Human Cell Strains Research Articles

Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.

Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.

Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining.

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)-deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs-deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs-deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs-deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.

Resveratrol inhibits lipogenesis of 3T3-L1 and SGBS cells by inhibition of insulin signaling and mitochondrial mass increase

Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

Apoptosis mechanism of taxol combined with resveratrol on human laryngeal carcinoma Hep-2 cells

Laryngeal cancer is one of the most common malignant tumors in the respiratory tumors, and its incidence ranks second highest in the respiratory tumors. Resveratrol (Res) is a kind of polyphenols, which can inhibit nucleotides can inhibit the growth of liver cancer cells, gastric cancer cells, pancreatic cells and other tumor cells by inhibiting ribonucleotide reductase in the cells. Taxol (Tax) is a kind of secondary metabolites of Taxus chinensis, which has anti-tumor activity for breast cancer, cervical cancer, ovarian cancer and other tumors by inhibiting cellular microtubule depolymerization. But at present the effects of resveratrol combined with taxol on human laryngeal carcinoma cell strain Hep-2 and their underlying molecular mechanisms are rarely reported. After human laryngeal cancer cell Hep-2 cells were processed with resveratrol (Res) and taxol (Tax), CCK-8 assay was used to evaluate the effect of these two herbs on the proliferation of cancer cells; AO/PI staining and JC-1 were used to detect Hep-1 cells apoptosis; the expression of Bax, Bcl-2, PARP, TRIB3, and XIAP genes was detected by real time quantitative PCR; the activity of caspase-3 and caspase-8 was determined with quantitative fluorescence method. The experimental results showed that compared with Tax, Res medication alone, joint group significantly enhanced inhibition of Hep-2 cells activity, decreased the dosage of Tax, increased the expression of Bax and PARP, TRIB3, reduced the expression of the Bcl-2 and XIAP, and promoted the activity of caspase-3 and caspase-8. The test results showed that compared with the single medication, combined group could significantly increase the inhibitory effect on Hep-2 cells, significantly reduce Tax dosage, increase expressions of Bax, PARP, TRIB3, reduce expressions of Bcl-2, XIAP, and promote activity of caspase-3, caspase-8. This indicated apoptosis of human laryngeal carcinoma cell strain Hep-2 may be induced with Res, Tax, and the combination of these two herbs by mitochondria pathway. It provides valuable clue for further research on combination of Res and Tax for the treatment of laryngeal cancer, and expanding the combined application of Res and Tax.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Pigeon RIG-I Function in Innate Immunity against H9N2 IAV and IBDV.

Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and mice) and waterfowl RIG-I (ducks and geese) are essential for type I interferon (IFN) synthesis during AIV infection. Like ducks, pigeons are also susceptible to infection but are ineffective propagators and disseminators of AIVs, i.e., “dead end” hosts for AIVs and even highly pathogenic avian influenza (HPAI). Consequently, we sought to identify pigeon RIG-I and investigate its roles in the detection of A/Chicken/Shandong/ZB/2007 (H9N2) (ZB07), Gansu/Tianshui (IBDV TS) and Beijing/CJ/1980 (IBDV CJ-801) strains in chicken DF-1 fibroblasts or human 293T cells. Pigeon mRNA encoding the putative pigeon RIG-I analogs was identified. The exogenous expression of enhanced green fluorescence protein (EGFP)-tagged pigeon RIG-I and caspase activation and recruitment domains (CARDs), strongly induced antiviral gene (IFN-β, Mx, and PKR) mRNA synthesis, decreased viral gene (M gene and VP2) mRNA expression, and reduced the viral titers of ZB07 and IBDV TS/CJ-801 virus strains in chicken DF-1 cells, but not in 293T cells. We also compared the antiviral abilities of RIG-I proteins from waterfowl (duck and goose) and pigeon. Our data indicated that waterfowl RIG-I are more effective in the induction of antiviral genes and the repression of ZB07 and IBDV TS/CJ-801 strain replication than pigeon RIG-I. Furthermore, chicken melanoma differentiation associated gene 5(MDA5)/ mitochondrial antiviral signaling (MAVS) silencing combined with RIG-I transfection suggested that pigeon RIG-I can restore the antiviral response in MDA5-silenced DF-1 cells but not in MAVS-silenced DF-1 cells. In conclusion, these results demonstrated that pigeon RIG-I and CARDs have a strong antiviral ability against AIV H9N2 and IBDV in chicken DF-1 cells but not in human 293T cells.

The c-Jun N-terminal kinase inhibitor SP600125 inhibits human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that causes severe diseases in congenitally infected newborns and immunocompromised patients. Currently, no vaccine is available to prevent HCMV infection. Anti-viral drugs are limited by their side effects and drug resistance. In this study, by performing a medium-sized, anti-HCMV chemical screening, we identified SP600125, CC-401, and the c-Jun N-terminal kinase (JNK) inhibitor VIII, three structurally different small molecule JNK inhibitors that effectively inhibited HCMV replication in cultured human fibroblasts (HFs). SP600125 showed its potential by inhibiting the viral replication of a HCMV laboratory strain in HFs and a HCMV clinical strain in human retinal pigment epithelial cells. Knockdown of JNK expression by RNA interference significantly impaired HCMV replication, mimicking the effect of the chemical inhibitors on virus infection. Mechanistically, SP600125 affects a very early step of the viral life cycle. Viral binding, entry, and the delivery of viral DNA into the cells were not inhibited by the compound. Instead, it suppressed the transcription of the immediate-early viral genes IE1/2 and the accumulation of their gene products. IE1/2 are among the first genes expressed after viral entry, and they are the master regulators of late phase viral gene expression. Consistent with this notion, the expression of other viral genes was also reduced after SP600125 treatment. We propose that JNK inhibitors have the potential to become a new class of anti-HCMV drug candidates, and JNK is a feasible target for the development of anti-HCMV drugs.

Прояв факторів патогенності у біоплівкоутворюючих та неформуючих біоплівку штамів Staphylococcus epidermidis

A comparative study of the manifestation of pathogenicity factors: hemolytic, lipase, letsytinase activity and ability to adhere in 20 film-forming and 17 non-film-forming strains of S. epidermidis. Studying pathogenicity factors of the film-forming strains it was found that complete hemolysis and lipase activity shown was by all the film-forming strains of S. epidermidis, letsytinase activity was observed in 80%. Among the non-film-forming strains complete hemolysis and lipase activity were observed in 89% and letsytinase - 71%. Researched non-film-forming and film-forming strains of S. epidermidis showed the ability to adhere to buccal epithelial cells of humans. Found that all the film-forming strains of S. epidermidis were hight level adgesion, the highest IAM was equal to 11,84. It was found that among non-film-forming strains of S. epidermidis were low-, medium- and hight level adgesion. IAM of non-film-forming strains of S. epidermidis is 3 times lower compared to the IAM of the film-forming strains of human epithelial cells and was 3.2.

Effects of HMGB1 on proliferation and apoptosis of human brain glioma CD133 cells.

To understand the effects of HMGB1 recombinant lentivirus vector on proliferation and apoptosis on CD133 cells after transfecting a human glioma cell strain CD133. The CD133 human glioma cell strain cultured in vitro was transfected with HMGB1 lentiviral vector and empty vector, respectively, while CD133 cells cultured normally were used as the blank control. Changes in HMGB1 protein level were detected with western blot hybridization; apoptosis was detected with a flow cytometer; the cell proliferation was analysed with MTT method. The results from the western blot hybridization indicated that the expression level of HMGB1 protein in the group infected with HMGB1 recombinant over-expressed lentiviral vector increased significantly (p<0.05) compared with the blank control and CD133 cells transfected with the empty vector. The absorbance value of cell proliferation in the group infected with HMGB1 over-expressed lentiviral vector increased gradually on days 1, 2, 3, 4 and 5, but the difference was significant on day 2 after infection and later compared with the blank control group and negative control group (p<0.05). Based on detection with a flow cytometer, the apoptosis rate of CD133 cells transfected with HMGB1 over-expressed viral vector was significantly higher than that in the blank control group and negative control group (p<0.05). The lentiviral expression vector has very high transfection efficiency in human glioma cell line CD133 and the infection is able to significantly up-regulate the expression and activation of HMGB1. Over-expression of HMGB1 is able to inhibit proliferation of tumor cells and promote apoptosis (Tab. 2, Fig. 6, Ref. 30).

Stable cellular senescence is associated with persistent DDR activation.

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.

EZH2 elevates the proliferation of human cholangiocarcinoma cells through the downregulation of RUNX3.

To investigate the impact of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on the proliferation and apoptosis of human cholangiocarcinoma cells as well as its related mechanisms. Immunohistochemistry and Western blot analyses were used to examine the expression of EZH2 in 40 cases of human cholangiocarcinoma tissues and four strains of human cholangiocarcinoma cells. The influence of EZH2 on cell growth and apoptosis were assessed by knockdown experiments, and a xenograft experiment in nude mice was performed to evaluate the impact of siEZH2 on the tumorigenicity of tumor cells. The correlation of EZH2, clinic pathological features and overall survival rates was also analyzed. EZH2 was highly expressed in human cholangiocarcinoma tissues and cells. Silencing of EZH2 could significantly reduce the methylation level of RUNX3 DNA in human cholangiocarcinoma cells and improve its protein expression as well as inhibit cell proliferation, induce apoptosis and slow down the growth of tumor in nude mice. In addition, the expression of EZH2 was associated with the tumor stage, lymph node positivity and poor prognoses. Overexpression of EZH2 can promote the proliferation of cholangiocarcinoma cells and inhibit their apoptosis. It is associated with poor prognoses in patients with cholangiocarcinoma. Therefore, EZH2 could be a potential clinical therapeutic target for the treatment of cholangiocarcinoma.

Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene.

quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58-836 for CYP1A1, 336-5587 for CYP1B1 and 5943-40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251-13234 for CYP1A1, 4133-57078 for CYP1B1 and 4456-55887 for NQO1. There were 3.5 (mean, range 0.2-15.8) BPdG adducts/10(8) nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Propagation of rhinovirus-C strains in human airway epithelial cells differentiated at air-liquid interface.

Rhinovirus-C (RV-C) were discovered recently using molecular methods. Classical methods failed to detect them since they could not grow in standard cell culture. The complete genome sequences of several RV-C strains are now available but there is little information about their biological characteristics. HRV-C were first grown in organ culture, and more recently, we developed a system for culturing RV-C strains in differentiated epithelial cells of human airway at air-liquid interface (ALI). These cultures supported efficient replication of RV-C strains as determined by quantitative RT-PCR. This system has enabled study of the biological characteristics of RV-C strains, including quantitative research.

Resolvin D1 inhibits multiple pro-inflammatory signaling pathways in primary human lung cells exposed to diverse inflammatory stimuli (IRM9P.721)

Abstract Resolution of inflammation is a bioactive process involving a new genus of lipid molecules called “specialized pro-resolving lipid mediators” (SPMs), mainly derived from dietary ω-3 polyunsaturated fatty acids, which actively antagonize inflammatory signaling pathways and promote resolution. Although it is broadly known that SPMs inhibit inflammation, the specific pathways and targets of many SPMs remained to be discovered. Here, we investigated the mechanism by which the SPM resolvin D1 (RvD1) inhibits pro-inflammatory signaling pathways using two distinct stimuli; IL-1β, which acts primarily via its own receptor, and poly(I:C), a double-stranded viral RNA mimetic that acts primarily through toll-like receptor (TLR)3. RvD1 inhibited production of pro-inflammatory mediators including IL-6 and IL-8, both primary human lung fibroblast and epithelial cell strains, when stimulated with either IL-1β or poly(I:C). RvD1 also blocked phosphorylation of ERK1/2 and translocation of NF-κB p65 in cells treated with both stimuli. Importantly, we found that RvD1 inhibited phosphorylation of TAK1, an upstream effector protein that regulates both the MAP-kinase and NF-κB pathways. These results demonstrate that RvD1 targets multiple pro-inflammatory signaling pathways and may have significant therapeutic potential in complex inflammatory diseases including pulmonary fibrosis, asthma and chronic obstructive pulmonary disease.

Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence

Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation.

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.

Effects of mesenchymal stem cells from human gastric cancer on proliferation and invasion of human gastric cancer cell strain SGC-7901

Objective To investigate effects of the mesenchymal stem cells isolated and cultured from human gastric cancer (hGC-MSCs) on proliferation and invasion of human gastric cancer cell strain SGC-7901.Methods The condition medium of hGC-MSCs (MSC-CM) was isolated,MSC-CM was applied on SGC-7901 cells at different concentrations (20％,40％ and 60％),the changes of proliferation and invasion were tested by methylthiazol tetrazolium (MTT) and Transwell chambers respectively; the expressions of proliferating cell nuclear antigen (PCNA) and matrix metalloproteinases 9 (MMP-9) were detected by Real-time polymerase chain reaction (PCR),Western blotting and Immunocytochemistry.Results MTT results showed that the proliferation of SGC-7901 was significantly inhibited by MSC-CM and the inhibition rate was positively correlated with MSC-CM concentration and reaction time (P ＜ 0.05).The transwell chambers results revealed that cell invasion was significantly decreased after cells were dealed with the MSC-CM,penetrating cells in the control group and the MSC-CM groups at different concentrations (20％,40％,60％) were 75.71 ± 7.32,63.68 ± 7.12,54.29 ± 6.97 and 41.36 ± 3.29 (P ＜ 0.05),respectively.Real-time PCR results showed that MSC-CM could down-regulate the expression of PCNA and MMP-9 (P ＜ 0.01).Western blot revealed the expression of PCNA in MSC-CM groups at different concentrations (20％,40％,60％) were down-regulated to (73.99 ± 16.71) ％,(51.56 ± 12.18) ％ and (40.11 ±9.37)％,MMP-9 (74.35 ±19.12)％,(3.59 ±10.41)％ and (29.58 ±7.93)％,Immunocytochemistry revealed the average absorbance of PCNA in MSC-CM groups at different concentrations (20％,40％,60％) were 0.201 ±0.049,0.185 ± 0.031,0.154 ± 0.019 and 0.128 ± 0.012,MMP-9 were 0.182 ± 0.032,0.164 ± 0.028,0.136 ± 0.017 and 0.098 ± 0.008 (all P ＜ 0.05).Conclusion Proliferation and invasion of SGC-7901 were inhibited by hGC-MSCs and the mechanism may be related to the reduction of PCNA and MMP-9 expression. Key words: Gastric cancer; Mesenchymal stem cells; Proliferating cell nuclear antigen; Matrix metalloproteinases

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Influenza viruses are global pathogens that infect approximately 10–20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.