Inhibitory effect of parvovirus H—1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability.in vitro of QGY-7703,a cultured human hepatoma cell line,and on the formation and growth of its tumors in nude mice was studied.With higher multiplicity of infection(MOI) of H-1 given,survival of the QGY-7703 cells was found to be decreased.H-1 DNA amplification level at 30h postinfection(p.i.) was detected to be 7.4 times higher than that at 2h by dispersed cells assay,while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells(new-born human kidney cell line,NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay.The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2h postinfection was observed to by prevented in 2 proups with given MOI 25 and 50.The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20d latent period.It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.     

Reversion of malignancy in human gastric cancer MKN—45 cells through the transfection of transforming growth factor—β type Ⅱ receptor gene

Human gastric cancer MKN-45 cells which are resistant to TGF-β growth inhibition and possess TGF-β type I and type Ⅲ receptors,but not type Ⅱ receptors,have been used as a model system to reconstitute these cancer cells with TGF-β RII cDNA.The results of these experiments indicated that the reexpression of TGF-β RII gene in MKN-45 cells can restore their sensitivity to TGF-β growth inhibition,decrease their growth rate,reduce their cloning efficiency in soft agar and tumorigenicity in nude mice in stable transfectants,in comparison with their control MKN-45 cells.Among different RII transfectants,their difference in the changes of these parameters,as a result of the regain of autocrine negative growth control by TGF-β,is roughly proportional to their level of expression of transfected RII mRNA.From these data,it is concluded that the inactivation of TGF-β RII gene is related to the escape of growth control by TGF-β in MKN-45 cells.The importance of the study of the interplay of TGF-β and its receptor system in the negative growth control of gastric cancer,and possibly also of other cancers,is discussed.     

Early Pattern of Epstein-Barr Virus Infection in Gastric Epithelial Cells by “Cell-in-cell”

Epstein-Barr virus(EBV)is an important human dsDNA virus,which has been shown to be associated with several malignancies including about 10%of gastric carcinomas.How EBV enters an epithelial cell has been an interesting project for investigation."Cell-in-cell"infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells.The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction.The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells(GES-1).The infection situation was observed under fluorescence and electron microscopies.Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors.The results demonstrated that EBV could get into gastric epithelial cells by"cell-in-cell"infection but not fully successful due to the host fighting.IL-1β,IL-6 and IL-8 played prominent roles in the cellular response to the infection.The activation of NF-κB and HSP70 was also required for the host antiviral response.The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electropo-rated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targetin     

Serum-free culture of fractionated bovine bronchial epithelial cells

Summary Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factorβ1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1μM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.     

Role of deficient type III interferon-lambda production in asthma exacerbations

Rhinoviruses are the major cause of asthma exacerbations, and asthmatics have increased susceptibility to rhinovirus and risk of invasive bacterial infections. Here we show deficient induction of interferon-lambdas by rhinovirus in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of rhinovirus-induced asthma exacerbation and virus load in experimentally infected human volunteers. Induction by lipopolysaccharide in asthmatic macrophages was also deficient and correlated with exacerbation severity. These results identify previously unknown mechanisms of susceptibility to infection in asthma and suggest new approaches to prevention and/or treatment of asthma exacerbations.     

Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model

Background

In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.

Methods

Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID₅₀. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.

Results

The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.

Conclusion

Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.     

Asthma, viruses, and nitric oxide

Over the last two decades there has been a worldwide increase in the morbidity and mortality associated with asthma, a chronic inflammatory disease of the airways. There is a growing body of evidence that suggests there is an association between upper respiratory viral infections, particularly rhinovirus infections, and asthma exacerbations. Virally induced airways hyperreactivity has been associated with elevated numbers of inflammatory cells in the bronchial mucosa. Upon virus infection, respiratory epithelial cells produce proinflammatory cytokines, including IL-6, IL-8, RANTES, and GM-CSF, which could contribute to the increased inflammatory cell recruitment noted in the airways. Whether or not a viral infection triggers an asthma attack may depend upon many factors, including the types of inflammatory cells recruited to the airways, the viral load, and variations in the host antiviral response. There is evidence to support the idea that eosinophils from asthmatic and symptomatic atopic subjects may be primed to respond to chemotactic cytokines produced by infected epithelial cells. Rhinovirus infections may therefore enhance airway eosinophilia in asthmatics, leading to airway hyperresponsiveness and impaired pulmonary function. Nitric oxide is a potent inhibitor of both rhinovirus-induced cytokine production and viral replication and may play an important role in the host response to viral infections. Based upon these observations, we speculate that nitric oxide donors may represent a novel therapeutic approach for the treatment of rhinovirus infections and viral exacerbations of asthma.     

Role of xanthine oxidase activation and reduced glutathione depletion in rhinovirus induction of inflammation in respiratory epithelial cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-kappaB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.     

Epidermal growth factor receptor signaling to Erk1/2 and STATs control the intensity of the epithelial inflammatory responses to rhinovirus infection

Rhinovirus infection is the most common cause of acute exacerbations of inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, where it provokes steroid refractory and abnormally intense neutrophilic inflammation that can be life threatening. Epidermal growth factor receptor (EGFR) expression correlates with disease severity and neutrophil infiltration in these conditions. However, the role of EGFR signaling in rhinovirus infection is unknown. We measured the key determinants of neutrophilic inflammation interleukin (IL)-8 and ICAM-1 in rhinovirus (RV16 serotype)-infected bronchial epithelial cells, BEAS-2B. RV16 infection stimulated IL-8 and ICAM-1 expression, which was further elevated (2-fold) by transient up-regulation of EGFR levels. Detection of viral RNA by quantitative real time PCR confirmed that enhanced expression was not associated with increased viral replication. EGFR ligands (epiregulin, amphiregulin, and heparin-binding epidermal growth factor) were induced by RV16 infection, and inhibition of metalloproteases responsible for ligand shedding partially suppressed this response. The EGFR inhibitor AG1478, completely blocked IL-8 and ICAM-1 expression to basal levels, as did the specific Erk1/2 inhibitor U0126. The p38 mitogen-activated protein kinase inhibitor SB203580 blocked IL-8 secretion but not ICAM-1 expression, whereas the PI3K inhibitor wortmannin was ineffective in both responses. Kinase inactive K721R EGFR, which is selectively deficient in STAT signaling, reversed RV16 responses associated with EGFR overexpression. In conclusion, RV16 infection rapidly promotes induction of EGFR ligands and utilizes EGFR signaling to increase IL-8 and ICAM-1 levels. These results suggest that targeting EGFR may provide a selective therapy that dampens neutrophil-driven inflammation without compromising essential antiviral pathways mediated by pathogen recognition receptors such as TLR3.