CD4+ and CD8+ T cells producing Th1 and Th17 cytokines are involved in the pathogenesis of autoimmune orchitis

Experimental autoimmune orchitis (EAO) is a useful model to study chronic testicular inflammation and infertility. EAO is characterized by severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. We previously reported an increase in CD4+ and CD8+ effector T cells in the testes of rats with EAO. Since cytokine patterns determine T cell effector functions, in the present work we analyzed the cytokines expressed by these cells during disease development. By flow cytometry, we detected an increase in the number of tumor necrosis factor-α (TNF) and interferon -γ (IFNG)-producing CD4+ T cells in the testis at EAO onset. As the severity of the disease progressed, these cells declined while CD8+ T cells producing TNF and IFNG increased, with the predominance of IFNG expression. As a novel finding, we identified by immunofluorescence CD4+ interleukin 17 (IL17)+ and CD8+ IL17+ cells in the testes of EAO rats, with CD4+ and CD8+ T cells predominating at the onset and in the chronic phase of EAO respectively. Moreover, IL17 (western blot) and IL23 content (ELISA) increased in EAO, with maximum levels in the chronic phase. These results suggest the involvement of CD4+ T helper (Th) 1 and Th17 subsets as co-effector cells governing EAO onset, as well as the central contribution of CD8+ T cells producing Th1 and Th17 cytokines in the maintenance of chronic inflammation. The expression of T-bet and RORγt (western blot) in the testis over the course of disease also supports the presence of Th1 and Th17 cells in the testes of EAO rats.     

Urinary trypsin inhibitor reduces inflammatory response in kidney induced by lipopolysaccharide

Human urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used in Japan as a drug for patients with acute inflammatory disorders such as septic shock and pancreatitis. Lipopolysaccharide (LPS) triggers the sepsis syndrome by activating monocytes to produce proinflammatory cytokines, including tumor necrosis factor alpha (TNFalpha), which potently stimulate the activation of neutrophils. The inhibitory mechanism of UTI on the systemic inflammatory response induced by the intraperitoneal injection of LPS in the kidney is unclear. This study was undertaken to examine the inhibitory effects of UTI on renal injury associated with the systemic inflammatory response induced by LPS stimulation, with emphasis on systemic TNFalpha and the activation of neutrophils in rat kidney. The systemic inflammatory response syndrome was induced by LPS treatment. Serum and renal TNFalpha, renal cytokine-induced neutrophil chemoattractant-1 (CINC-1) and myeloperoxidase (MPO) levels, as well as renal function after LPS stimulation, were evaluated. UTI (50,000 U/kg) inhibited LPS-induced increases in the serum and renal tissue levels of TNFalpha, as well as the renal tissue levels of CINC-1 and MPO after LPS stimulation. UTI (50,000 U/kg) also inhibited the production of serum TNFalpha associated with the systemic inflammatory response syndrome induced by LPS stimulation, thereby attenuating neutrophil infiltration into renal tissues and subsequent neutrophil-mediated renal injury. These findings may have important implications in understanding the biologic functions of UTI. UTI may prove useful in protecting against acute renal injury associated with a systemic inflammatory response.     

Radioadaptive response induced by alpha-particle-induced stress communicated in vivo between zebrafish embryos

We report data demonstrating that zebrafish embryos irradiated by alpha particles can release a stress signal into the water, which can be communicated to the unirradiated zebrafish embryos sharing the same water medium and thereby inducing a radioadaptive response in these unirradiated zebrafish embryos. The effects of radiation on the whole embryos were studied through quantification of apoptotic signals at 24 h post fertilization through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. In these experiments, dechorionated embryos were irradiated and then partnered with two other groups of unirradiated embryos, namely the bystander group (no more further treatments) and adaptive group (subjected to a further challenging dose) of embryos. The adaptive group of embryos were then separately further irradiated with a challenging dose. The results show that the number of apoptotic signals for the adaptive group is smaller than that for the corresponding control group, while that for the bystander group is larger than that for the corresponding control group. These suggest that the stress communicated in vivo between the irradiated zebrafish embryos and those unirradiated embryos sharing the same medium will induce radioadaptive response in the unirradiated embryos.     

藏蒲公英多糖纳米乳剂对小鼠外周血T淋巴细胞亚群CD4+、CD8+的影响

研究藏蒲公英多糖对小鼠免疫功能的调节作用.采用流式细胞术检测健康小鼠在灌胃0.1 ml质量分数0.85％生理盐水、0.1 ml质量分数1％藏蒲公英多糖水溶液和0.1 ml质量分数1％藏蒲公英多糖纳米乳剂情况下,外周血T淋巴细胞亚群CD4+、CD8+数量的动态变化.结果发现,藏蒲公英多糖和藏蒲公英多糖纳米乳剂均能够显著提高小鼠外周血T淋巴细胞亚群CD4+、CD8+水平,降低T淋巴细胞亚群CD4+、CD8+比值,尤以藏蒲公英多糖纳米乳剂效果最为显著.     

T lymphocyte proliferative capacity and CD4+/CD8+ ratio in primiparous and pluriparous lactating cows

T cells play a central role in specific immunity; their populations and phenotypes could be affected by number of lactation in high-yielding dairy cows. To investigate the effects of parity on the dynamics of T lymphocytes, lymphoproliferative capacity, T lymphocyte subsets and CD4+/CD8+ ratio were studied in peripheral blood of primiparous and pluriparous dairy cows during mid-late lactation. A non-radioactive technique was also adapted for a detailed lymphoproliferation assay. Compared with the primiparous cows, the pluriparous cows exhibited weaker lymphoproliferative activity, larger number of CD4+ cells and substantially greater CD4+/CD8+ ratio in their blood circulation. The increase of the CD4+/CD8+ ratio in the blood of pluriparous dairy cows was mainly due to the rise in the proportion of CD4+ cells and decline in the proportion of CD8+ cells. This increase of the CD4+/CD8+ ratio coincided with the decrease of mitogen-induced proliferation capacity of T lymphocytes. Of four lymphocyte divisions or generations during the lymphoproliferation assay, maximal lymphocyte proliferation capacity at generation 3 in primiparous cows was markedly greater than in pluriparous cows. With an alternatively safer, faster and more reproducible assay (compared with 3H-thymidine scintillation assay) we showed for the first time that aging in dairy cows leads to a decreased mitogen-induced lymphoproliferation and disturbed proportion between CD4+ and CD8+ T cells. This CD4+-CD8+ imbalance together with diminished lymphoproliferative capacity may lead to a weaker T cytotoxic-mediated immunity and increased susceptibility to infectious diseases in pluriparous lactating cows. Our study also emphasizes further application of the methods in farm animals.     

Rates of arsenopyrite oxidation by oxygen and Fe(III) at pH 1.8-12.6 and 15-45 degrees C

The oxidation rate of arsenopyrite by dissolved oxygen was measured using a mixed flow reactor at dissolved O2 concentrations of 0.007-0.77 mM, pH 1.8-12.6, and temperatures of 15-45 degrees C. As(III) was the dominant redox species (>75%) in the experimental system, and the As(III)/As(V) ratio of effluent waters did not change with pH. The results were used to derive the following rate law expression (valid between pH 1.8 and 6.4): r = 10((-2211 +/- 57)T) (mO2)(0.45 +/- 0.05), where r is the rate of release of dissolved As in mol m(-2) s(-1) and T is in Kelvin. Activation energies (Ea) for oxidation of arsenopyrite by 02 at pH 1.8 and 5.9 are 43 and 57 kJ/mol, respectively, and they compare to an Ea value of 16 kJ/mol for oxidation by Fe(III) at pH 1.8. Apparent As release rates passed through a minimum in the pH range 7-8, which may have been due to oxidation of Fe2+ to hydrous ferric oxide (HFO) with attenuation of dissolved As onto the freshly precipitated HFO.     

Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.     

Contraception in mice immunized with recombinant zona pellucida subunit 3 proteins correlates with Th2 responses and the levels of interleukin 4 expressed by CD4+ cells

The immune responses and contraceptive effect in mice were tested following immunization with purified recombinant zona pellucida (ZP) proteins produced using a vaccinia (v) virus T7 mammalian expression system. Female BALB/c and CBA mice were immunized with recombinant mouse (m) ZP3 (vmZP3) or pig (p) ZPC (vpZPC) using Freund's adjuvants and boosted three times. Fertility and mean litter size were significantly reduced in groups of BALB/c mice immunized with recombinant vmZP3 and vpZPC compared with controls treated with Freund's adjuvants alone. In CBA mice, fertility and mean litter size were significantly reduced in groups of animals immunized with vmZP3 but not with vpZPC compared with the controls. Most infertile animals treated with vmZP3 and a single infertile BALB/c mouse treated with vpZPC lacked mature follicles in the ovaries, whilst no abnormalities were detected in the remaining vpZPC treated, fertile vmZP3 treated and control mice. All mice (both fertile and infertile) immunized with vmZP3 and vpZPC produced IgG antibodies, but the levels of total IgG, IgG1 and IgG2a did not correlate with infertility. All BALB/c and CBA mice immunized with vmZP3 and vpZPC showed greater delayed type hypersensitivity responses in the footpads after challenge with their respective antigens than controls, but these did not differ between the fertile and infertile mice. There was, however, a significant correlation between infertility and the levels of the Type 2 T helper cell (Th2) cytokine interleukin 4 produced by CD4+ cells from vmZP3 immunized mice in response to stimulation with vmZP3 and this did not apply to the levels of the Type 1 T helper cell (Th1) cytokine interferon gamma or the general proliferation response. The results support the conclusion that induction of Th2 responses in individual mice determines whether infertility develops in response to immunization with zona pellucida proteins.     

离子注入以及响应面法选育L-组氨酸产生菌

以一株枯草芽孢杆菌Y1为出发菌株,以90个单位,每单位注入量为2.5×1013/cm3的N+进行诱变。经筛选和摇瓶发酵培养最终获得一株产酸量较高遗传性状稳定的突变株Y-217(6-MPR,transketolase-,histidase-),产酸量为2.57g/L,由原先的1.76g/L增长到2.57g/L。在单因素实验的基础上,利用响应面法对菌株的发酵条件进行进一步优化,得出最佳发酵条件为:豆饼粉14g/L,葡萄糖10g/L,硫酸铵1.5g/L,玉米粉11.2g/L,KH2PO4 1.5g/L,Mg SO4·7H2O 1.8g/L,碳酸钙5g/L,优化后菌株的产酸能力由2.57 g/L增长到4.61g/L.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

Stiffness alterations of single cells induced by UV in the presence of nanoTiO2

Nanocrystalline titanium dioxide (nanoTiO2) has been reported to generate reactive oxygen species (ROS) under UV illumination. In our studies, changes in mechanical properties of human skin fibroblasts, exposed to the oxidative stress induced in the presence of nanoTiO2 and UV light, were studied using atomic force microscopy (AFM). The exposure of cells to the action of ROS was performed at low TiO2 concentration (4 microg/mL) and under illumination with low-intensity UVA (8 and 20 mW/cm2) or UVC (0.1 mW/ cm2). AFM measurements of the cell stiffness were carried out immediately after exposure of cells to the oxidative stress. The data suggest that under illumination with low-intensity UVA nanoTiO2 generates ROS, which, in turn, damage cellular and subcellular structures. This process was detected by AFM as a marked drop in the cellular stiffness of ca. 30-75%, which occurred rapidly, in the time frame of 1 min. The photo-oxidative stress inducing the decrease of cell stiffness was cancelled in the presence of a well-established antioxidant, beta-carotene. The results highlight the sensitivity of AFM to detect early changes in mechanical properties of cells exposed to oxidative stress.     

山莴苣苦素改善FFA诱导的HepG2细胞脂肪变性及氧化应激的研究

目的:研究山莴苣苦素对游离脂肪酸(FFA)诱导的HepG2细胞脂肪变性的改善作用,并初步探讨其可能作用机制.方法:观察山莴苣苦素对HepG2细胞活力的影响.采用FFA诱导培养HepG2细胞,构建脂肪变性模型(NAFLD体外模型),并用山莴苣苦素干预72h后,检测各组细胞内脂滴数量、甘油三酯(TG)和总胆固醇(TC)含量...     

Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis

The corpus luteum is a transient endocrine gland specializing in the production of progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of fetal bovine serum for 24 or 48 h. Under serum starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with hematoxylin after being subjected to serum starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic DNA. Apoptosis of serum-deprived luteal cells was dependent upon caspase activation. Serum starvation induced cleavage of poly (ADP-ribose) polymerase (PARP), suggesting that caspase-3 had been activated under the stress of withdrawal of growth factors. This was confirmed by cleavage of full-length procaspase-3. Finally, the fact that serum starvation promoted the cleavage of full-length procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.     

Apoptotic effects of cooked and in vitro digested soy on human prostate cancer cells

Previous laboratory and animal studies reported that soy isoflavones were major bioactive compounds in soy to exert chemoprotection of prostate cancer. However, these studies cannot reflect the realistic effects that soy may induce through diets, and little is known about the bioavailability of isoflavones from whole soy food and their bioactivities after cooking and digestion. In this study, cooking and in vitro digestion were used to prepare soy extracts and the effects of cooking and digestion on the isoflavone contents and bioactivities of the whole soy extracts were examined. The cooking procedure generally increased the amount of daidzin, genistin and daidzein, but decreased that of genistein. Digestion process significantly lowered contents of daidzin and genistin in 60min cooked sample, while increased the contents of daidzin and daidzein and decreased the content of genistein in the uncooked sample. Antioxidant activities of soy extracts increased after cooking and in vitro digestion, while no consistent increase of the four soy isoflavones was determined. The apoptotic effects of soy extracts on both LNCaP and C4-2B cells were generally in a dose-dependent manner. Compared to purified single isoflavones, cooked and digested soy were more effective on induction of prostate cancer cell apoptosis, which indicated synergistic interactions between various bioactive compounds in the whole soy.     

Amelioration of scopolamine induced cognitive dysfunction and oxidative stress by Inonotus obliquus - a medicinal mushroom

The present study was aimed to investigate the cognitive enhancing and anti-oxidant activities of Inonotus obliquus (Chaga) against scopolamine-induced experimental amnesia. Methanolic extract of Chaga (MEC) at 50 and 100 mg kg (-1)doses were administered orally for 7 days to amnesic mice. Learning and memory was assessed by passive avoidance task (PAT) and Morris water maze (MWM) test. Tacrine (THA, 10 mg kg (-1), orally (p.o)) used as a reference drug. To elucidate the mechanism of the cognitive enhancing activity of MEC, the activities of acetylcholinesterase (AChE), anti-oxidant enzymes, the levels of acetylcholine (ACh) and nitrite of mice brain homogenates were evaluated. MEC treatment for 7 days significantly improved the learning and memory as measured by PAT and MWM paradigms. Further, MEC significantly reduced the oxidative-nitritive stress, as evidenced by a decrease in malondialdehyde and nitrite levels and restored the glutathione and superoxide dismutase levels in a dose dependent manner. In addition, MEC treatment significantly decreased the AChE activity in both the salt and detergent-soluble fraction of brain homogenates. Further, treatment with MEC restored the levels of ACh as did THA. Thus, the significant cognitive enhancement observed in mice after MEC administration is closely related to higher brain anti-oxidant properties and inhibition of AChE activity. These findings stress the critical impact of Chaga, a medicinal mushroom, on the higher brain functions like learning and memory.     

芸薹种UGMS标记及抗根肿病基因连锁标记的定位

为定位芸薹种抗根肿病基因的连锁标记,利用芸薹种单一基因微卫星（UGMS）标记及基因组SSR和抗根肿病（clubroot resistance,CR）基因的紧密连锁分子标记,构建了结球白菜59-1×芜菁（CR）WJ04遗传连锁图谱,对芜菁WJ04和结球白菜59-1进行根肿病抗性鉴定和连锁位点分析。结果表明：抗根肿病芜菁自交系WJ04对来自我国根肿病主要疫区的4个根肿菌生理小种2、4、7和10均具有显性抗性,而59-1则均表现为感病。UGMS标记在大白菜和芜菁亚种间的多态性比率为30.1%,低于基因组SSR的50.8%;图谱覆盖长度为1 116.2cM,包含分布在10条连锁群的59个UGMS、72个基因组SSR和4个分别与CR基因Crr1、Crr2、Crr3和CRb连锁的标记。     

Differential effects in mammalian cells induced by chemical mixtures in environmental biota as profiled using infrared spectroscopy

Environmental contaminants accumulate in many organisms and induce a number of adverse effects. As contaminants mostly occur in the environment as mixtures, it remains to be fully understood which chemical interactions induce the most important toxic responses. In this study, we set out to determine the effects of chemical contaminants extracted from Northern Gannet (Morus bassanus) eggs (collected from the UK coast from three sampling years (1987, 1990, and 1992) on cell cultures using infrared (IR) spectroscopy with computational data handling approaches. Gannet extracts were chemically analyzed for different contaminants, and MCF-7 cell lines were treated for 24 h in a dose-related manner with individual-year extracts varying in their polybrominated diphenyl ether (PBDE) to polychlorinated biphenyl (PCB) ratios. Treated cellular material was then fixed and interrogated using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy; resultant IR spectra were computationally analyzed to derive dose-response relationships and to identify biomarkers associated with each contaminant mixture treatment. The results show distinct biomarkers of effect are related to each contamination scenario, with an inverse relationship with dose observed. This study suggests that specific contaminant mixtures induce cellular alterations in the DNA/RNA spectral region that are most pronounced at low doses. It also suggests alterations in the "biochemical-cell fingerprint" of IR spectra can be indicative of mixture exposures.     

酱香型白酒醇类活性成分抗幽门螺杆菌损伤胃黏膜上皮细胞活性的研究

为研究酱香型白酒醇类活性成分(active alcohols,AAs)抑制幽门螺杆菌(Helicobacter pylori)损伤胃黏膜上皮(GES-1)细胞活性及潜在的机制,检测了AAs抑制H.pylori的最小半数抑菌浓度(50％minimun inhibitory concentration,MIC50)及苯乙醇...