中性氧化铝)的质量比为1比4;2洗脱剂乙腈的用量为6mL。以Symmetry C18色谱柱为分离柱,以甲醇-水(1+1)混合液为流动相进行分离。采用荧光检测,激发波长为365nm,发射波长为435nm。黄曲霉毒素B1、B2、G1和G2在一定的质量浓度范围内与其峰面积呈线性关系,方法的检出限(3S/N)在0.05~0.15μg·L-1之间,测定下限(10S/N)在0.15~0.50μg·L-1之间。以空白样品为基体进行加标回收试验,所得回收率在88.3%~96.8%之间,测定值的相对标准偏差(n=6)在4.3%~6.8%之间。     

Association of ADIPOR1 polymorphisms with bone mineral density in postmenopausal Korean women

Adiponectin may affect bone through interactions with two known receptors, adiponectin receptors (ADIPOR) 1 and 2. We examined the association between polymorphisms of ADIPOR1 and ADIPOR2 and bone mineral density (BMD) in postmenopausal Korean women. Six polymorphisms in ADIPOR1 and four polymorphisms in ADIPOR2 were selected and genotyped in all study participants (n = 1,329). BMD at the lumbar spine and femur neck were measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment and the occurrence of non-vertebral fractures examined using self-reported data. P values were adjusted for multiple testing using Bonferroni correction (P(corr)). ADIPOR1 rs16850799 and rs34010966 polymorphisms were significantly associated with femur neck BMD (P(corr) = 0.036 in the dominant model; P(corr) = 0.024 and P(corr) = 0.006 in the additive and dominant models, respectively). Subjects with the rare allele of each polymorphism had lower BMD, and association of rs34010966 with BMD showed a gene dosage effect. However, ADIPOR2 single nucleotide polymorphisms and haplotypes were not associated with BMD at any site. Our results suggest that ADIPOR1 polymorphisms present a useful genetic marker for BMD in postmenopausal Korean women.