Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.     

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.     

Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells

Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.     

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.     

A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine,Sphingosine-1-Phosphate,and Heme of Human Lung

Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH.     

Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome

It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-alpha, IFN-gamma, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.     

High-volume ventilation induces pentraxin 3 expression in multiple acute lung injury models in rats

Pentraxin 3 (PTX3) is an acute-phase protein, which can be produced by a variety of tissue cells at the site of infection or inflammation. It plays an important role in innate immunity in the lung and in mediating acute lung injury. The aim of this study was to determine the effect of mechanical ventilation on PTX3 expression in multiple lung injury models. Male Sprague-Dawley rats were challenged with intravenous injection of lipopolysaccharide (LPS) or hemorrhage followed by resuscitation (HS). The animals were then subjected to either relatively higher (12 ml/kg) or lower (6 ml/kg, positive end-expiratory pressure of 5 cmH(2)O) volume ventilation for 4 h. High-volume ventilation significantly enhanced PTX3 expression in the lung, either alone or in combination with LPS or hemorrhage. A significant increase of PTX3 immunohistochemistry staining in the lung was seen in all injury groups. The PTX3 expression was highly correlated with the severity of lung injury determined by blood gas, lung elastance, and wet-to-dry ratio. To determine the effects of HS, LPS, or injurious ventilation (25 ml/kg) alone on PTX3 expression, another group of rats was studied. Injurious ventilation significantly damaged the lung and increased PTX3 expression. A local expression of PTX3 induced by high-volume ventilation, either alone or in combination with other pathological conditions, suggests that it may be an important mediator in ventilator-induced lung injury.     

Cell-based tissue engineering for lung regeneration

Emphysema is a chronic lung disease characterized by alveolar enlargement and tissue loss. Tissue engineering represents an attractive potential for regeneration of several organ systems. The complex three-dimensional architectural structure of lung parenchyma requiring connections of alveolar units to airways and the pulmonary circulation makes this strategy less optimistic. In the present study, we used Gelfoam sponge as a scaffold material, supplemented with fetal rat lung cells as progenitors, to explore the potential application of cell-based tissue engineering for lung regeneration in adult rats. After injection into lung parenchyma, the sponge showed porous structures similar to alveolar units. It did not induce severe local inflammatory response. Fetal lung cells in the sponge were able to survive in the adult lung for at least 35 days, determined by CMTMR [5-(and-6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine] labeling. Proliferation of cells within the sponge was demonstrated in vivo by bromodeoxyuridine (BrdU) labeling. Cells formed "alveolar-like structures" at the border between the sponge and the surrounding lung tissue with positive immunohistochemical staining for epithelial and endothelial cells. Neovascularization of the sponge was demonstrated with India ink perfusion. The sponge degraded after several months. This study suggests that cell-based tissue engineering possesses the potential to regenerate alveolar-like structures, an important step towards our ultimate goal of lung regeneration.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.