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PubMed2006

In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.

PURPOSE: The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro. METHODS: Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure. RESULTS: Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05). CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.

Adult↗
PubMed1981

Ultrastructural and chemical studies on wall-deficient forms, spheroplasts and membrane vesicles from Mycobacterium aurum.

Wall-deficient forms of Mycobacterium aurum were prepared by agitating the cells during exponential growth with D-cycloserine, glycine, lysozyme, EDTA and LiCl for approximately the time of three cell divisions (18 h). Wall-deficient forms were then converted to spheroplasts by gentle stirring with lysozyme and EDTA in a Tris/HCl buffer containing sucrose until all the cells appeared spherical by phase contrast microscopy. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles. Ultrastructural and chemical properties of the spheroplasts and membrane vesicles are described. The spheroplasts were susceptible to lysis by 0.25% (w/v) sodium dodecyl sulphate and were permeable to certain enzyme substrates.

Anti-Bacterial Agents↗
PubMed1991

Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle.

Vacuoles of Saccharomyces cerevisiae were visualized by phase-contrast microscopy. Visualization was enhanced by adding polyvinylpyrrolidone. Vacuolar segregation during the cell cycle was analysed in 42 individual cells of strain X2180 by time-lapse photomicrography. Within 15 min of bud emergence, more than 80% of the cells contained a vacuolar segregation structure in the form of either a tubule or an alignment of vesicles. The structure emerged from one point of the mother vacuole, then elongated and moved into the bud in a few minutes. The vacuolar segregation structure disappeared, usually within 20 min, before nuclear migration, leaving a separate vacuole in the bud. To test the generality of this observation several strains were grown in the presence of the vacuolar vital dye fluorescein isothiocyanate. The bud size was used to measure progress in the cell cycle. All strains formed vacuolar segregation structures in cells with small buds, although with variations in duration and timing in the cell cycle. In the presence of nocodazole vacuolar segregation occurred normally, thus, microtubules seem not to be essential in this process.

Cell Cycle↗
PubMed2005

Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system.

The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.

Animals↗
PubMed1986

Effect of 8-methoxypsoralen plus long-wave ultraviolet (PUVA) radiation on mast cells. II. In vitro PUVA inhibits degranulation of rat peritoneal mast cells induced by compound 48/80.

Rat peritoneal mast cells incubated with a histamine liberator, compound 48/80, showed a significantly reduced capacity for releasing histamine following in vitro treatment with 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 1-5 J/cm2 of long-wave ultraviolet (UVA) irradiation (PUVA). No remarkable inhibition in histamine release was observed in the cells treated with 8-MOP only. Irradiation with 5 J/cm2 of UVA alone exerted an inhibitory effect on histamine release, to a lesser extent than PUVA. PUVA irradiation did not bring any decrease in cell viability or any spontaneous release of histamine from irradiated cells as shown by phase-contrast microscopy and by histamine assay, respectively. These results suggest that PUVA treatment may cause a noncytotoxic disturbance at mast cell membranes or on surface receptors, leading to a decreased capacity for secreting chemical mediators.

Animals↗
PubMed2004

Plaque development in relation to the periodontal condition and bacterial load of the saliva.

AIM: To investigate the influence of the oral bacterial load on plaque development in various groups of periodontitis patients and in healthy subjects. MATERIAL AND METHODS: This study included subjects with a healthy periodontium, a healthy reduced periodontium after treatment, an inflamed reduced periodontium after treatment and untreated periodontitis. At the start of the study, subjects were instructed to rinse with 10 ml reduced transport fluid (RTF) for 10 s in order to evaluate the oral bacterial load. The microbiological evaluation included anaerobe culture and phase-contrast microscopy. Next, the amount of plaque and the clinical condition were evaluated. Thereafter, all supragingival plaque was removed and patients were instructed to refrain from all oral hygiene procedures for 19 h. Subsequently, the rinsing procedure and the evaluation of the amount of plaque were repeated. RESULTS: The amount of plaque that developed in 19 h was significantly higher in the untreated periodontitis group as compared with the two healthy groups. In case of an inflamed reduced periodontium, sites with deep pockets developed more plaque in 19 h than sites with shallow pockets. The number of bacteria present in the rinsing samples of the two inflamed groups was considerably higher than of the two periodontally healthy groups. A significant correlation was found between the bleeding index at intake and the plaque index at 19 h. No correlations were found between gingival recession and the bacterial counts at intake, and the plaque index at 19 h. CONCLUSION: The present findings support the concept that the periodontal condition is the dominating factor in relation to the rate of plaque formation. The number of bacteria present in the oral cavity as ascertained by means of a rinsing sample does not seem to play a role.

Adult↗
PubMed1975

Langerhans cells in tissue cultures of guinea-pig epidermal cells.

Dispersed cell cultures of guinea-pig epidermis have been maintained for up to 36 days. The progress of these cultures has been studied by phase contrast microscopy, the adenosine triphosphatase reaction, and cinephotomicrography. Langerhans cells have been identified and their behaviour studied, particularly in relation to keratinocytes.

Adenosine Triphosphatases↗
PubMed1991

Increase of lipid fluidity and suppression of proliferation resulting from liposome uptake by human keratinocytes in vitro.

The in vitro effects of liposomes on HaCaT human keratinocytes were studied with regard to their uptake, lipid fluidity and proliferation of the cells. Oligolamellar liposomes, prepared from soya bean phospholipids, had a mean size of 150 mm and consisted predominantly of phosphatidylcholine (83%) and phosphatidylethanolamine (10%) and the fatty acids comprised mainly linoleic acid (66%) or other unsaturated fatty acids. After 6 and 24 h of incubation with 1 and 0.1% w/v of liposomal lipids, phase-contrast microscopy revealed marked cytoplasmic vacuolization of the cells. Keratinocytes treated with the liposomes contained aggregations of multilaminated lipid material without delimiting cell membranes. The cellular lipid fluidity (reciprocal of diphenylhexatriene fluorescence polarization P-value) correlated with liposomal concentration and incubation time. A significant elevation of lipid fluidity (P less than 0.05) was observed with 1 and 0.1% liposomes after 1 h of incubation (81.8 +/- 4.7 and 95.7 +/- 1.2% of control P value) and for 0.01% liposomes after 3 h (96.2 +/- 1.5%). Maximum fluidity occurred after 48 h of exposure to 1% liposomes (42.1 +/- 3.1%). Exposure to liposomal lipids for 24 and 48 h resulted in suppressed cell proliferation with 50% inhibition concentrations (IC50), being 0.06% for incorporation of [3H]-thymidine. 0.08% for [14C]-amino-acid incorporation and greater than 1% for protein content per well after 24 h of exposure. The cells were able to proliferate and lipid fluidity returned to normal within 7 days following discontinuation of incubation with liposomal lipids.

Cell Division↗
PubMed2005

Long-term culture of adult murine epidermal keratinocytes.

BACKGROUND: Long-term cultures of epidermal cells from mouse skin have been notoriously difficult to establish. OBJECTIVES: To develop a modified serum-free medium and technique for long-term culture of adult mouse epidermal keratinocytes. METHODS: Epidermal cells from trypsin-treated adult mouse dorsal and ventral skin were grown on type I collagen-coated dishes without feeder layers in a serum-free medium supplemented with only 10 ng mL(-1) epidermal growth factor (EGF) and 10(-10) mol L(-1) cholera toxin (CT). RESULTS: After removing coexisting fibroblasts several times, we were able to obtain almost pure basal epidermal keratinocytes. Our technique supports sustained multiplication of mouse basal keratinocytes for more than 100 population doublings, and they retained the capacity to undergo terminal differentiation when given the appropriate stimulus. The epithelial nature of these cultivated cells was demonstrated both by phase-contrast microscopy and by immunostaining with antikeratin antibodies. EGF and CT, which have been reported to accelerate the cellular growth rate, were essential for successful long-term cultivation during multiple passages. CONCLUSIONS: Our technique is very simple. It provides a useful and suitable model for investigations of growth, differentiation and skin remodelling in vitro.

Animals↗
PubMed1980

The Ultra-Flo 100 platelet counter: a new approach to platelet counting.

The Clay Adams Ultra-Flo 100 whole blood platelet counter is a semiautomated instrument. The count is made on dilute whole blood by the detection of comparatively small current changes induced by the cells suspended in a conducting diluent as they flow thrugh an orifice. Alarm systems are incorporated in the instrument to detect sample irregularities due to microcytosis, and large and small platelets. The results of this evaluation are given, and confirm that the results using the Ultra-Flo 100 compare very favourably with those obtained using phase contrast microscopy.

Blood Platelets↗
PubMed1995

Microbial alterations in supragingival dental plaque in response to a triclosan-containing dentifrice.

A total of 325 subjects were entered into a double-blind, stratified 2-treatment clinical study that examined the effects of a dentifrice containing 0.3% triclosan, 2% Gantrez copolymer and 0.243% sodium fluoride on supragingival dental plaque and gingivitis. A subset of 159 subjects including 72 men and 87 women participated in the microbiological component of this study, which was designed to detect shifts in supragingival bacterial species in response to triclosan. Subjects were divided into two groups: one performed normal oral hygiene with the triclosan/copolymer dentifrice and a control group used a placebo dentifrice without triclosan. At baseline, 3 and 6 months during treatment and at 6, 12, 18 and 24 weeks post-treatment, supragingival dental plaque was collected from the buccal and lingual surfaces of the 4 first molar teeth and assayed for: 1) bacterial morphotypes by phase-contrast microscopy; 2) select bacterial groups and bacterial species by culture; and 3) target periodontal pathogens including Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis and Prevotella intermedia by immunofluorescence microscopy. There were few statistically significant differences between treatment groups in any of the 90 microbiological parameters measured at the 7 different time points. The control group demonstrated significantly higher levels of cultivable Neisseria and higher proportions at the 3-month treatment period of P. gingivalis-infected subjects and mean cells. After 6 months of treatment, the triclosan group demonstrated higher levels of fusiforms. Analysis of triclosan resistance data failed to detect a shift towards increased proportions of bacteria resistant to triclosan, and both treatment groups demonstrated triclosan-resistant strains, predominantly Veillonella dispar. This study confirms the microbiological safety of triclosan-containing dentifrices and suggests that continued use can be associated with beneficial alterations in the bacterial composition of supragingival dental plaque.

Actinomyces↗
PubMed1980

Spontaneous and polyamine-induced formation of filamentous polymers from soluble fibronectin.

Fibronectin is a high-molecular-weight glycoprotein present in a soluble form in plasma and in other body fluids and as insoluble protein in connective tissue matrix. This study reports that soluble fibronectin is polymerized into filamentous structures and that polyamines stimulate this process and precipitate fibronectin. Fibronectin purified from human plasma under non-denaturing conditions appeared after negative staining as non-globular extended structures in the electron microscope. During storage of purified fibronectin at +4 degrees C, in particular a low ionic strength, increasing amounts of the protein appeared as protein filaments. These filaments had a diameter of 2--3 nm and a length of up to several micrometers. The filaments also formed bundles of variable thickness, apparently through lateral association. These structures could also be visualized by phase-contrast microscopy. Polyamines, at a concentration of 1--5 mM and at a low ionic strength, induced a rapid, extensive polymerization of fibronectin into filamentous structures. The effect increased in the order putrescine less than spermidine less than spermine. Polyamine-induced precipitation of fibronectin was reversible upon removal of the polyamine. Fibronectin secreted by normal and by malignant cells could be fairly selectively precipitated from the culture medium with polyamines. The observed filamentous polymers of soluble fibronectin resemble the filamentous fibronectin-containing pericellular structures in fibroblast cultures and may provide a model for studies on the deposition of fibronectin in matrix form.

Chromatography, Agarose↗
PubMed1996

The leukocytes of the roughtail gecko Cyrtopodion scabrum: a bright-field and phase-contrast study.

The morphology of the peripheral blood leukocytes of the roughtail gecko, Cyrtopodion scrabum, is carefully described in Wright-Giemsa and toluidine-blue-stained blood films, and in the living condition by phase-contrast microscopy, using supravitally stained preparations. Mature eosinophils, basophils and small lymphocytes commonly occur in the blood, while monocytes are rarely seen. In addition, macrophages are occasionally encountered, but neutrophils cannot be observed. Developmental stages in eosinophil and basophil differentiation can be seen. This study serves as a basis for the cytochemical localization of substances within these blood cells.

Animals↗
PubMed1995

Co-expression of fibroblastic, histiocytic and smooth muscle cell phenotypes on cultured adherent cells derived from human palatine tonsils: a morphological and immunocytochemical study.

Adherent cells derived from human palatine tonsils were isolated and cultivated. Exponentially growing adherent cells (TAC) were observed by phase-contrast microscopy and transmission electron microscopy. Immunocytochemical studies were also performed. TAC were composed of relatively monotonous cells with polygonal or spindle shapes and high proliferative activity. In addition to the development of rough endoplasmic reticulum and lysosomes, the TAC possessed a moderate amount of pinocytotic vesicles and a few microfilaments. All of the TAC strongly expressed fibroblastic markers and partial monocyte/macrophage markers, such as beta-subunit of prolyl 4-hydroxylase (DAKO-fibroblast), lysozyme, anti-alpha-1-antichymotrypsin (alpha ACT), and CD68 (KP-1, EBM/11). It was noted that, as the TAC were cultured for a longer period, they gradually increased the reactivity with the monoclonal antibody PG-M1. Furthermore, the TAC expressed myocytic phenotype, such as alpha-smooth muscle actin (alpha SMA) with various intensity. Moreover, as to extracellular matrix, TAC stained for collagen type I, collagen type III, laminin, and fibronectin. Collagen type IV was weakly positive. The results presented here showed that the TAC expressed three different phenotypes of fibroblasts, histiocytes and smooth muscle cells at the same time. The monoclonal antibody raised against the TAC reacted strongly with the subendothelial pericytes and/or smooth muscle cells in the extrafollicular area in human tonsils. The present results also suggested that the origin of the TAC was probably subendothelial pericytes and/or smooth muscle cells of the microvasculatures in the tonsil.

Adult↗
PubMed1998

Kartagener's syndrome: a re-visit with Chinese perspectives.

The objective of this study was to evaluate the clinical, radiological investigation profiles, and ciliary function and ultrastructure in Chinese patients with Kartagener's syndrome (presence of dextrocardia, sinusitis and bronchiectasis). All patients with dextrocardia were assessed for the presence of sinusitis and bronchiectasis in our hospital network. Patients identified with Kartagener's were assessed when they were at steady state for their bronchiectasis. Seven cases (4 males; mean age 34.9 years) were identified and systematically reviewed. The mean 24 h sputum volume was 26.6 +/- 32.77 mL/day and the patients suffered from a mean of 2.9 exacerbations/year. Nasal symptoms (anosmia in one, obstruction in six and persistent discharge in three patients) were common. Only two cases (1 M) were married and both had normal fertility. Lung function assessment showed a mean FEV1/FVC of 83.3 +/- 38.78/86.5 +/- 36.72 (% predicted) with little reversibility. High resolution computerized tomography (HRCT) revealed bronchiectactic involvement of the lower lobes in seven and middle lobe/lingula in four cases. Assessment of alpha-1-anti-trypsin, aspergillus precipitins, auto-antibodies and serology for Pseudomonas pseudomallei was normal. Sputum culture yielded Pseudomonas aeruginosa in three, Haemophilus influenzae in three and commensals in one case. Phase contrast microscopy assessment of respiratory cilia, obtained by brushing the inferior turbinate, revealed that most of the mucosa was unciliated. The mean ciliary beat frequency was 5.2 +/- 6.76 Hz (range 0-13.7; normal range 12-18 Hz). Four patients had immotile cilia whilst the rest had normal ciliary movement. Transmission electron microscopy showed the absence of dynein arms in four patients. The results of this study show that patients with Kartagener's syndrome may have normal ciliary ultrastructure and the absence of dynein arms is not necessarily associated with ciliary immotility. The presence of ciliary immotility might have prognostic value as these patients appear to have more active bronchiectasis. Our experience on this series should help clinicians in the investigation and management of these patients.

Adolescent↗
PubMed1981

Identification of beta-adrenergic receptor binding sites in rat brain microvessels, using [125I]iodohydroxybenzylpindolol.

Brain microvessels were prepared from rat cerebral cortex. The purity was confirmed by phase-contrast microscopy and by the measurement of an enzymatic marker, gamma-glutamyltranspeptidase. The microvessel preparation was subjected to radioreceptor assay using a 125I-labelled beta-adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80% microgram per tube. It was saturated at 200 pM IHYP concentration. The KD value calculated by Scatchard analysis was 69.4 +/- 9.9 pM. The maximum binding (Bmax) was 107 +/- 4 fmol/mg protein. The binding reached equilibrium within 30 min and was dissociated by addition of (-)-propranolol. The inhibitory effects of isomers of propranolol and isoproterenol on this binding showed that (-)-isomers were two orders of magnitude more potent than the (+)-isomers. Other neurotransmitters did not affect IHYP binding. The characteristics of the binding, saturability, high affinity, reversibility and stereospecificity, suggest tha IHYP is bound to beta-adrenergic receptor sites located on brain microvessels.

Animals↗
PubMed1979

Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.

Animals↗
PubMedDate not supplied

Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture.

We report here the in vitro isolation of Cajal-like interstitial cells from human inactive mammary-gland stroma. Primary cell cultures examined in phase-contrast microscopy or after vital methylene-blue staining revealed a cell population with characteristic morphological phenotype: fusiform, triangular or polygonal cell body and the corresponding (very) long, slender, moniliform cytoplasmic processes. Giemsa staining pointed out the typical knobbed aspect of cell prolongations. Immunofluorescence (IF) showed, like in situ immunohistochemistry, that Cajal-type cells in vitro (primary cultures), expressed c-kit/CD117 and vimentin. In conclusion, the images presented here reinforce our previous hypothesis that human mammary glands have a distinct population of Cajal-like cells in non-epithelial tissue compartments.

Breast↗