Monolayer Cultures Of Epithelial Cells Research Articles

Potential Contribution of Cell Adhesion Molecule 1 to the Binding of SARS-CoV-2 Spike Protein to Mouse Nasal Mucosa

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first infects the host nasal mucosa, where the viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the mucosal cells. This study aimed at searching host cell surface molecules that could contribute to the infection in two views; abundance on host cells and affinity to the spike protein. Since the nasal mucosa is lined by respiratory and olfactory epithelia, and both express an immunoglobulin superfamily member cell adhesion molecule 1 (CADM1), whether CADM1 would participate in the spike protein binding was examined. Immunohistochemistry on the mouse nasal cavity detected CADM1 strongly in the olfactory epithelium at cell-cell contacts and on the apical surface but just faintly in the respiratory epithelium. In contrast, ACE2 was detected in the respiratory, not olfactory, epithelium. When mice were administered intranasally with SARS-CoV-2 S1 spike protein and an anti-CADM1 ectodomain antibody separately, both were detected exclusively on the olfactory, not respiratory, epithelium. Then, the antibody and S1 spike protein were administered intranasally to mice in this order with an interval of 1 h. After 3 h, S1 spike protein was detected as a protein aggregate floating in the nasal cavity. Next, S1 spike protein labeled with fluorescein was added to the monolayer cultures of epithelial cells exogenously expressing ACE2 or CADM1. Quantitative detection of fluorescein bound to the cells revealed that S1 spike protein bound to CADM1 with affinity half as high as to ACE2. Consistently, docking simulation analyses revealed that S1 spike protein could bind to CADM1 three-quarters as strongly as to ACE2 and that the interface of ACE2 was similar in both binding modes. Collectively, intranasal S1 spike protein appeared to prefer to accumulate on the olfactory epithelium, and CADM1 was suggested to contribute to this preference of S1 spike protein based on the molecular abundance and affinity.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes.

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Regulation of Enteroendocrine Cell Networks by the Major Human Gut Symbiont Bacteroides thetaiotaomicron.

Gut microbes have critical roles in maintaining host physiology, but their effects on epithelial chemosensory enteroendocrine cells (EEC) remain unclear. We investigated the role that the ubiquitous commensal gut bacterium Bacteriodes thetaiotaomicron (Bt) and its major fermentation products, acetate, propionate, and succinate (APS) have in shaping EEC networks in the murine gastrointestinal tract (GIT). The distribution and numbers of EEC populations were assessed in tissues along the GIT by fluorescent immunohistochemistry in specific pathogen free (SPF), germfree (GF) mice, GF mice conventionalized by Bt or Lactobacillus reuteri (Lr), and GF mice administered APS. In parallel, we also assessed the suitability of using intestinal crypt-derived epithelial monolayer cultures for these studies. GF mice up-regulated their EEC network, in terms of a general EEC marker chromogranin A (ChrA) expression, numbers of serotonin-producing enterochromaffin cells, and both hormone-producing K- and L-cells, with a corresponding increase in serum glucagon-like peptide-1 (GLP-1) levels. Bt conventionalization restored EEC numbers to levels in SPF mice with regional specificity; the effects on ChrA and L-cells were mainly in the small intestine, the effects on K-cells and EC cells were most apparent in the colon. By contrast, Lr did not restore EEC networks in conventionalized GF mice. Analysis of secretory epithelial cell monolayer cultures from whole small intestine showed that intestinal monolayers are variable and with the possible exclusion of GIP expressing cells, did not accurately reflect the EEC cell makeup seen in vivo. Regarding the mechanism of action of Bt on EECs, colonization of GF mice with Bt led to the production and accumulation of acetate, propionate and succinate (APS) in the caecum and colon, which when administered at physiological concentrations to GF mice via their drinking water for 10 days mimicked to a large extent the effects of Bt in GF mice. After withdrawal of APS, the changes in some EEC were maintained and, in some cases, were greater than during APS treatment. This data provides evidence of microbiota influences on regulating EEC networks in different regions of the GIT, with a single microbe, Bt, recapitulating its role in a process that may be dependent upon its fermentation products.

Production of progenitor cells from primary human epithelial cell monolayer cultures.

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

3D Model Replicating the Intestinal Function to Evaluate Drug Permeability.

Animal models are essential in drug development but present many concerns in the practical and ethical sense. To avoid the unnecessary use of animals other models are used in the beginning of any scientific discovery, the in vitro models. The relevance of in vitro cell based culture models for studying intestinal drug absorption and transcytosis during early stages of drug development is undeniable. Several in vitro co-culture models have been described for this purpose, however excluding the integration of the complex intestinal architecture and neglecting different physiological mechanisms involved in the drug transport. 2-D cell cultures are the current standard, but despite their widely use, they no longer are considered the most trustworthy in vitro models since they do not mimic many aspects that happen in vivo. The simulation of a complete microenvironment capable of mimicking the intestinal mucosa requires therefore further investigation, particularly focused in addressing the abovementioned unmet needs. 3D models came as bridge between the in vitro and in vivo models. These models are proven to be influential of the drug effect in cells, being the most adequate to mimic the live tissue especially in drug development. Supported by the great amount of studies using simple and reductionist co-culture monolayers, and pushing forward an innovative model previously reported by our group, the present study aims to describe a sophisticated and highly reproducible in vitro 3D co-culture intestinal model. Here, the components are assembled in a multistage process into Transwell filters by co-culturing human colon carcinoma Caco-2 and mucus-producing HT29-MTX cells over a layer of collagen embedding intestinal myofibroblasts (CCD-18Co). The 3D co-culture intestinal model described herein represents a particularly powerful and versatile tool that recapitulates the intestinal functioning regarding mucus production, tightness of the different cell types, and the 3D architecture, bridging the gap between simple monolayer cultures of epithelial cells and the complex in vivo physiological conditions. Importantly, it shows tremendous potential in predicting intestinal absorption of orally administered drugs when delivered alone, or encapsulated into micro- and nanosystems, the current leading force of pharmaceutical technology research.

Unsaturated glycoceramides as molecular carriers for mucosal drug delivery of GLP-1

The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short- or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides.

Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining IgA transcytosis across Transwells. IgA transcytosis required induction of polymeric immunoglobulin receptor (pIgR) expression, which could be stimulated by a combination of LPS and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that TNFα, IL-1β, IL-17 and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFNγ however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12)N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.

Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

ATP and the Response to Aldosterone

It has been known for many years that adenosine 5′-triphosphate (ATP) production is necessary for the stimulation of sodium reabsorption by aldosterone, a hormone that plays a critical role in regulating sodium homeostasis. However, the exact mechanism whereby aldosterone stimulates sodium reabsorption--and the precise role of ATP in mediating its effects--has been unclear. Using scanning ion conductance microscopy to image the apical membranes of A6 renal epithelial cells in monolayer culture, Gorelik et al. observed that aldosterone produced morphological changes consistent with contraction of clusters of cells. This contraction, which resembled that elicited by hypoosmotic stress of the basolateral surface, was associated with loss of the normal architecture of the microvilli. The progressive recruitment of contracted cells paralleled an increase in Na + permeability of the epithelial sodium channel (ENaC, measured as an amiloride-sensitive increase in transepithelial conductance, G t ENaC ). However, aldosterone-mediated contraction was not sensitive to amiloride but was sensitive to phosphatidylinositol 3-kinase (PI3K) inhibition (which also blocked the increase in conductance). Moreover, patch-clamp analysis of contracted and noncontracted cells revealed that only contracted cells showed ENaC activity. Aldosterone also stimulated basolateral release of ATP, and the nonhydrolyzable ATP analog ATPγS stimulated both G t ENaC and cell contraction. In contrast, trapping free ATP by adding glucose and hexokinase to the basolateral solution eliminated the effects of aldosterone on conductance, as did treatment with a P1 receptor antagonist. Thus, the authors propose that aldosterone stimulates the release of ATP, which, through a purinergic mechanism, stimulates autocrine and paracrine contraction, leading to reorganization of the apical membrane and activation of apical ENaC in the early phase of aldosterone action. J. Gorelik, Y. Zhang, D. Sánchez, A. Shevchuk, G. Frolenkov, M. Lab, D. Klenerman, C. Edwards, Y. Korchev, Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited. Proc. Natl. Acad. Sci. U.S.A. 102 , 15000-15005 (2005). [Abstract] [Full Text]

Secretion of monocyte chemotactic protein-1 by human uterine epithelium directs monocyte migration in culture

To determine whether primary human uterine epithelial cells in culture are able to influence monocyte chemotaxis and to establish whether the causal agent of chemotaxis is monocyte chemotactic protein (MCP)-1. Tissue culture study. University medical center. Women aged 23 to 53 years who were undergoing hysterectomy (n=7). Primary human endometrial epithelial cells were acquired from surgical specimens and grown to confluence and high transepithelial resistance. Conditioned media from epithelial cultures were analyzed for the presence of MCP-1 and for capacity to affect monocyte chemotaxis using the THP-1 monocyte line. Antibody neutralization of conditioned media was used to establish the role of MCP-1 in chemotaxis. Assay of conditioned media for MCP-1, quantitative measurement of monocyte chemotaxis to conditioned media, and inhibition of chemotaxis by antibody neutralization of MCP-1. Primary endometrial epithelial cells in monolayer culture secrete MCP-1 to both the apical and basolateral compartments. Monocyte chemotactic protein-1 was identified as the primary agent of monocyte chemotaxis by antibody neutralization. These findings suggest that biologically active MCP-1 is secreted into both the uterine lumen as well as the underlying stroma and that it mediates the presence of monocytes, macrophages, and other immune cells in the uterine endometrium.

The Epstein-Barr virus-encoded LMP2A and LMP2B proteins promote epithelial cell spreading and motility.

The frequent expression of latent membrane proteins LMP2A and LMP2B in Epstein Barr virus (EBV)-associated tumors suggests that these proteins play a role in EBV-induced epithelial cell growth transformation. Expression of LMP2A and LMP2B had no effect on the morphology of squamous epithelial cells in monolayer culture, but their expression was associated with an increased capacity to spread and migrate on extracellular matrix. Although the mechanisms by which LMP2A and LMP2B promote cell spreading and motility are unclear, the use of selective pharmacological inhibitors has established a role for tyrosine kinases in this phenotype but ruled out contributions of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase/mitogen-activated protein kinase, and protein kinase C. The ability of LMP2B to induce a phenotype that is virtually indistinguishable from that of LMP2A suggests that regions of the LMP2 protein in addition to the cytosolic amino terminus are capable of inducing phenotypic effects in epithelial cells. Thus, rather than serving to modulate the activity of LMP2A, LMP2B may directly engage signaling pathways to influence epithelial cell behavior such as cell adhesion and motility.

The epitheliome: agent-based modelling of the social behaviour of cells

We have developed a new computational modelling paradigm for predicting the emergent behaviour resulting from the interaction of cells in epithelial tissue. As proof-of-concept, an agent-based model, in which there is a one-to-one correspondence between biological cells and software agents, has been coupled to a simple physical model. Behaviour of the computational model is compared with the growth characteristics of epithelial cells in monolayer culture, using growth media with low and physiological calcium concentrations. Results show a qualitative fit between the growth characteristics produced by the simulation and the in vitro cell models.

Oxysterols from human bile induce apoptosis of canine gallbladder epithelial cells in monolayer culture.

Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol, and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. Because the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol +/- various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphological analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by gas chromatography/mass spectrometry, whereas release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot analysis. Compared with cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (P < 0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one, and 5beta-cholestan-3-one (all P < 0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.

Human Amniotic Epithelial Cells Possess Hepatocyte-like Characteristics and Functions

Hepatocyte transplantation is expected to become a novel method for treatment of liver disease. However, many questions remain regarding this approach, especially concerning donor cells. To evaluate whether human amniotic epithelial cells can be used as a cell source for hepatocyte transplantation, hepatic gene expression and functions of human amniotic epithelial cells were analyzed. Reverse transcription-polymerase chain reaction analysis demonstrated that human amniotic epithelial cells expressed albumin, alpha(1)-antitrypsin, and other hepatocyte-related genes. Cultivated human amniotic epithelial cells demonstrated albumin production, glycogen storage, and albumin secretion consistent with the hepatocyte gene expression profile. In organ culture, the amnion secreted 30-fold larger amounts of albumin than human amniotic epithelial cells in monolayer culture. Moreover, in organ culture the amnion also secreted alpha(1)-antitrypsin. Following transplantation into mice, the amnion survived and secreted albumin. These observations suggest that transplantation of human amniotic epithelial cells and/or amnion could be novel therapeutic strategy for treatment of hepatic diseases, including alpha(1)-antitrypsin deficiency.

The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures.

Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.

Paracrine mediators of mechanotransduction in lung development.

The process of normal fetal lung development is dependent on "mild" tissue distension (approximately 3 mm Hg) by fluid, resulting in the production of pulmonary surfactant which is necessary for survival at the time of birth. The mechanical "stretching" of lung tissue triggers a cellular differentiation cycle, in part by stimulating the expression and production of cell phenotype-specific soluble cytokines. Pulmonary cytokines regulate differentiation and metabolic function of neighboring cells. For example, tonic stretching of type II alveolar epithelial cells in monolayer culture stimulates the expression and production of the differentiation factor parathyroid hormone-related peptide (PTHrP), which is released by type II cells and specifically binds to its receptor on contiguous fibroblasts, stimulating the "second messenger" cyclic AMP. Tonic distension of cultured type II cells increases PTHrP production, and distension of fibroblasts in monolayer culture increases their PTHrP responsiveness, suggesting that stretching couples and coordinates the production and receptor-mediated action of PTHrP. These data provide a mechanistic basis for the previously observed hand-in-glove spatial pattern of PTHrP and the PTHrP receptor (PTHrPR) in developing terminal airways. PTHrP stimulates specific differentiated functions of fetal lung fibroblasts by: 1) augmenting glucocorticoid binding; 2) increasing metabolic activities directly related to surfactant synthesis, such as lipoprotein lipase elaboration and triglyceride uptake rate; 3) stimulating cytokines, such as interleukins 6 and 11, that can act in a retrograde fashion on epithelial cells; 4) thereby increasing the synthesis of surfactant phospholipids and surfactant-associated proteins, closing this stretch-mediated cell-cell interactive loop. Experimental interruption of this mechanism at any of these steps blocks the spontaneous maturation of the lung in vitro, as evidenced by the inhibition of surfactant production.

Paracrine Mediators of Mechanotransduction in Lung Development

The process of normal fetal lung development is dependent on "mild" tissue distension (approximately 3 mm Hg) by fluid, resulting in the production of pulmonary surfactant which is necessary for survival at the time of birth. The mechanical "stretching" of lung tissue triggers a cellular differentiation cycle, in part by stimulating the expression and production of cell phenotype-specific soluble cytokines. Pulmonary cytokines regulate differentiation and metabolic function of neighboring cells. For example, tonic stretching of type II alveolar epithelial cells in monolayer culture stimulates the expression and production of the differentiation factor parathyroid hormone-related peptide (PTHrP), which is released by type II cells and specifically binds to its receptor on contiguous fibroblasts, stimulating the "second messenger" cyclic AMP. Tonic distension of cultured type II cells increases PTHrP production, and distension of fibroblasts in monolayer culture increases their PTHrP responsiveness, suggesting that stretching couples and coordinates the production and receptor-mediated action of PTHrP. These data provide a mechanistic basis for the previously observed hand-in-glove spatial pattern of PTHrP and the PTHrP receptor (PTHrPR) in developing terminal airways. PTHrP stimulates specific differentiated functions of fetal lung fibroblasts by: 1) augmenting glucocorticoid binding; 2) increasing metabolic activities directly related to surfactant synthesis, such as lipoprotein lipase elaboration and triglyceride uptake rate; 3) stimulating cytokines, such as interleukins 6 and 11, that can act in a retrograde fashion on epithelial cells; 4) thereby increasing the synthesis of surfactant phospholipids and surfactant-associated proteins, closing this stretch-mediated cell-cell interactive loop. Experimental interruption of this mechanism at any of these steps blocks the spontaneous maturation of the lung in vitro, as evidenced by the inhibition of surfactant production.

Immunobead and density gradient purification of Paget cells: in vitro studies of proliferation.

Previous studies using primary monolayer cultures of epithelial cells from the involved epidermis of patients with mammary and extramamary Paget's disease investigated whether Paget cells proliferate as other malignant cells do. Although epithelial monolayers from the involved skin were maintained for approximately 45 days, no permanent cell lines were established. The proportion of carcinoembryonic antigen (CEA)-positive cells did not increase in the long-term cultures. Herein, we report studies of whether there is a real reduction of Paget cell numbers or if this is merely a decrease in the expression of CEA by the cells. Furthermore, we investigated whether Paget cells survive longer when cultured free from any potential inhibitory keratinocytes or other epidermal cells. Skin samples were obtained from one patient with mammary Paget's disease and three with extramammary Paget's disease; epidermal cells were cultured in vitro. An enrichment of Paget cells was carried out from the cultured epidermal cells by combining an antiepithelial membrane antigen monoclonal antibody, binding to immunobeads, and density gradient centrifugation in Nycodenz. The separated cells were re-cultured in Keratinocyte-SFM serum-free media. The proportion of CEA-positive cells did not increase in the culture, and the purified cells did not show any increase in survival times compared to the non-purified cultured cells. These results suggest that the decrease of CEA-positive cells noted during culture results from a decline in expression of CEA in the Paget cells. Paget cells in the involved epidermis do not proliferate significantly and thus differ from many other malignant cells.