Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signalregulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.One of the primary functions of the airway epithelium is to provide a protective barrier against inhaled environmental toxins and airborne pathogens, and airway epithelial cells express a number of intrinsic factors that serve to minimize invasion by infectious agents and to promote repair processes following injury (1, 2). A newly discovered NADPH oxidase homolog, dual oxidase (Duox), 3 has been recently identified within the tracheobronchial epithelium (3-6) and is thought to contribute to innate epithelial host defense based on structural similarities with the phagocyte NADPH oxidase system (7,8). Of the two known Duox isoforms, Duox1 is primarily expressed within the ciliated epithelium and believed to be responsible for epithelial H 2 O 2 production after cell stimulation (5), whereas Duox2 has been localized to salivary or submucosal glands, thus constituting a functional host defense system with co-localized lactoperoxidase (4, 5). Other than recent observations in Drosophila (9), a direct role for Duox1/2 in host defense has not yet been directly demonstrated, and other functions of airway epithelial Duox1 have been postulated (6, 10).Both Duox isozymes contain two EF-hand Ca 2ϩ -binding domains and are activated by Ca 2ϩ -mobilizing stimuli (11, 12). A critical mechanism of Ca 2ϩ -mediated signaling within epithelial cells after bacterial infection or mechanical or oxidative injury involves the activation of purinergic receptors at the cell surface by release of ATP (13-15). ATP-mediated autocrine or paracrine signaling is known to regulate diverse processes involved in host defense, including anion transport, ciliary function, and mucin expression (16 -19), and some of these events have recently been associated with Duox1 act...

Unenhanced helical CT accurately determines the presence or absence of ureterolithiasis in patients with acute flank pain. CT precisely identifies stone size and location. When ureterolithiasis is absent, other causes of acute flank pain can be identified. In most cases additional imaging is not required.

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A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na

Acute lung inflammation and injury were induced by intranasal instillation of lipopolysaccharide (LPS) in normal and type 2 nitric oxide synthase (NOS2)-deficient (NOS2 Ϫ/Ϫ ) C57BL/6 mice. LPS-induced increases in extravasated airway neutrophils and in lung lavage fluid of TNF-␣ and macrophage inflammatory protein-2 were markedly lower in NOS2 Ϫ/Ϫ than in wild-type mice, indicating that NOS2-derived nitric oxide (NO⅐) participates in inflammatory cytokine production and neutrophil recruitment. Instillation of LPS also increased total lung lavage protein and induced matrix metalloproteinase-9 and mucin 5AC, as indexes of lung epithelial injury and/or mucus hyperplasia, and increased tyrosine nitration of lung lavage proteins, a marker of oxidative injury. All these responses were less pronounced in NOS2 Ϫ/Ϫ than in wild-type mice. Inhibition of NOS activity also suppressed production of TNF-␣ and macrophage inflammatory protein-2 by LPS-stimulated mouse alveolar MH-S macrophages, and this was restored by NO⅐ donors, illustrating involvement of NO⅐ in macrophage cytokine signaling. Oligonucleotide microarray (GeneChip) analysis of global lung gene expression revealed that LPS inhalation induced a range of transcripts encoding proinflammatory cytokines and chemokines, stress-inducible factors, and other extracellular factors and suppressed mRNAs encoding certain cytoskeletal proteins and signaling proteins, responses that were generally attenuated in NOS2 Ϫ/Ϫ mice. Comparison of both mouse strains revealed altered expression of several cytoskeletal proteins, cell surface proteins, and signaling proteins in NOS2 Ϫ/Ϫ mice, changes that may partly explain the reduced responsiveness to LPS. Collectively, our results suggest that NOS2 participates in the acute inflammatory response to LPS by multiple mechanisms: involvement in proinflammatory cytokine signaling and alteration of the expression of various genes that affect inflammatory-immune responses to LPS.

The airway epithelium provides a protective barrier against inhaled environmental toxins and microorganisms, and epithelial injury initiates a number of processes to restore its barrier integrity, including activation of matrix metalloproteinases such as MMP-9 (92-kD gelatinase B). Airway epithelial cells continuously produce nitric oxide (NO), which has been linked to cell migration and MMP-9 regulation in several cell types, but the importance of epithelial NO in mediating airway epithelial repair or MMP-9 activation is unknown. Using primary or immortalized human bronchial epithelial cells, we demonstrate that low concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which was associated with increased localized expression and activation of MMP-9. In addition, in HBE1 cells that were stably transfected with inducible NOS (NOS2), to mimic constitutive epithelial NOS2 expression in vivo, NOS inhibition decreased epithelial wound repair and MMP-9 expression. The stimulatory effects of NO on epithelial wound repair and MMP-9 expression were dependent on cGMPmediated pathways and were inhibited by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase. Inhibition of cGMP-dependent protein kinase (PKG) attenuated NO-mediated epithelial wound closure, but did not affect MMP-9 expression. However, pharmacologic MMP inhibition and siRNA knockdown of MMP-9 expression demonstrated the contribution of MMP-9 to NO-mediated wound closure. Overall, our results demonstrate that NOS2-derived NO contributes to airway epithelial repair by both PKG-dependent and -independent mechanisms, and involves NO-dependent expression and activation of MMP-9.