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PubMed1996

Isolation and in vitro propagation of human choroidal fibroblasts. Morphology and characterization of the cultures.

Subretinal ingrowth of choroidal fibroblasts is of importance in several clinical settings such as age-related macular degeneration or after traumas such as laser treatment and infection. In the present investigation we describe a method for isolation and propagation of human choroidal fibroblasts in vitro. Outgrowth from the outseeded choroid was studied by means of phase contrast microscopy, scanning electron microscopy and by immunohistochemical methods. The secondary cultures were found to contain almost only fibroblasts (more than 95% of the cells), some contaminating retinal pigment cells were also found. Choroidal fibroblasts contribute to the fibrovascular subretinal scarring process, and our cell culturing system seems to be a suitable tool for studying factors regulating the behavior of these cells in vitro.

Aged↗
PubMed1988

Clinical and microbiological effects of root debridement in periodontal furcation pockets.

The aim of the present study was to investigate longitudinally over 52 weeks the clinical and microbiological effects of plaque control and root debridement at molar furcation sites. The results were compared with changes at non-molar sites. 24 non-molar sites and 31 grade II molar furcation sites with probing depth greater than or equal to 5.0 mm were monitored in 11 patients. Clinical measurements consisted of plaque scores, probing depths, and changes in probing attachment level. Microbiological monitoring was carried out with phase-contrast microscopy and anaerobic culturing. The debridement resulted in improvement in probing measurements and microbiological counts for both groups of sites. A slightly less favorable clinical response was noted for molar furcation sites. Higher post-operative microbiological counts were found throughout the 52-week observation period for molar furcation sites. Sites with probing attachment loss showed higher microbial counts and higher proportions of spirochetes, black pigmented colony forming units (CFU), and Bacteroides gingivalis CFU than sites with probing attachment gain. Individual site analysis, however, demonstrated marked variations of the microbiological counts at the different postoperative time points. In the few available sites undergoing probing attachment loss, no apparent association between target micro-organisms and periodontal deterioration was observed.

Adult↗
PubMed1995

Microbiological features of gingivitis in pubertal children.

The subgingival microflora of 42 pubertal children (aged 12-15 years) and 18 young adults (aged 21-25 years) was investigated by anaerobic culture and phase contrast microscopy. Motile rods, spirochetes and Prevotella intermedia were elevated proportionately in pubertal children with gingivitis (median GI > or = 1); however, no statistically significant differences in enumerated organisms on selective media were observed between pubertal children and young adults. These organisms were positively correlated with the index of bleeding on probing and the gingival index of pubertal children. Pubertal children were divided into 3 distinct subject clusters according to the similarity of subgingival microbial features, and the subjects who were then selected for the gingivitis group were distributed into clusters 1 and 2. The proportions of motile rods, P. intermedia and Eikenella corrodens were significantly higher in cluster 2 than in cluster 1. These results suggest that these 2 species and motile rods which differentiated features of clusters, are useful for screening of high-risk subjects for worsening of inflammation.

Actinomyces↗
PubMed1995

Periodontal pathogens on polytetrafluoroethylene membrane for guided tissue regeneration inhibit healing.

This study determined the microbial composition of the apical parts of the expanded polytetrafluoroethylene membrane surfaces facing the gingiva and the tooth in guided tissue regeneration. Microbial and clinical features of 2-to-3 wall periodontal bony defects treated with membranes with and without concomitant use of systemic Augmentin therapy were also determined. 18 patients with 18 study sites participated. 9 patients received systemic 500 mg Augmentin 1 h prior to surgery, and 500 mg TID for 8 days thereafter. 9 patients received no systemic antimicrobial therapy. Microbiological examination was performed 1 h prior to surgery, at the time of membrane removal at week 6, and at 6 months post-surgery. Microbial morphotypes, total viable counts, and the occurrence of selected microbial species were determined by phase-contrast microscopy, selective and non-selective culture, and DNA probes. Study sites were examined for probing pocket depths and attachment levels. At baseline, no microbial or clinical parameter showed statistical differences between groups. At 6 months, the Augmentin group demonstrated a significantly higher (P = 0.032; Student t-test) mean probing attachment gain (36.5% of potential gain to the cemento-enamel junction) than the 9 control patients (22.4% of potential gain). At the time of removal, membranes in the Augmentin group showed significantly fewer organisms than membranes in the control group (52.2 x 10(6) versus 488.6 x 10(6)). Sites free of pathogens on the membrane surface toward the tooth gained the most clinical attachment, even in the presence of various pathogens on the gingiva-facing membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1981

Cytotoxic effects of dental cements on two cell culture systems.

Four dental cements and one bis-GMA composite were tested in cultures of human periodontal ligament fibroblasts and mouse 3T3 fibroblasts. It was found that these cell types reacted to the cements with different intensities, and that there were differences in evaluation by phase contrast microscopy and succinic dehydrogenase histochemistry.

Animals↗
PubMed1988

Reaction of fibroblasts to various dental casting alloys.

The cytotoxicity of a series of dental casting alloys in the as-cast and polished condition was determined with cell culture techniques involving phase contrast microscopy to examine cell morphology and the succinic dehydrogenase histochemical reaction to measure any ring of inhibition of Balb/c 3T3 cellular respiration around alloys. Crown and bridge casting alloys and a nickel- and a cobalt-base alloy were biocompatible in the polished condition, but less so in the as-cast condition. The only two exceptions were casting alloys containing 50-60 wt% Cu. Porcelain-fused-to-metal alloys were biocompatible in either the as-cast or polished condition. This direct contact method appeared satisfactory for evaluating biocompatibility of dental casting alloys, especially since these materials are in contact with gingival tissues.

Alloys↗
PubMed1990

Microbial patterns in pooled subgingival plaque samples from young adults with advanced marginal periodontitis.

Different bacterial profiles of subgingival plaque have been described for different clinical categories of marginal periodontitis. In the present investigation the subgingival microflora was studied in young adults with advanced marginal periodontitis. From 12 patients pooled subgingival plaque samples from 5 advanced stages of diseased sites were examined by direct differential phase-contrast microscopy and by cultivation on enriched and selective media. The proportions of the following genera and species were calculated: black-pigmented Bacteroides sp., B. gingivalis, Fusobacterium nucleatum, Veillonella sp., Actinobacillus actinomycetemcomitans, Capnocytophaga sp., Eikenella corrodens, Campylobacter sp., and Candida sp. Spirochete identification was carried out ultrastructurally. Calculated percent minimum similarity levels between the individuals revealed that each patient harbored its characteristic cultivable subgingival microflora different from the other individuals. The spirochetes seemed to constitute the subgingival bacterial group with the smallest variation as certain morphotypes were dominating in almost all individuals.

Adult↗
PubMed2005

Expression of type IV collagen and laminin at the interface between epithelial cells and fibroblasts from human periodontal ligament.

The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.

Adolescent↗
PubMed1981

Characterization of a human epithelial cell line with special reference to its ultrastructure.

A commercially available cell line (NCTC 2544) originating from presumably normal human skin was chosen as an in vitro model system for subsequent studies of the effects of different agents on human epithelial cells. The cell line, therefore, was ultrastructurally and otherwise characterized at intervals by techniques which allow standardized controls of the model. The cell line was classified as epithelial both by phase contrast microscopy and transmission electron microscopy. The cells were polygonal, fully developed desmosomes were demonstrated and no extracellular filamentous material was observed. Specific epidermal markers like keratohyalin granules or keratinosomes were not demonstrated. Comparison of the nuclear morphology of cells from 1, 3 and 6 days old cultures revealed that only minor changes took place. The same was true for cytoplasmic features. Thus this cell line has a well-defined ultrastructural morphology. Flow cytometry studies showed the cell line to be tetraploid, with no sub-populations with other ploidies. Surface antigens were typed by a microcytotoxicity assay using 12 different antisera. The electrophoretic patterns of the isoenzymes lactate dehydrogenase and glutathione reductase were human. The cells did not form colonies in soft agar. Though several parameters indicate that this cell line is not a HeLa cell contaminant, this possibility cannot, however, be totally excluded.

Antigens, Surface↗
PubMed1990

Binding of bacteria to carbohydrates immobilized on beads to demonstrate the presence of cell-associated hemagglutinins in Vibrio cholerae.

We describe a phase contrast microscopy method for direct observation of classical and El Tor vibrios to agarose beads containing covalently attached L-fucose or D-mannose. Binding of the vibrios to L-fucose beads was found to correlate with fucose-sensitive agglutination of human O erythrocytes, while binding of bacteria to beads with D-mannose was consistent with mannose-sensitive agglutination of chicken erythrocytes. Furthermore, vibrios expressing both fucose and mannose-sensitive hemagglutinins adhered equally to L-fucose and D-mannose-containing beads. Because this procedure is neither subject to biological variations in different populations of erythrocytes nor affected by other factors known to interfere with hemagglutination tests, it offers a suitable, more robust and specific alternative to detect functional adhesins in Vibrio cholerae and other bacteria.

Bacteria↗
PubMed2003

Establishment and characterization of a human ovarian granulosa tumor cell line (HSOGT).

We successfully established a novel ovarian granulosa tumor cell line (HSOGT). The tumor tissue of the ovary was derived from a 25 year-old Japanese woman under her consent. The cell line was maintained for over 14 months, subcultured more than 73 times, and had a population doubling time of 18.9 hours. Phase contrast microscopy displayed a pavement-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy and the mode was 83; many marker chromosomes were observed. The HSOGT was also successfully xenotransplanted into nude mice. The cell line produced estradiol and has preserved some characters of granulosa cells with stable growth in vitro. We firmly believe that this cell line will be a most useful tool for endocrinological investigation of human granulosa cells.

Adult↗
PubMed1992

Anterior capsule opacification: a cell culture model.

We cultured the human lens epithelial cells with attaching anterior lens capsule using surgically removed capsular flaps obtained in intraocular lens implantation. The capsular flap was placed with cell side down in a small culture well. In phase contrast microscopy, an anti-meniscus plate was placed on the bathing medium to avoid optical aberration. The human lens epithelial cells showed outgrowth which ceased after 3 to 4 weeks of incubation. The cells under the capsule lost distinctive cell margin. The cells outgrew from the capsular edge both onto the anterior capsular surface and the well bottom. The outgrowth length and Bromodeoxyuridine uptake indicated a low proliferative potency of the human lens epithelial cells.

Cataract↗
PubMed1977

Calcium and stimulus-secretion coupling in the mast cell: stimulant and inhibitory effects of calcium-rich media on exocytosis.

1. Isolated rat peritoneal mast cells incubated in Ca-free media for 2 h, with or without EDTA, and observed by phase-contact microscopy, became ;bubbled' in appearance when subsequently exposed to media rich in calcium (16-110 mM).2. Electron microscopy showed the response to be ;compound' exocytosis of the sort elicited by conventional mast cell secretagogues such as antigen (in sensitized cells) and 48/80.3. The response to Ca was inhibited by withdrawing glucose and adding dinitrophenol and was thus energy-dependent.4. Mg in similarly high concentration had no such stimulant effect on Ca-deprived cells, and excess Ca stimulated only after Ca deprivation.5. It is suggested that Ca deprivation may increase the permeability of the plasma membrane of the mast cell thereby allowing some Ca, when subsequently introduced in high concentration, to penetrate and activate exocytosis; and the results are considered further support for the postulated mediator function of Ca in stimulus-secretion coupling.6. Two inhibitory effects of calcium in high concentration were detected: (a) suppression of migration or expulsion of granules from the exocytotic pits within the cellular domain; and (b) diminished sensitivity to 48/80.

Animals↗
PubMed1992

In vitro and in vivo antibacterial activities of E1077, a novel parenteral cephalosporin with a broad antibacterial spectrum.

E1077 is a new injectable cephalosporin with a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria, including staphylococci and Pseudomonas aeruginosa. The in vitro activities of E1077 against clinical isolates of methicillin-susceptible Staphylococcus aureus (MIC of E1077 for 90% of the strains tested [MIC90], 0.78 microgram/ml) and methicillin-resistant S. aureus (MIC90, 50 micrograms/ml) were similar to those of cefpirome and flomoxef. Against Enterococcus faecalis (MIC90, 6.25 micrograms/ml), E1077 was the most active of the drugs tested and four times more active than cefpirome. The MIC90S of E1077 for streptococci, Haemophilus influenzae, and Neisseria gonorrhoeae ranged from 0.05 to 0.78 microgram/ml; E1077 was similar in activity to cefpirome. E1077 inhibited 90% of most species of the family Enterobacteriaceae at concentrations of less than or equal to 1.56 micrograms/ml, with the exception of Serratia marcescens and Proteus vulgaris (12.5 micrograms/ml). The activity of E1077 against P. aeruginosa (MIC90, 6.25 micrograms/ml) was comparable to that of ceftazidime. In vivo activity was evaluated with systemic infections in mice. E1077 showed a protective effect against systemic infections by gram-positive or gram-negative bacteria, as reflected by its in vitro activity. The protective effects of E1077 were higher than those of cefpirome against S. aureus and P. aeruginosa infections and similar to those of cefpirome against other bacterial infections. Morphological studies using differential interference and phase-contrast microscopy showed that low concentrations of E1077 caused swelling of S. aureus and spheroplast and bulge formation in P. aeruginosa. In general, the antibacterial profile of E1077 is similar to that of cefpirome.

Animals↗
PubMed1970

Effect of miracil D on marker frequency ratio and cytotoxicity in Bacillus subtilis.

When Miracil D was added to mid-log phase Bacillus subtilis cells, the rate of growth decreased immediately, and the turbidity of the culture began to decrease within 15 to 20 min after addition of the drug. At this stage, ghostlike cells were observed under phase-contrast microscopy. The viable count also began to decrease rapidly after 15 min in the presence of the drug, and within 60 min there was a 10,000-fold reduction in viability. Incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) proceeded normally up to 20 min of exposure to the drug, after which incorporation ceased. Cells which were prelabeled with (3)H-thymidine and exposed to the drug released labeled DNA into the medium after 15 to 20 min. Results of transformation analyses with donor DNA from cells grown in the presence of Miracil show a decrease with time in the ratio of origin markers to terminus markers. Electron micrographs of sectioned cells grown for 5, 10, and 15 min in the presence of Miracil D show profound cytotoxic effects. The most striking aspect of such cells is the very condensed appearance of the nucleoid. In those cells about to divide, the nucleoid appears not to be properly separating, being strung out in the region of the developing septum. Finally, the mesosome is very poorly defined or entirely absent in Miracil-treated cells.

Adenine↗
PubMed1972

Growth, cell division, and fragmentation in a species of Flexibacter.

Flexibacter FS-1, a gram-negative gliding bacterium was grown in liquid culture as long (over 100-mum) filaments. The filaments possessed a triple-track wall which resembled that found in other gram-negative bacteria. Although phase-contrast microscopy indicated that the long filaments were nonseptate, electron microscopy revealed three or four septa along the length of each filament. The septa contained lysozyme-sensitive, electron-opaque material, presumed to be peptidoglycan, sandwiched between cell membranes. The outer triple track wall was not part of the septum. Mesosomes were seen in various areas of the cell and frequently were observed attached to septa in different stages of completion. Studies of the organism in slide culture revealed that individual filaments grew in an exponential fashion and divided in the middle despite the long length and multiseptate condition. When the temperature of a liquid culture growing exponentially with a generation time of 90 minutes was shifted from 30 to 35 C, the filaments fragmented into three or four shorter cells within 120 min. The short cells continued to grow exponentially at 35 C at approximately the same rate as at 30 C. When the culture was shifted back to 30 C, the cells immediately stopped dividing and began to elongate. After a period of 2 to 3 hr, cell division resumed. It is suggested that the shift-up in temperature induced the completion of the cross wall (centripetal growth of the triple-track wall) and cell separation at the sites of previously formed septa, whereas the shift-down in temperature caused a transient inhibition of cross-wall formation but not of growth. Fragmentation was inhibited by sodium azide but took place despite the inhibition of protein synthesis by chloramphenicol or the inhibition of deoxyribonucleic acid synthesis by mitomycin C.

Azides↗
PubMed1973

Morphologica and structural changes during the yeast-to mold conversion of Phialophora dermatitidis.

The details of the morphological and structural events occurring during yeast-to-mold conversion of the human pathogenic fungus Phialophora dermatitidis as seen by phase-contrast microscopy and electron microscopy are described and illustrated. Budding yeasts growing exponentially were observed to have thin walls and a cytoplasm exhibiting the characteristics of rapidly growing cells including numerous mitochondria, abundant ribosomes, few vacuoles, and little accumulation of storage material. In contrast, thick-walled yeasts were characterized by less apparent or significantly fewer mitochondria and ribosomes and the presence of considerable amounts of storage materials. Microscope observations of yeast-to-mold conversion revealed that only thick-walled yeasts having prominent lipid bodies in their cytoplasm converted to hyphal forms. Typically, the thick-walled yeast formed two to a number of moniliform hyphal cells which in turn often produced true hyphae. The results indicated that yeasts of P. dermatitidis must acquire spore-like characteristics by becoming thick-walled and by accumulating considerable endogenous substrate reserves before they convert and produce hyphae.

Cell Wall↗
PubMed1973

Use of cobalt as a mitochondrial vital stain to study cytoplasmic exchange in matings of the basidiomycete Schizophyllum commune.

Differential labeling of mates, based on the selective uptake of cobalt by mitochondria of one of the partners, has been used to determine visually by means of phase-contrast microscopy whether transfer of mitochondria occurs after hyphal fusion. Compatible and incompatible matings of the tetrapolar basidiomycete, Schizophyllum commune, were studied. Transfer was detectable in common-A, common-AB, and fully compatible matings. It was not detectable in common-B matings

Basidiomycota↗