Preparation of Primary Bovine Corneal Epithelial Cell Cultures for Use in Virological Investigations

Primary and secondary cell cultures in sufficient quantity for use in virological investigations were prepared and were found suitable for growth of animal viruses.     

Phenotypic Responses of Differentiated Asthmatic Human Airway Epithelial Cultures to Rhinovirus

ObjectivesHuman airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.MethodsAir-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.ConclusionsALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.     

Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient

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Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO₂} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.     

Laminin Receptor 37/67LR Regulates Adhesion and Proliferation of Normal Human Intestinal Epithelial Cells

Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO₂, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.     

Characterization of Butyrate Uptake by Nontransformed Intestinal Epithelial Cell Lines

Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon. Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells. Uptake of ¹⁴C-BT by IEC-6 cells was (1) time- and concentration-dependent; (2) pH-dependent; (3) Na⁺-, Cl⁻- and energy-dependent; (4) inhibited by BT structural analogues; (5) sensitive to monocarboxylate transporter 1 (MCT1) inhibitors; and (6) insensitive to DIDS and amiloride. IEC-6 cells express both MCT1 and Na⁺-coupled monocarboxylate transporter 1 (SMCT1) mRNA. We conclude that ¹⁴C-BT uptake by IEC-6 cells mainly involves MCT1, with a small contribution of SMCT1. Acute exposure to ethanol, acetaldehyde, indomethacin, resveratrol and quercetin reduced ¹⁴C-BT uptake. Chronic exposure to resveratrol and quercetin reduced ¹⁴C-BT uptake but had no effect on either MCT1 or SMCT1 mRNA levels. Uptake of ¹⁴C-BT by FHC cells was time- and concentration-dependent but pH-, Na⁺-, Cl⁻- and energy-independent and insensitive to BT structural analogues and MCT1 inhibitors. Although MCT1 (but not SMCT1) mRNA expression was found in FHC cells, the characteristics of ¹⁴C-BT uptake by FHC cells did not support either MCT1 or SMCT1 involvement. In conclusion, uptake characteristics of ¹⁴C-BT differ between IEC-6 and FHC cells. IEC-6 cells demonstrate MCT1- and SMCT1-mediated transport, while FHC cells do not.     

Effect of Configuration of the Inhibitors on the Mode of Binding to the Enzyme,Thermolysin

Abstract

Preferred conformations of the competitive inhibitors glycyl-L-phenylalanine and glycyl-D-phenylalanine and their mode of binding to thermolysin have been studied. The difference in configuration is shown to affect significantly the mode of binding to thermolysin. Gly-D-Phe prefers to enter the active site in the global minimum conformation whereas Gly-L-Phe may enter in a higher energy conformation. Moreover, D-enantiomer is shown to have a better fit than the L-counterpart in the active site.     

Culturing the Unculturable: Human Coronavirus HKU1 Infects,Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.About 335 new or emerging infectious diseases have been identified since 1940 (23), and while many threaten human health, the global economy, and national security, respiratory pathogens are of particular public health concern. Using modern methods, several previously unknown viruses have been identified, including respiratory pathogens (1, 18, 27, 54, 57), yet research remains restricted to prevalence and disease association studies since a virus culture system is oftentimes lacking. Immortalized tissue culture cells are adapted to growth in laboratory conditions and, as such, display altered gene expression patterns, which may not be optimal for the replication of fastidious viruses. Primary cell-differentiated culture models provide alternative in vitro model systems closer in nature to the in vivo host tissue environment for infection studies and amplification of pathogens for further characterization. Here, we use an in vitro model of human ciliated airway epithelial cell cultures (HAE) that mimic the properties of the cartilaginous airway epithelium (17) to culture the previously unculturable human coronavirus HKU1 (HCoV-HKU1).Coronaviruses are important pathogens of humans and animals, causing a range of symptoms depending on the host. Following the severe acute respiratory syndrome (SARS)-CoV epidemic, several new strains of human coronaviruses were identified by molecular techniques, including HCoV-NL63, identified in the Netherlands from an infant with bronchiolitis (54), and HCoV-HKU1, identified in an adult patient with severe pneumonia in Hong Kong (57). HCoV-NL63 has been demonstrated to infect and replicate in both conventional immortalized cells and human ciliated airway cell cultures, producing sufficient amounts of virus for characterization studies of viral replication and pathogenesis and the successful development of an infectious clone (3, 13, 22, 41). In contrast, little is known about HCoV-HKU1, as no in vitro replication model has been identified to date, limiting further investigations of the virus.Clinical isolates of previously isolated human coronaviruses have been adapted to replicate in standard transformed cell culture; for example, SARS-CoV and HCoV-NL63 replicate efficiently in epithelial monkey kidney cells (VeroE6 and LLC-MK2), HCoV-OC43 in BHK21 cells, and HCoV-229E in MRC5 cells (14, 24, 35, 47, 54, 59). Despite the successful amplification of these human coronaviruses in cell lines, all attempts to date to culture a clinical isolate of HCoV-HKU1 have failed. No HCoV-HKU1 genomic replication was observed after inoculation of standard cell lines previously utilized for virus propagation, including RD (human rhabdomyosarcoma cells), HRT-18 (colorectal adenocarcinoma cells), HEp-2 (human epithelial carcinoma cells), MRC-5 (human lung fibroblast cells), A549 (human lung epithelial adenocarcinoma cells), Caco2 (human colorectal adenocarcinoma cells), Huh-7 (human hepatoma cells), B95a (marmoset B-lymphoblastoid cells), mixed neuron-glia culture, LLC-MK2 (rhesus monkey kidney cells), FRhK-4 (rhesus monkey kidney cells), BSC-1 (African green monkey kidney cells), Vero E6 (African green monkey kidney cells), MDCK (Madin-Darby canine kidney cells), I13.35 (murine macrophage cells), and L929 (murine fibroblast cells) (57).Here, we use human ciliated airway epithelial cell cultures to successfully propagate HCoV-HKU1 for the first time in vitro. In this culture model, HCoV-HKU1 genome copy numbers increased by several logs over the initial three-day incubation period and electron micrograph, Northern blot, and immunofluorescence data confirmed HKU1 replication in HAE and that ciliated cells were the preferential target for virus infection, the same cell type infected by all human coronaviruses tested so far in these model systems.     

E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells

E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5^th hour and receded by the 7^th hour. A second alteration followed at the 13^th hour. Treatment with CSC caused a significant initial shift already by the 1^st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5^th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.     

Epithelial WNT Ligands Are Essential Drivers of Intestinal Stem Cell Activation

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Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.