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.     

石参提取物诱导MKN45细胞凋亡的初步研究

通过石参提取物作用于人胃癌MKN-45细胞,检测其对细胞活性的影响并探讨其作用机制。利用MTT法检测石参提取物对细胞活性的影响;采用AO/EB双荧光染色检测石参提取物对细胞形态的影响;使用JC-1荧光探针检测线粒体膜电位变化;利用Western blot检测石参提取物对细胞凋亡内源性通路的信号蛋白表达。结果表明浓度为20、30mg/mL的石参提取物能够抑制MKN-45细胞的活性,使细胞形态发生改变;10、20、30mg/mL的石参提取物可引起细胞线粒体膜电位下降,并引起Bcl-2的表达量下降,Bax、细胞色素c和caspase-3的表达量增加,PARP被剪切。提示石参提取物可通过线粒体途径诱导MKN-45细胞凋亡。      

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.     

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.     

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.     

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.     

Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells

Scope: Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables such as broccoli. The ability of SFN to inhibit histone deacetylase (HDAC) enzymes may be one mechanism by which it acts as a chemoprevention agent. The ability of a chemopreventive agent to specifically cause cytotoxicity in cancer and not normal cells is an important factor in determining its safety and clinical relevance. Methods and results: We characterized the effects of SFN in normal (PrEC), benign hyperplasia (BPH1) and cancerous (LnCap and PC3) prostate epithelial cells. We observed that 15 μM SFN selectively induced cell cycle arrest and apoptosis in BPH1, LnCap and PC3 cells but not PrEC cells. SFN treatment also selectively decreased HDAC activity, and Class I and II HDAC proteins, increased acetylated histone H3 at the promoter for P21, induced p21 expression and increased tubulin acetylation in prostate cancer cells. HDAC6 over‐expression was able to reverse SFN‐induced cyotoxicity. In PrEC cells, SFN caused only a transient reduction in HDAC activity with no change in any other endpoints tested. The differences in sensitivity to SFN in PrEC and PC3 are likely not due to differences in SFN metabolism or differences in phase 2 enzyme induction. Conclusion: SFN exerts differential effects on cell proliferation, HDAC activity and downstream targets in normal and cancer cells.     

钙离子与pH诱导鲤鱼卵肽纳米颗粒的形成

探讨钙离子和p H诱导鲤鱼卵肽（Carp egg peptide,CEP）形成纳米颗粒的条件及稳定纳米颗粒空间结构的作用力。通过浊度检测初步筛选CEP纳米颗粒形成条件,用粒径分析和电镜观察方法确认纳米颗粒的形成。通过次级键破坏实验分析稳定纳米颗粒空间结构的作用力,并通过红外光谱和圆二色谱分析CEP纳米颗粒形成后的空间结构变化。结果表明,当p H6.0⁹.0时,CEP能与一定浓度的钙离子结合形成粒径20¹00 nm的纳米颗粒,疏水作用和静电作用是稳定纳米颗粒的主要作用力,CEP形成纳米颗粒后伴随着β折叠的形成。CEP能够作为生物模板在温和条件下合成肽-钙纳米颗粒。      

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.     

Volunteer study and serum protein profiling to understand inflammatory response induced by Satsuma mandarin

It has been observed that consumption of a certain amount of Satsuma, lychee, and longan often caused a symptom characterized by dry or sore throat, gum swelling and even mouth ulcer, which significantly impaired the life quality of a large population. We define the adverse reaction to Satsuma as Satsuma-induced syndrome (SIS). Volunteers were assigned to oral Satsuma challenge in an open manner. The results showed that SIS was characterized with symptoms affecting the throat, oral cavity, face, gastrointestinal system and eye either individually or in combination. A comparative proteomic study was performed to investigate the differences of serum proteins in the Post-SC (after Satsuma challenge) and Pre-SC (before Satsuma challenge) serum samples of 15 volunteers with severe SIS. Ten proteins were identified to be differentially expressed (P < 0.05). Of these, levels of complement component C9 precursor were elevated significantly in the Post-SC serum samples and were further verified by enzyme-linked immunosorbent assay, indicating that the complement system may be activated and plays a significant role in inflammatory response. Meanwhile, serum samples were subjected to immobilized metal affinity capture (IMAC3) protein chip surfaces and tested by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The data were analyzed by Ciphergen ProteinChip Software. A diagnostic model was constructed to discriminate the SIS from normal samples, using principal component analysis. A total of 50 detected biomarkers were found to be different with statistical significance (P < 0.05). The multivariate logistic analysis demonstrates a complete distinction between the two groups. Our findings suggest that these assays may provide potential biomarkers for the diagnosis of SIS.     

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.     

邻-（2,3,4,5,6-五氟苄基）羟胺盐酸盐衍生-HPLC法测定果蔬中甲醛含量

通过邻-（2,3,4,5,6-五氟苄基）羟胺盐酸盐与甲醛衍生,建立了测定果蔬中甲醛含量的高效液相色谱法。样品在温度为70℃超声波条件下直接提取衍生30 min,经离心纯化后液相色谱检测,外标法定量。优化的色谱条件为:Eelipse XDB-C₁₈柱（4.6 mm×250 mm,5μm）;流动相为乙腈和水（70∶30,v/v）,流速1.0 m L/min,柱温40℃,检测波长210 nm。结果表明,该方法的检出限可达到0.114 mg/kg,在0.57⁵7 mg/kg范围内呈良好的线性关系,平均回收率为82.3%⁹4.1%,相对标准偏差为4.0%⁷.1%（n=6）。该方法样品前处理简便,稳定性好,检测限低,适合果蔬中甲醛的快速定量检测。      

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.