Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction

Cellular mechanisms contributing to renewal of terminal bronchioles remain poorly defined. Our previous studies identified pollutant-resistant Clara cell secretory protein (CCSP)-expressing stem cells that localize to the neuroepithelial body (NEB) and contribute to renewal of the proximal bronchiolar epithelium. However, activation of NEB-associated stem cells is unlikely to contribute to renewal of terminal bronchiolar epithelium because of the paucity of NEBs at this location. Goals of this study were to determine the location and properties of cells contributing to renewal of terminal bronchioles after Clara cell depletion. Pollutant-resistant CCSP-expressing cells were identified that localized to the bronchoalveolar duct junction (BADJ) and contribute to restoration of a phenotypically diverse epithelium. CCSP-expressing cells comprise the predominant proliferative population in initial terminal bronchiolar repair and include a population of label-retaining cells suggesting that they maintain characteristics of a stem cell population. Furthermore, immunohistochemical co-localization studies involving CCSP and the NEB-specific marker calcitonin gene-related peptide indicate that BADJ-associated CCSP-expressing stem cells function independently of NEB microenvironments. These studies identify a BADJ-associated, NEB-independent, CCSP-expressing stem cell population in terminal bronchioles and support the notion that regiospecific stem cell niches function to maintain epithelial diversity after injury.     

In vitro degradation of silk fibroin

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg/ml Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.     

Ultrastructure of Ehrlichia canis

The ultrastructure of Ehrlichia canis was examined in both pulmonary mononuclear cells and in monocytes cultured from an infected dog. The cytoplasmic inclusions, or morulae, of E. canis consisted of a membrane-lined vacuole-containing elementary bodies which varied in size and number. The elementary bodies were bound by two trilamellar membranes. The organism shared morphological properties of both the genus Rickettsia and genus Chlamydia.     

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.     

Fast implementation of the single scatter simulation algorithm and its use in iterative image reconstruction of PET data

In positron emission tomography (PET), scatter correction is usually performed prior to image reconstruction using a more or less exact model of the scatter processes. These models require estimates of the true activity and object density distributions of the imaged object. The problem is that these estimates are computed from measured data and, therefore, already contain scattered events. The purpose of this work was to overcome this problem by incorporating scatter characteristics directly into the process of iterative image reconstruction. This could be achieved by an optimized implementation of the single scatter simulation (SSS) algorithm, which results in a significant speed-up of the scatter estimation procedure. The scatter simulation was then included in the forward projection step of maximum likelihood image reconstruction. The results demonstrate that this approach leads to a more exact estimation of the scatter component which cannot be obtained by a simple sequential data processing strategy.     

4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.     

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le a antigen

Several lines of evidence indicate that sialosyl Le a , tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for a1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le a tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le a and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le a -negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metas-tases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le a antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.© Kluwer Academic Publishers 1998     

Effects of stimulant medications on the EEG of children with Attention-Deficit/Hyperactivity Disorder Predominantly Inattentive type.

Stimulant medications are the most commonly-used treatments for Attention-Deficit/Hyperactivity Disorder (ADHD) in North America and Australia, although it is still not entirely known how these medications work. This study investigated the effects of stimulant medications on the EEG of children with the Inattentive type of ADHD. An initial EEG was recorded during an eyes-closed resting condition and Fourier transformed to provide absolute and relative power estimates for the delta, theta, alpha and beta bands. Theta/alpha and theta/beta ratios were also calculated. Subjects were placed on a 6-month trial of a stimulant and a second EEG was recorded at the end of the trial. Subjects were included in this study only if they showed a good clinical response during the trial. The unmedicated ADHD group had significantly greater absolute and relative theta, less relative alpha, and higher theta/alpha and theta/beta ratios than the control group. The stimulant medications resulted in a normalisation of the EEG, with changes in the theta, alpha and beta bands being most evident. These results suggest that stimulants act to increase cortical arousal in children with ADHD, normalising their EEG.     

TGFbeta1 enhances formation of cellular Abeta/apoE deposits in vascular myocytes

Brain injury increases the risk of Alzheimer's disease (AD) through unknown mechanisms. We studied deposition of amyloid-beta protein (Abeta) in cells exposed to transforming growth factor beta1 (TGFbeta1), a cytokine that regulates cell metabolism during brain injury, and apolipoproteinE (apoE), the major lipid transporter in the brain. The studies were conducted by using brain vascular smooth muscle cells that are engaged in beta-amyloidosis in vivo and produce Abeta in cell culture. We found that cell treatment with TGFbeta1 together with apoE4 strongly increased the amount of cellular Abeta. The intracellular Abeta co-localized with apoE but not with TGFbeta, similarly as in vascular beta-amyloid. Some cellular Abeta/apoE deposits increased in size and persisted in culture even after the TGFbeta1 and apoE4 were removed. The appearance of cellular deposits of Abeta was associated with increased production of the amyloid-beta precursor protein and cellular retention of its mature form. The results suggest that the concomitant presence of apoE and TGFbeta1 can trigger vascular beta-amyloidosis by inducing intracellular formation of stable Abeta/apoE deposits.