Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy.     

Application of percoll density gradients in studies of the adhesion ofStreptococcus pyogenes to human epithelial cells

A new assay was used to study the adhesion ofStreptococcus pyogenes strains to epithelial cells. [³H]thymidine-labeled bacteria were incubated with standardized preparations of epithelial cells collected from oral-pharyngeal surfaces of human volunteers. The mixtures were then centrifuged in 50% Percoll to form a density gradient. Epithelial cells with attached bacteria formed a band near the top of the tube, whereas unattached bacteria were located near the bottom. The epithelial cells were collected on membrane filters, and the number of adherent bacteria was then determined by scintillation counting.The abilities of M-protein-positive (M⁺) and M-protein-negative (M^–) strains ofS. pyogenes to attach to human pharyngeal, buccal, and tongue epithelial cells were compared. The results obtained confirmed the significant difference previously shown to exist between the attachment of M⁺ and M^– strains to human epithelial cells. M⁺ strains ofS. pyogenes exhibited a much greater ability to bind to pharyngeal epithelial cells than did M^– variants. Also, M⁺ strains were bound in higher numbers to pharyngeal epithelial cells than to buccal or tongue epithelial cells. The adhesion ofS. pyogenes strains to epithelial cells was time dependent, and a significant increase in the adhesion of M⁺ strains occurred after 3–4 h of exposure of the bacteria to epithelial cells.The adsorption ofS. pyogenes strains to epithelial cells was described by a Langmuir isotherm. With this model, the number of binding sites and the affinities of the streptococci for epithelial cells were estimated. Significantly higher numbers of binding sites were calculated to be present on pharyngeal epithelial cells for M⁺ strains ofS. pyogenes than on buccal cells. However, the affinity of the organisms was similar for both types of cells.Adsorption of M⁺ strains to human pharyngeal epithelial cells was inhibited by certain galactosides and fucose, but not by glucose or xylose. This suggests that saccharide moities play a role in the binding of M⁺ strains ofS. pyogenes to human pharyngeal epithelial cells.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Cultivation of chicken proventricular epithelial cells and their potential for differentiation

Epithelial cells of chicken proventriculus (glandular stomach) differentiate into two types; luminal and glandular epithelial cells. The molecules regulating the differentiation of proventricular epithelial cells are not well understood. As the first step in screening the molecular determinants involved in the cell differentiation process, we tried to establish an in vitro culture system for isolated proventricular epithelial cells. Various basal media, growth factors and sera were tested. The medium that supports well the proliferation of epithelial cells was composed of Ham's F12 as the basal medium with epidermal growth factor (10 μg/mL), insulin (10 μg/mL), cholera toxin (1 μg/mL) and bovine pituitary extract (100 μg/mL). Fetal calf serum stimulated cell proliferation 1.7-fold, while horse serum was rather toxic. Proventricular epithelial cells proliferated for 3 days, but began to die out within 1 week of culture. Cultured epithelial cells never expressed embryonic chicken pepsinogen (ECPg), a marker gene of glandular epithelial cells, or maintained ECPg expression. The capacity for ECPg expression in cultured epithelial cells was analyzed by recombination with the proventricular mesenchyme and ECPg was detected in epithelial cells cultured up to 3 days. We concluded therefore, that epithelial cells keep the capacity for ECPg expression for 3 days of cultivation and proventricular mesenchymal cells are required for the actual expression of the ECPg gene.     

A simple process for the isolation of epithelial cells from bacteria-contaminated samples using anchoring molecules

A simple and efficient tool to isolate epithelial cells from bacteria-contaminated samples has been developed using two different microparticles functionalized with chemical molecules. The epithelial cells could be captured simply by biocompatible anchors for membranes (BAM), consisting of poly(ethylene glycol) functionalized with oleyl-chain-conjugated NHS (N-hydroxysuccinimide) on glass microparticles, whereas bacteria were adsorbed on 3-aminopropyltrimethoxysilane (ATPS)-functionalized magnetic microparticles. In the case of samples highly contaminated with bacteria, epithelial cells were not isolated successfully by both of the single BAM- and antibody-functionalized microparticles. Therefore, serial isolation steps of these two different chemical functionalized microparticles were introduced. The concentration of bacteria was decreased dramatically by using APTS-functionalized magnetic particles prior to the isolation of epithelial cells by BAM microparticles. With these serial processes, successful isolation of epithelial cells was achieved from bacteria-contaminated epithelial samples. The applicability of this method was verified with bacteria-contaminated intestinal samples biopsied from a BALB/C mouse for primary cell cultivation.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Epstein-Barr virus infection of polarized epithelial cells via the basolateral surface by memory B cell-mediated transfer infection

Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b–negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

The effect of N‐acetylcysteine on chloride efflux from airway epithelial cells

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N‐acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S‐nitrosoglutathione) previously has been shown to be able to promote Cl^? efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl^? efflux from CF and non‐CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37°C. The effect of NAC on Cl^? transport was measured by Cl^? efflux measurements and by X‐ray microanalysis. Cl^? efflux from CFBE cells was stimulated by NAC in a dose‐dependent manner, with 10 mM NAC causing a significant increase in Cl^? efflux with nearly 80% in CFBE cells. The intracellular Cl^? concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl^? efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.     

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.     

Binding selectivity ofStreptococcus pyogenes and M-protein to epithelial cells differs from that of lipoteichoic acid

The ability of M-protein-positive (M⁺) and M-protein-negative (M^–) strains (including an M^– mutant lacking the structural gene for M-protein) ofStreptococcus pyogenes to attach to human pharyngeal, buccal, and tongue epithelial cells was compared. We observed that M⁺ strains ofS. pyogenes attached in significantly higher numbers to human pharyngeal epithelial cells than to human buccal or tongue cells. M^– strains did not exhibit high-level binding to any type of epithelial cell. Also, the adhesion of an M⁺ and an M^– strain ofS. pyogenes was low to all types of rat epithelial cells tested. The apparent differences in the surface components between human pharyngeal and buccal epithelial cells were confirmed by studies utilizing radiolabeled lectins.Ulex europaeus lectin with a specificity for fucosyl residues, andTriticum vulgaris lectin with a specificity for N-acetyl glucosamine and N-acetyl neuraminic acid residues, bound in higher amounts to human pharyngeal cells than to buccal cells. Pretreatment of pharyngeal epithelial cells with microgram quantities of highly purified type 6 M-protein or miligram quantities of lipoteichoic acid (LTA) derived fromS. pyogenes decreased the subsequent attachment of the organism. However, the binding specificities of³H-LTA were different from those of intact streptococci;³H-LTA bound comparably to human pharyngeal, buccal, and tongue epithelial cells, and it bound in higher quantities to rat epithelial cells. Also, although the adsorption ofS. pyogenes cells to pharyngeal cells was inhibited by the presence of fucose and galactose, these sugars had little effect on the binding of³H-LTA to epithelial cells. In contrast, the high adhesion of M⁺ strains but not M^– mutants to pharyngeal cells suggested that M-protein may play an important role. This possibility was supported by the observation that³H-labeled purified type 6 M-protein bound in higher concentrations to human pharyngeal epithelial cells than to human buccal cells. Furthermore, human pharyngeal epithelial cells were estimated to contain larger numbers of binding sites for M-protein than buccal cells, whereas the affinity of M-protein was similar to both cell types. These adsorption parameters are similar to those previously established for intact streptococcal cells.     

Lymphocytes Accelerate Epithelial Tight Junction Assembly: Role of AMP-Activated Protein Kinase (AMPK)

The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-α. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Autocrine/paracrine action of oxytocin in pig endometrium

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.     

The cell cycle of cultured iris epithelial cells: Its possible role in cell-type conversion

Cell cycle parameters were estimated in primary cultures of iris epithelial cells, obtained from explanted dorsal and ventral irises of adult newts (Notophthalmus viridescens). No significant difference was found between parameters of dorsal and ventral iris epithelial cell cultures. Compared with the total cell cycle time of iris epithelial cells in situ in the pathway of conversion, that of cultured iris epithelial cells is longer by a factor of 1.88. The results support the working hypothesis that the basic requirement for conversion of iris epithelial cells into lens cells is the passage of a definite number of cell cycles instead of the inductive influence of neural retina.     

Adherence of Candida albicans to buccal and vaginal epithelial cells: ultrastructural observations

The adherence of Candida albicans to human buccal and vaginal epithelial cells was studied by transmission electron microscopy. Adherence to epithelial cells was confirmed by both a radiometric assay as well as direct microscopic examination of stained cell preparations. Ultramicroscopic preparations revealed that yeast cells were closely appressed to epithelial cell surfaces and were often partially enclosed within phagocyticlike invaginations of the epithelial cells. A murine model of vaginitis caused by C. albicans was also used to study adherence to epithelial cells and to follow the course of colonization. Ultramicroscopic preparations of murine vaginal tissue revealed that within 2 h postinfection, yeast cells could be seen adhering to epithelial cells. At 6 h postinfection, hyphae and yeast cells were not only found on the epithelial cell surface but also within the submucosal tissue. When observed on the epithelial cell surface, Candida cells were either attached to host cells, or when infected tissue was stained with ruthenium red, Candida cells were observed on the epithelial surface embedded within an electron-dense matrix. Fungal elements were abundant in the submucosa at 24 h postinfection and were still observed on the epithelial cell surface; all of this was accompanied by an inflammatory response.     

The Secretome of Alginate-Encapsulated Limbal Epithelial Stem Cells Modulates Corneal Epithelial Cell Proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Localization of myosin and actin in ocular nonmuscle cells

Summary Myosin and actin were localized by indirect immunofluorescence microscopy using specific antibodies prepared in rabbits against highly purified gizzard myosin and actin. A strong fluorescence staining with both antibodies was observed in rat corneal epithelial cells, anterior lens epithelial cells, rod inner segments, and in rat and frog pigment epithelial cells. The immunohistochemical localization of myosin in corneal epithelial cells was further supported by the electrophoretic and immunological identification of smooth muscle type myosin heavy chain in pure corneal epithelial abrasions. Electron-microscopic observations revealed a clear correlation between staining with actin antibodies and the presence of numerous thin cytoplasmic filaments (50–80 Å in diameter). The functional and biochemical nature of 90–110 Å filaments occurring in corneal and lens epithelial cells, as well as the ultrastructural localization of myosin in ocular nonmuscle cells under study remains obscure.     

Escherichia coli lipopolysaccharide induces alveolar epithelial cell stiffening

IntroductionApplication of lipopolysaccharide (LPS) is a widely employed model to mimic acute respiratory distress syndrome (ARDS). Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited only to P. aeruginosa LPS. Considering that LPS from different bacteria could promote a specific mechanical response in epithelial cells, we aim to assess the effect of E. coli LPS, widely employed as a model of ARDS, in the biomechanics of alveolar epithelial cells.MethodsYoung’s modulus (E) of alveolar epithelial cells (A549) was measured by atomic force microscopy every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (100 μg/mL). The percentage of cells presenting actin stress fibers (F-actin staining) was also evaluated. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point.ResultsApplication of LPS induced significant increase in E after 20 min (77%) till 60 min (104%) in comparison to controls. Increase in lung epithelial cell stiffness induced by LPS was associated with a higher number of cells presenting cytoskeletal remodeling.ConclusionsThe observed effects of E. coli LPS on alveolar epithelial cells suggest that this widely-used LPS is able to promote a quick formation of actin stress fibers and stiffening cells, thereby facilitating the disruption of the pulmonary epithelial barrier.