Inhibitory effect of some flavonoids on tumor necrosis factor-alpha production in lipopolysaccharide-stimulated mouse macrophage cell line J774.1

Certain naturally occurring flavonoids affect immunoregulatory activities in vitro and in vivo against cytokine production. Since tumor necrosis factor (TNF)-alpha is one of the major inflammatory cytokines, the effects of various dietary flavonoids on TNF-alpha production in lipopolysaccharide (LPS)-stimulated J774.1 cells were evaluated in vitro. Flavones, flavonols, and chalcone are the most potent inhibitors of production of TNF-alpha. Flavanone, naringenin, anthocyanidin, pelargodinin, and cyanidin exhibit moderate inhibitory activity. In contrast, genistein isoflavone displays weak inhibition, while eriodictyol flavanone is inactive. It is clear that the double bond between carbons 2 and 3 and the ketone group at position 4 of flavonoids are necessary for potent inhibitory effect. The difference in inhibitory action appears to depend on the categorized subclass of flavonoids.     

Syntheses of tetrahydroisoquinoline derivatives that inhibit NO production in activated BV-2 microglial cells

Seventeen tetrahydroisoquinoline derivatives were designed, synthesized and evaluated for inhibition of NO production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 5a, 9c and 11a potently attenuated NO production by >60%, and 5a and 11a inhibited BH4 production by >48% at 100 microM. In particular, N-ethylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (11a) reduced NO production by 64% and tetrahydrobiopterin (BH4) production by 49%. Introducing longer alkyl component at C1 or N2 position led to attenuation of the inhibitory effect. It is possible that 11a inhibits NO production by blocking BH4-dependent dimerization of newly synthesized iNOS monomers.     

环磷酸腺苷在川芎嗪促Caco-2细胞系分泌中的作用

目的研究川芎嗪(TMP)在Caco-2细胞系中作用的信号转导途径.方法运用细胞培养,破膜和短路电流(Isc)技术,信号转导途径中的激动剂和阻断剂研究TMP作用的信号转导途径.结果Forskolin (AC激动剂)和IBMX(PDE抑制剂)预处理后显著抑制了TMP在Caco-2细胞系上引起的Isc.MDL-12330A(AC抑制剂) 预处理后也明显抑制TMP的作用.结论TMP通过环磷酸腺苷-蛋白激酶A(cAMP-PKA)系统引起Caco-2细胞系的分泌作用.     

An avian cell line designed for production of highly attenuated viruses

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.     

孕酮对生殖支原体诱导单核细胞产生前炎症细胞因子的影响

目的:探讨孕酮(P4)对生殖支原体(Mg)诱导人单核细胞产生前炎症细胞因子IL-1β,IL-8和TNF-α的影响。方法:以人单核细胞系THP-1为研究对象,用不同浓度的P4孵育24 h后,再加入热灭活的Mg刺激,采用酶联免疫吸附试验(ELISA)检测3种前炎症细胞因子的产生情况,MTT检测细胞活性。结果:Mg能诱导THP-1细胞产生IL-1β、TNF-α和IL-8,P4处理后能增强IL-1β和IL-8的量,但能抑制TNF-α的产生。结论:P4对Mg诱导的IL-1β、IL-8和TNF-α的产生具有不同调控作用。因此,其在早产预防中的使用并非抑制细胞因子的产生。     

Supplementation of omega 3 fatty acids improves oxidative stress in activated BV2 microglial cell line

Abstract

Many reports have shown promising beneficial effects of long-chain polyunsaturated fatty acids (L-PUFAs) of the omega 3 series in several brain diseases. In the present study, we tested the hypothesis that omega 3 fatty acids supplement reduced pro-inflammatory functions in vitro and in vivo. We demonstrated that a supplement rich in PUFAs (SRP) increased cell viability in a dose-dependent manner suggesting its protective role against lipopolysaccharide (LPS)-induced cell death in BV2 microglial cell line. In the same cultures, the supplement rich in PUFAs reduced the reactive oxygen species (ROS) and nitric oxide (NO) production. A most prominent target for ROS management is the family of peroxisome proliferator-activated receptors (PPARs). The co-treatment with SRP and LPS increased significantly the nuclear immunoreactivity of PPAR-γwhen compared the LPS treatment alone. Moreover, the chronic administration of the SRP in rats, increased the immunoreactivity of the PPAR-γ1 protein confirming its potential neuroprotective effect.     

Lipopolysaccharide-stimulated nitric oxide production and inhibition of cell proliferation is antagonized by ethanol in a clonal macrophage cell line.

Both chronic and acute ethanol exposure have been shown to be cytotoxic and also to disrupt normal cell function or responses in a variety of cell types. Macrophage function has specifically been shown to be disrupted by chronic ethanol exposure by mechanisms that have not been elucidated. It is known that exposure of macrophages to lipopolysaccharide (LPS) from gram-negative bacteria will decrease the number of cells. Since increased exposure to endotoxin is often associated with chronic alcoholism, this may be one mechanism to account for loss of macrophages in alcoholic patients. The loss of macrophages, as a consequence of endotoxin treatment, appears to be linked to cell activation and, in particular, LPS-stimulated synthesis of nitric oxide which has been suggested to cause an increase in apoptosis. Ethanol also increases apoptosis in some cell types but, in general, ethanol inhibits activation of macrophages. Thus, the overall effect on cell numbers and cell proliferation elicited by treating macrophages concomitantly with ethanol and LPS depends on the balance between inhibiting LPS-mediated activation and the actions of ethanol. The interaction between ethanol and LPS was investigated in a macrophage cell line (RAW 264.7 cells) by measuring nitric oxide production and cell proliferation. A 24-h exposure to ethanol (100 mM) decreased [3H]-thymidine incorporation significantly. LPS treatment elicited a concentration-dependent decrease in [3H]-thymidine incorporation at LPS concentrations of 0.1 ng/ml to 1000 ng/ml and stimulated nitric oxide production at concentrations above 1 ng/ml. LPS-stimulated nitric oxide production was inhibited by ethanol (20 to 100 mM) and the nitric oxide synthesis inhibitor, N(G)Nitro-L-arginine methyl L-NAME) ester (100 and 500 microM). However, LPS-inhibited [3H]-thymidine incorporation was not be totally reversed by ethanol- or L-NAME-treatment. A direct correlation between nitric oxide production and inhibition of cell proliferation could not be demonstrated. However, it appears that ethanol and LPS do affect some common mechanism(s) in this cell line.     

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency.Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log₁₀(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 10⁶ cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log₁₀(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.     

Molecular epidemiology, population genetics, and pathogenic role of Helicobacter pylori

Helicobacter pylori infection is linked to various gastroduodenal diseases; however, only approximately 20% of infected individuals develop severe diseases. Despite the high prevalence of H. pylori infection in Africa and South Asia, the incidence of gastric cancer in these areas is much lower than in other countries. Furthermore, the incidence of gastric cancer tends to decrease from north to south in East Asia. Such geographic differences in the pathology can be explained, at least in part, by the presence of different types of H. pylori virulence factors, especially cagA, vacA, and the right end of the cag pathogenicity island. The genotype of the virulence genes is also useful as a tool to track human migration utilizing the high genetic diversity and frequent recombination between different H. pylori strains. Multilocus sequence typing (MLST) analysis using seven housekeeping genes can also help to predict the history of human migrations. Population structure analysis based on MLST has revealed seven modern population types of H. pylori, which derived from six ancestral populations. Interestingly, the incidence of gastric cancer is closely related to the distribution of H. pylori populations. The different incidence of gastric cancer can be partly attributed to the different genotypes of H. pylori circulating in different geographic areas. Although approaches by MLST and virulence factors are effective, these methods focus on a small number of genes and may miss information conveyed by the rest of the genome. Genome-wide analyses using DNA microarray or whole-genome sequencing technology give a broad view on the genome of H. pylori. In particular, next-generation sequencers, which can read DNA sequences in less time and at lower costs than Sanger sequencing, enabled us to efficiently investigate not only the evolution of H. pylori, but also novel virulence factors and genomic changes related to drug resistance.     

某雷管生产线项目职业病危害预评价

目的通过对某雷管生产线可能存在的职业病危害因素进行预评价,提出有效的防护对策和建议。方法根据国家相关标准,采用类比法和经验法相结合的原则进行评价。结果可比性较高的类比企业检测结果显示:铅尘和苯无超标,噪声最高为88.7 dB(A),超标率为59.26%。结论本项目属于严重职业病危害项目,如果做好个人防护,可以预防职业病的发生。从职业病防治角度分析,项目建设可行。     

Glycan analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin

Mammalian cell culture processes are commonly used for production of recombinant glycoproteins, antibodies and viral vaccines. Since several years there is an increasing interest in cell culture-based influenza vaccine production to overcome limitations of egg-based production systems, to improve vaccine supply and to increase flexibility in vaccine manufacturing. With the switch of the production system several key questions concerning the possible impact of host cell lines on antigen quality, passage-dependent selection of certain viral phenotypes or changes in hemagglutinin (HA) conformation have to be addressed to guarantee safety and efficiency of vaccines. In contrast to the production of recombinant glycoproteins, comparatively little is known regarding glycosylation of HA, derived from mammalian cell cultures. Within this study, a capillary DNA-sequencer (based on CGE-LIF technology), was utilized for N-glycan analysis of three different influenza virus strains, which were replicated in six different cell lines. Detailed results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Strong host cell but also virus type and subtype dependence of HA N-glycosylation was found. Clear differences were already observed, by N-glycan fingerprint comparison. Further structural investigations of the N-glycan pools revealed that host cell dependence of HA N-glycosylation was mainly related to minor variations of the (monomeric) constitution of single N-glycans. To some extent, shifts in the N-glycan pool composition regarding the proportion of different N-glycan types were observed. In contrast to this, a principal switch of the N-glycan type attached to HA was observed when comparing different virus types (A and B) and subtypes (H1N1 and H3N2).     

Role of putrescine in cell proliferation in a colon carcinoma cell line

OBJECTIVES: Putrescine, the precursor for higher polyamine biosynthesis, is necessary for cell growth in mammals. Ornithine decarboxylase (ODC) activity and polyamine production are increased in neoplastic cells. Using colon cancer cell line derived from a tumor with high metastatic potential (CT-26), our objective was to study the effect of exogenous putrescine on ODC regulation, polyamine metabolism, and cell proliferation. METHODS: Cells cultured with fetal calf serum were exposed to 100, 550, and 1000 microM putrescine for 24 h. RESULTS: Intracellular free putrescine, determined by high-performance liquid chromatography, showed a statistically significant increase in exposed cells compared with controls and a significant correlation with levels of the metabolite present in the medium (r = 0.93; P < 0.001). Bromodeoxyuridine incorporation into newly synthesized DNA, a marker of cell proliferation, showed a statistically significant increase in the three putrescine groups as opposed to the control group. In samples with added aminoguanidine, significant increases in DNA synthesis were observed in the 550- and 1000-microM putrescine groups as opposed to the control group. Spermidine and spermine intracellular contents in all three putrescine-treated groups remained below control levels. No statistical differences in ODC enzymatic activity or ODC mRNA content were observed. Newly incorporated putrescine stimulated colon tumor cell growth. CONCLUSIONS: Because neither enhanced conversion into the higher polyamines nor aminoguanidine inhibition of proliferation was observed, we suggest that this effect can be attributed to the putrescine molecule itself.     

Plasma levels of folates, ascorbate, vitamin B6 and riboflavin in children.

Plasma vitamin levels of approximately 125 children admitted to the Queens Child Psychiatric Center were determined in a controlled study, with respect to medication and dietary conditions. The age of these children varied from 4 to 16 years. The data present the mean values with respect to the age and racial origin. There are no significant variations in the mean levels of these vitamins in terms of the age etc. of the subjects. As such the data indicate the levels in children and are comparable to the data for adults.     

某光盘生产线职业病危害因素检测结果分析

目的通过对某可录类光盘生产线职业病危害因素的识别与分析,确定职业病危害的关键控制点。方法采用现场职业卫生学调查和职业危害因素检测检验方法进行分析。结果可录类光盘生产线主要存在的职业病危害因素为苯及苯系物、丙烯酸丁酯和噪声。检测结果表明,5个工种作业工人接触苯、甲苯、二甲苯、丙烯酸丁酯浓度均低于国家职业接触限值;对6个接触噪声的工种进行了噪声等效声级测定,作业工人接触的8h等效声级均未超过职业接触限值的规定。结论可录类光盘生产线存在的职业病危害的防治应从职业卫生管理、严格佩戴个人防护用品以及职业健康监护等方面着手综合采取切实可行的防护设施或措施。     

亚砷酸钠对HaCaT细胞增殖周期及ROS生成影响

目的 探讨亚砷酸钠(NaAsO₂)对人皮肤角质形成细胞(HaCaT)增殖、细胞周期及活性氧(ROS)生成影响。方法 以0、25、50μmol/L NaAsO₂作用于HaCaT细胞6 h后,以Alamar Blue还原法检测细胞增殖水平;以流式细胞仪检测细胞周期及ROS水平。结果 25、50μmol/L NaAsO₂组细胞增殖水平分别为(93.5±1.18)%、(82.9±2.64)%,明显低于对照组(100±0.51)%(P<0.05);G2/M期细胞数分别为(19.15±0.29)%、(18.12±1.35)%,明显高于对照组(15.24±0.04)%(P<0.05);ROS水平分别为(128.61±6.23)%、(152.92±7.25)%,明显高于对照组(100.00±3.45)%(P<0.05)。结论 NaAsO₂作用可引起HaCaT细胞ROS含量增高,同时G2/M期细胞数增多,但G2/M期细胞比例增高并不是由细胞增殖作用所引起。     

Pathogenic role of cardiac mast cell activation/degranulation,TNF-alpha, and cell death in acute drug-related fatalities

Background

Intravenous injection of narcotic stimulants affects many cellular functions relevant for the pathophysiological mechanisms of heart failure. There is considerable evidence that mast cells (MCs), TNF-α, and cell death play crucial roles in the pathogenesis and progression of cardiac arrest. In this study, we examined and compared the participation of MCs, TNF-α, apoptosis and necrosis in the heart of drug-related fatalities and the victims of sudden death due to the cardiac failure or aortic dissection.

Methods and results

Serum level of postmortem tryptase was determined in all study subjects that consisted of 50 autopsy cases: 30 drug overdose fatalities which were further divided into two groups with high and low level of tryptase, and 20 cases of sudden natural death (SND). The distribution profile of cardiac infiltrated-MCs and production patterns of TNF and C9 (necrotic marker) were investigated immunohistochemically. In situ-detection of apoptosis with TUNEL was applied to the heart sections. The level of tryptase was elevated (>45 μg/L) in the drug fatalities but remained below the cut-off value in SND. In the myocardium of overdose victims, MC-infiltration and degranulation were significantly increased as well as production of myocytic TNF-α compared with the SND cases. The expressions profile of myocytic TNF varied between the groups. Apoptotic myocytes were seen more frequently in the SND group while necrosis was more evident in the heart of drug-related fatalities.

Conclusion

Mast cells are recruited and activated in the heart of drug-associated deaths and the myocytes are the main source of TNF-α with the ability of different production patterns. The high degree of MC degranulation and the elevated levels of tryptase together with the pathological changes in heart of drug-related victims resemble that of the anaphylactic deaths as demonstrated in our previous study.