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PubMed1959

Fine structure of cellular inclusions in measles virus infections.

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mmicro in diameter, the strands seen in the inclusions cannot be fully formed virus.

HeLa Cells↗
PubMed1982

Hormonally induced changes in the cytoskeleton of human thyroid cells in culture.

Serially cultivated thyroid follicular cells are not active in hormone synthesis but retain a thyrotropin-responsive adenylate cyclase. The exposure of such cells to thyrotropin leads to an increase in the concentration of intracellular cAMP and a drastic change in morphology including a total cytoplasmic arborization. The present communication describes these changes at the cytoskeletal level using a cell line derived from a human functioning thyroid adenoma. Phase contrast microscopy showed that the cytoplasmic arborization was preceded by a total disappearance of stress fibers, visible within 20 min of exposure. Small marginal membrane ruffles could also be seen. These morphological changes could also be induced by the addition of dibutyryl cAMP. The action of both thyrotropin and dibutyryl cAMP was potentiated by theophylline. High voltage electron microscopy of whole mounted cells confirmed the loss of stress fibers (microfilament bundles). In addition, thyrotropin treatment led to an uneven redistribution of the cytoplasmic ground substance and to changes in the organization of the microtrabecular lattice. Stereo images demonstrated numerous minute surface ruffles. The thyrotropin-induced arborization was reversible even in the presence of thyrotropin. After 24 h of treatment, cells had flattened and then contained very straight and condensed microfilament bundles. The results thus demonstrate that thyrotropin induces a disintegration of microfilament bundles in human, partially dedifferentiated, follicular cells and that this effect to all appearances is caused by cAMP, the second messenger in thyrotropin action. The relation of this event in partially dedifferentiated cells to the effect of thyrotropin in the intact thyroid gland is unclear. The fact that several other cultured hormone-responsive cells round up or become arborized in conjunction with an increase in cAMP levels implies that cAMP may be a major factor in the disassembly of microfilament bundles in these cells.

Bucladesine↗
PubMed1975

Inhibition of multiplication of Toxoplasma gondii by human monocytes exposed to T-lymphocyte products.

The multiplication of Toxoplasma gondii was quantitated in human monocytes in vitro by phase-contrast microscopy. Toxoplasma multiplication was identical in monocytes from subjects byt was significantly inhibited in cells from both sources if the monocytes were preincubated with immune lymphocytes and toxoplasma monocytes were preincubated with immune lymphocytes and toxoplasma antigen. Supernates prepared from toxoplasma-immune lymphocytes incubated with toxoplasma antigen were also effective in inducing in monocytes the capacity to inhibit toxoplasma multiplication. Supernative acitivty was evident after lymphocytes and antigen were incubated for as little as 15 min. The instruction of monocytes was also repid and reversible. Monocytes were fully induced to inhibit toxoplasma multiplication after a 2 h exposure to an active supernate, but they lost their inhibitory capacity on culture in vitro for 48 h in the absece of immune cells or their products. The lymphocytes particupating in the monocyte induction were identified as t cells. The in vitro stimulation of monocytes appeared to exhibit some specificity, since no inhibition of toxopreotein derivative and lymphocytes from tuberculin-positive subjects, concanavalin a-stimulated lymphocytes, or their supermates. Supernates which induced monocytes to inhibit toxoplasma multiplication did not influence parasite growth in HeLa cells.

Adult↗
PubMed1978

The interaction in vitro of Pneumocystis carinii with macrophages and L-cells.

A model was developed for studying the interaction between Pneumocystis, rat-derived cells, and humoral factors. Pneumocystis were obtained in large quantity by bronchial lavage of steroid-treated rats. The trophozoite was the predominant form obtained, and it could readily be recognized by phase contrast microscopy. Organisms maintained a typical morphology for at least 3 days in culture, and 10-20% took up radiolabeled nucleotides. Pneumocystis readily adhered to cell surfaces in a similar manner in alveolar macrophages from steroid-treated or normal rats, mouse peritoneal macrophages, and L-cells. Adherent organisms were not interiorized to a significant degree in the absence of antipneumocystis serum. After addition of rabbit antipneumocystis serum, rapid interiorization of organisms occurred from the surface of macrophages but not L-cells. Organisms appeared to be promptly destroyed within macrophages after interiorization. Persisting or multiplying intracellular forms were not seen. Antipneumocystis serum did not morphologically alter Pneumocystis. These observations suggest a role for antibody and mononuclear phagocytes during the immune response to Pneumocystis.

Animals↗
PubMed2005

Cell detection in phase-contrast images used for alpha-particle track-etch dosimetry: a semi-automated approach.

A novel alpha-particle irradiator has recently been developed that provides the ability to characterize cell response. The irradiator is comprised of a collimated, planar alpha-particle source which, from below, irradiates cells cultured on a track-etch material. Cells are imaged using phase-contrast microscopy before and following irradiation to obtain geometric information and survival rates; these can be used with data from alpha-particle track images to assess cell response. A key step in this process is determining cell location within the pre-irradiation images. Although this can be done completely by a human observer, the number of images requiring analysis makes the process time-consuming and tedious. To reduce the potential human error and decrease user interaction time, a semi-automated, computer-aided method of cell detection has been developed. The method employs a two-level adaptive thresholding technique to obtain size and position information about potential cell cytoplasms and nuclei. Proximity and geometry-based thresholds are then used to mark structures as cells. False-positive detections from the automated algorithm are due mostly to imperfections in the track-etch background, camera effects and cellular residue. To correct for these, a human observer reviews all detected structures, discarding false positives. When analysing two randomly selected cell dish image databases, the semi-automated method detected 92-94% of all cells and 94-97% of cells with a well-defined cytoplasm and nucleus while reducing human workload by 32-83%.

Algorithms↗
PubMed2003

Formation of hydroxyapatite-mediated three-dimensional structures by mouse fibroblasts in response to physical stimulation.

Hydroxyapatite (HAP) ceramics are widely used as implant materials for periodontal bone defects because of their excellent biocompatibility. We demonstrated that physical stimulation, that is, (1). mechanical stimuli or (2). laser irradiation, causes HAP-mediated C3H10T1/2 mouse fibroblasts to form three-dimensional tissue-like structures. Trypsinized 10T1/2 cells were cultured simultaneously with 200 HAP granules on a rotator for 7 days in mechanical stimulation experiments. The cells were later transferred to a regular incubator. Cell reactions were observed by phase-contrast microscopy. The formation of three-dimensional structures around the HAP granules was observed in the third week of cultivation after stimulation. In laser irradiation experiments, trypsinized cells were irradiated with 1, 5, and 16 J/cm(2) at a wavelength of 1000 nm and cultured with 200 HAP granules for 10 weeks. The formation of three-dimensional structures, like those observed in the mechanical stimulation experiments, was observed in the third week after irradiation. The formation of these structures was most frequent at 1 J/cm(2), and the frequency of formation of these structures gradually decreased as the irradiation dose was increased. These results indicate that physical stimuli may stimulate cell proliferation, leading to the repair of damaged tissue. These results also indicate that mouse fibroblasts do not form these three-dimensional structures without HAP and that HAP alone is not sufficient to stimulate the formation of three-dimensional structures.

Animals↗
PubMed1999

Evaluation of Sysmex UF-100 urine flow cytometer vs chamber counting of supravitally stained specimens and conventional bacterial cultures.

We evaluated the Sysmex UF-100 urine flow cytometer (TOA Medical Electronics, Kobe, Japan) with 269 uncentrifuged urine specimens by comparing it with Sternheimer staining and particle counting in 1-microL disposable chambers with both brightfield and phase-contrast microscopy (the reference method). Results of routine test strip analysis, sediment microscopy (182 specimens), and bacterial culture (204 specimens) were also available. Detection of urinary WBCs and RBCs was highly reliable with the UF-100 compared with manual chamber counting (r = .98 and .88, respectively). Identification of bacteria was equal to that with visual microscopy of uncentrifuged specimens; sensitivity was 55%, and specificity 90%, compared with bacterial cultures at a cutoff of > 10(3) colony-forming units per milliliter. Renal damage was difficult to evaluate even with manual methods because of the low counts of renal tubular cells and casts; with standard manual Sternheimer-stained sediment analysis, sensitivity was 65% to 69% and specificity 66% to 91%, compared with the uncentrifuged chamber method at a cutoff of 3 and 10 particles per microliter, respectively. Renal damage was demonstrated with the UF-100 with a sensitivity of 26% to 69% and specificity 92% to 94%, compared with chamber counts. Automated urinalysis with the UF-100 urine flow cytometer offers considerable savings in time and labor. When high sensitivity is needed, visual microscopic review should be performed to detect renal disease.

Autoanalysis↗
PubMed1994

Levels of workplace exposure.

A brief description was presented of the evolution of workplace exposure monitoring. Measurements began in the 1930s and developed through particle counting to the introduction of the membrane filter, light microscope method in the mid-1960s. It is important to be aware that most data from the past were collected in relation to workplace control. The membrane filter phase contrast microscope method provides an index of exposure. Fibres with diameters less than 0.2 microns are unlikely to be counted. The method is not capable of analysing individual fibre types and consequently all fibres are counted. There have been several changes to the detail of the membrane filter method. The possible effect of these changes were presented. The Asbestos International Association (AIA) has implemented a fibre counting performance trial and also an annual return of workplace monitoring data. A WHO project is in progress with the objective of harmonizing the membrane filter method for fibre counting of all fibre types using phase contrast microscopy.

Asbestosis↗
PubMed1990

Antibacterial effect of meropenem and imipenem on Proteus mirabilis.

Phase-contrast microscopy, killing-curves and turbidimetric growth-curves were used in a comparative study of the antibacterial effects of a new carbapenem, meropenem (SM 7338) and imipenem on five strains of Proteus mirabilis. Despite the low MIC (0.2 mg/l) of imipenem for the five strains included in our study, the MBC remained relatively high (4.4 mg/l). During the first few hours of incubation, imipenem induced large lemon-shaped cells while the turbidity increased without substantial changes in culture viability. Later, most of the cell-wall deficient bacteria generated small spheroplasts until the antibiotic concentration exceeded 32 times the MIC. The MIC of meropenem was lower (0.03 mg/l) with an MBC (0.08 mg/l) very close to the MIC. Meropenem also induced large bodies but these cell-wall deficient bacteria did not generate small round bodies as observed with imipenem. In conclusion, imipenem produced in strains of Pr. mirabilis an amdinocillin-like change in cell morphology, responsible for the discrepancies observed between MIC and MBC. This effect was not observed with meropenem.

Imipenem↗
PubMed1996

In-vitro model of uterine leiomyomas: formation of ball-like aggregates.

To clarify the biological characteristics of uterine leiomyomas, cells explanted and cultured from uterine leiomyomas and from normal myometrial tissue were observed by time-lapse cinemicrography and phase-contrast microscopy. The histological characteristics were evaluated by electron microscopy and immunofluorescence microscopy, and these observations revealed significant differences. By time-lapse cinemicrography, the cells cultured from leiomyomas and myometrium differed in their behaviour. Cells from the myometrium started to grow in parallel with the cell's major axis and formed topographically uniform hills and valleys by day 21 of culture. In contrast, the cells from leiomyomas started to grow irregularly, as if having no contact inhibition, and formed ball-like aggregates of cells by day 21 of culture. The aggregates resembled the nodules of leiomyoma in vivo. Ultrastructurally, cells from both leiomyomas and myometrium had typical features of smooth muscle. Immunofluorescently, a different distribution of alpha-smooth muscle actin-positive filaments and different staining of cellular fibronectin and N-cadherin between the cells from leiomyomas and myometrium were observed, which may contribute in part of the different behaviour of the cells. Given that the explant cell culture system resembles the features of uterine leiomyomas in vivo, this suggests that it can be used as an in-vitro model.

Antibodies↗
PubMed2005

Anti-endothelial cell antibodies from lupus patients bind to apoptotic endothelial cells promoting macrophage phagocytosis but do not induce apoptosis.

OBJECTIVE: Anti-endothelial cell antibodies (AECA) have been reported to induce apoptosis. We investigated the induction of apoptosis by these autoantibodies and their involvement in the removal of apoptotic cells. METHODS: AECA isolated from patients with active systemic lupus erythematosus (SLE) were incubated with human umbilical vein endothelial cells (HUVECs). AECA-positive sera were identified using a cell-based ELISA. Apoptosis was measured by morphology and phosphatidylserine externalization using flow cytometry with fluorescein isothiocyanate (FITC)-conjugated annexin V. Flow cytometry was used to investigate AECA binding to apoptotic cells using FITC-conjugated anti-human immunoglobulin G (IgG). Apoptotic endothelial cells were stained with a red dye (PKH26) and co-cultured with macrophages, and phagocytosis was visualized under phase contrast microscopy. RESULTS: AECA from patients with SLE did not induce apoptosis compared with normal IgG (nIgG) at any time point, as assessed by morphology (at 24 h, P = 0.167) or phosphatidylserine externalization (at 24 h, P = 0.098). However, there was increased binding of AECA to apoptotic endothelial cells (48.8 +/- 11.9 compared with 25.8 +/- 6.7% AECA binding to freshly isolated cells, P< 0.001). These opsonized endothelial cells showed greater phagocytosis by macrophages (mean phagocytic index 24.9 +/- 4.5%) when cells opsonized with nIgG were compared with AECA (34.8 +/- 3.4% n = 5, P = 0.01). CONCLUSION: In conclusion, AECA bind to apoptotic endothelial cells but do not induce endothelial cell apoptosis. Macrophage phagocytosis is increased by opsonization of apoptotic endothelial cells by AECA, a proinflammatory mechanism of cell removal.

Adolescent↗
PubMed1999

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (</= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality.

Actins↗
PubMed2001

In vitro effects of antiviral agents on human keratocytes.

PURPOSE: To study the effects of antiviral agents on human keratocytes in vitro. METHODS: Cultured human keratocytes were incubated with either ganciclovir, idoxuridine, trifluridine, or cidofovir at concentrations from 0.0001 to 10 mg/mL. Phase-contrast microscopy and XTT (sodium [2,3-bis [2-methoxy-4-nitro-5-sulphophenyl]-2h-tetrazolium-5-carboxanilide, inner salt) colorimetric assay were performed after 24, 48, and 72 hours of incubation. RESULTS: When adjustments were made for time of incubation and concentration, trifluridine reduced cell viability significantly more than ganciclovir, idoxuridine, and cidofovir (p<0.001, three-way analysis of variance). There was significant time-and dose-dependent reduction of cell viability (p<0.001) with trifluridine and cidofovir. After a 72-hour incubation with ganciclovir or idoxuridine, cell viability was reduced as compared with 24- and 48-hour incubation (p<0.001); only the effects of the highest concentration tested (1.0 mg/mL) were significantly different from those of the lower concentrations (p<0.002). At a concentration of 1.0 mg/mL, trifluridine and cidofovir produced moderate to severe signs of cytotoxicity, whereas ganciclovir and idoxuridine displayed much less severe morphologic signs. CONCLUSIONS: Our results indicate that antiviral agents may have both time- and concentration-related toxic effects on stromal keratocytes. These findings may impact the selection of the most appropriate antiviral drug when it is needed to treat infections involving the corneal stroma.

Antiviral Agents↗
PubMed2003

In vitro toxicity of netilmicin and ofloxacin on corneal epithelial cells.

PURPOSE: To evaluate the in vitro cytotoxic effect of the aminoglycoside antibiotic netilmicin on rabbit corneal epithelial cells (SIRC) compared with ofloxacin, a commonly used fluoroquinolone ocular antibiotic. METHODS: SIRC cell cultures were incubated for 8 to 72 h in the presence and absence of netilmicin (1.5, 3, and 6 mg/mL) and equal concentrations of ofloxacin. Cell viability in treated and untreated SIRC cells was measured by both neutral red and MTT colorimetric assays at 8, 24, and 72 h, whereas changes in cell morphology were examined at 8, 24, 48, and 72 hours by the use of phase-contrast microscopy. RESULTS: Netilmicin, at all tested concentrations, failed to alter SIRC cell viability or morphology. In contrast, all concentrations of ofloxacin caused statistically significant dose- and time-dependent reductions in cell viability even after 8 h. After 72 h there was complete loss of cell viability. Morphologic examination of SIRC cells after 8 h of incubation with ofloxacin revealed that the fluoroquinolone antibiotic, at all concentrations, produced large numbers of dead cells, compromised intercellular contacts, and altered general morphology. After 48 h the cell monolayer was observed to be completely destroyed. CONCLUSION: Netilmicin, at the concentrations used, is an antibiotic devoid of obvious cellular toxicity and may also be considered as a suitable first-choice drug in the treatment of those pathologies that compromise the integrity of the ocular surface.

Animals↗
PubMed2004

The effect of endogenous nitric oxide on cholinergic ciliary stimulation of human nasal mucosa.

OBJECTIVES/HYPOTHESIS: Endogenous nitric oxide (NO) production by inducible nitric oxide synthase is enhanced in the nasal ciliated respiratory tract epithelium of patients with allergic rhinitis. Recent experimental data have suggested endogenous NO to be strongly involved in the complex regulation of ciliary activity, the driving force of the mucociliary transport system. The authors investigated the effect of endogenous NO on acetylcholine-stimulated ciliary activity of human nasal mucosa. STUDY DESIGN: In vitro study. METHODS: Cultures of human nasal mucosa explants were incubated with tumor necrosis factor-alpha and bacterial lipopolysaccharides to enhance endogenous NO production. Expression of inducible NO synthase was morphologically demonstrated by immunohistochemistry. Ciliary beat frequency was determined by phase-contrast microscopy of ciliated epithelium, using a computerized photoelectric technique. Stimulation experiments were performed in vitro with acetylcholine and N(G)-nitro-l-arginine methyl ester (L-NAME), a NO synthase inhibitor. RESULTS: Upregulation of inducible NO synthase in the respiratory tract epithelium after stimulation with tumor necrosis factor-alpha and lipopolysaccharide was visualized by immunohistochemical analysis. Experimental inhibition of enhanced endogenous NO production by 10 mol/L L-NAME significantly reduced baseline ciliary beat frequency from 8.6 +/- 0.2 to 7.8 +/- 0.2 Hz (P < .05). Cholinergic ciliary stimulation above baseline by 10 mol/L acetylcholine was not significantly different before (11.5%) or after (10.8%) blocking of endogenous NO production. CONCLUSION: Taken together, the study results suggest that baseline ciliary activity depends on endogenous NO production but that the extent of cholinergic ciliary stimulation is independent of endogenous NO production. The combination of the two effects may improve nasal mucociliary clearance of inhaled allergens in patients with allergic rhinitis.

Acetylcholine↗
PubMed1998

Heat-induced apoptosis in human glioblastoma cell line A172.

OBJECTIVE: Hyperthermia has been clinically applied to some types of brain tumors. However, the detailed mechanisms of this growth inhibition are not clear. The effect of mild hyperthermia on cultured human glioblastoma cell line, A172, was studied. METHODS: A172 cells were heat treated (43-44.5 degrees C) for 1 hour in the growing phase. Cell viability was assessed by trypan blue dye exclusion assay. The presence of apoptosis was determined by the morphological changes observed using phase contrast microscopy and nuclear changes observed using HOECHST 33342 stain. For the evaluation of cellular deoxyribonucleic acid fragmentation, the TUNEL method was used. The expression of p53 and bax proteins was evaluated by Western blot, and the bax messenger ribonucleic acid was detected by Northern blot. RESULTS: Heat treatment induced cell death in time- and temperature-dependent manners. The nuclear staining with HOECHST 33342 demonstrated morphological changes consistent with apoptosis. The TUNEL stain also demonstrated damages in the deoxyribonucleic acid. These morphological changes were accompanied by the accumulation of p53 protein, bax protein, and messenger ribonucleic acid. CONCLUSION: These results indicate that mild hyperthermia induces apoptosis in A172 glioblastoma cells.

Apoptosis↗
PubMed1996

Does the HMG-CoA reductase inhibitor pravastatin influence nucleation of cholesterol crystals in supersaturated model bile?

OBJECTIVE: To assess whether the presence in bile of HMG-CoA reductase inhibitors, which are secreted predominantly into the bile, influences biliary lithogenicity. DESIGN: Physiologic biliary concentrations of the hydrophilic HMG-CoA reductase inhibitor pravastatin were added to supersaturated model bile (cholesterol saturation index 1.4) and vesicles, the latter with and without the concomitant addition of the nucleation-promoting bile salt taurodeoxycholate. OUTCOME MEASURES: Nucleation time, defined as the number of days after which cholesterol monohydrate crystals are visible by phase contrast microscopy in filtered samples of model bile and vesicles, was assessed. RESULTS: The addition of pravastatin 0.01-1 mg/ml did not influence the nucleation time of supersaturated model bile (mean nucleation time without pravastatin: 8.3 +/- 2.2 days (SD), with pravastatin 1 mg/ml 9.3 +/- 0.4 days and pravastatin 0.01 mg/ml 7.6 +/- 2.3 days). The addition of similar concentrations of pravastatin to vesicle fractions alone did not influence nucleation time (> 20 days), nor could it prevent the nucleation-promoting effect of taurodeoxycholate (nucleation time with or without pravastatin 1 day). CONCLUSION: The results from this in-vitro study indicate that the presence of pravastatin in bile may not influence gallbladder bile lithogenicity. It can be hypothesized that this also applies to other HMG-CoA reductase inhibitors.

Anticholesteremic Agents↗