Fetal Lung Cells Research Articles

<i>Stachybotrys chartarum</i> (<i>atra</i>) spore extract alters surfactant protein expression and surfactant function in isolated fetal rat lung epithelial cells, fibroblasts and human A549 cells

Moulds, notably Stachybotrys chartarum (atra), are constant contributors to air pollution particularly to air quality in buildings. The spores themselves or their volatile organic products are present in variable amounts in almost all environments, particularly in buildings affected by flooding. These moulds and products can account for the sick building syndrome and have been tied to such occurrences as the outbreak of pulmonary hemosiderosis and hemorrhage in infants in Cleveland, Ohio. The present study was designed to investigate the effects of S. chartarum extracts on surfactant protein expression, surfactant quality and cell survival in the developing lung. S. chartarum extracts were incubated with cultures of several cell types; isolated fetal lung type II cells and fetal lung fibroblasts, and human lung A549 cells, a continuously growing cell line derived from surfactant producing type II alveolar cells. MTT formazan assays were employed to test cell viability. The synthesis and release of the predominant surfactant protein A (SP-A), which is involved in the regulation of surfactant turnover and metabolism, and surfactant protein B (SP-B) involved in shuttling phospholipids between surfactant subcompartments was also assessed. Antibodies to these proteins and western blotting results were used to assess the quantity of protein produced by the various cell types. A novel approach utilizing captive bubble surfactometry was employed to investigate the quality of surfactant in terms of surface tension and bubble volume measurements. Electron microscopy was used to examine changes in cellular structure of control and S. chartarum-treated cells. Results of the study showed that exposure to the S. chartarum extracts had deleterious effects on fetal lung epithelial cell viability and their ability to produce pulmonary surfactant. S. chartarum extracts also induced deleterious changes to the developing fetal lung cells in terms of expression of SP-A and SP-B as well as to the surface tension reducing abilities of the pulmonary surfactant. Ultrastructurally, spore toxin associated changes were apparent in the isolated lung cells most notably in the lamellar bodies of fetal rat lung alveolar type II and human A549 cells. This study has demonstrated the potential damage to surfactant production and function which may be induced by inhaling S. chartarum toxins.

First molecular characterization of visna/maedi viruses from naturally infected sheep in Turkey

Recent worldwide serological and genetic studies of small ruminant lentiviruses (SRLV) have led to the description of new genotypes and the development of new diagnostic tests. This study investigated the detection and molecular characterization of visna/maedi virus (VMV) infection in serum and blood samples from pure and mixed sheep breeds acquired from different regions in Turkey using ELISA and PCR techniques. The prevalence of VMV was 67.8 % by ELISA and/or LTR-PCR with both assays showing a medium level of agreement (kappa: 0.26; ± 0.038 CI). Positivity of VMV in sheep increased according to the age of the animal, although PCR positivity was higher than ELISA in young individuals. Phylogenetic analysis of 33 LTR sequences identified two distinct clades that were closely related to American and Greek LTR sequences. Phylogenetic analysis of 10 partial gag gene sequences identified A2, A3, A5, A9, A11 subtypes of genotype A SRLVs. In vitro culture of all isolates in fetal sheep lung cells (FSLC) showed a slow/low phenotype causing less or no lytic infection compared with infection with the WLC-1 American strain characterized by a rapid/highly lytic phenotype. Phylogenetic analysis revealed that Turkish VMV sequences preceded the establishment of American or Greek strains that were associated with the migration of sheep from the Middle East to Western Europe several centuries ago. This is the first study that describes Turkish VMV sequences with the molecular characterization of LTR and gag genes, and it strongly suggests that SRLV-genotype A originated in Turkey.

Pulmonary Epithelial Apoptosis in Fetal down Syndrome: Not Higher than Normal

Children with Down syndrome (DS) are at high risk for acute lung injury (ALI). Pulmonary epithelial apoptosis is an important factor in the pathophysiology of ALI. Whether the risk of ALI in DS is associated with a high level of pulmonary epithelial apoptosis is not known. We hypothesized that the percentage of apoptotic epithelial cells is higher in DS than in control lungs. Lung tissue sections from autopsies of 21 fetuses with DS and 12 controls were stained with antibodies against the epithelial marker pan-cytokeratin (CK) and apoptosis marker activated caspase-3 (aC3). Spectral imaging software was used to quantify the mean percentage of pixels that showed colocalization of CK and aC3. Mean (standard deviation [SD]) gestational age in weeks was 18.7 (1.4) in DS and 18.9 (2.0) in controls (P = 0.67). The mean (SD) percentage of CK-positive pixels was 27.2% (4.7%) in DS compared to 27.1% (6.2%) in controls (P = 0.97). The median (interquartile range [IQR]) percentage of CK-positive pixels that showed colocalization of aC3 was 0.16% (0.18%) in DS compared to 0.27% (0.24%) in controls (P = 0.45). The mean (SD) number of CK-positive pixels increased from 22.5% (5.2%) to 30.4% (4.6%) with the appearance of saccular morphology in controls but not in DS (P = 0.01). The percentage of apoptotic epithelial cells in DS fetal lungs does not differ from that in controls. However, we did find a difference in the development of epithelial structures between DS and controls that may be associated with anomalies in alveolar development found at birth in DS.

Abstract 429: The high mobility group A1 oncogene enhances cellular reprogramming to a pluripotent stem-like cell

Abstract The molecular mechanisms that enable cancer cells to metastasize are poorly understood, although emerging evidence indicates that transcriptional networks required for stem cell properties during embryogenesis are co-opted by cancer cells during metastatic progression. To elucidate the molecular underpinnings of “stemness” in cancer and normal development, we investigated transcriptional networks and epigenetic alterations during the induction of pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes proteins that bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. Our group first discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive tumors in transgenic mice. Recent studies also identified HMGA1 as a key transcription factor enriched in human embryonic stem (hESCs) cells, induced pluripotent stem cells (iPSCs), refractory leukemia, and high-grade/poorly differentiated cancers arising from diverse tissues. Together, these findings are consistent with the hypothesis that HMGA1 drives an undifferentiated, stem cell-like state during malignant transformation and normal development. To further investigate the role of HMGA1 in the stem cell state, we assessed its function in the derivation of iPSCs using multiple approaches. Here, we demonstrate for the first time that HMGA1 significantly enhances the reprogramming of somatic cells (bone marrow-derived mesenchymal stem cells, fetal lung cells, or mononuclear blood cells) into iPSCs together with OCT4, SOX2, KLF4, and cMYC (OSKM) using a retroviral or episomal, non-integrating approach. When hESCs are induced to differentiate, HMGA1 expression falls and parallels that of other pluripotency factors, such as OCT4, NANOG, and SOX2. We also found that forced expression of HMGA1 blocks differentiation of hESCs. To determine how HMGA1 induces a stem cell state, we assessed gene expression and epigenetic changes. During the reprogramming process, HMGA1 induces the expression of pluripotency and cancer genes, including SOX2, LIN28, and cMYC, while in hESCs, knock-down of HMGA1 results in repression of these genes. In addition, NANOG and OCT4 are repressed in hESCs following knock-down of HMGA1. By chromatin immunoprecipitation, HMGA1 binds to the promoters of SOX2, LIN28, and cMYC in vivo in hESCs. Moreover, HMGA1 is associated with decreased promoter methylation of select stem cell genes early in reprogramming. These findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks and epigenetic remodeling that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in poorly differentiated, stem-like cancers or exploited in regenerative medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 429. doi:1538-7445.AM2012-429

Abstract 4699: A siRNA high-throughput screening identified Wee1 as determinant of Chk1 inhibitor sensitivity

Abstract Targeting Chk1 protein kinase is a valid anti cancer strategy to enhance the cytotoxic effects of radio and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. The effect of Chk1 inhibition on a wide panel of cancer cell lines has been herein conducted by using a specific and selective Chk1 inhibitor PF-00477736. The results showed that 2 cancer cell lines out of 16 analyzed (the ovarian cancer cell line OVCA-432 and the breast cancer cell line SK-BR3) were very sensitive to PF-00477736 treatment. To identify genes in synthetic lethality with Chk1, which may justify the sensitivity to Chk1 inhibitor of some cancer cell lines, a high-throughput screening using a siRNA library directed against 719 human protein kinases, in combination with either scramble or Chk1 siRNA, was undertaken. The ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibition, was used to perform the screening. After 2 independent screenings and the validation procedure using specific esiRNAs, and the three siRNAs of each target (used in the screening as a pool), the Wee1 tyrosine kinase was identified as the most significant gene in synthetic lethality with Chk1. Treatment with both PF-00477736 and a Wee1 inhibitor (MK-1775) confirmed the remarkable synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate). Same results have been obtained with AZD-7762 Chk1 inhibitor. Isogenic cellular systems differing only for the status of p53 (the colon carcinoma cancer cell lines HCT-116 p53 +/+ and p53 -/- and the ovarian cancer cell lines A2780 and A2780/E6) showed a comparable and strong synergistic effect of the combined treatment. Interestingly, in the normal fetal human lung cell line MRC-5 the effect of the combined treatment was additive. Time lapse experiments and an evaluation of the ability of cells to form colonies, suggested that cells treated with the two inhibitors almost completely lost the ability to proliferate and failed to perform a proper cell division. A detailed molecular analysis showed that the combined treatment causes cells to undergo premature mitosis before the end of DNA replication and with damaged DNA, leading to a massive cell death by apoptosis. Taken together these preclinical data address a new promising combined targeted anticancer strategy that deserves clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699. doi:1538-7445.AM2012-4699

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Bovine Herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ∼80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ∼50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.

Antibacterial activity, cytotoxicity and chemical constituents of Hydnora johannis roots

In Sudan, the roots of Hydnora johannis (Hydnoraceae) are traditionally used for the treatment of dysentery, diarrhea, cholera and swelling tonsillitis. The ethnomedicinal value of H. johannis was investigated through phytochemical study, in vitro antibacterial activity and preliminary cytotoxic tests. Determination of total phenols, flavonoids and proanthocyanidins was carried out using spectrometric methods. The antibacterial activity of the water and ethanolic extracts was determined using the microdilution method. Pure compounds were isolated from the ethyl acetate extract by chromatographic methods and their structures were established by spectroscopic methods. Cytotoxicity assay was performed against selected human mouth epidermoid carcinoma cell line (KB), and non-cancer human fetal lung cell line (MRC-5). Both water and ethanol (70%) extracts were found to contain the same amount of total phenols and proanthocyanidins, whereas the level of flavonoids was higher in the ethanol extract. The water extract was found to possess antibacterial activity against Enterococcus faecalis (MIC value of 16 μg/mL), Bacillus subtilis, B. cereus and Staphylococcus aureus (MIC values of 64 μg/mL) but not against bacteria mainly responsible for diarrhea. This leads to the suggestion that, the mode of action of water extract which is rich in tannins was not connected to their inhibition to the diarrhea bacteria but to their action on the digestive tract. Reduction in potency of the water and ethanol (70%) extracts when fractionated was observed. The ethyl acetate fraction obtained from fractionation of ethanol extract possessed only activity against the two strains of S. aureus with MIC values of 128 μg/mL. In addition, six compounds were isolated from the ethyl acetate extract as cirsiliol (3′,4′,5-trihydroxy-6-7-dimethoxy flavone) ( 1), trans 3′5-dihydroxy-4′7-dimethoxydihydroflavonol ( 2), oleic acid ( 3), vanillin (4-hydroxy-3-methoxybenzaldehyde) ( 4), protocatechuic acid (3,4 dihydroxy benzoic acid) ( 5) and dl catechin (trans (+) 2-(3,4-dihydroxyphenyl-3,4-dihydro-2H-1-benzopyran-3,5,7-triol)) ( 6). Four compounds from the ethyl acetate extract were also identified by GCMS as stigmasterol ( 7), oleic acid ( 3), myristic acid ( 8), and palmitic acid ( 9). Little cytotoxicity is reported against the cell lines used. Thus, the safety of this plant in the traditional medicine should be verified by much further testing, including in vivo experiments and clinical studies.

Endothelial Differentiation by Multipotent Fetal Mouse Lung Mesenchymal Cells

During fetal lung development, cells within the mesenchyme differentiate into vascular endothelia. This process of vasculogenesis gives rise to the cells that will eventually form the alveolar capillary bed. The cellular mechanisms regulating lung vasculogenesis are poorly understood, partly due to the lack of experimental systems that model this process. Here, we have developed and characterized a novel fetal mouse lung cell model of mesenchymal to endothelial differentiation. Using mesenchymal cells from the lungs of embryonal day 15 Immortomice, we show that endothelial growth media containing fibroblast growth factor-2 and vascular endothelial growth factor can stimulate formation of vascular endothelial cells in culture. These newly formed endothelial cells retain plasticity, as removing endothelial growth media causes loss of vascular markers and renewed formation of α-smooth muscle actin positive stress fibers. Cells with the highest Flk-1 expression differentiated into endothelia more efficiently. Individual mesenchymal cell clones had varied ability to acquire an endothelial phenotype. These fetal lung mesenchymal cells were multipotent, capable of differentiating into not only vascular endothelia, but also osteogenic and chondrongenic cell lineages. Our data establish a cell culture model for mesenchymal to endothelial differentiation that could prove useful for future mechanistic studies in the process of vasculogenesis both during normal development and in the pathogenesis of pulmonary vascular disease.

Enhanced In Vivo Function of Bioartificial Lungs in Rats

More than 11 million Americans live with chronic lung disease; in search for an alternative to donor organs, we attempted to regenerate lungs based on perfusion decellularized lung scaffolds that can be transplanted similar to a donor organ. Cadaveric rat lungs were decellularized by detergent perfusion. Resulting scaffolds were mounted in bioreactors and seeded with endothelial and fetal lung cells. Biomimetic organ culture was maintained for 7 days. Resulting bioartificial left lungs were transplanted in orthotopic position after left pneumonectomy in rats. Cadaveric left lung transplants and pneumonectomies served as controls. Blood gas analyses, compliance testing, and fluoroscopies were performed on postoperative days 1, 7, and 14. Lungs were removed for final analysis on day 14. Perfusion decellularization of cadaveric lungs yielded acellular scaffolds with intact architecture and matrix composition. Alveolar volumes, number, and size were comparable in bioartificial and native lungs, as were gas exchange, vital capacity and compliance in vitro. After using improved graft preservation and postoperative weaning protocols, animals could be fully recovered, and bioartificial lung constructs provided oxygenation as long as 7 days at levels comparable to cadaveric lung transplants. Compliance, gas exchange, and radiographic appearance gradually declined over the subsequent 7 days owing to progressive graft consolidation and inflammation. Perfusion decellularization of cadaveric lungs yields intact scaffolds that can be seeded with cells to generate bioartificial lung grafts. After orthotopic transplantation, grafts are perfused by the recipient's circulation, ventilated through the recipient's airway and provide gas exchange in vivo for 7 days.

Differential Expression of MMP-2 and -9 and their Inhibitors in Fetal Lung Cells Exposed to Mechanical Stretch: Regulation by IL-10

Abnormal remodeling of the extracellular matrix (ECM) has been implicated in the pathogenesis of bronchopulmonary dysplasia. However, the contribution of lung parenchymal cells to ECM remodeling after mechanical injury is not well defined. The objective of these studies was to investigate in vitro the release of MMP-2 and -9 and their respective inhibitors TIMP-2 and -1, and to explore potential regulation by IL-10. Mouse fetal epithelial cells and fibroblasts isolated on E18-19 of gestation were exposed to 20% cyclic stretch to simulate lung injury. MMP-2 and MMP-9 activity were investigated by zymography and ELISA. TIMP-1 and TIMP-2 abundance were analyzed by Western blot. We found that mechanical stretch increased MMP-2 and decreased TIMP-2 in fibroblasts, indicating that excessive stretch promotes MMP-2 activation, expressed as the MMP-2/TIMP-2 ratio. Incubation with IL-10 did not change MMP-2 activity. In contrast, mechanical stretch of epithelial cells decreased MMP-9 activity and the MMP-9/TIMP-1 ratio by 60-70%. When IL-10 was added, mechanical stretch increased the MMP-9/TIMP-1 ratio by 50%. We conclude that mechanical stretch differentially affects MMP-2/9 and their inhibitors in fetal lung cells. IL-10 modulates MMP-9 activity through a combination of effects on MMP-9 and TIMP-1 levels.

Bovine Immunodeficiency Virus as a potentiating cofactor for the experimental Bovine Leukemia Virus infection in sheep

Coinfection with bovine retroviruses Bovine Leukemia Virus (BLV) and Bovine Immunodeficiency Virus (BIV) is currently noted in the naturally infected cattle. The effect of BIV as a cofactor for the BLV infectivity is still unknown. In our study we examined the impact of BIV on the BLV in experimentally infected sheep. Four sheep were inoculated with Foetal Bovine Lung cells (FBL) infected with the R29 isolate of BIV and the other four, mock-infected, received uninfected FBL cells. Two weeks later all animals were inoculated intravenously with 5x105 of PBMC of cow persistently infected with BLV. Blood samples were collected every 4 week up to 40th week after inoculation (a.i.). From 4th up to 40th week a.i. antibody titer to BLV in dually infected sheep were almost 1.0 log10 higher than those in BLV group. The BLV infectivity, quantified by BLV-induced syncytia, revealed that the numbers of syncytia in BLV/BIV inoculated animals were higher then those in sheep inoculated with BLV alone. BLV proviral load, determined by a qPCR, increased in the doubly infected animals from 16th to 28th and between 36th and 40th week a.i. Finally semiquantitative RT-PCR disclosed significant increment of BLV tax mRNA at 36th and 40th weeks a.i. in BIV/BLV inoculated sheep. In conclusion these findings confirmed that BIV infection enhance expression of BLV infection in experimentally inoculated sheep.

The role of transforming growth factors beta1 and beta3 in pre- and post-natal pulmonary surfactant development

The aim was to explore the pulmonary surfactant regulatory effect of TGF-β1 and TGF-β3 during pre- and post-natal porcine development. Pigs on embryonic day 99 (E94) (term = 114 days) and 1-h (D0) and 15-day (D20) neonates were killed to obtain whole lungs. DSPC (disaturated phosphatidylcholine) was separated from other phospholipids, and chemical methods were used to determine the amounts of DSPC, TPL (total phospholipids) and TP (total protein) in BALF (bronchoalveolar lavage fluid). TPL was elevated at E94. DSPC in TPL was significantly higher in the D20 group than in the E94 group. Reductions in TP correlated with developmental age. The levels of TGF-β1 and TGF-β3 mRNA were determined by RT (reverse transcription)-PCR and Northern blot. The expression of TGF-β1 mRNA was low at E94, increased at D0 and then decreased at D20. The expression of TGF-β3 was high at E94, reduced at D0, and then elevated at D20. We further examined the effect of exogenously administered TGF-1 on the expression of SPs (surfactant proteins) and cytidine triphosphorylate: CCT (phosphocholine cytidylyltransferase) activity in porcine fetal lung cells cultured for 5 days. The results indicated that TGF-β1 inhibited the expression of all three SPs (SP-A, SP-B and SP-C) and CCT activity, but did not alter the expression level of SP-D transcripts. We conclude that TGF-1 inhibits the expression of surfactant components. The alterations of TGF-β3 seem to partly explain the pulmonary surfactant changes observed in development, but this result needs further investigation.

Bovine ISG15: an antiviral and inducible protein in BIV infected fetal bovine lung cells

Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.

The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung

Pulmonary neuroendocrine cells (PNECs) are the first cell type to differentiate within the primitive airway epithelium, suggesting a possible role in lung development. The differentiation of PNECs in fetal lung is governed by proneural genes such as the mammalian homolog of the achaete–scute complex (Mash-1) and a related transcription factor, hairy and enhancer of split1 (Hes-1). We examined the expression of Mash-1 and a downstream transcription factor Prox-1 in the developing mouse lung of wild-type and respective knockout mouse models. During early stages (embryonic day 12, E12) of development, only some PNECs expressed Mash-1 and Prox-1, but by E15, all PNECs coexpressed both transcription factors. PNECs failed to develop in Mash-1 but not in Prox-1-null mice, indicating that Mash-1 is essential for the initiation of the PNEC phenotype, whereas Prox-1 is associated with the development of this phenotype. As lung develops within a low O2 environment (fetal euoxia, pO2 ∼20 to 30 mm Hg), we examined the effects of hypoxia on PNEC differentiation. Organ cultures of fetal mouse lungs at E12 and E16 were maintained under either 20% O2 (normoxia, Nox) or 5% O2 (hypoxia, Hox) and were examined every 24 h for up to 6 days in culture. In E12 explants, Hox enhanced branching morphogenesis and increased cell proliferation, but PNEC numbers and Mash-1 expression were significantly reduced. This effect could be reversed by switching the explants back to Nox. In contrast, Hox had no apparent effect on Hes-1 expression. Similarly, Hox had no effect on airway branching, PNEC numbers, or Mash-1 expression in E16 explants, indicating locked-in developmental programming. We suggest that during early stages of lung development, pO2 concentration in concert with neurogenic gene expression modulates PNEC phenotype. Thus, disturbances in intrauterine pO2 homeostasis could alter the functional maturation of the PNEC system and hence be involved in the pathogenesis of various perinatal pulmonary disorders.

Genome Wide Profiling of USF1 and USF2 Occupancy in Human Primary Erythroid Cells.

Abstract 1456Poster Board I-479USF1 and USF2 are ubiquitously expressed basic helix-loop-helix leucine zipper proteins that participate in a large number of biologic processes. USF1 and USF2 bind DNA as homodimers or heterodimers, typically binding E box consensus motifs. One role of USF proteins is functioning as transcription factors. Although ubiquitously expressed, they regulate expression of many cell-type and developmental-stage specific genes, such as hepcidin in hepatocytes and surfactant protein A in fetal lung cells. Another role of USF proteins is in the maintenance of chromatin architecture in barrier insulator elements, such as the well characterized 5'HS4 insulator element in the chicken beta-globin locus. In mammalian erythroid cells, USF1 and USF2 participate in the regulation of beta-globin transcription, interacting both at hypersensitive site 2 (HS2) of the beta-globin locus control region (LCR) and at the beta-globin promoter. Depletion of USF proteins leads to decreased beta-globin production. We hypothesize that in addition to beta-globin, USF proteins are important for regulation of many erythroid expressed genes. To address this hypothesis, chromatin immunoprecipitation with antibodies against USF1 and USF2 was coupled with ultra high throughput, massively parallel sequencing (Illumina Solexa sequencing, ChIP-seq) to generate a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells. To generate cells for ChIP and mRNA expression profiling, human CD34+ cells isolated from peripheral blood were cultured in serum free media with erythropoietin to induce erythroid differentiation. After 14 days in culture, FACS analysis was used to confirm cells were positive for both CD 71 and glycophorin A (the R3/R4 stage of erythroid development). mRNA transcript analyses were performed using Illumina human V6-2 expression arrays and quantitative real time RT-PCR. ChIP-seq experiments for USF1 and USF2 were done in duplicate and only binding sites present in both ChIP-seq replicates were included in data analyses. A total of 20450 USF1 and 21128 USF2 sites of occupancy were identified. Co-localization of USF1 and USF2 was common, with 16739 sites binding both USF1 and USF2 (81.9% of USF1 sites and 79.2% of USF2 sites). In an analysis of a subset of erythroid expressed focus genes, USF binding was associated with active transcription. In agreement with previous studies, there was binding of USF proteins in the beta-globin LCR, and beta-globin promoter. USF binding most commonly occurred close to annotated genes, with 48.5% of USF1 sites, 44.6% of USF 2 sites and 53.0% of sites of USF1-USF2 co-localization located within 1 kb of a transcription start site (TSS), supporting the role of USF proteins as a transcription factor in these locations. A small, but significant, number of USF binding sites were located in intergenic regions > 100 kb from any annotated TSS. (1206 USF1, 1408 USF2, and 776 USF1-USF2). Interestingly, at sites of intergenic binding, USF1 and USF2 were much less likely to co-localize, (64% of USF1 and 55% of USF2 sites), implying that the USF proteins serve a different function at these remote binding sites than at sites of binding in close proximity to a TSS. USF proteins can bind DNA in an E-box dependent or independent manner. The Weeder Algorithm (Pavesi, Bioinformatics, 2001) was used to determine the most common binding motifs for USF1 and USF2. Over-represented motifs at sites of USF1 and USF2 binding were similar, with the most common sequences being a canonical E-box, CACGTG, as well as the related sequences ACGTGA and TCACGT. This genome-wide map of USF binding correlated with mRNA expression data indicates that USF proteins serve several different, important functions throughout the human genome and support the hypothesis that USF proteins participate in the regulation of many erythroid-expressed genes. DisclosuresNo relevant conflicts of interest to declare.

Stimulation of 11β-HSD1 expression by IL-1β via a C/EBP binding site in human fetal lung fibroblasts

Proinflammatory cytokines, just like glucocorticoids (GCs), have been reported to upregulate 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression in many cell types. This concerted regulation of 11β-HSD1 by interleukin-1β (IL-1β) and GCs is in marked contrast to their antagonistic effects on inflammation. Further, the molecular mechanisms underlying the induction of 11β-HSD1 by IL-1β are not very well understood. In this study, we demonstrated that IL-1β dramatically stimulated 11β-HSD1 expression and enzyme activity as well as promoter activity including the -64bp fragment upstream to the transcription start site in human fetal lung fibroblasts (HFL-1). Nucleotide mutations of the proximal CCAAT box within this region abolished the induction of 11β-HSD1 promoter activity by IL-1β. Western blotting analysis demonstrated that IL-1β induced the expression of C/EBPβ dramatically while C/EBPα was barely detectable in HFL-1 cells. Global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression plasmid (CMV500-A-C/EBP) significantly attenuated the induction of 11β-HSD1 by IL-1β, whereas over-expression of C/EBPβ enhanced the expression of 11β-HSD1. Chromatin immunoprecipitation assay revealed the recruitment of C/EBPβ to the promoter region containing the C/EBP binding site. In conclusion, IL-1β induces the expression of 11β-HSD1 mRNA in the fetal lung tissue through mechanisms that involve C/EBPβ binding to the promoter. This impact of IL-1β on the expression of 11β-HSD1 in human fetal lung cells may explain the alternate mechanism for the lung maturation that appears to occur when there is a risk of premature delivery of the fetus due to the presence of infection.

Mycophenolic acid impairs growth and differentiation of rat embryos in vitro at subtherapeutic concentrations

Cadmium is a well-known teratogen in laboratory animals and a widespread environmental pollutant. The frequencies of sister chromatid exchanges (SCEs), nucleolar organizing regions (NORs) and chromosomal aberrations were analysed in maternal bone marrow and fetal liver and/or lung cells of mice, following maternal treatment with cadmium chloride, on gestational days 8 through 10. The embryotoxic effects and morphological changes on day 18 fetuses were also studied.Cadmium chloride is readily transferred across the placenta and significant levels were detected in both the placenta and fetus. No significant changes in the frequencies of SCEs or NORs in maternal and fetal cells were observed following exposure to cadmium chloride. Fetal tissues showed mitotic inhibition at the highest dose levels (8.4 and 11.4 mg/kg, b.w.). Maternal treatment with cadmium chloride increased embryonic resorptions and fetal lethality, as well as reduced placental weight; however, it did not produce significant chromosomal changes except at the highest dose level (11.4 mg/kg).

ISG15 expression in response to double-stranded RNA or LPS in cultured Fetal Bovine Lung (FBL) cells

Fetal bovine lung (FBL) cells are used in the culture of viruses which infect cattle and ISG15 plays a role in innate immunity against viral infections. However, whether the expression of ISG15 gene can be induced in FBL cells is still unknown. In this work, the expression of ISG15 in cultured FBL cells was detected after stimulated with poly I:C or LPS. Real-time PCR analyses revealed that the transcript of ISG15 can be induced by poly I:C or LPS. The increased expression of free ISG15 was confirmed via Western blotting. Furthermore, immunofluorescence assays demonstrated that IRF-3 was translocated from the cytoplasm to the nucleus in the FBL cells treated with poly I:C. Chromatin immunoprecipitation assays showed that IRF-3 can bind to the promoter of the bISG15 gene. To demonstrate IRF-3 can promote the expression of bISG15, we establish a luciferase-reporter system of bovine ISG15 gene in 293 T cells. The luciferase assay showed that the over-expression of bovine IRF-3 could activate the promoter of bISG15 gene. Taken together, these results suggest that the expression of bISG15 can be induced in FBL cells stimulated with poly I:C or LPS, and IRF-3 may play a role in inducing the expression of ISG15 in FBL cells.