Lef-1 controls cell cycle progression in airway basal cells to regulate proliferation and differentiation.

The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine.

Repeat Dosing of AAV2.5T to Ferret Lungs Elicits an Antibody Response That Diminishes Transduction in an Age-Dependent Manner.

Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl- currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.

Association of colorectal polyps and cancer with low-dose persistent organic pollutants: A case-control study.

BACKGROUND: Low-dose persistent organic pollutants (POPs) have recently been linked to immunosenescence, a key mechanism in carcinogenesis, as well as many aging-related chronic diseases. Since feces are the main excretion route of POPs, the large intestine is a potential target organ for these pollutants. We performed a case-control study to evaluate whether exposure to low-dose POPs is related to the risk of colorectal polyps and cancer. METHODS: A total of 277 participants were recruited from one hospital: 99 cancer patients, 102 polyp patients, and 76 control subjects. As typical examples of POPs, we measured the serum concentrations of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). RESULTS: Across the tertiles of the summary measure of POPs, the adjusted odds ratios (ORs) of colorectal polyps and cancer were 2.8 (1.2-6.8) (Ptrend = 0.01) and 3.0 (1.0-8.8) (Ptrend = 0.02), respectively, for subjects in the highest tertile. When OCPs and PCBs were analyzed separately, OCPs were linked to an increased risk of both polyps and cancer; the adjusted ORs were 2.3 (0.9-5.7) (Ptrend = 0.05) for polyps and 3.6 (1.1-11.8) (Ptrend< 0.01) for cancer. However, PCBs were only significantly associated with a high risk of polyps but not cancer; the adjusted OR was 2.8 (1.2-6.6) (Ptrend = 0.01). CONCLUSION: Chronic exposure to low-dose POPs may be associated with an increased risk of colorectal polyps and cancer. Our findings suggest the carcinogenic potential of strong lipophilic chemical mixtures such as POPs which are accumulated in adipose tissue, released to circulation, and eliminated through feces.

Biological synthesis and characterisation of silver nanoparticles using Pseudomonas geniculata H10 for pharmaceutical activity.

In the present study, silver nanoparticles (SNPs) were synthesised for the first time using Pseudomonas geniculata H10 as reducing and stabilising agents. The synthesis of SNPs was the maximum when the culture supernatant was treated with 2.5 mM AgNO3 at pH 7 and 40°C for 10 h. The SNPs were characterised by field emission scanning electron microscopy-energy-dispersive spectroscopy, transmission electron microscopy, dynamic light scattering, X-ray diffraction and UV-vis spectroscopy. Fourier transform infrared spectroscopy indicated the presence of proteins, suggesting they may have been responsible for the reduction and acted as capping agents. The SNPs displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 28.301 µg/ml) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate (IC50 = 27.076 µg/ml) radical scavenging activities. The SNPs exhibited a broad antimicrobial spectrum against several human pathogenic Gram-positive and Gram-negative bacteria and Candida albicans. The antimicrobial action of SNPs was due to cell deformation resulting in cytoplasmic leakage and subsequent lysis. The authors' results indicate P. geniculata H10 could be used to produce antimicrobial SNPs in a facile, non-toxic, cost-effective manner, and that these SNPs can be used as effective growth inhibitors in various microorganisms, making them applicable to various biomedical and environmental systems. As far as the authors are aware, this study is the first to describe the potential biomedical applications of SNPs synthesised using P. geniculata.

Improved biosynthesis of silver nanoparticles using keratinase from Stenotrophomonas maltophilia R13: reaction optimization, structural characterization, and biomedical activity.

In the present study, keratinase from Stenotrophomonas maltophilia R13 was used for the first time as a reducing agent for the eco-friendly synthesis of AgNPs. The keratinase produced by strain R13 was responsible for the reduction of silver ions and the subsequent formation of AgNPs. Maximum AgNP synthesis was achieved using 2 mM AgNO3 at pH 9 and 40 °C. Electron microscopy and dynamic light scattering analysis showed AgNPs were spherical and of average diameter ~ 8.4 nm. X-ray diffraction revealed that AgNPs were crystalline. FTIR indicated AgNPs were stabilized by proteins present in the crude enzyme solution of strain R13. AgNPs exhibited a broad antimicrobial spectrum against several pathogenic microorganisms, and the antimicrobial mechanism appeared to involve structural deformation of cells resulting in membrane leakage and subsequent lysis. AgNPs also displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 0.0112 mg/ml), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate radical scavenging (IC50 = 0.0243 mg/ml), and anti-collagenase (IC50 = 23.5 mg/ml) activities.

Development of a minipig physical phantom from CT data.

Quantification of pathological progression of radiation-induced injury is essential in development of treatment methods, and a proper animal model is necessary for relevant radiological and medical studies. A minipig is a current animal model selected because of its similarities to humans in anatomy and pathology. In the present study, a minipig physical phantom was developed using computed tomography (CT) data. For dosimetry purposes, the minipig physical phantom was constructed on a slice-by-slice basis, with an array of holes to accommodate dosimeters. The phantom is constituted of three major organs, i.e. bone, lung, and remaining soft tissue, and the organs are clearly distinguishable on each 20-mm-thick axial slice. The quality of the tissue-equivalent (TE) substitutes was analyzed in terms of the atomic compositions and Hounsfield units (HUs). The density (in g/cm3) and effective atomic number of TE substitutes for the bone, lung, and soft tissue are 1.4 and 7.9, 0.5 and 10.0, and 1.0 and 5.9, respectively. Although the TE substitutes have slightly different physical properties, we think the phantom is acceptable because the HU values of the TE substitutes lie in the HU range of real tissues.

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes.

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

Postoperative pain and patient-controlled epidural analgesia-related adverse effects in young and elderly patients: a retrospective analysis of 2,435 patients.

In this retrospective study, data of 2,435 patients who received fentanyl and ropivacaine-based patient-controlled epidural analgesia (PCEA) for pain relief after elective surgery under general or spinal anesthesia were reviewed. Differences in postoperative pain, incidence of patient-controlled analgesia (PCA)-related adverse effects, and risk factors for the need for rescue analgesics for 48 hours postsurgery in young (age 20-39 years) and elderly (age ≥70 years) patients were evaluated. Although there were no significant differences in postoperative pain intensity between the two groups until 6 hours postsurgery, younger patients experienced greater postoperative pain intensity compared with older patients 6-48 hours postsurgery. While younger patients exhibited greater incidence of numbness, motor weakness, and discontinuation of PCA postsurgery, elderly patients exhibited greater incidence of hypotension, nausea/vomiting, rescue analgesia, and antiemetic administration. Upon multivariate analysis, low fentanyl dosage and history of smoking were found to be associated with an increased need for rescue analgesia among younger patients, while physical status classification III/IV and thoracic surgery were associated with a decreased need for rescue analgesia among the elderly. Discontinuation of PCA was more frequent among younger patients than the elderly (18.5% vs 13.5%, P=0.001). Reasons for discontinuation of PCA among young and elderly patients, respectively, were nausea and vomiting (6.8% vs 26.6%), numbness or motor weakness (67.8% vs 11.5%), urinary retention (7.4% vs 8.7%), dizziness (2.2% vs 5.2%), and hypotension (3.1% vs 20.3%). In conclusion, PCEA was more frequently associated with numbness, motor weakness, and discontinuation of PCA in younger patients and with hypotension, nausea/vomiting, and a greater need for rescue analgesics/antiemetics among elderly patients. Therefore, in order to minimize the adverse effects of PCEA and enhance pain relief, different PCEA regimens and administration/prevention strategies should be considered for young and elderly patients.

Clinical significance of thrombocytosis before preoperative chemoradiotherapy in rectal cancer: predicting pathologic tumor response and oncologic outcome.

BACKGROUND: Thrombocytosis is considered an adverse prognostic factor in various malignancies. However, the clinical significance of thrombocytosis in rectal cancer patients is unknown. We investigated the predictive value of thrombocytosis for pathologic tumor response to preoperative chemoradiotherapy (CRT) and oncologic outcomes in patients with rectal cancer. METHODS: A total of 314 patients who underwent preoperative CRT and subsequent rectal resection for rectal cancer were retrospectively evaluated at two tertiary institutions. Univariate and multivariate analyses of the clinical parameters were performed to identify markers predictive of a pathologic complete response (pCR). The Kaplan-Meier method was used to estimate 3-year disease-free and overall survival rates. RESULTS: Sixty-nine patients (22 %) had thrombocytosis before CRT, which significantly correlated with a large tumor size and advanced tumor depth. Thirty-nine patients (12.4 %) achieved a pCR. In the multivariate analyses, a platelet count of <370,000/µl (odds ratio 5.483; 95 % confidence interval, 1.271-23.653; P = 0.023) and a carcinoembryonic antigen (CEA) level of <5 ng/dl (odds ratio, 3.084; 95 % confidence interval, 1.291-7.368; P = 0.011) were identified as independent predictive factors for a pCR. Patients with pretreatment thrombocytosis had lower 3-year disease-free (P = 0.037) and overall survival (P = 0.001) rates than patients with normal pretreatment platelet counts. CONCLUSIONS: Thrombocytosis is a negative predictive factor for a pCR and has an adverse impact on survival in rectal cancer. The predictive value of this easily available clinical factor should not be underestimated, and better therapeutic strategies for these tumors are required.

Genetic alterations in intrahepatic cholangiocarcinoma as revealed by degenerate oligonucleotide primed PCR-comparative genomic hybridization.

Intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm of the biliary epithelium,is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of ICC are not well understood and only a few cytogenetic studies of ICC have been published. Recently, technique of degenerate oligonucleotide primed (DOP)-PCR comparative genomic hybridization (CGH) permits genetic imbalances screening of the entire genome using only small amounts of tumor DNA. In this study chromosomal aberrations in 33 Korean ICC were investigated by DOP-PCR CGH. The common sites of copy number increases were 20q (67%), 17 (61%), 11q11-q13 (42%), 8p12- qter (39%), 18p (39%), 15q22-qter (36%), 16p (36%), 6p21 (30%), 3q25-qter (27%), 1q41-qter (24%), and 5p14-q11.2 (24%). DNA amplification was identified in 16 carcinomas (48%). The frequent sites of amplification were 20q, 17p, 17q23-qter, and 7p. The most frequent sites of copy number decreases were 1p32-pter (21%) and 4q (21%). The recurrent chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of ICC.

Effects of p53 or p27 overexpression on cyclooxygenase-2 gene expression in head and neck squamous cell carcinoma cell lines.

BACKGROUND: Although cyclooxygenase-2 (COX-2) has been suggested to play an important role in carcinogenesis, the effects of tumor suppressors on COX-2 gene expression and the combined antitumor effects of tumor suppressors and COX-2 inhibitors have rarely been investigated. METHODS: The effects of p53 or p27 gene transfer on COX-2 expression by adenoviral vector and the combined effects of p53 or p27 gene transfer and COX-2 inhibitor exposure on the proliferation of cancer cells were investigated in head and neck squamous cell carcinoma (HNSCC) cell lines. RESULTS: Overexpression of p53 markedly downregulated the transcription of COX-2, but the overexpression of p27 did not affect COX-2 levels in HNSCC cell lines. The combined antitumor effects of p53 or p27 gene transfer and of a COX-2 inhibitor (NS 398) were mainly at least additive in terms of the inhibition of cell proliferation and cell cycle arrest and additive in terms of apoptotic induction. CONCLUSIONS: These results suggest that the overexpression of p53 could exert antitumor effects, at least in part, through the suppression of COX-2 gene expression, whereas growth suppression by the overexpression of p27 probably occurs by mechanisms other than the downregulation of COX-2 expression. In addition, the administration of COX-2 inhibitors, as an adjunct to p53 or p27 gene therapy, could offer a new strategy of cancer treatment and prevention.

Chromosomal aberrations in Korean nonsmall cell lung carcinomas: degenerate oligonucleotide primed polymerase chain reaction comparative genomic hybridization studies.

Chromosomal aberrations were investigated in 48 Korean nonsmall cell carcinomas of the lung (NSCLC), by degenerate oligonucleotide primed polymerase chain reaction comparative genomic hybridization. These included 16 adenocarcinomas, 27 squamous cell carcinomas (SCCs), and 5 large-cell carcinomas. The common sites of copy number increases were 3q26 approximately qter (23 cases, 48%); 8q23 approximately qter (46%); 20q13.1 (42%); 1q42 approximately qter (38%); 3q25 (38%); 21q22 (38%); and 22q13 (38%). DNA amplification was identified in 19 carcinomas (40%), and the frequent sites of amplification were 8q24 (seven cases), 3q26 (seven cases), and 3q27 (seven cases). The frequently under-represented chromosomal regions were Yq (38%), 4q25 approximately q26 (23%), and 4q31 (23%). In particular, gains of 3q26 approximately qter (74%), 15q (56%), and 19q (59%) and loss of 13q22 approximately q31 (26%) were more frequently detected in SCCs of the lung. These nonrandom aberrations can serve as starting points for the identification of potential oncogenes/tumor suppressor genes related to the tumorigenesis of Korean NSCLC.