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PubMed1993

[What is the practical attitude toward isolated microscopic hematuria?].

Practical attitude towards isolated microscopic hematuria. The finding of isolated asymptomatic microhematuria usually raises questions about the need to perform further, invasive investigations. Phase contrast microscopy of the urine sediment is a sensitive, noninvasive method that provides information on the glomerular or non glomerular origin of hematuria, as well as on its grade. This analysis, however, must be performed by an experienced technician under standard conditions. The presence of dysmorphic (i.e. glomerular) erythrocytes indicates a glomerular disease if the count is higher than 10 erythrocytes per microliter but is considered physiological if the count is below this number. In these two cases, no further investigation will be undertaken if all criteria for isolated microhematuria are verified. Conversely, isomorphic erythrocytes reveal a non glomerular origin of hematuria, which may indicate a serious urological disease. In this case, further investigations (e.g. ultrasound, urine cytology) are recommended, taking into account the age of the patient.

Erythrocyte Count↗
PubMed1993

Expression of type I and type III procollagen by lens epithelial cells.

PURPOSE: To identify the types of procollagen that are synthesized by cultured bovine lens epithelial cells and to determine what types of post-translational modifications are involved in procollagen biosynthesis and what patterns of procollagen gene expression occur in these cells. METHODS: The epithelial nature of the cultured cells was confirmed with transmission electron and phase contrast microscopy. To label collagen, primary or secondary monolayer cultures were pulsed with [3H] proline for various periods of time. Procollagens were identified by immunoprecipitation with specific antibodies, pepsin digestion, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procollagen messenger RNA was identified by Northern blot analysis using specific DNA probes. RESULTS: In the normal lens epithelium, cells are attached to each other by tight junctions punctuated with desmosomes. After the proteins were labeled, they were immunoprecipitated with antibodies specific to collagen precursors and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to further confirm their identification as types I and III procollagen or processed forms of these proteins. The secretion of procollagen was inhibited by treatment with 2,2'-dipyridyl and bands of underhydroxylated procollagen were observed in the cell layer after electrophoresis. The sizes of messenger RNA observed were 6.9 kb and 5.4 kb for type alpha 1(I), 5.0 kb for type alpha 2(I), and 5.3 kb for the type III transcripts. CONCLUSIONS: This is the first report that lens epithelial cells express fibrillar procollagen genes, making the lens capsule a unique ocular basement membrane.

Animals↗
PubMed1996

In vitro effects of aminoglycosides and fluoroquinolones on keratocytes.

PURPOSE: Commonly used fluoroquinolones are reported to have less of an effect than aminoglycosides on corneal epithelial cells. The purpose of this study was to assess the effects of these antibiotics on stromal keratocytes in vitro. METHODS: Cultured rabbit keratocytes were incubated with various concentrations of gentamicin, tobramycin, ofloxacin, norfloxacin, and ciprofloxacin. Evaluations were performed by means of phase-contrast microscopy and [3H]thymidine uptake assay after 24 hours and 48 hours of incubation with the drug (concentrations from 3 to 0.003 mg/ml). RESULTS: At a concentration of 3 mg/ml, all three fluoroquinolones inhibited keratocyte proliferation significantly more than either aminoglycoside after 24 hours (P<0.001) and after 48 hours (P<0.001). In contrast to the aminoglycosides, all three fluoroquinolones induced a dose- dependent inhibition of proliferation after 24 hours. Even at the lowest concentration (0.003 mg/ml), ofloxacin and norfloxacin inhibited keratocyte proliferation significantly (P=0.001) compared to control after 24 hours. Concentrations of fluoroquinolones ranging from 0.09 to 0.24 mg/ml produced a 50% inhibition of proliferation, a level of inhibition not observed with any tested concentration of aminoglycosides. After 24 hours, all three fluoroquinolones, but neither of the aminoglycosides, showed moderate to severe signs of cytotoxicity at a concentration of 3 mg/ml. CONCLUSIONS: Relative effects of fluoroquinolones and aminoglycosides on epithelial cells and stromal keratocytes appear to be different. This might have an impact on choosing the optimal antibiotic drug to be applied prophylactically in clinical situations in which the epithelium is absent, such as after photorefractive keratectomy or chemical burn.

Aminoglycosides↗
PubMed1996

Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro.

Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-I beta (IL-1 beta) stimulated endothelial calls in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1 beta for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: alpha v beta 3 after 1 hour of IL-1 beta stimulation; E-selectin after 4 hours of IL-1 beta stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1 beta stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes.

Amino Acid Sequence↗
PubMed1996

[Morphological response of urinary bacteria to ceftibuten].

UNLABELLED: The morphological response of urinary Gram-negative bacteria to ceftibuten (CETB) was investigated in four patients with urinary tract infections (one patient: acute uncomplicated cystitis, three patients: chronic complicated urinary tract infection). The daily dose of CETB was 400 mg administered orally and the durations were 3 days for the acute uncomplicated cystitis case and 5 days for the chronic complicated urinary tract infection cases. In the four patients, changes in urinary CETB concentrations, viable bacterial counts, and morphology of the bacteria were studied after the initial administration. The urinary concentrations of CETB were 7.38-60.3 micrograms/ml at one hour after the first administration. The urinary viable cell counts were 1-5 x 10(7) cells/ml before administration, but they were reduced to 1-3 x 10(3) cells/ml at one hour after the first oral administration. Morphological changes of bacteria: Under phase contrast microscopy, filamentous debris and spherical bacteria with severe body damage were observed at 30 minutes after the first administration. By transmission electron microscopy, the cell wall of the filamentous bacteria showed a number of projections with formation of vacuolar structures in the space between the cell wall and the irregularly-shaped cytoplasm. According to the Japanese UTI criteria, the efficacy rate was 100% in the 4 patients. Neither side effect nor abnormal laboratory result was observed in any patient. CONCLUSION: The morphological changes of the bacteria suggested that CETB, in vivo, bounds not only penicillin-binding protein (PBP)-3 but also PBP-1.

Ceftibuten↗
PubMed1997

Flow cytometric determination of endocytosis of viable labelled Legionella pneumophila by Acanthamoeba palestinensis.

Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palestinensis) has been studied using flow cytometry. L. pneumophila cells were labelled with CM-DiI, a lipophilic fluorescent probe under conditions that did not modify viability. Coculturing the bacteria with amoebae was accompanied by rapid endocytosis; after 5 min, 90% of the amoebae had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba increased. Moreover, the number of ingested bacteria was found to be dependent on the size of the amoeba. The validity of the internalization analyzed by flow cytometry was confirmed by observation using epifluorescence and phase contrast microscopy. CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determination of bacteria endocytosis by free-living amoeba.

Acanthamoeba↗
PubMed1997

[Differentiation between glomerular and non-glomerular erythrocyturia: what is the value of differential microhematuria diagnosis?].

A few years ago, a new and simple method has been proposed to help guiding the investigation of microhematuria. This method which consists in quantifying the percentage of deformed polymorphous erythrocytes in the urinary sediment using phase contrast microscopy allows to distinguish glomerular from non-glomerular erythrocytes. In this paper, we have reviewed the recent literature concerning this approach and have discussed the conclusions according to our own experience based on the analysis of 147 patients presenting with microhematuria. Our results demonstrate that this technique is still limited by the difficulty to obtain well-defined cut-off values which effectively differentiate renal from urologic diseases. Thus, only extreme results showing either the total absence or the presence of a very high percentage of dismorphic erythrocytes appear to be helpful for the physician. Despite the introduction of this new method, many patients with microhematuria are insufficiently investigated.

Diagnosis, Differential↗
PubMed1998

Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye.

PURPOSE: Transforming growth factor-beta has been shown to induce cataractous changes in rat lenses. This study assesses the relative cataractogenic potential of TGF-beta1, TGF-beta2, and TGF-beta3 and their expression patterns in the rat eye. METHODS: Lens epithelial explants and whole lenses from weanling rats were cultured with TGF-beta1, TGF-beta2, or TGF-beta3 at concentrations ranging from 0.025 ng/ml to 4 ng/ml for 3 to 5 days. Cataractous changes were monitored daily by phase contrast microscopy and by immunofluorescent detection of cataract markers alpha-smooth muscle actin and type I collagen. Expression of TGF-beta was studied by immunofluorescence and in situ hybridization on eye sections from neonatal and weanling rats. RESULTS: All three isoforms induced morphologic changes in lens epithelial explants and cultured lenses that are typically associated with human subcapsular cataract. Transforming growth factor-beta2 and TGF-beta3 were approximately 10 times more potent than TGF-beta1. All three isoforms were expressed in the eye in spatially distinct but overlapping patterns. Transforming growth factor-beta1 and TGF-beta2 and their mRNA were detected in most ocular tissues, including the lens. Although TGF-beta3 was immunolocalized in lens epithelium and fibers and in other ocular tissues, its mRNA was detected only in the retina and choroid. CONCLUSIONS: All three isoforms of TGF-beta are potentially available to lens cells and have the potential to induce cataractous changes. The results suggest that TGF-beta activity is normally tightly regulated in the eye. Activation of TGF-beta in the lens environment, such as may occur during injury, in wound healing, or in pathologic conditions may contribute to cataractogenesis in vivo.

Actins↗
PubMed1998

Early events in the development of neuronal polarity in vitro are altered by ethanol.

Among the neuropathological effects of prenatal exposure to ethanol is the disruption of neuromorphogenesis. The effects of ethanol on early events in the development of axons and dendrites were studied using cultured embryonic rat hippocampal neurons, which develop in vitro in a stereotypical sequence of events that mimics their development in vivo. During the first 24 hr in culture, hippocampal neurons attach to the substrate and develop into one of three stages identified by phase-contrast microscopy: (i) neurons having lamellipodia and no processes (stage 1); (ii) neurons developing minor processes (<40 microm) that subsequently become the cell's axon or dendrites (stage 2); or (iii) polarized neurons with at least one axon (process with length > or =40 microm) (stage 3). Exposure to ethanol (300 mg/dl or 800 mg/dl) in the culture medium resulted in an increase in both the number of minor processes per neuron and the number of stage 3 neurons having more than the typical single axon. In addition, ethanol exposure significantly altered the proportion of neurons in the three early stages of development at 18 to 24 hr in vitro, without affecting overall neuron survival. With ethanol, there was a smaller proportion of neurons in the first stage of development, and a greater proportion of polarized stage 3 neurons. These findings suggest that ethanol alters the normal establishment of neuronal polarity, disrupting mechanisms that ensure the formation of the appropriate number of processes and that regulate the timing of process outgrowth.

Animals↗
PubMed1999

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma.

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies.

Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, M18, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody M18 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.

Antibodies, Monoclonal↗
PubMedDate not supplied

In-plane contributions to phase contrast in intermittent contact atomic force microscopy.

Contrast in the phase response of intermittent-contact atomic force microscopy (IC-AFM) reveals in-plane structural and mechanical properties of polymer monolayers. This result is unexpected, as IC-AFM has previously only been considered as a probe of out-of-plane properties. Until now, AFM measurements of nanoscale in-plane properties have employed contact mode techniques. In-plane property measurements are possible with intermittent contact AFM because there is a small but significant component of tip motion parallel to the sample surface. This in-plane component of tip displacement is virtually universal in AFM, implying that oscillating-tip techniques generally are sensitive to in-plane material properties. We present a simple Hertzian model of intermittent-contact AFM that includes such an in-plane displacement.

Journal Article↗
PubMed1955

Studies on the structure of muscle. III. Phase contrast and electron microscopy of dipteran flight muscle.

1. The flight muscles of blowflies are easily dispersed in appropriate media to form suspensions of myofibrils which are highly suitable for phase contrast observation of the band changes associated with ATP-induced contraction. 2. Fresh myofibrils show a simple band pattern in which the A substance is uniformly distributed throughout the sarcomere, while the pattern characteristic of glycerinated material is identical with that generally regarded as typical of relaxed vertebrate myofibrils (A, I, H, Z, and M bands present). 3. Unrestrained myofibrils of both fresh and glycerinated muscle shorten by not more than about 20 per cent on exposure to ATP. In both cases the A substance migrates during contraction and accumulates in dense bands in the Z region, while material also accumulates in the M region. It is proposed that these dense contraction bands be designated the C(z), and C(m) bands respectively. In restrained myofibrils, the I band does not disappear, but the C(z) and C(m) bands still appear in the presence of ATP. 4. The birefringence of the myofibrils decreases somewhat during contraction, but the shift of A substance does not result in an increase of birefringence in the C(z) and C(m) bands. It seems therefore that the A substance, if it is oriented parallel with the fibre axis in the relaxed myofibril, must exist in a coiled or folded configuration in the C hands of contracted myofibrils. 5. The fine structure of the flight muscle has been determined from electron microscopic examination of ultrathin sections. The myofibrils are of roughly hexagonal cross-section and consist of a regular single hexagonal array of compound myofilaments the cores of which extend continuously throughout all bands of the sarcomere in all states of contraction or relaxation so far investigated. 6. Each myofilament is joined laterally with its six nearest neighbours by thin filamentous bridges which repeat at regular intervals along the fibre axis and are present in the A, I, and Z, but not in the H or M bands. When stained with PTA, the myofilaments display a compound structure. In the A band, a lightly staining medullary region about 40 A in diameter is surrounded by a densely staining cortex, the over-all diameter of the myofilament being about 120 A. This thick cortex is absent in the I and H bands, but a thinner cortex is often visible. 7. It is suggested that the basic structure is a longitudinally continuous framework of F actin filaments, which are linked periodically by the lateral bridges (possibly tropomyosin). The A substance is free under certain conditions to migrate to the Z bands to form the C(z) bands. The material forming the C(m) bands possibly represents another component of the A substance. The results do not clearly indicate whether myosin is confined to the A bands or distributed throughout the sarcomere.

Actin Cytoskeleton↗
PubMed1989

Intermediary structures during membrane fusion as observed by cryo-electron microscopy.

Lipidic phases, containing 'lipidic particles' (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcho lin e and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed 'directly' by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles.

Calcium↗
PubMed2001

Problems with platelet counting in thrombocytopenia. A rapid manual method to measure low platelet counts.

Because most automated platelet counters cannot be relied on in thrombocytopenia, clinicians face a problem when decision making is based on platelet counts. Therefore we evaluated a visual platelet counting method from a blood smear with white blood cells (WBCs) as reference (PCW = platelet count based on WBC). Platelet counting for 74 thrombocytopenic (<120 x 10(9)/L) children was performed with PCW and with an automated counter (impedance principle); both methods were compared with evaluation by phase-contrast microscopy as the standard method. The PCW correlated well with the phase-contrast microscopy evaluation (y = -0.38 + 1.01x, r2 = 0.99). For platelet counts <20 x 10(9)/L the maximal deviation was 2 x 10(9)/L. The correlation between automated counts and the standard method was poor. The regression was y = 9.63 + 0.94x, r2 = 0.86. For platelet counts <20 x 10(9)/L the maximal deviation was 37 x 10(9)/L; on average, 7 x 10(9)/L platelets were counted in excess when compared with the standard method. PCW, in contrast to the automated impedance method, discriminated platelets from nonplatelet particles such as debris, fragments of red blood cells (hemolytic-uremic syndrome [HUS]) and of blast cells, and identified platelets of abnormal size. In addition, the appearance ofplatelets, WBCs, and RBCs gave clues to the etiology of thrombocytopenia, such as leukemia, infection, HUS, familial macrothrombocytopenia, and immune thrombocytopenia. PCW is a fast, reliable platelet counting method requiring less experience than the phase-contrast method. Visual evaluation from a stained smear clearly differentiates platelets and nonplatelet particles in contrast to most automated counters. In addition, the original smear can be preserved and reevaluated.

Blood Platelets↗
PubMed1981

Further light microscopic studies on morphology and development of Pneumocystis carinii.

In order to add more advance in light microscopic investigation of P. carinii, phase contrast microscopy partly followed by wet giemsa stain and semiultrathin section of the lungs embedded in JB-4 plastic were studied. In phase contrast microscopy, small and large sized trophozoites of P. carinii were clearly recognized. Although movement of trophozoite was not found, rhythmic movement of intracystic bodies with filopodia was often seen in mature cyst. Those living organisms were then directly stained with Giemsa by infiltrating under the coverglass. Thus the organism could be investigated both in unstained and stained conditions. It is noticed with interest that 8 intracystic bodies seem to fill up the cavity of cyst when cell division is completed, then they liberate and become independent into spherical bodies, followed by banana-shaped or amoeboid forms with motility. An emphasis was done that semiultrathin section made from JB-4 plastic embedded lungs was quite useful for investigation of P. carinii infection. Several sizes of mononuclear thin-walked trophozoites, mature and immature cysts, and empty cysts were more clearly distinguished than any other light microscopical method ever reported.

Animals↗
PubMed1983

Cytoplasmic granules in exfoliated buccal epithelial cells.

The cytoplasmic granules in exfoliated epithelial cells of healthy cheek mucosae of seven subjects were studied by means of phase-contrast microscopy, electron microscopy and histochemistry. An electron microscopic technique was used to study specific granules which had been identified with the light microscope. Phase-contrast microscopy revealed that the cytoplasmic granules are present in unfixed material. Ultrastructurally they consist mostly of discrete, dense granular bodies. Also seen were collections of electron-dense masses, collections of organelles and organelles mixed with granular bodies and electron-dense masses. Histochemically they stain like keratohyalin. No lipids were detected. We propose that the discrete bodies are normal keratohyalin granules and the electron-dense masses are granules in stages of disintegration. When the granules break down they become mixed with organelles. The keratohyalin granules are the darkly-stained granules seen microscopically, and the disintegrating granules and collections of organelles the weakly-stained, indistinct cytoplasmic granules.

Adult↗
PubMedDate not supplied

Use of phase imaging in atomic force microscopy for measurement of viscoelastic contrast in polymer nanocomposites and molecularly thick lubricant films.

Phase contrast microscopy, using an atomic force microscope, is used to detect and quantify changes in composition across polymer nanocomposites and molecularly thick lubricated surfaces. The technique takes advantage of the contrast in viscoelastic (viscous energy dissipation) properties of the different materials across the surface. Some materials, especially polymers, are found to display viscoelastic behavior. For such materials, the strain response lags the stress by a phase angle that is characteristic of the material. In tapping (or intermittent contact) mode, phase angle contrast is found to be highly dependent on vibration amplitude and mean tip-to-sample distance (setpoint). Phase angle contrast seems to be a stronger function of viscoelastic properties at relatively high vibration amplitude and low mean tip-to-sample distance. In this regime the effects of sample deformation, and thus viscoelastic properties, are dominant. In these contrast images, low phase angle corresponds to materials with low viscoelastic properties. This technique was used to find fairly reproducible phase angle contrast for polyethylene terephthalate (PET) films with embedded ceramic particles, metal particle (MP) magnetic tape, and Si(100) with a nonuniform Z-15 lubricant film. Very little correlation is found between phase angle images and friction force images for PET films with embedded ceramic particles and MP tape; phase angle images give information that cannot be obtained from topography or friction images. A numerical vibration model verifies that viscoelastic properties are dominant for high vibration amplitude and low mean tip-to-sample distance. For these conditions, the model also verifies that low phase angle corresponds to low viscoelastic properties.

Journal Article↗