In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells.

The interaction between Treponema hyodysenteriae and isolated swine intestinal epithelial cells or mouse adrenal cells in culture was examined. Studies were performed in which treponemes were incubated with each type of anomal cell in an atmosphere of 5% CO2 in air. Coincubation was terminated at various time intervals, and the percentage of treponemal attachment evaluated by light microscopy. The extent of attachment was dependent on both incubation time and temperature. The mechanism of attachment to the animal cell surface was examined by scanning electron microscopy. Interaction of the parasite with the host cell did not appear to alter cellular morphology or result in changes of the cell surface at the site of attachment. Preference for a cellular site of attachment was not found.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

The attachment to human buccal epithelial cells by Candida albicans: An in vitro kinetic study using concanavalin A

The early in vitro kinetics of Candida albicans attachment to human buccal epithelial cells was studied with the aid of an adhesion assay and solutions of concanavalin A (Con A), a lectin which is capable of inhibiting yeast adhesion. Various saccharides and putative receptor analogues were also tested. Solutions of each single reagent were added to tubes containing aliquots of mucosal cells and germinated yeasts at the beginning of a 1-hour incubation period (time O) or at 10 minute intervals during the assay. The number of yeasts attached to 200 mucosal cells was subsequently determined microscopically. Yeast adhesion remained constant following addition of phosphate-buffered saline (PBS) at time 0 or at any time thereafter. However, addition of Con A at 0, 10 or 20 minutes of incubation decreased adhesion significantly to 38%, 45% and 63% of control values. This inhibitory effect dwindled as time of incubation prior to lectin addition increased and Con A could not inhibit adhesion significantly after twenty minutes. Results obtained with Con A using live germinated yeasts were similar to those obtained with formalin-killed C. albicans. The other reagents tested failed to decrease adhesion significantly. These included the putative receptor analogues fibronectin, N-acetyl-d-glucosamine and d-galactose, and several non-specific saccharides such as -d-methylglucopyranoside, d-ribose and d-xylose. It is suggested that in vitro attachment to human mucosal cells by C. albicans is inhibitable up to a defined point in time by a lectin with affinity for mannosecontaining surface moieties, but becomes non-reversible thereafter. This experimentally-observed irreversibility is independent of yeast cell viability.     

Biocompatibility testing of an experimental fluoride releasing resin using human gingival epithelial cells in vitro

Summary Cell culture is a valuable method of evaluating the biocompatibility of new dental materials. The purpose of this study was to compare the in vitro biocompatibility of an experimental fluoride composite resin with fluoride and non-fluoride-releasing materials currently available. The dental materials tested were: MQ Silicate (silicate cement), KETAC-CEM and FUJI (type II glass ionomer cements), VISIO DISPERS (a light-cured, nonfluoridated, microfilled composite resin), and FR-17 (an experimental fluoride-releasing composite resin). The Smulow-Glickman (S-G) human, gingival epithelial cell line, which exhibits semidifferentiated characteristics, was used in the study as a test system. Biocompatibility was quantified by counting the viable cells per unit area remaining after 24 and 48 h at two radial distances from cured specimens immersed in the cell culture medium. The test materials were observed to be most toxic to cells nearest the materials. A Time-Distance Cytotoxicity Index (TDCI) was calculated to relate the percentage of dead cells to viable cells at each diffusion distance for each exposure time compared to a nontoxic control. The relative toxicity ranking of the materials tested based on the TDCI was VISIO DISPERS (91%), FUJI (82%), FR-17 (30%), MQ Silicate (23%), and KETAC-CEM (10%), which exhibited the least toxicity. The cytotoxicity of the experimental resin FR-17 was within the range of cytotoxicity of currently accepted restorative materials. this study was supported in part by grant R01-DE04749 from the National Institutes of Health, Bethesda, MD, to H. R. R., and by grant no. S07-RR05704-13 from the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, awarded to the Louisiana State University School of Dentistry.     

Sulfatide mediates attachment of Pseudomonas aeruginosa to human pharyngeal epithelial cells

Pseudomonas aeruginosa infections are particularly common in people with cystic fibrosis and despite regular treatment with antibiotics, lung damage due to chronic infection with P. aeruginosa remains the major cause of death in those patients. In order to initiate an infection, P. aeruginosa needs contact with the respiratory epithelial surface and by means of its adhesins i.e., fimbria, hemagglutinins,etc., it recognizes and adheres to the corresponding epithelial receptors. We treated P. aeruginosa strains isolated from sputum of cystic fibrosis patients with several glycolipids such as sulfatide, sulfated ganglioside mixture (GM1a, GD1b, GT1b), asialo-GM1 and galactocerebrosides to determine their effect on attachment with pharyngeal epithelial cells. Sulfated ganglioside mixture and sulfatide inhibited the attachment of P. aeruginosa significantly, whereas asialo-GM1, Gal-Cer and sodium sulfite had no effect on attachment inhibition. This finding suggests that sulfated glycoconjugates found in the extracellular matrix, in mucus and on the surface of epithelial cells of human trachea and lung mediates attachment of P. aeruginosa.     

Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.     

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.     

Intracellular degradation of Fusobacterium nucleatum in human gingival epithelial cells

The role of Fusobacterium nucleatum in oral health and disease is controversial. We have previously shown that F. nucleatum invades gingival epithelial cells. However, the destiny of the internalized F. nucleatum is not clear. In the present study, the intracellular destiny of F. nucleatum and its cytopathic effect on gingival epithelial cells were studied. The ability of F. nucleatum and seven other oral bacterial species to invade immortalized human gingival epithelial (HOK-16B) cells were compared by confocal microscopy and flow cytometry. F. nucleatum had the highest invasive capacity, comparable to that of Porphyromonas gingivalis, a periodontal pathogen. Confocal microscopic examination revealed colocalization of internalized F. nucleatum with endosomes and lysosomes. Examination by transmission electron microscopy revealed that most intracellular F. nucleatum was located within vesicular structures with single enclosed membranes. Furthermore, F. nucleatum could not survive within gingival epithelial cells and had no cytopathic effects on host cells. Interestingly, endosomal maturation played a role in induction of the antimicrobial peptides human beta defensin (HBD)-2 and -3 by F. nucleatum from gingival epithelial cells. F. nucleatum is destined to enter an endocytic degradation pathway after invasion and has no cytopathic effect on gingival epithelial cells, which may cast new light on the role of F. nucleatum in the pathogenesis of periodontitis.     

Ultrastructure of the human enamel organ

Summary The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated.Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions.A parallel differentiation of stratum intermedium — external enamel epithelium, and the ameloblast layer is demonstrated.The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.     

Defining conditions to promote the attachment of adult human colonic epithelial cells

Summary An improved method for the attachment and growth of normal human colonic epithelial cells from minute 1 to 3-mm³ biopsies has been developed. This yields four times as many cultured cells per biopsy than older methods, with a success rate of 97% in a series of 29 biopsies. Fetal bovine serum was eliminated from the medium, the medium pH was decreased to 6.7, the oxygen tension in the incubator was decreased to 3%, and the NCTC 168 medium was supplemented with ethanolamine, phosphoethanolamine, hydrocortisone, ascorbic acid, transferrin, glutamine, insulin, epidermal growth factor, pentagastrin, and deoxycholic acid. The best substrate for cell attachment was a mixture of ungelled collagen I and bovine serum albumin. This substrate was better than the identical mixture with fibronectin added, fibronectin alone, a thin gelatin film, collagen IV with or without fibronectin, and basement membrane preparations from four different cell lines.     

Ultrastructure of surface epithelial cells of the normal human rectum

Luminal surface epithelial cells, excluding a few endocrine cells of the normal human rectum, were studied electron microscopically and 5 types of cells were recognized with special reference to some structure containing mucous substances. Principal-1 cells showing few tiny vesicles and Principal-2 cells containing some tiny vesicles seemed to belong to the absorptive cell group. Vesicle cells having numerous tiny vesicles, and Columnar mucous cells accompanied by numerous tiny vesicles and some round or oval mucous vacuoles, seemed to be labelled as of the secretory cell group. The common features of the epithelial columnar cells, except for the Goblet cell, were columnar shape, microvilli whose length and density had considerable variation, glycocalyceal bodies around the microvilli, and thick surface coat. Goblet cells were characterized by a goblet shape which was expanded by numerous mucous droplets. It is of special interest that 4 different types of columnar epithelial cells are recognized on the luminal surface of the normal human rectum, and that Vesicle cells and Columnar mucous cells are first observed on the luminal surface of the large intestine. Similar epithelial cells have only been reported in the crypt of the large intestine and not on the luminal surface.     

Role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells

The goal of this study was to determine the role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells. Strain 2951 and its P(k) mutant strain 2951 galE were used in this study. This study suggests that the P(k) epitope of LOS is not an adhesin for M. catarrhalis, but plays a crucial role by its surface charge in the initial stage of attachment.     

Initial attachment ofCandida albicans cells to buccal epithelial cells

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) ofCandida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion ofC. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer ofC. albicans responsible for adhesion to host cells.     

Micropatterning of nanoengineered surfaces to study neuronal cell attachment in vitro

Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell-cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, "comparison chips," comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A2 (sPLA2), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure-function relationships.     

Binding of epithelial cells to lectin-coated surfaces

Summary Epithelial cells may relate to their basement membrane substrates via lectin-like interactions. In a model system for study of this type of interaction, lectin-coated bacteriological plastic petri dishes were presented as substrates for epithelial cell adhesion. Of 21 lectins tested by mixed agglutination against two epithelial cell types, Madin-Darby canine kidney (MDCK), and human embryonic kidney cells (HEK), nine gave less than 5% rosettes and 12 gave 5 to 50% rosettes. Wheat germ agglutinin (WGA) andGeodia cydonium lectin gave the highest percentage of rosettes. Wheat germ agglutinin was readily adsorbed to plastic surfaces and maintained specificity in binding interactions. Both MDCK and HEK cells attached as well to WGA coated petri dishes as to conventional tissue culture dishes. Furthermore, both spread over the lectin-coated surfaces. The MDCK cells grew to confluence and could be subcultured and maintained indefinitely on such surfaces, although WGA in solution was toxic to the cells in concentrations as low as 0.1 to 1.0 μg/ml. Cell attachment to WGA coated dishes was blocked by cycloheximide only if the cells had been preincubated with the inhibitor for several hours. Cell attachment was not inhibited by pretreatment of cells with neuraminidase. Precoating cells with WGA blocked binding to both WGA-coated surfaces and untreated tissue culture dishes. Cells attached to WGA-coated dishes could not be readily dislodged by trypsin-EDTA for the first 2 h after subculture. By 4 h, attachment was again trypsin sensitive, suggesting that the cells synthesized a trypsin-sensitive material that was laid down between the cell surface and the WGA-coated dish. Regeneration of trypsin sensitivity was not blocked by cycloheximide. This work was supported by Research Grant AG01986 from the National Institutes of Health, Bethesda, Maryland.     

Anin vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. Thein vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca²⁺. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killedEscherichia coli. These reductions in toxicity were presumably due to the binding of the cat onic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.Abbreviations ANOVA analysis of variance - [Ca²⁺]i calcium concentration in internal medium of liposomes - DMEM Dulbecco's modified Eagle medium - EDTA ethylenediamine tetraacetic acid - FBS fetal bovine serum - Hepes N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid - HGF-1 human gingival fibroblast cell line - HSD honestly significant differences - KB cell line derived from a human epidermoid carcinoma in the mouth - LDH lactic acid dehydrogenase - NADH nicotinamide adenine dinucleotide, reduced form - NR neutral red - NR₅₀ concentration inhibiting neutral red uptake by 50% - PBS phosphate-buffered saline - SEM standard error of the mean - S-G Smulow-Glickman human gingival epithelial cell line