Multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type C.

Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIß (PI4K-IIIß) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.

Discovery and optimization of C-2 methyl imidazopyrrolopyridines as potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2.

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

IL-13 and epidermal growth factor receptor have critical but distinct roles in epithelial cell mucin production.

Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13-induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-alpha. However, there was constitutive release of TGF-alpha from normal human bronchial epithelial cells, and inhibition of TGF-alpha or EGFR reduced both constitutive and IL-13-induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production.

A distinctive alveolar macrophage activation state induced by cigarette smoking.

RATIONALE: Macrophages are believed to play a central role in emphysema based largely on data from mouse models. However, the relevance of these models to smoking-related lung disease in humans is uncertain. OBJECTIVES: We sought to comprehensively characterize the effects of smoking on gene expression in human alveolar macrophages and to compare these with effects seen in transgenic mouse models of emphysema. METHODS: We used DNA microarrays with genomewide coverage to analyze alveolar macrophages from 15 smokers, 15 nonsmokers, and 15 subjects with asthma (disease control). Selected gene expression changes were validated by polymerase chain reaction and ELISA. Expression changes were compared with those identified by microarray analysis of interleukin-13-overexpressing and integrin-beta6-deficient mice, which both develop emphysema. MEASUREMENTS AND MAIN RESULTS: All 15 smokers shared a common pattern of macrophage gene expression that distinguished them from nonsmokers, a finding not observed in subjects with asthma. We identified 110 genes as differentially expressed in smokers despite using conservative statistical methods. Matrix metalloproteinase 12, a proteinase that plays a critical role in mouse models, was the third most highly induced gene in smokers (ninefold, p < 0.0001). However, most changes in smokers were not reflected in mouse models. One such finding was increased osteopontin expression in smokers (fourfold, p = 0.006), which was confirmed at the protein level and correlated with the degree of airway obstruction. CONCLUSIONS: Smoking induces a remarkably consistent and distinctive pattern of alveolar macrophage activation. These studies identify aspects of mouse models that are directly relevant to human smokers and also reveal novel potential mediators of smoking-related diseases.

Dissecting asthma using focused transgenic modeling and functional genomics.

BACKGROUND: Asthma functional genomics studies are challenging because it is difficult to relate gene expression changes to specific disease mechanisms or pathophysiologic features. Use of simplified model systems might help to address this problem. One such model is the IL-13/Epi (IL-13-overexpressing transgenic mice with STAT6 expression limited to epithelial cells) focused transgenic mouse, which isolates the effects of a single mediator, IL-13, on a single cell type, the airway epithelial cell. These mice develop airway hyperreactivity and mucus overproduction but not airway inflammation. OBJECTIVE: To identify how effects of IL-13 on airway epithelial cells contribute to gene expression changes in murine asthma models and determine whether similar changes are seen in people with asthma. METHODS: We analyzed gene expression in ovalbumin allergic mice, IL-13-overexpressing mice, and IL-13/Epi mice with microarrays. We analyzed the expression of human orthologues of genes identified in the mouse studies in airway epithelial cells from subjects with asthma and control subjects. RESULTS: In comparison with the other 2 models, IL-13/Epi mice had a remarkably small subset of gene expression changes. Human orthologues of some genes identified as increased in the mouse models were more highly expressed in airway epithelial cells from subjects with asthma than in controls. These included calcium-activated chloride channel 1, 15-lipoxygenase, trefoil factor 2, and intelectin. CONCLUSION: The combination of focused transgenic models, DNA microarray analyses, and translational studies provides a powerful approach for analyzing the contributions of specific mediators and cell types and for focusing attention on a limited number of genes associated with specific pathophysiologic aspects of asthma.

Aspergillus antigen induces robust Th2 cytokine production, inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2.

BACKGROUND: Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma. METHODS: To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response. RESULTS: We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines. CONCLUSION: We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.

Differential gene expression by integrin beta 7+ and beta 7- memory T helper cells.

BACKGROUND: The cell adhesion molecule integrin alpha 4 beta 7 helps direct the migration of blood lymphocytes to the intestine and associated lymphoid tissues. We hypothesized that beta 7+ and beta 7- blood memory T helper cells differ in their expression of genes that play a role in the adhesion or migration of T cells. RESULTS: RNA was prepared from beta 7+ and beta 7- CD4+ CD45RA- blood T cells from nine normal human subjects and analyzed using oligonucleotide microarrays. Of 21357 genes represented on the arrays, 16 were more highly expressed in beta 7+ cells and 18 were more highly expressed in beta 7- cells (>/=1.5 fold difference and adjusted P < 0.05). Several of the differentially expressed transcripts encode proteins with established or putative roles in lymphocyte adhesion and chemotaxis, including the chemokine receptors CCR9 and CCR10, the integrin alpha 4 subunit, L-selectin, KLRB1 (CD161), NT5E (CD73), LGALS1 and LGALS2 (galectin-1 and -2), and RGS1. Flow cytometry was used to determine whether differences in levels of transcripts encoding cell surface proteins were associated with differential expression of those proteins. Using this approach, we found that surface expression of KLRB1, LAIR1, and NT5E proteins was higher on beta 7+ memory/effector T cells than on beta 7- cells. CONCLUSIONS: Memory/effector T cells that express integrin beta 7 have a distinct pattern of expression of a set of gene transcripts. Several of these molecules can affect cell adhesion or chemotaxis and are therefore likely to modulate the complex multistep process that regulates trafficking of CD4+ memory T cell subsets with different homing behaviors.

Spotted long oligonucleotide arrays for human gene expression analysis.

DNA microarrays produced by deposition (or 'spotting')of a single long oligonucleotide probe for each gene may be an attractive alternative to other types of arrays. We produced spotted oligonucleotide arrays using two large collections of approximately 70-mer probes, and used these arrays to analyze gene expression in two dissimilar human RNA samples. These samples were also analyzed using arrays produced by in situ synthesis of sets of multiple short (25-mer) oligonucleotides for each gene (Affymetrix GeneChips). We compared expression measurements for 7344 genes that were represented in both long oligonucleotide probe collections and the in situ-synthesized 25-mer arrays. We found strong correlations (r = 0.8-0.9) between relative gene expression measurements made with spotted long oligonucleotide probes and in situ-synthesized 25-mer probe sets. Spotted long oligonucleotide arrays were suitable for use with both unamplified cDNA and amplified RNA targets, and are a cost-effective alternative for many functional genomics applications. Most previously reported evaluations of microarray technologies have focused on expression measurements made on a relatively small number of genes. The approach described here involves far more gene expression measurements and provides a useful method for comparing existing and emerging techniques for genome-scale expression analysis.