Effects of vitamin A-deficiency and inflammation on the conducting airway epithelium of Syrian golden hamsters

The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48-55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6-7 fold in the epithelium lining the lobar bronchus (p less than 0.0002) and the bronchioles (p less than 0.0001), and the proportions of ciliated cells were decreased (p less than 0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that was not reversed when vitamin A was restored to the diet.     

Effects of vitamin A-deficiency and inflammation on the conducting airway epithelium of Syrian golden hamsters

The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48–55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6–7 fold in the epithelium lining the lobar bronchus (p< 0.0002) and the bronchioles (p< 0.0001), and the proportions of ciliated cells were decreased (p<0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that wasnot reversed when vitamin A was restored to the diet. This is contribution no. 2911 from the Pathobiology Laboratory     

Histogenesis and morphogenesis of epidermoid metaplasia in hamster tracheal organ explant culture

The formation of epidermoid metaplasia was studied in hamster tracheal epithelium in long-term serum-free organ explant culture. Explants were cultured up to 5 weeks in CMRL 1066 with antibiotics and amphotericin B. At 3 weeks there were rare small foci of epidermoid metaplasia and they became larger and more numerous at 4 and 5 weeks. Three dimensional reconstructions from serial sections demonstrated that the small deep-seated foci were discrete and did not reach the epithelial surface, whereas the larger foci were expansive and involved the full thickness of the explant epithelium. Each small focus consisted of a few swollen electron-lucent basal cells attached to the basal lamina, covered by a layer of flattened electron-dense secretory cells which formed a tight-fitting cap over the basal cells. The altered secretory cells displayed moderately well-developed rough endoplasmic reticulum and tonofilament bundles. During the early stages of formation the deep-seated metaplastic foci were completely covered by a layer of normal appearing cuboidal to low-columnar secretory and ciliated cells. Expansion of the metaplastic foci occurred by addition of flattened, electron-dense secretory cells to the cap so that multiple layers of altered secretory cells covered a core of basal cells, analogous to the structure of an onion. The secretory cells became cornified and with time the foci broke through the columnar mucociliary surface layer. In well-advanced foci, the uppermost cornified squames (metaplastic secretory cells) exfoliated into the tracheal lumen. The study emphasizes similarities and differences between the morphogenesis and histogenesis of epidermoid metaplasia in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies

All stages of regeneration in hamster tracheal epithelium were studied following a denuding mechanical injury. At 1 h all the cells had sloughed from the wound site leaving a bare and sometimes disrupted basal lamina. Viable cells at the wound margins rapidly changed shape, flattened and migrated to cover the denuded lesion by 12 h. In addition, epithelial cells that remained viable demonstrated sublethal changes that included the rapid discharge of mucous granules from secretory cells, internalization of cilia by ciliated cells and evidence of heterophagy in both cell types. By 24 h a wave of epithelial cell divisions occurred, primarily by secretory cells. This produced a multilayered epidermoid metaplasia that was best developed at 48 h. The metaplastic epithelium was largely composed of cells with both secretory (mucous granules) and epidermoid (tonofilament bundles and numerous desmosomes) characteristics. The peroxidase-antiperoxidase (PAP) method demonstrated a few keratin-positive cells in the wound as early as 12 h post-wounding and keratin was demonstrated in more cells by 24 h. All cells in the metaplastic wound epithelium were keratin-positive by 48 h. Following 48 h some of the most superficial keratinized cells sloughed from the epithelium and the keratin content of the remaining cells began to decline. At 72 h pre-ciliated and pre-secretory cells were seen in the wound. Pre-ciliated cells were characterized by an abundant electron-lucent cytoplasm, large pale nucleus, filiform apical microvilli and evidence of ciliogenesis, similar to that seen during fetal development. Pre-ciliated cells often contained apical mucous granules, apparently carried over from the parent secretory cells. With the appearance of these columnar cells the normal mucociliary morphology was restored in small wounds by 120 h, but some persistent epidermoid metaplasia remained in the large wounds through 168 h post-wounding. These data provide further evidence for the important role of secretory cells in the histogenesis of epidermoid metaplasia and the regeneration of normal morphology following injury. The implications of these findings in understanding the histogenesis of other lesions in the tracheo-bronchial epithelium are discussed.     

Vitamin A deprivation in hamsters. Correlations between tracheal epithelial morphology and serum/tissue levels of vitamin A

The effects of vitamin A deprivation on the tracheal epithelium of young hamsters were investigated. Colchicine was administered 6 h prior to death to induce metaphase arrest, thus making it possible to quantify the mitotic rates of basal cells and secretory (mucous) cells in the epithelium. Blood samples were taken from all hamsters, and liver samples from some, in order to measure serum and tissue levels of vitamin A. Age-matched controls were compared with the following groups of hamsters maintained on a vitamin A deficient diet: pre weight plateau animals (those gaining weight), weight plateau-early weight loss animals (those maintaining approximately the same weight for 3 or 4 days, followed in some cases by a loss of weight for 3 or 4 days), and prolonged weight loss animals (those showing a loss of weight for 5 or more days). Four week old hamsters in a pre weight plateau had undetectable amounts of vitamin A in their livers and declining levels in their serum, whereas 4 1/2 week old hamsters still gaining weight had barely detectable levels of vitamin A in their serum. Nevertheless, the tracheal epithelium of these animals was not different from controls in appearance, proportions of different cell types, mitotic rates of secretory and basal cells, or in the number of cells per millimeter of basement membrane (cell density). Vitamin A was undetectable in the serum and livers of hamsters in the weight plateau-early weight loss stage. At this time the tracheal epithelium showed minimal morphological change, with small focal areas of epidermoid metaplasia in some animals. The tracheas of animals in early weight loss were smaller than tracheas in the control group, and there was a trend towards an increase in the number of epithelial cells per millimeter basement membrane. Cell types in the minimally changed epithelium appeared nearly normal, but there was an increase in the proportion of basal cells, and an absence (or near absence) of division in both basal and secretory cells. Tracheal rings from hamsters in the prolonged weight loss stage were lined by a cornifying metaplastic epidermoid epithelium. Our findings demonstrate that barely detectable levels of vitamin A in the serum are sufficient to maintain normal growth and differentiation of hamster tracheal epithelium (late pre weight plateau stage). When vitamin A serum levels fall below detectable limits the animals enter the weight plateau-early weight loss stage. This stage is accompanied by an inhibition of tracheal epithelial cell growth, although nearly normal cellular differentiation is maintained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of epithelium on mucus secretion from feline tracheal submucosal glands

We studied the effect of airway epithelium on mucus secretion by use of an isolated tracheal submucosal gland preparation reported previously (J. Appl. Physiol. 60: 1237-1247, 1986). Mucus glycoconjugate release from submucosal glands of feline trachea was examined using [3H]glucosamine as a mucus precursor. Isolated glands showed significantly higher secretory responses to cholinergic, alpha-, and beta-adrenergic agonists and dibutyryladenosine 3',5'-cyclic monophosphate (average 400% of control) than the conventional tracheal mucosal explants, which contained epithelium and submucosal tissues in addition to submucosal glands (average 160% of control). The addition of isolated epithelium depressed the secretory response of isolated glands to the same level as that of tracheal explants. However, the supernatant from isolated epithelium failed to inhibit secretory responses to methacholine in isolated glands, suggesting that the epithelium-derived inhibitory factor to secretion may be short-lived. Leukotriene D4 antagonist (FPL 55712), cyclooxygenase and/or lipoxygenase inhibitors (indomethacin or BW 755C) caused no significant change in the inhibitory action of epithelium, suggesting that the inhibition is not due to arachidonic acid metabolites. The newly found secretory inhibitory action of epithelium is of particular interest in the pathogenesis of hypersecretion associated with epithelial damage.     

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. II. A histomorphological study

The effects of all-trans retinol and cigarette smoke condensate (CSC) on tissue morphology and cellular differentiation were investigated in vitamin A-deprived tracheal epithelium cultured in vitamin A-and serum-free hormone-supplemented medium. Physiological retinol concentrations prevented the development of hyperplasia and squamous metaplasia with or without keratinization, and induced differentiation to mucous cells. Squamous metaplastic foci with keratinization were observed during 12 days of culture with low retinol concentrations and with dimethylsulfoxide (DMSO) which was accompanied by an increased number of basal and indeterminate cells. CSC induced a dose-related hyperplasia and irregularly shaped foci of squamous metaplasia with atypical epithelial proliferation. In non-metaplastic epithelium, CSC exposure increased the number of ciliated cells. Hyperplasia and squamous metaplasia were inhibited if the tracheal rings were first treated with retinol followed by CSC exposure, or if the tracheas were simultaneously treated with retinol and CSC. CSC-exposure prior to retinol treatment induced similar histomorphological alterations as CSC alone.     

Histogenesis of benzo(a)pyrene-induced lesions in tracheal explants

Cytokinetic and histogenic alterations associated with the development of benzo(a)-pyrene (BP) induced epidermoid metaplasia were studied in tracheal explants derived from normal hamsters. Treatment of the explants with BP induced hyperplasia in both the basal and mucous cells. The hyperplasia of the basal cells persisted throughout the duration of the experiment whereas the hyperplasia of the mucous cells subsided between 7 and 10 days after treatment. This was accompanied by stimulation of ciliated cell differentiation and aberrant ciliogenesis which was not limited to the surface cells since some basal cells were observed differentiating into ciliated cells. Subsequently, the differentiation of basal cells into mucous cells was inhibited. Instead, the basal cells differentiated into metaplastic cells. With the progression of the lesions, the mucociliary surface layer was sloughed into the lumen due to the population pressure from the underlying actively proliferating metaplastic cells and their subsequent epidermoid differentiation. Approximately 50% of the explants exhibited focal areas of squamous metaplasia at 7 days after the treatment and extensive epidermoid metaplasia was present in approximately 90% of the explants at 10 days. These results support the hypothesis that BP induced epidermoid metaplasia of tracheal explants originates from the basal cells.     

Effects of B-retinyl acetate on human breast epithelium in explant culture

Explants of human breast (obtained aseptically from ten women ages 18-37) were examined and the vitamin A compound B-retinyl acetate was tested (at 3 X 10(-5) and 3 X 10(-6) M) for its effects on the fine structure and growth of the epithelium. In the absence of B-retinyl acetate, cells growing out from the explant (outgrowth cells) underwent squamous metaplasia, began to accumulate many intermediate filaments (tonofilaments), and revealed large desmosomes after 2 weeks in culture. In the presence of either concentration of B-retinyl acetate, the epithelial cells were largely prevented from undergoing squamous metaplasia. The glandular epithelium inside the explant maintained a typical secretory appearance for 2 weeks in control cultures (without the retinoid) and then began to show increased numbers of lysosomes and a loss of secretory granules. These glandular epithelial cells did not undergo squamous metaplasia, but they contained increased numbers of intermediate filaments. In contrast, glandular epithelium cultured in either concentrations of B-retinyl acetate appeared secretory for as long 6 weeks in culture, and intermediate filaments were not obvious. Autoradiographs demonstrated that both concentrations of B-retinyl acetate inhibited cell division in the outgrowth epithelium and in the (internal) glandular epithelium. The ability of B-retinyl acetate to reverse squamous metaplasia in the outgrowth cells was also tested. Outgrowth cells reversed to a more normal ductlike appearance after 6 weeks culture in standard medium followed by only 1 week in 20 micrograms/ml B-retinyl acetate. After 7 weeks in standard medium and 1 week in 10 micrograms/ml B-retinyl acetate, the cells showed a partial reversal of the squamous metaplasia.     

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium. An immunohistomorphological study using monoclonal antibodies to keratins

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.     

Effects of vitamin A-deprivation on hamster tracheal epithelium. A quantitative morphologic study

The effects of vitamin A-deprivation on the tracheal epithelium were studied in 35-day old hamsters that had been raised since birth on a vitamin A-deficient diet. Colchicine and 3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by 3HTdR labeling indices and mitotic rates. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphologic change. The mitotic rates and labeling indices were reduced 3 to 4-fold in basal cells and 14-fold in mucous cells (analyzed as percent of total number of each cell type) compared with controls. Thus, replication of mucous cells was more inhibited by lack of vitamin A, than replication of basal cells. The disparate hypoplasia of basal cells and mucous cells in epithelium showing minimal change, resulted in a relative increase in the proportion of basal cells and a relative decrease in the proportion of mucous cells, which could be erroneously interpreted as "basal cell hyperplasia". Proportions of preciliated and ciliated cells were also decreased compared to controls. At foci of stratification and epidermoid metaplasia, cell replication rates were increased over controls and more than 70% of all mitotic activity was associated with "non-basal" cells. Genesis of these lesions was coincident with cell death and cell loss. The histogenesis of stratification and epidermoid metaplasia was characterized. Morphological evidence indicated that these lesions were closely related histogenetically and were composed, for the most part, of altered mucous cells which expressed dual phenotypes i.e. keratinization and mucus synthesis.     

Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.     

Tracheal Dysplasia Precedes Bronchial Dysplasia in Mouse Model of N-Nitroso Trischloroethylurea Induced Squamous Cell Lung Cancer

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5⁺/K14⁺ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP⁺) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP⁺ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5⁺ basal cells in the trachea, and altered bronchial mitotic population from CCSP⁺ to K5⁺ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.     

Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury

Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases.     

Histochemical localization of carbonic anhydrase in the trachea of the guinea pig

 Tissue specimens from guinea pigs were examined using an enzyme-histochemical reaction to explore the presence of carbonic anhydrase (CA) activity in the trachea. CA activity was detected in a group of morphologically distinct epithelial cells, in goblet cells, and in glands of the tracheal mucosa. The epithelial cells showing CA activity were distributed singly and sparsely throughout the entire trachea. These cells showed a wide morphological variability and were clearly different from those forming the pseudostratified ciliated epithelium. Their number was higher in sections closer to the tracheal bifurcation than in those near the larynx. Although the nature of these cells is unknown, based on their morphological and histochemical characteristics and their distribution, they may represent a specialized chemoreceptor. To our knowledge, this is the first report of CA localized in tracheal epithelial cells. Accepted: 6 March 1996     

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

Summary The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.     

Evolution and testosterone content of the epididymis during the annual cycle of the lizard Lacerta vivipara

The lizard epididymis provides a model for studying the control, by testosterone, of a secretory activity related to the physiology of spermatozoa. To evaluate seasonal changes and to establish chronological correlations between the structure of the epididymis and its testosterone content, lizards (Lacerta vivipara) were killed between March (emergence) and October (retreat). The epididymal tissue was examined histologically and assayed for testosterone content. Ten stages of development were defined, mainly on the basis of the epithelial structure and the morphological features of secretory activity. Degeneration of the epithelium after the breeding period and its subsequent renewal also were considered. Increased epithelial height and secretory activity coincided with a progressive rise of the testosterone level, and a severe atrophy followed a sudden reduction of blood testosterone. Reorganization of the epithelium takes place when testosterone is at its lowest level, and the hormonal dependency of this stage is questionable. This study confirms in vivo, during a sexual cycle, experimental evidence previously obtained concerning testosterone's control of the secretory activity of the lizard epididymis.     

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro‐proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro‐proliferative effects of ANW on fibroblasts are dependent on insulin‐like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC₅₀ of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki‐67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.     

Biosynthesis of respiratory-tract mucins. A comparison of canine epithelial goblet-cell and submucosal-gland secretions

1. A modified canine tracheal organ culture system was used to investigate differences between mucous secretions of epithelial goblet cells and the submucosal glands. 2. Denuded explants were prepared by removing goblet, ciliated and basal cells from the surface epithelium leaving an intact basement membrane and viable submucosa. 3. Denuded explants actively incorporated radioactive precursors into secreted macromolecules when cultured in medium 199 containing label. 4. Chromatography on Bio-Gel A-150m and electrophoresis on 1% agarose gels indicated that epithelial goblet cell secretions were relatively more sulphated than submucosal glandular secretions. 5. The glandular structures were shown to respond to a parasympathomimetic agent.     

A primary culture model of differentiated murine tracheal epithelium

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.