A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

Dectin-1 plays a deleterious role in high fat diet-induced NAFLD of mice through enhancing macrophage activation.

The innate immune response and inflammation contribute to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). Dectin-1 is a pathogen recognition receptor in innate immunity. In this study, we investigated the role of Dectin-1 in the pathogenesis of NAFLD. We first showed that Dectin-1 expression was significantly elevated in liver tissues of patients with NASH. NAFLD was induced in mice by feeding high fat diet (HFD) for 24 weeks. At the end of treatment, mice were sacrificed, and their blood and liver tissues were collected for analyses. We showed HFD feeding also increased liver Dectin-1 levels in mice, associated with macrophage infiltration. Either gene knockout or co-administration of a Dectin-1 antagonist laminarin (150 mg/kg twice a day, ip, from 16th week to 24th week) largely protected the livers from HFD-induced lipid accumulation, fibrosis, and elaboration of inflammatory responses. In primary mouse peritoneal macrophages (MPMs), challenge with palmitate (PA, 200 µM), an abundant saturated fatty acid found in NAFLD, significantly activated Dectin-1 signaling pathway, followed by transcriptionally regulated production of pro-inflammatory cytokines. Dectin-1 was required for hepatic macrophage activation and inflammatory factor induction. Condition media generated from Dectin-1 deficient macrophages failed to cause hepatocyte lipid accumulation and hepatic stellate activation. In conclusion, this study provides the primary evidence supporting a deleterious role for Dectin-1 in NAFLD through enhancing macrophage pro-inflammatory responses and suggests that it can be targeted to prevent inflammatory NAFLD.

MD2 deficiency prevents high-fat diet-induced AMPK suppression and lipid accumulation through regulating TBK1 in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most predominant form of liver diseases worldwide. Recent evidence shows that myeloid differentiation factor 2 (MD2), a protein in innate immunity and inflammation, regulates liver injury in models of NAFLD. Here, we investigated a new mechanism by which MD2 participates in the pathogenesis of experimental NAFLD. METHODS: Wild-type, Md2-/- and bone marrow reconstitution mice fed with high-fat diet (HFD) were used to identify the role of hepatocyte MD2 in NAFLD. Transcriptomic RNA-seq and pathway enrich analysis were performed to explore the potential mechanisms of MD2. In vitro, primary hepatocytes and macrophages were cultured for mechanistic studies. RESULTS: Transcriptome analysis and bone marrow reconstitution studies showed that hepatocyte MD2 may participate in regulating lipid metabolism in models with NAFLD. We then discovered that Md2 deficiency in mice prevents HFD-mediated suppression of AMP-activated protein kinase (AMPK). This preservation of AMPK in Md2-deficient mice was associated with normalized sterol regulatory element binding protein 1 (SREBP1) transcriptional program and a lack of lipid accumulation in both hepatocytes and liver. We then showed that hepatocyte MD2 links HFD to AMPK/SREBP1 through TANK binding kinase 1 (TBK1). In addition, MD2-increased inflammatory factor from macrophages induces hepatic TBK1 activation and AMPK suppression. CONCLUSION: Hepatocyte MD2 plays a pathogenic role in NAFLD through TBK1-AMPK/SREBP1 and lipid metabolism pathway. These studies provide new insight into a non-inflammatory function of MD2 and evidence for the important role of MD2 in NALFD.

Poly(ADP-ribose) polymerase-1 inhibitor ameliorates dextran sulfate sodium-induced colitis in mice by regulating the balance of Th17/Treg cells and inhibiting the NF-κB signaling pathway.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a critical role in inflammatory pathways. The PARP-1 inhibitor, 5-aminoisoquinolinone (5-AIQ), has been demonstrated to exert significant pharmacological effects. The present study aimed to further examine the potential mechanisms of 5-AIQ in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Colitis conditions were assessed by changes in weight, disease activity index, colon length, histopathology and pro-inflammatory mediators. The colonic expression of PARP/NF-κB and STAT3 pathway components was measured by western blot analysis. Flow cytometry was used to analyze the proportion of T helper 17 cells (Th17) and regulatory T cells (Tregs) in the spleen. Western blot analysis and reverse transcription-quantitative PCR were employed to determine the expression of the transcription factors retinoic acid-related orphan receptor and forkhead box protein P3. The results demonstrated that 5-AIQ reduced tissue damage and the inflammatory response in mice with experimental colitis. Moreover, 5-AIQ increased the proportion of Treg cells and decreased the percentage of Th17 cells in the spleen. Furthermore, following 5-AIQ treatment, the main components of the PARP/NF-κB and STAT3 pathways were downregulated. Collectively, these results demonstrate that the PARP-1 inhibitor, 5-AIQ, may suppress intestinal inflammation and protect the colonic mucosa by modulating Treg/Th17 immune balance and inhibiting PARP-1/NF-κB and STAT3 signaling pathways in mice with experimental colitis.

Seroprevalence, Risk Factors, and Genotypes of Toxoplasma gondii in Free-Range Chickens Intended for Human Consumption in China.

Toxoplasma gondii is a protozoan with worldwide distribution that infects birds and mammals, including humans. The consumption of free-range chicken meat is a common practice in many parts of the world. However, little information is available concerning the molecular prevalence and genotypes of T. gondii infection in free-range chickens intended for human consumption in China. In this study, a total of 1360 serum samples were collected from food markets in Hunan province of China for detecting T. gondii antibodies by indirect hemagglutination assay. In addition, 650 brain tissues were also collected to investigate T. gondii DNA by amplification of B1 gene with a seminested polymerase chain reaction (PCR), and the positive DNA samples were typed at 10 genetic markers using multilocus PCR-restriction fragment length polymorphism. Antibodies to T. gondii were detected in 457 of the examined serum samples (33.6%; 95% confidence interval [CI]: 31.1-36.1), and 72 DNA samples (11.1%; 95% CI: 8.6-13.4) were positive for the T. gondii B1 gene. In this study, region and age of free-range chickens were shown to be risk factors for T. gondii infection (p < 0.01). Two genotypes (ToxoDB#9 and ToxoDB#52) were identified from two samples with complete genotyping results. Our study revealed a high prevalence of T. gondii infection in free-range chickens intended for human consumption in Hunan province, suggesting that recommendations to consumers should be made, especially in some regions of China where consumption of undercooked chicken meat is common. This is the first genetic characterization of T. gondii in free-range chickens intended for human consumption in Hunan province, China, and also the first report of genotype ToxoDB#52 in China.

Target-controlled propofol infusion with or without bispectral index monitoring of sedation during advanced gastrointestinal endoscopy.

BACKGROUND AND AIM: Target-controlled infusion (TCI) uses averaged pharmacokinetic datasets derived from population samples to automatically control the infusion rate. Bispectral index (BIS) technology non-invasively measures levels of consciousness during surgical procedures. We compared the efficacy and safety of propofol TCI with or without BIS monitoring for sedation during advanced gastrointestinal endoscopy. METHODS: This prospective study enrolled 200 patients who were premedicated with midazolam 2 mg and alfentanil 0.4 mg before undergoing advanced gastrointestinal endoscopy. The initial target blood concentration of propofol was set at 1.0 µg/mL, and adjustments of 0.2 µg/mL were made as necessary to maintain moderate-to-deep sedation. Patients were randomized to either the BIS-blind group and evaluated for depth of anesthesia by monitoring scores of 1-2 on the Modified Observer's Assessment of Alertness/Sedation scale (n = 100) or to the BIS-open group and monitored by BIS scores of 60-80 (n = 100). The primary outcome was the total amount of propofol required to maintain anesthesia. Secondary outcomes were sedation-induced adverse events, recovery, and quality of sedation (endoscopist and patient satisfaction). RESULTS: The mean propofol infusion rate was significantly higher in patients not monitored by BIS scores than in those who were (5.44 ± 2.12 vs 4.76 ± 1.84 mg/kg/h; P = 0.016). Levels of satisfaction were higher for endoscopists who used BIS monitoring than in those who did not. CONCLUSIONS: Mean infusion rates were higher in propofol TCI without BIS monitoring compared with propofol TCI with BIS during advanced gastrointestinal endoscopy. Endoscopists expressed satisfaction with BIS monitoring.

Variation of 24 plasma amino acid metabolite levels in patients with gastric cancer / 第二军医大学学报

Objective To study whether plasma amino acid metabolites in patients with gastric cancer can be used as biomarkers for the diagnosis of gastric cancer. Methods The levels of 24 kinds of plasma amino acids were detected by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), and were compared between patients with gastric cancer and normal controls, between patients with different stages of gastric cancer, and between gastric cancer patients before and after operation. Results The levels of 18 kinds of plasma amino acids including alanine (Ala), glycine (Gly) and glutamic acid (Glu) in gastric cancer patients were significantly lower than those in the normal controls, while the levels of valine (Val), arginine (Arg) and serine (Ser) were significantly higher than those in the normal controls (all P0.05); there were no significant differences in symmetric dimethylarginine (SDMA), kynurenine (Kyn) or hippuric acid (HA) levels between the two groups (P0.05). The plasma level of Arg in patients with III-stage of gastric cancer was significantly higher than that in the-Ⅱstage, while the plasma glutamine (Gln), Glu, methionine (Met) and phenylalanine (Phe) levels were significantly lower than those in the-Ⅱstage (all P0.05). The levels of plasma leucine (Leu), Arg and citrulline (Cit) in patients after operation were significantly lower than those before operation, while the plasma Gln, lysine (Lys), Glu and Phe levels were significantly higher than those before operation (all P0.05). Conclusion Amino acids metabolites in plasma of patients with gastric cancer such as Gln and Arg play important roles in the early prediction of gastric cancer.

Three kinds of mesenchymal stem cells:differentiating into neuron-like cells / 中国组织工程研究

BACKGROUND:Stem cells,characterized with convenient collection,wide sources and great potential,have been developed in the cell therapy.The most commonly used stem cells include bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.OBJECTIVE:To compare the biological changes in bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells after co-cultured with nerve cells.METHODS:Human bone marrow mesenchymal stem cells,human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro.The morphological changes of mesenchymal stem cells differentiated into nerve cells were observed by co-culture with Transwell culture plates.The expression of neuron-specific enolase was detected after induction.RESULTS AND CONCLUSION:In the co-culture system,these three kinds of mesenchymal stem cells gradually formed a star-like shape and were interconnected.Moreover,all the cells were positive for neuron-specific enolase.These findings reveal that the microenvironment provided by nerve cells can induce and promote the differentiation of three kinds of mesenchymal stem cells into neuron-like cells.

Correlation between HLA-DB1 genes and susceptibility to echinococcosis in Tibetan population in Tibetan Plateau / 中国血吸虫病防治杂志

Objective To determine the susceptibility genes and resistance genes in HLA-DRB1 alleles in Tibetan patients with cystic and alveolar hydatid diseases,so as to provide the references for the research of the genetic characteristics and infec-tion mechanism of Tibetan hydatid diseases. Methods The case control method was applied. The Tibetan patients with cystic and alveolar hydatid diseases(63 and 73 cases respectively)in Yushu and Guoluo Tibetan Autonomous Prefecture,and unrelat-ed healthy people(60 cases)in this area were selected as the study subjects. The polymerase chain reaction-sequence based typ-ing(PCR-SBT)technique was applied for genotyping of HLA-DRB1,and the comparison of the gene frequency. Results The frequency of HLA-DRB1*04 in the alveolar/cystic echinococcosis group was lower than that in the control group(χ2 =4.71, 4.31,both P<0.05). Conclusion HLA-DRB1*04 genotypes may be associated with the resistance of cystic and alveolar echi-nococcosis and its resistance genes.

Combined chromium and magnesium decreases insulin resistance more effectively than either alone.

BACKGROUND AND OBJECTIVES: Peroral supplementation with trivalent-chromium (Cr) or magnesium (Mg) has been shown to improve insulin resistance (IR). The objective of this study was to determine whether combined peroral supplementation with Cr and Mg improves IR more effectively than Cr or Mg alone. METHODS AND STUDY DESIGN: Subjects (n=120, age range 45-59 years old) and diagnosed with IR were randomly divided into four groups and monitored for a period of 3 months: group 1 (the placebo control group), group 2 (160 µg/d Cr), group 3 (200 mg/d Mg), and group 4 (160 µg/d Cr plus 200 mg/d Mg). Fasting blood glucose (FBG), fasting insulin (FIns), erythrocyte Cr and Mg content, and glucose-transporter-4 (GLUT4) and glycogen-synthase-kinase-3ß (GSK3ß) mRNA levels in activated T-lymphocytes were measured, and insulin resistant index (IRI) was calculated. RESULTS: Significant decreases between the baseline and study conclusion values of FBG (0.37 mmol/L, p<0.01), FIns (2.91 µIU/mL, p<0.01) and IRI (0.60, p<0.01) were observed in group 4, but not groups 1-3. Similarly, compared with baseline, significant changes in GLUT4 (2.9-fold increase, p<0.05) and GSK3ß (2.2-fold decrease, p<0.05) mRNA levels in activated T-lymphocyte were observed at the study's conclusion in group 4, but not in groups 1-3. CONCLUSIONS: Our results indicate that combining peroral supplementation with Cr and Mg improves IR more effectively than Cr or Mg alone, and this may be attributable to increased induction and repression, respectively, of GLUT4 and GSK3ß expression.

miRNA-103: molecular link between insulin resistance and nonalcoholic fatty liver disease.

AIM: To investigate the associations between miRNA-103 (miR-103) and insulin resistance and nonalcoholic fatty liver disease (NAFLD). METHODS: Serum samples were collected from 50 NAFLD patients who were overweight or obese (NAFLD group) and from 30 healthy subjects who served as controls (normal control group). Quantitative polymerase chain reaction was used to detect expression of miR-103. Fasting plasma glucose, fasting insulin, and triglyceride (TG) levels were measured. Homeostasis model assessment was used to evaluate basal insulin resistance (HOMA-IR). Patient height and weight were measured to calculate body mass index (BMI). RESULTS: Compared with the normal control group, higher serum levels of miR-103 were expressed in the NAFLD group (8.18 ± 0.73 vs 4.23 ± 0.81, P = 0.000). When P = 0.01 (bilateral), miR-103 was positively correlated with HOMA-IR (r = 0.881), TG (r = 0.774) and BMI (r = 0.878), respectively. miR-103, TG and BMI were all independent factors for HOMA-IR (ß = 0.438/0.657/0.251, P = 0.000/0.007/0.001). miR-103, TG, BMI and HOMA-IR were all risk factors for NAFLD (odds ratio = 2.411/16.196/1.574/19.11, P = 0.009/0.022/0.01/0.014). CONCLUSION: miR-103 is involved in insulin resistance and NAFLD, and may be a molecular link between insulin resistance and NAFLD and a therapeutic target for these disorders.

Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation / 生物医学工程学杂志

Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.

Progress in understanding of molecular mechanisms of pathogenesis and drug-resistance in ovarian clear cell adenocarcinoma / 肿瘤

Ovarian clear cell adenocarcinoma (OCCA) is a special epithelial ovarian cancer with distinct clinical and molecular characteristics from other subtypes. What may contribute to the pathogenesis of OCCA are specific gene mutation in AT-rich interactive domain 1A (ARID1A) and phosphatidyl inositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK 3 CA) as well as aberrant phosphatidyl inositol-3-hydroxy kinas (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathways. Within the pathways, some specifically expressed molecules, such as hepatocyte nuclear factor-1β (HNF-1β), insulin-like growth factor-binding protein 1 (IGFBP-1) and Napsin A, could be used as potential diagnostic signatures for OCCA. The underlying mechanisms of poor prognosis and resistance to conventional platinum-based chemotherapy are closely related to tumor angiogenesis, low proliferation capacity, DNA repair system and high expression of Stathmin. Developing optimized therapeutic strategies aiming at novel targets, such as mTOR, would provide new insight in the treatment of OCCA.

Selenium dioxide inducing apoptosis of cervical cancer cells by regulating apoptosis-related proteins / 重庆医学

Objective To investigate the inducing effects of selenium dioxide(SeO2 ) on the apoptosis in human cervical carcino-ma cell line Hela and its influence on the expression of apoptosis-related proteins caspase-3 and P53 .Methods Hela cells were trea-ted with different concentrations of SeO2 for 24 h in vitro ;the morphological changes of Hela cells were observed by the optical mi-croscope;the influence of SeO2 on the cell proliferation and vitality was examined by the MTT assay ;the flow cytometry was em-ployed to detect the cell apoptosis rate ;the expressions of caspase-3 and P53 proteins in Hela cells were determined by the Western blot analysis .Results Under the optical microscopy ,SeO2 generated the obvious influence on the cell growth morphology ,a large number of cells became rounded and shrunken ,and lost the normal form ,while the adherence cell number was evidently decreased and the proliferation was slowed down ;the MTT results showed that SeO2 markedly inhibited the cell proliferation and viability in a dose-dependent manner ,in which ,the cell apoptosis rates induced by the 0 ,1 .875 ,3 .750 ,7 .500 ,15 .000 and 30 .000 μmol/L con-centrations of SeO2 were 3 .12% ,30 .56% ,33 .42% ,37 .50% ,45 .43% and 69 .38% respectively ,which revealing the obviously in-creasing trend;the Western blot assay revealed that SeO2 could up-regulate the caspase-3 and P53 levels ,and reached the peak value at the concentration of 7 .500μmol/L .Conclusion SeO2 could induce the cervical cancer cell apoptosis possibly by up-regulating the expressions of caspase-3 and p53 in Hela cells .

Regulatory effects of lanthanum chloride on the activation of nuclear factor kappa B inhibitor kinase beta induced by tumor necrosis factor alpha / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).</p><p><b>METHODS</b>(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.</p><p><b>RESULTS</b>(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).</p><p><b>CONCLUSIONS</b>LaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.</p>

Construction of TK Gene-deleted PRV SH StrainContaining a Single LoxP Site / 中国生物工程杂志

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

A statin-based inhibitor of lymphocyte function antigen-1 protects against ischemia/reperfusion-induced leukocyte adhesion in the colon.

1. Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti-inflammatory effects to the statins. LFA703 is a novel statin-derived compound, which potently inhibits lymphocyte function antigen-1 (LFA-1, CD11a/CD18) but does not affect HMG-CoA reductase activity. 2. The objective of this study was to examine the anti-inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)-induced leukocyte-endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy. 3. First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA-1 and ICAM-1 abolished leukocyte adhesion, whereas inhibition of VLA-4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose-dependently reduced LFA-1-dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 microm. 4. I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA-1 significantly reduced I/R-induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R-provoked leukocyte rolling was insensitive to inhibition of LFA-1 function. 5. Administration of 30 mg kg-1 of LFA703 decreased reperfusion-induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA-1-dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA-1 on circulating leukocytes. 6. This study demonstrates that LFA-1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin-based synthetic compound has the capacity to abolish LFA-1-dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R-induced tissue injury, such as organ transplantation, trauma and major surgery.