Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.
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We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function. (+info)
SP-A enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection.
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Surfactant protein A (SP-A) is a member of the collectin family of host defense molecules expressed primarily in the epithelial cells of the lung. To determine the role of SP-A in pulmonary adenoviral infection, SP-A-deficient (SP-A -/-) mice were intratracheally infected with a replication-deficient recombinant adenovirus, Av1Luc1. Lung inflammation was markedly increased in SP-A -/- compared with SP-A +/+ mice and was associated with increased hemorrhage and epithelial cell injury. Polymorphonuclear cells in bronchoalveolar lavage fluid (BALF) were increased in SP-A -/- mice after administration of adenovirus. Coadministration of adenovirus and purified human SP-A ameliorated adenoviral-induced lung inflammation in SP-A -/- mice. Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta were increased in BALF of SP-A -/- mice. Likewise, TNF-alpha, IL-6, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein-1, and MIP-2 mRNAs were increased in lung homogenates from SP-A -/- mice 6 and 24 h after viral administration. Clearance of adenoviral DNA from the lung and uptake of fluorescent-labeled adenovirus by alveolar macrophages were decreased in SP-A -/- mice. SP-A enhances viral clearance and inhibits lung inflammation during pulmonary adenoviral infection, providing support for the importance of SP-A in antiviral host defense. (+info)
Clinical characteristics of acute viral lower respiratory tract infections in hospitalized children in Seoul, 1996-1998.
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This study was performed to investigate the etiologic agents, age distribution, clinical manifestations and seasonal occurrence of acute viral lower respiratory tract infections in children. We confirmed viral etiologies using nasopharyngeal aspirates in 237 patients of the ages of 15 years or younger who were hospitalized for acute lower respiratory tract infection (ALRI) from March 1996 to February 1998 at Samsung Seoul Hospital, Seoul, Korea. The overall isolation rate was 22.1%. The viral pathogens identified were adenovirus (12.7%), influenza virus type A (21.1%), -type B (13.9%), parainfluenza virus type 1 (13.5%), -type 2 (1.3%), -type 3 (16.0%) and respiratory syncytial virus (21.5%). The occurrence of ALRIs was highest in the first year of life, although parainfluenza virus type 1 infection occurred predominantly in the second year of life and influenza virus caused illnesses in all age groups. The specific viruses are frequently associated with specific clinical syndromes of ALRI. The respiratory agents and associated syndromes frequently have characteristic seasonal patterns. This study will help us to estimate the etiologic agents of ALRI, and establish a program for the prevention and treatment. An annual nationwide survey is necessary to understand the viral epidemiology associated with respiratory illnesses in Korea. (+info)
Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia.
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Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia. (+info)
CCSP deficiency does not alter surfactant homeostasis during adenoviral infection.
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Clara cell secretory protein (CCSP) deficiency in mice is associated with increased susceptibility to pulmonary inflammation after hyperoxia or viral infection. Because adenoviral exposure perturbs pulmonary surfactant homeostasis in vivo, we hypothesized that CCSP deficiency would influence surfactant metabolism after pulmonary infection. Alveolar and total lung saturated phosphatidylcholine pool sizes were similar in CCSP-deficient [CCSP(-/-)] and wild-type [CCSP(+/+)] mice before and 7 days after intratracheal administration of adenovirus. Radiolabeled choline and palmitate incorporation into saturated phosphatidylcholine was similar, and there was no alteration by previous infection 7 days before the incorporation measurements. Furthermore, CCSP deficiency did not influence clearance of [(14)C]dipalmitoylphosphatidylcholine and (125)I-labeled recombinant surfactant protein C. Increased persistence of alveolar capillary leak was observed in CCSP(-/-) mice after adenoviral infection. Surfactant lipid homeostasis was not influenced by CCSP before or after administration of adenovirus to the lung. Persistence of alveolar capillary leak in CCSP(-/-) mice after adenovirus provides further evidence for the role of CCSP in the regulation of pulmonary inflammation. (+info)
An outbreak of acute respiratory disease caused by Mycoplasma pneumoniae and adenovirus at a federal service training academy: new implications from an old scenario.
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Outbreaks of Mycoplasma pneumoniae and adenovirus have been reported in military institutions for several decades. During a recent outbreak in a federal service training academy, we performed an epidemiological and laboratory investigation to better characterize and control the outbreak. Of 586 students responding to a questionnaire, 317 (54%) reported having a respiratory illness during the outbreak period. Among 42 students who underwent complete laboratory testing, 24 (57%) had evidence of M. pneumoniae infection, 8 (19%) had evidence of adenovirus infection, and 4 (10%) had evidence of both. Polymerase chain reaction testing of oropharyngeal swabs revealed more acute M. pneumoniae infections (57% positive) than did serology or culture. Multivariate analysis revealed that visiting the campus health clinic >3 times for a nonrespiratory condition, such as injury, was a significant risk factor for illness among freshmen early in the course of the outbreak, whereas having an ill roommate was a risk factor throughout the duration of the outbreak. (+info)
Roles of E2F1 in mesangial cell proliferation in vitro.
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Roles of E2F1 in mesangial cell proliferation in vitro. BACKGROUND: The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. METHODS: To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. RESULTS: E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. CONCLUSIONS: In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins. (+info)
The distribution and kinetics of polyomavirus in lungs of intranasally infected newborn mice.
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The primary cell types that sustain polyomavirus (Py) replication following intranasal infection as well as the nature of the host cellular response to Py were unknown. As this is an essential and specific site for virus entry, it seems likely that viral gene function must be adapted to these mucosal tissues. Using immunohistochemistry and in situ hybridization, we determined the cell types in the lung that support Py gene expression and replication following intranasal inoculation of newborn mice within 24 h of birth. Lungs were collected daily from days 1 to 10 postinfection for Py DNA and early T antigen analysis and for histological examination by H&E staining, using methods that preserve the delicate newborn lung architecture. Viral DNA was present in increasing quantities from 2 to 6 dpi in a subset of the Clara cells lining the inner lumen of the bronchi and bronchioles, while T antigen expression was present in a majority of the cells in the bronchi and bronchiole lumen. A distinct and transient pattern of hyperplasia was observed among the cells expressing T antigen and was present from 3 through 6 dpi. Py DNA-containing cells exfoliated into the bronchiole lumen and alveolar ducts, but Py T antigen was not detected in these cells. Py DNA was first detected at 2 dpi, increased through 6 dpi, and abruptly declined through 9 dpi at which time there was no sign of viral DNA in the lungs by in situ hybridization. An unusual infiltration of neutrophils began before the presence of exfoliated cells or Py replication and continued for 2-3 days and was followed by a lymphocytic infiltration at 8-10 dpi lasting 2-3 days. Neither the hyperplasia nor the neutrophil infiltration occurred following infection with the MOP1033 MT-Ag or RB1 LT-Ag mutants of Py. In addition, both the neutrophil infiltration and the transient hyperplasia are in stark contrast to the heavy macrophage infiltration that follows infection of lungs with mouse adenovirus. Thus it appears that Py elicits a distinct host response pattern not seen with other DNA viral infections. (+info)