Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes. In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks. In rats, in vivo intraportal administration of a recombinant Ad containing the E. coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection. Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks. Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

firmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.

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Various volatile organic compounds (VOCs) in breath exhaled by patients with lung cancer, healthy controls, and patients with lung cancer who underwent surgery for resection of cancer were analyzed by gas condenser-equipped gas chromatography-mass spectrometry (GC/MS) for development of an exhaled breath monitoring prototype system involving metal oxide gas sensors, a gas condenser, and gas chromatography columns. The gas condenser-GC/MS analysis identified concentrations of 56 VOCs in the breath exhaled by the test population of 136 volunteers (107 patients with lung cancer and 29 controls), and selected four target VOCs, nonanal, acetoin, acetic acid, and propanoic acid, for use with the condenser, GC, and sensor-type prototype system. The prototype system analyzed exhaled breath samples from 101 volunteers (74 patients with lung cancer and 27 controls). The prototype system exhibited a level of performance similar to that of the gas condenser-GC/MS system for breath analysis.

Chronic obstructive pulmonary disease (COPD) is characterized by both chronic inflammation in the airway and systemic inflammation; however, the molecular mechanism of COPD has not been fully elucidated. By measuring microRNA (miRNA) expression in the plasma of COPD subjects, we aimed to identify the clinical relevance of plasma miRNA levels in these patients. Blood samples were obtained from COPD patients and age-matched normal controls. We initially produced plasma miRNA expression profiles using TaqMan low-density array screening. For further validation, individual qRT-PCRs were performed in 40 COPD patients and 20 healthy subjects. TaqMan low-density array screening showed that 9 miRNAs (miR-29b, miR-483-5p, miR-152, miR-629, miR-26b, miR-101, miR-106b, miR-532-5p and miR-133b) were significantly downregulated in the plasma from COPD patients when compared with normal smokers. Among these miRNAs, we focused on miR-106b. A reduction in the plasma miR-106b levels was evident in COPD ex-smokers and COPD current smokers compared with levels in smokers. There was a negative correlation between the plasma miR-106b level and the duration of disease since diagnosis in COPD ex-smokers and the duration of smoking in COPD current smokers. These findings support the concept that progressive reduction in the plasma miR-106b level may reflect persistent and systemic changes even after the discontinuation of smoking in COPD patients. miR-106b may therefore play an important role in the pathogenesis of COPD.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by scattered fibrotic lesions in the lungs. The pathogenesis and genetic basis of IPF remain poorly understood. Here, we show that a homozygous missense mutation in SFTPA1 caused IPF in a consanguineous Japanese family. The mutation in SFTPA1 disturbed the secretion of SFTPA1 protein. Sftpa1 knock-in (Sftpa1-KI) mice that harbored the same mutation as patients spontaneously developed pulmonary fibrosis that was accelerated by influenza virus infection. Sftpa1-KI mice showed increased necroptosis of alveolar epithelial type II (AEII) cells with phosphorylation of IRE1α leading to JNK-mediated up-regulation of Ripk3. The inhibition of JNK ameliorated pulmonary fibrosis in Sftpa1-KI mice, and overexpression of Ripk3 in Sftpa1-KI mice treated with a JNK inhibitor worsened pulmonary fibrosis. These findings provide new insight into the mechanisms of IPF in which a mutation in SFTPA1 promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF.

To examine a possible protective effect of exogenous glial cell line-derived neurotrophic factor (GDNF) gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 1 day before 90 minutes of transient middle cerebral artery occlusion (MCAO) in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-injected group at 24 hours after the transient MCAO was significantly smaller than that of vehicle- or Ad-LacZ-treated group. Enzyme-linked immunosorbent assay (ELISA) for immunoreactive GDNF demonstrated that GDNF gene products in the Ad-GDNF-injected group were higher than those of vehicle-treated group at 24 hours after transient MCAO. Immunoreactive GDNF staining was obviously detected in the cortex around the needle track just before or 24 hours after MCAO in the Ad-GDNF group, whereas no or slight GDNF staining was detected in the vehicle group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 hours after transient MCAO were markedly reduced by the Ad-GDNF group. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO in association with the reduction of apoptotic signals.

The utility of replication-deficient recombinant adenovirus vector-mediated transfer and expression of the alpha 1-antitrypsin (alpha 1AT) cDNA to peritoneal mesothelial tissues was evaluated as a means of delivering alpha 1AT to the systemic circulation. Preliminary studies with Ad.RSV beta gal, an adenovirus vector expressing the Escherichia coli lacZ gene (beta-galactosidase), showed that intraperitoneal injection of 10(9) plaque-forming units (pfu) to cotton rats resulted in beta-galactosidase activity in mesothelial cells lining the peritoneal cavity. After intraperitoneal administration of 10(9) pfu of Ad alpha 1AT (an adenovirus vector containing the human alpha 1AT cDNA), human alpha 1AT was detectable in serum for up to 24 days, with a maximal level of 3.4 micrograms/ml at 4 days. Expression of the exogenous gene was localized to the peritoneal mesothelium as PCR analyses detected no evidence of expression of the exogenous gene in any other tissues evaluated. Anti-adenovirus vector antibodies were detectable in serum after intraperitoneal administration of the recombinant vectors, including antibodies with neutralizing activity. Repeat administrations of adenovirus vectors to the peritoneal cavity at 1 wk and 1 mo after the initial dose failed to show gene expression, but repeat administration 3 mo after demonstrated measurable gene transfer and expression. Together these observations suggest replication-deficient adenovirus-mediated gene transfer to the peritoneal mesothelium offers a promising means to transfer alpha 1AT to the systemic circulation, although immunity induced against the adenovirus may limit frequent repetitive dosing.