Contribution of emphysema and small airways in COPD.

BACKGROUND: The contribution and role of emphysema and small airways disease in causing expiratory airflow limitation in COPD is controversial. METHODS: We obtained high-resolution thin-section 2-mm CT scans of the lung for emphysema grading and lung function in 116 consecutively seen COPD outpatients with fixed expiratory airflow limitation. In this group, inflated whole lung(s) were subsequently obtained in 24 patients (23 autopsy, 1 surgery) for morphologic studies and results compared with lung CT. Airway histologic condition was studied in 17 of the 24 patients. RESULTS: There was fair to weak negative correlation between CT emphysema score and either FEV1/FVC percent (r = -0.51, p = 0.001) or FEV1 percent predicted (r = -0.31, p = 0.001). In only 24 of the 81 patients (30%) with FEV1 less than 50% predicted, the CT emphysema score was 60 or more, indicating severe emphysema. In the 24 patients studied, there was a good correlation (r = 0.86, p = 0.001) between CT and pathologic grade of emphysema. While respiratory bronchioles (RBs) and membranous bronchioles (MBs) demonstrated marked morphologic abnormalities, there was a weak correlation with emphysema grade (for RB, r = 0.36, p = 0.16; for MB, r = 0.41, p = 0.10) or with FEV1 percent predicted (for RB, r = -0.21, p = 0.42; for MB, r = -0.28, p = 0.28). There was no correlation between emphysema and FEV1 percent predicted (r = -0.13, p = 0.54). CONCLUSIONS: High-resolution CT lung scans are an in vivo surrogate to quantitate moderate to severe morphologic emphysema. Emphysema does not appear to be primarily responsible for severe expiratory airflow limitation in most patients with severe COPD. There was no correlation between severity of small airway histologic condition and emphysema or FEV1 percent predicted. The causes of the lesions responsible for small airways obstruction need to be identified.

Effect of adenovirus E1A on ICAM-1 promoter activity in human alveolar and bronchial epithelial cells.

In previous studies we demonstrated that the E1A DNA and proteins of group C adenovirus are present in excess in the lungs of patients with chronic obstructive pulmonary disease (COPD). Because adenovirus EIA gene products are known to regulate the expression of many genes by interacting with cellular transcription factors, we postulated that E1A enhances the production of inflammatory mediators and exacerbates the inflammatory process in smokers' lungs. We reported that LPS-induced ICAM-1 expression in A549 cells is upregulated by E1A. In the current study we investigated whether this regulation is mediated through the ICAM-1 promoter. A549 cells and primary human bronchial epithelial (HBE) cells were transiently cotransfected with a plasmid containing the ICAM-1 enhancer-promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene (pBS-CAT-P) and either a plasmid carrying the adenovirus 5 E1A gene (pE1Aneo) or a control plasmid (pneo). To compare the effect of transient versus stable E1A expression on the activity of this promoter, we also transiently transfected stable E1A-expressing A549 cells with pBS-CAT-P. Transient cotransfection of pE1Aneo and pBS-CAT-P had no effect on basal ICAM-1 promoter activity in A549 or HBE cells. After stimulation of A549 cells with TNF-alpha, IFN-gamma, or LPS, promoter activity was increased by two- to threefold in the presence of adenovirus EIA. In HBE cells, on the other hand, E1A repressed the ICAM-1 promoter after stimulation with IFN-gamma and LPS with little change after TNF-alpha stimulation. In stable E1A transfectants, ICAM-1 promoter activity was 2 to 2.5 times higher than in control transfectants with or without stimulation with TNF-alpha or LPS. These findings suggest that EIA can modulate the activity of the ICAM-1 promoter in lung epithelial cells and this modulation is different in cells of alveolar origin compared to bronchial epithelial cells.

P63 expression in lung carcinoma: a tissue microarray study of 408 cases.

p63 is a recently discovered member of the p53 family that has been shown to be important in the development of epithelial tissues. p63 may also play a role in squamous cell carcinomas of the lung, head and neck, and cervix, and its expression is increased in these tumors. The purpose of this study was to investigate the expression of p63 in a broad spectrum of histologic types of lung tumors. A total of 441 cases of primary lung tumors with follow-up data were identified, and the paraffin-embedded tissue blocks were used to construct a duplicate core tissue microarray. After review of the tissue cores, 408 cases, consisting of 123 squamous cell carcinomas, 93 adenocarcinomas, 68 large cell carcinomas, 68 classic carcinoids, 31 atypical carcinoids, 11 large cell neuroendocrine carcinomas, and 14 small cell carcinomas, were adequate for analysis. Immunohistochemistry was performed at 2 different laboratories using monoclonal antibody 4A4 to detect the expression of p63, using different staining protocols. p53 expression was also studied with immunohistochemistry using monoclonal antibody DO-7. Kaplan-Meier curves were plotted to compare the survival of p63-expressing versus nonexpressing tumors. A large proportion of squamous cell carcinomas expressed p63 (96.9%), most showing strong positive nuclear immunoreactivity. Expression in other nonsmall cell lung cancers was also present. Thirty percent of adenocarcinomas and 37% of large cell carcinomas showed p63 expression. In the neuroendocrine tumors, an increasing proportion of tumors stained for p63 as tumor grade increased; 1.9% of classic carcinoids, 30.8% of atypical carcinoids, 50% of large cell neuroendocrine carcinomas, and 76.9% of small cell carcinomas were positive. Approximately half of the positively staining neuroendocrine cases showed strong staining. Expression of p63 was of prognostic significance in neuroendocrine tumors (P < 0.0001), with higher-grade tumors more likely to express p63. Correlation between p63 and p53 expression was not observed (P = 0.18) in nonsmall cell lung cancer; however, a significant correlation between the 2 markers was found in neuroendocrine tumors (P < 0.0001). p63 staining was repeated with a different staining protocol, yielding similar results overall but a lower percentage of positive cases (34.2% vs. 48.4% of tumors positive). In conclusion, p63 expression is consistently expressed in squamous cell carcinoma in the lung, but is also expressed in a subset of adenocarcinomas and large cell carcinomas. Pulmonary neuroendocrine tumors also show p63 staining in some instances, particularly in higher-grade tumors, and the majority of small cell carcinomas are p63-positive. These results suggest that p63 may be involved in oncogenesis in a broader range of tumors than was previously thought.

Comparison of the neutral red and methylene blue assays to study cell growth in culture.

The neutral red and methylene blue in vitro cytotoxicity assays were compared under a variety of conditions using normal human ovarian epithelial cells to determine whether either assay is superior for studying cell growth. The results were standardized against a DNA spectrofluorometric assay. Although the assays were equivalent in reflecting cell number, each has specific advantages: while neutral red discriminates between viable and dead cells, the methylene blue assay is more sensitive and easier to perform.

Evaluation of small sample cDNA amplification for microdissected airway expression profiling in COPD.

Small airway obstruction and emphysematous destruction account for the airflow limitation that defines chronic obstructive pulmonary disease (COPD). While laser capture microdissection (LCM) allows gene expression studies in small airways separately from the surrounding parenchyma, tissue size limits the number of genes examined. The present study evaluates the Clontech SMART amplification to test the hypothesis that this amplification provides RNA in sufficient quantity and quality to evaluate large numbers of genes in airways < 2 mm diameter obtained by LCM. Commercial reference RNA was amplified 200-fold and the expression levels of 51 genes relative to the unamplified RNA had a correlation coefficient of 0.84. For two pairs of RNA preparations (commercial placenta versus commercial lung; lung sections prepared for LCM from GOLD 0 (at risk for COPD) versus GOLD 2 (moderate disease) patients linear regression of Delta Ct's (delta cycle thresholds) of unamplified versus amplified RNA gave correlation coefficients of R = 0.95. In RNA from microdissected small airways, expression patterns in all GOLD classes of COPD severity were very similar between unamplified and amplified RNA. We conclude that SMART amplification provides cDNA sufficient for studying large numbers of genes even in laser-captured small airways and this cDNA maintains the relative expression found in corresponding unamplified RNAs.

Light-controlled Leaf Expansion in Peas Grown under Different Light Conditions.

Several photosystems control leaf expansion in Alaska peas (Pisum sativum). Phytochrome is known to control expansion in dark-grown peas. But plants exposed briefly to red light are insensitive to phytochrome, an insensitivity that is itself phytochrome-produced. Leaf expansion in these plants is promoted by 440 or 630 nm of light (probably mediated by protochlorophyll). Plants grown in white fluorescent light required simultaneous exposure to high intensity blue and yellow light for promotion of leaf expansion. Since these results parallel studies on light-controlled inhibition of stem elongation, shoot growth as a whole is coordinated by these photosystems. Such coordination might be a mechanism of plant competition for light.

Control of Leaf and Stem Growth in Light-grown Pea Seedlings by Two High Irradiance Responses.

The control exerted by light on leaf and stem growth in light-grown Alaska pea seedlings was studied during the main photoperiod. Two high irradiance responses were observed. The action spectrum for one had a single sharp peak at 600 nanometers. The action spectrum for the other showed a broad peak between 440 and 470 nanometers. These two light responses must be activated simultaneously for any inhibition of stem growth or promotion of leaf growth. Both action spectra may be explained in terms of the high irradiance response of phytochrome.

Somatostatin concentrations in the pancreatic-intestinal tissues of the sea lamprey, Petromyzon marinus L., at various periods of its life cycle.

1. Somatostatin concentrations were measured in homogenates of the pancreas-intestinal tissues from each period of the life cycle of Petromyzon marinus using radioimmunoassay. 2. Levels were very low in larva (4.0 pg/mg wet weight) and in the first three stages of metamorphosis, but increased from stage 4 onwards and reached a high in upstream-migrating adults (210.0 ng/mg). 3. These data correlate well with our previous morphological and immunohistochemical observations on the morphogenesis of somatostatin-containing D-cells during the life cycle and indicate that the increased concentration of hormone accompanies the development of the endocrine pancreas in lampreys.

Changes in photosensitive stem growth in intact peas following irradiation.

Etiolated pea seedlings given a short red-light pretreatment followed by 30 hr of darkness no longer showed a typical red-light inhibition of internode elongation. The induction of phytochrome-insensitive growth was itself mediated by phytochrome, since far-red light reversed the effect of the short red-light pretreatment. Peas grown in white light showed a similar insensitivity to red light. However, in this instance the phytochrome system exerted some control over internode elongation since far-red light promoted growth slightly, and this effect was red-reversible.The loss of sensitivity to red light was correlated with a decrease in the amount of spectrophotometrically assayable phytochrome. However, the loss of phytochrome occurred in a relatively short time compared to the period necessary to attain maximal insensitive growth (2 hr versus about 30 hr). Also, after the red-light and dark pretreatment, although 40% of the original amount of phytochrome remained, red light had no effect on elongation. Neither loss of phytochrome nor loss of red-light sensitive growth was observed at 0 to 1 degrees .

Light-controlled Stem Elongation in Pea Seedlings Grown under Varied Light Conditions.

There appears to be an orderly transition from one photosensitive state to another in light-controlled stem elongation in the garden pea, Pisum sativum L. cv. Alaska. Stem elongation in dark-grown plants is known to be phytochrome-controlled. However, seedlings are insensitive to phytochrome after a red light pretreatment. An action spectrum for inhibition in these seedlings has peaks at 440 and 620 nm. Protochlorophyll is suggested as the photoreceptor. If these red pretreated seedlings are further exposed to white light, sensitivity to 440 to 620 nm light is lost. Promotion by blue-green light is the only photoresponse shown by these seedlings. Inhibition of completely white light-grown seedlings required simultaneous exposure to high intensity blue light and 600 nm light.

Morphogenesis and distribution of the endocrine pancreas in adult lampreys.

The endocrine pancreas of larval lampreys appears as islets of cells isolated in the submucosa and those both continuous with, and within, the gut epithelium at the intestinal-oesophageal-bile duct junction. The islets, and occasionally follicles, are composed of only insulin-secreting (B) cells. During metamorphosis, the bile duct either completely degenerates or its epithelium transforms into a caudal endocrine pancreas while a cranial pancreas appears as a specialization and expansion of the larval pancreas. Immunocytochemistry and histochemistry demonstrates that there is a wide variation in the distribution of the pancreatic tissue in adults of lamprey species, and this variation may result from interspecific differences in morphogenetic events at metamorphosis. Despite species variability in its distribution, the endocrine pancreatic tissue in all adult lampreys is composed of equal numbers of B cells and somatostatin-secreting (D) cells, but there are no glucagon-secreting (A) cells. Immunocytochemistry reveals that B and D cells of the caudal pancreas differentiate from cells of the larval bile duct during metamorphosis of the sea lamprey,Petromyzon marinus.

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L.

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B- and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.

Growth of normal human ovarian surface epithelial cells in reduced-serum and serum-free media.

The human ovarian surface epithelium (OSE) is believed responsible for over 85% of ovarian cancers, yet little is known about the normal biology of these cells. To date, culture of OSE has only been reported in media with high serum supplements. We have developed two media, one with less than 1% of serum (OSEM-1) and the other comprised of highly purified and defined materials (OSEM-2), which allow us to study OSE under relatively defined conditions. By substituting 0.05% of Pedersen's fetuin for 15% fetal bovine serum (FBS) with Medium 199/MCDB105 basal medium, the cell numbers reached 50 to 60% of those in the presence of 15% FBS over 7 days. However, over several weeks, the total number of population doublings achieved were comparable to those in 15% FBS. Addition of insulin, transferrin, ethanolamine, lipoic acid, and phosphatidylcholine to the medium with Pedersen's fetuin (OSEM-1) enhanced growth up to 20% more than in their absence. Supplementation of M199/105 with highly purified (> 99%) fetuin, alpha 2-macroglobulin, and hydrocortisone resulted in a defined medium (OSEM-2) that permitted 1 to 2 doublings/7 days. In addition, cells maintained a more normal, epithelial-like morphology in culture for a longer period in the presence of Pedersen's or purified fetuin than in M199/105/15% FBS, thus increasing the number of morphologically normal cells available for experimentation. Addition of 0.05% Pedersen's fetuin to M199/105 in the presence of 6 to 8% FBS resulted in levels of growth equivalent to those in M199/105/15% FBS alone. We are now able to study the effects of various compounds on the growth and differentiation of OSE under defined conditions, and have reduced the requirement for FBS to produce large numbers of OSE cells.

Development of the adult endocrine pancreas during metamorphosis in the sea lamprey, Petromyzon marinus L. I. Light microscopy and autoradiography.

The results of this study were found to support the hypothesis put forth by Boenig (Z. Mikrosk-Anat. Forsch., 17:125-184, 1929) that the caudal pancreas of adult lamprey develops from the epithelium of the extrahepatic common bile duct in that the bile duct cells were found to undergo a great proliferation during the early stages of metamorphosis, with a large number of the cells incorporating 3H-thymidine. If the bile duct degenerated as suggested by Barrington (The Biology of Lampreys. Academic Press, London, pp. 135-169, 1972), this uptake would not be expected. The cranial pancreas was determined to develop in a similar manner to the larval islets, with formation of the islets taking place within the intestinal/diverticular epithelium. The newly formed islets would migrate into the surrounding connective tissue. During the later stages of metamorphosis a small number of cells was found to incorporate the tritiated thymidine within mature islets.

Development of the adult endocrine pancreas during metamorphosis in the sea lamprey, Petromyzon marinus L. II. Electron microscopy and immunocytochemistry.

The development of the adult endocrine pancreas was followed throughout metamorphosis in the sea lamprey using electron microscopy and immunocytochemistry. It was discovered that the caudal pancreas develops from the larval extrahepatic common bile duct through the process of transdifferentiation (dedifferentiation/redifferentiation). Early in metamorphosis the bile duct epithelial cells possess large vacuoles, resembling autophagic vacuoles, containing recognizable cell material. There is a loss of the large bundles of intermediate filaments characteristic of the larval bile duct epithelium. These same cells are then seen to contain granules immunoreactive for insulin. Pancreatic islets develop within the base of the bile duct epithelium from these transdifferentiated cells and migrate into the surrounding connective tissue to form the caudal pancreas. The cranial pancreas was found to develop from the epithelia lining the developing adult diverticulum and anterior intestine in a similar fashion as those in the larva. The second cell type to appear in either portion of the developing pancreas is similar to the third cell type of the adult: cells immunoreactive for somatostatin do not appear until late in metamorphosis in either region.

Immunodetection of adenoviral E1A proteins in human lung tissue.

The adenoviral E1A proteins possess the ability to associate with the DNA binding domains of a number of transcription factors, and in this manner promiscuously to activate a wide variety of genes. The present study was designed to determine whether this protein is expressed in human lungs where nonlytic adenoviral infection has been demonstrated. Lung tissue from 12 patients harboring trace amounts of viral DNA were examined along with A549 cells infected with adenovirus 5 and uninfected Graham 293 (G293) cells as controls. Immunohistochemical staining was used to identify E1A proteins. The control studies examined both types of cultured cells either grown on coverslips, as cryosections of cells embedded in blocks, and or as formalin-fixed, paraffin-embedded sections. E1A proteins were detected in all three control preparations in both types of cells and were detected in the nucleus of adenovirus 5-infected A549 cells 4 h postinfection. Generally, all preparations of infected A549 cells showed greater of staining than the corresponding preparation of G293 cells. Formalin-fixed, paraffin-embedded cells gave the best morphology. Immunolabeling for adenovirus E1A proteins was also demonstrated in six of 12 paraffin-embedded lung samples. We conclude that adenovirus E1A proteins are expressed in human lung tissue and speculate that they may contribute to the pathogenesis of chronic obstructive pulmonary disease by amplifying the airways inflammation associated with cigarette smoking.

Adenovirus E1A upregulates interleukin-8 expression induced by endotoxin in pulmonary epithelial cells.

Adenovirus E1A DNA and proteins are frequently detected in lungs of patients with chronic obstructive pulmonary disease. Because adenovirus E1A can regulate host gene expression by interacting with cellular transcription factors, we postulate that E1A enhances synthesis of inflammatory mediators. To examine this possibility, we measured the expression of inflammatory cytokines in E1A-producing A549 human pulmonary epithelial cells and control cells. Interleukin (IL)-8 mRNA was markedly induced by lipopolysaccharide (LPS) in E1A-producing cells but not in controls. IL-8 protein levels were elevated in parallel. In both cell types, monocyte chemotactic and activating factor mRNA induced by LPS was low, and transforming growth factor-beta 1 mRNA was not affected. IL-1 beta, IL-6, granulocyte macrophage colony-stimulating factor, and granulocyte colony-stimulating factor mRNAs were too low to show any effect of E1A. We conclude that the LPS responsiveness of A549 pulmonary epithelial cells is altered by adenoviral E1A by upregulating IL-8. We speculate that this mechanism may be important in the amplification of the inflammatory process of lungs to other irritants.

Adenovirus E1A gene dysregulates ICAM-1 expression in transformed pulmonary epithelial cells.

Previous studies from our laboratory demonstrated that adenovirus E1A DNA and proteins are detected in lungs of patients with chronic obstructive pulmonary disease (COPD). Since adenovirus E1A gene products are known to regulate the expression of many genes by interacting with cellular transcription factors, we postulate that E1A enhances the production of inflammatory mediators and exacerbates the inflammatory process in smokers' lungs. To examine this possibility, we transfected A549 human pulmonary epithelial cells with a plasmid carrying the adenoviral E1A gene and isolated stable transfectants expressing E1A proteins. These E1A-producing clones were tested for intercellular adhesion molecule-1 (ICAM-1) expression. As compared with parental cells or cells transfected with control plasmid, ICAM-1 expression was suppressed after IFN-gamma stimulation but markedly increased by LPS stimulation of E1A-positive cells. This LPS-mediated ICAM-1 induction was serum-dependent but the LPS receptor, CD14, was not detected on the surface of the E1A transfectants. We conclude that E1A proteins modulate ICAM-1 induction by inflammatory stimuli and render lung epithelial cells sensitive to LPS, and suggest that dysregulation of inflammatory mediator expression by adenoviral E1A could amplify the inflammatory process present in airways of smokers to produce COPD.

A comparison of endocrine pancreatic tissue in adults of four species of lampreys in British Columbia: a morphological and immunohistochemical study.

Histological techniques and immunohistochemistry with antisera against insulin and somatostatin were used in a study of the identification and the distribution of endocrine pancreatic tissue in adults of four species of lampreys in British Columbia. The fine structure of the pancreatic cells of one of the species was also examined. The four species (Lampetra ayresi, Lampetra tridentata, Lampetra macrostoma, and Lampetra richardsoni) all possess cranial and caudal pancreatic masses and an intervening intermediate cord, but there are variations in amount and deposition of the pancreatic tissue with respect to the alimentary canal and the liver which appear to be species specific. There is also some variation in the distribution of pancreatic tissue between L. richardsoni of different stream populations. A newly identified parasitic variety of L. richardsoni in Morrison Creek, called L. richardsoni var. marifuga, usually has no cranial pancreas but has an extensive intermediate cord. The distribution of pancreatic tissue in these lampreys is of relevance to taxonomy and to variations in development of the endocrine pancreas during lamprey metamorphosis. The pancreatic tissue of all species and var. marifuga is composed of cells which are immunoreactive to either insulin or somatostatin antisera. The fine structure of cells from the cranial and caudal pancreatic tissue from L. ayresi does not vary with saltwater and freshwater acclimation. Insulin-containing B cells and somatostatin-containing D cells are present in equal numbers in both pancreatic regions and the cells each have characteristic cytoplasmic granules. A third cell type is most abundant in the cranial pancreas but it is of unknown function.