George R. Jackson

Intrinsic Phenotypic Differences of Asthmatic Epithelium and Its Inflammatory Responses to Respiratory Syncytial Virus and Air Pollution

A substantial proportion of healthcare cost associated with asthma is attributable to exacerbations of the disease. Within the airway, the epithelium forms the mucosal immune barrier, the first structural cell defense against common environmental insults such as respiratory syncytial virus (RSV) and particulate matter. We sought to characterize the phenotype of differentiated asthmatic-derived airway epithelial cultures and their intrinsic inflammatory responses to environmental challenges. Air-liquid interface (ALI) cultures were generated from asthmatic (n = 6) and nonasthmatic (n = 6) airway epithelial cells. Airway tissue and ALI cultures were analyzed by immunohistochemistry for cytokeratin-5, E-cadherin, Ki67, Muc5AC, NF-κB, the activation of p38, and apoptosis. ALI cultures were exposed to RSV (4 × 10(6) plaque forming unit/ml), particulate matter collected by Environmental Health Canada (EHC-93, 100 μg/ml), or mechanically wounded for 24, 48, and 96 hours and basolateral supernatants analyzed for inflammatory cytokines, using Luminex and ELISA. The airway epithelium in airway sections of patients with asthma as well as in vitro ALI cultures demonstrated a less differentiated epithelium, characterized by elevated numbers of basal cells marked by the expression of cytokeratin-5, increased phosphorylation of p38 mitogen-activated protein kinase, and less adherens junction protein E-cadherin. Transepithelial resistance was not different between asthmatic and nonasthmatic cultures. In response to infection with RSV, exposure to EHC-93, or mechanical wounding, asthmatic ALI cultures released greater concentrations of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor, compared with nonasthmatic cultures (P < 0.05). This parallel ex vivo and in vitro study of the asthmatic epithelium demonstrates an intrinsically altered phenotype and aberrant inflammatory response to common environmental challenges, compared with nonasthmatic epithelium.

Novel technologies and an overall strategy to allow hazard assessment and risk prediction of chemicals, cosmetics, and drugs with animal-free methods.

Several alternative methods to replace animal experiments have been accepted by legal bodies. An even larger number of tests are under development or already in use for non-regulatory applications or for the generation of information stored in proprietary knowledge bases. The next step for the use of the different in vitro methods is their combination into integrated testing strategies (ITS) to get closer to the overall goal of predictive in vitro-based risk evaluation processes. We introduce here a conceptual framework as the basis for future ITS and their use for risk evaluation without animal experiments. The framework allows incorporation of both individual tests and already integrated approaches. Illustrative examples for elements to be incorporated are drawn from the session Innovative technologies at the 8th World Congress on Alternatives and Animal Use in the Life Sciences, held in Montreal, 2011. For instance, LUHMES cells (conditionally immortalized human neurons) were presented as an example for a 2D cell system. The novel 3D platform developed by InSphero was chosen as an example for the design and use of scaffold-free, organotypic microtissues. The identification of critical pathways of toxicity (PoT) may be facilitated by approaches exemplified by the MatTek 3D model for human epithelial tissues with engineered toxicological reporter functions. The important role of in silico methods and of modeling based on various pre-existing data is demonstrated by Altamiras comprehensive approach to predicting a molecules potential for skin irritancy. A final example demonstrates how natural variation in human genetics may be overcome using data analytic (pattern recognition) techniques borrowed from computer science and statistics. The overall hazard and risk assessment strategy integrating these different examples has been compiled in a graphical work flow.

Molecular Impact of Electronic Cigarette Aerosol Exposure in Human Bronchial Epithelium

Little evidence is available regarding the physiological effects of exposure to electronic cigarette (ECIG) aerosol. We sought to determine the molecular impact of ECIG aerosol exposure in human bronchial epithelial cells (HBECs). Gene-expression profiling was conducted in primary grown at air liquid interface and exposed to 1 of 4 different ECIG aerosols, traditional tobacco cigarette (TCIG) smoke, or clean air. Findings were validated experimentally with quantitative polymerase chain reaction and a reactive oxygen species immunoassay. Using gene set enrichment analysis, signatures of in vitro ECIG exposure were compared with those generated from bronchial epithelial brushings of current TCIG smokers and former TCIG smokers currently using ECIGs. We found 546 genes differentially expressed across the ECIG, TCIG, and air-exposed groups of HBECs (ANOVA; FDR qu2009<u2009.05; fold changeu2009> 1.5). A subset of these changes were shared between TCIG- and ECIG-exposed HBECs. ECIG exposure induced genes involved in oxidative and xenobiotic stress pathways and increased a marker of reactive oxygen species production in a dose-dependent manner. ECIG exposure decreased expression of genes involved in cilia assembly and movement. Furthermore, gene-expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users (qu2009<u2009.01). In summary, our data suggest that ECIG aerosol can induce gene-expression changes in bronchial airway epithelium in vitro, some of which are shared with TCIG smoke. These changes were generally less pronounced than the effects of TCIG exposure and were more pronounced in ECIG products containing nicotine than those without nicotine. Our data further suggest that the gene-expression alterations seen with the in vitro exposure system reflects the physiological effects experienced in vivo by ECIG users.

Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model.

Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well‐defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose‐dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose‐dependent increase in secretion of IL‐6, IL‐8, TNF‐α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well‐characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner.

Abstract 3173: Identification of miR-4423 as a primate-specific microRNA highly expressed in airway epithelium and associated with lung cancer

Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Our group has previously shown that epithelial gene expression is altered throughout the airway of smokers and that some of these changes are regulated by microRNAs. Moreover, we have previously identified gene expression differences in cytologically normal bronchial airway epithelial cells between smokers with and without lung cancer that can serve as an early diagnostic biomarker for lung cancer. Here, we use next-generation sequencing of small RNAs to identify novel microRNAs expressed in airway epithelium and associated with lung cancer. We identify miR-4423 as a primate-specific microRNA highly expressed in the airway epithelium. In vitro, the expression of miR-4423 increases as Normal Human Bronchial Epithelial cells are differentiated into mucociliary epithelium at an Air Liquid Interface, while its mRNA targets decrease in expression. Furthermore, the expression of miR-4423 is reduced in lung tumors and in the cytologically normal bronchial airway epithelium of smokers with lung cancer. In gain-of-function experiments, ectopic expression of miR-4423 in lung cancer cell lines resulted in reduced colony formation in soft agar. Taken together, these data support the power of next-generation sequencing in identifying novel cell type- specific transcripts and provides evidence that this newly characterized microRNA may play a role in promoting the differentiation and/or maintenance of airway epithelium, and can reduce anchorage-independent lung cancer cell growth. 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 3173. doi:1538-7445.AM2012-3173