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PubMed1979

Sarcomere motion in isolated cardiac cells.

Computerized image-analysis techniques have been employed to examine the sarcomere dynamics of isolated mammalian cardiac myocytes. The cells were prepared by perfusion of adult rabbit hearts with hyaluronidase-collagenase solutions; they exhibited phasic contractions in the presence of 10(-6) M Ca2+. The dissociated cells were visualized by phase microscopy and a video camera interfaced in a minicomputer. Digitized cell images were processed by an algorithm utilizing signal averaging and contrast enhancement to yield data showing individual sarcomere position and shortening vs. time, so that patterns of sarcomere activation could be observed in spontaneously contracting cells. Compared to records of whole-cell shortening and of striation displacement, computerized image analysis provided a much more faithful indication of time course and sequence of sarcomere shortening. Spontaneously contracting cells showed sequential sarcomere shortening beginning at one end and propagating longitudinally with a constant velocity, typically at 100--150 micron/s for beat rates of 40 min-1. Velocities of initial sarcomere shortening appeared to increase with elevated Ca2+. These observations are consistent with a regenerative mechanism of calcium-induced calcium release.

Animals↗

PubMed1986

Use of monoclonal antibodies to culture rat proximal tubule cells.

Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation.

Animals↗

PubMed1997

Developmental changes in flow-wave propagation velocity in embryonic chick vascular system.

We analyzed flow-wave propagation velocity in the early embryonic vascular system and its responses to acute alterations in circulating blood volume. Two 20-MHz pulsed Doppler velocimeters were positioned along the arterial system in stage 18 (n = 12), 21 (n = 10), and 24 (n = 11) chick embryos. Distance between the two measurement sites was measured by video-microscopy. Phase velocity was calculated using Fourier transform up to the fourth harmonics. Wave-front velocity was also calculated by threshold technique. In a subset of embryos at stage 24 (n = 10), circulating blood volume was acutely altered to change stroke volume. Mean phase velocity increased from 42.9 +/- 3.3 to 95.8 +/- 7.5 cm/s from stage 18 to 24 (P < 0.05 by analysis of variance), whereas wave-front velocity increased from 52.8 +/- 2.4 to 72.2 +/- 5.2 cm/s. Stroke volume and mean aortic pressure paralleled the changes in mean phase velocity and wave-front velocity in normal development and in response to changes in circulating blood volume. Thus developmental changes in wave-propagation velocity were consistent with changes in the size of the vascular system, pressure range, and elastic properties of the arterial wall during systemic vasculogenesis in the embryo.

Animals↗

PubMed1990

Human tracheobronchial submucosal gland cells in culture.

Cellular mechanisms regulating airway secretion, secretory products of individual airway cell types, and control of airway cell growth and differentiation are poorly understood. In order to aid studies of these questions, we have established a system for culturing human tracheobronchial submucosal gland cells. Gland acini were isolated by enzymatic disaggregation from submucosal tissue obtained postmortem from patients without pulmonary diseases and from patients with cystic fibrosis. In culture, acini attached to a collagen substratum, and gland cells proliferated and formed confluent monolayers which were homogeneous by phase microscopy. In contrast to cells of freshly disaggregated acini which expressed either serous or mucous gland cell secretory antigens, in culture virtually all cells (greater than or equal to 95%) concurrently expressed both antigens as assessed by immunocytochemical staining with serous and mucous cell-specific antibodies. Similarly, electron microscopy revealed cells with serous- or mucous-type secretory granules, and cells containing both types of granules. Cultures incorporated 35S into high (greater than 10(6) D) and lower (greater than 700 kD; 150 kD) molecular weight molecules. Cholinergic and adrenergic agonists increased release of radio-labeled secretions. These findings demonstrate that human tracheal gland cells in culture retain immunocytochemical, ultrastructural, and functional features of both differentiated serous and mucous gland cells. This culture system will be useful for studying the biology and pathology of human tracheobronchial submucosal gland cells.

Adolescent↗

PubMed1985

Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol. Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0). Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge. By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells. Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2. Cumulative suspension cells on ECM dishes were eight times controls. ECM influenced both hematopoietic progenitor cell proliferation and differentiation. Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05). There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points. This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment.

Animals↗

PubMed1981

Thrombocytopenia in neonatal infection.

Serial platelet counts by phase microscopy were done for three groups of neonates who were admitted to the Special Care Unit of the Miller Children's Hospital, Group 1 (78 neonates) was evaluated for septicemia. Group 2 (28 neonates) was randomly selected sick neonates whose working diagnosis was not septicemia. Group 3 (16 neonates) was clinically normal preterm neonates (28 to 36 weeks gestation). For group 1 and 2, platelet counts were done serially, at the time blood and cerebrospinal fluid cultures were obtained and then 12, 24, 48 and 72 hours later. For group 3, plasma counts were done on the second, seventh, fourteenth, twenty-first, and twenty-eighth day of life. Sixteen of the group 1 infants were found to have septicemia; ten of these 16 had thrombocytopenia (platelets less than 100,000/mm3). In group 2, five infants had thrombocytopenia, one because of isoimmune disease and four as a result of possible disseminated intravascular coagulation. Thrombocytopenia persisted for 1 to 10 days; platelet counts of group 3 were the same as those of older children and adults.

Female↗

PubMed2001

Application of a PCR assay to enhance the detection and identification of Tritrichomonas foetus in cultured preputial samples.

The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination. Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay. The objective of this study was to evaluate a previously described PCR assay for detecting T. foetus in cultured preputial material. The detection limits of the assay for T. foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined. Preputial samples were collected from 13 bulls uninfected with T. foetus. The PCR assay was able to detect 5 T. foetus organisms in the growth medium and the cultured preputial material. Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms. The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination. This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test. In addition, the assay could be used to confirm the identification of T. foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.

Animals↗

PubMed2005

A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites.

BACKGROUND: In the absence of intermediate animal hosts, the process of embryogenesis leading to fecundity of adult female filarial worms is very critical for persistence of these obligate parasites in human communities. Embryogenesis in adult female filarial parasites involves fertilization of eggs or oocytes by sperms and their subsequent development into motile microfilariae inside the uterine cavity of worms. Development of assays for monitoring embryogenesis in adult female worms is a critical requirement in filariasis research--filarial worms are known to harbour endosymbionts such as Wolbachia which play a significant role in fecundity. Tetracycline or doxycycline treatment of the infected hosts effectively eliminates the endosymbionts resulting in inhibition of embryogenesis in female worms. Currently, inhibition of embryogenesis in adult filarial worms can be monitored only by microscopic examination of in vitro harvested intrauterine stages. METHODS: Adult female filarial worms of bovine filarial parasites, Setaria digitata were collected from the peritoneum of infected animals and intrauterine stages were harvested in culture medium and were analyzed for forward and side scatter by flowcytometry using a BD FACS Calibur. Different populations were gated, sorted and identified by phase microscopy. Binding of biotinylated lectins to intra uterine stages was monitored using FITC labeled Avidin and monitored by flow cytometry of gated populations. Similarly, binding of antibodies in human filarial sera to intrauterine stages was monitored using FITC labeled anti-human immunoglobulins. RESULTS: The forward and side scatter for intrauterine stages delineated 3 distinct populations labeled as R1, R2 and R3. The three populations were sorted and identified to be a) fully stretched microfilariae, b) early and c) late developmental stages of eggs respectively. Lectins such as Wheat Germ agglutinin or Concanavalin-A were found to bind strongly to egg stages and less prominently to intra-uterine microfilariae. Similarly the binding of antibodies in filarial sera to the three intra-uterine stages could also be precisely quantified. CONCLUSION: The manuscript reports a novel flow cytometry based method to monitor progression of embryogenesis in adult filarial worms. Apart from relative quantification of different intra uterine developmental stages, the assay allows quantitative binding of lectins and antibodies to each of the intrauterine stages. It may now be possible to quantify levels of antibodies in infected and immune hosts to monitor anti-fecundity immunity in filariasis--the assay can thus be used as a powerful tool for drug development and in immunological studies in human and experimental filariasis.

Journal Article↗

PubMed1983

Alterations in cultured fibroblasts of sibs with an infantile form of a free (unbound) sialic acid storage disorder.

Cultured fibroblasts from two sibs with generalized hypertonia, hepatosplenomegaly, and psychomotor retardation within the first year of life were found to have unusual morphologic features. When examined by phase microscopy, the unstained and unfixed cells contained a large number of vacuolated structures whose gross appearance resembled that of a honeycomb in the cell cytoplasm. Electron microscopy studies, following fixation, showed the "honeycombing" to be the result of numerous, closely packed, cytoplasmic, membrane-bound vacuoles. In some of these structures the remains of fibrilogranular material could be detected. Biochemical analysis of crude sonicates of these cells revealed increased levels (4--7 x N) of an acid soluble component that reacted with thiobarbituric acid. Analysis of trimethylsilyl derivatives of this material by gas liquid chromatography and mass spectrometry showed it to be indistinguishable from sialic acid (N-acetylneuraminic acid). Quantitation of this material from the cells of one of the sibs after isolation on a Dowex column yielded 39.8 nmoles of free (unbound) sialic acid per mg protein whereas normal fibroblasts had 1--2 nmoles per mg. Bound sialic acid levels were at the upper limits of normal (24.8 versus 11--23 nmoles per mg protein). The concentration of cytidine monophosphate-sialic acid was normal. After incubation of the patient's fibroblasts with [3H]-N-acetylmannosamine for 72 h, there was a 7-fold increase (compared to normal fibroblasts) in the amount of radioactivity in free sialic acid present in the acid soluble fraction. The amount of labeled, bound sialic acid in the acid-insoluble pool, however, was the same in both patient and control fibroblasts.

Carbohydrate Metabolism, Inborn Errors↗

PubMed1988

The motility of human milk macrophages in collagen gels.

The hypothesis that the diminished motility of human milk leukocytes is due to a decrease in adherence was tested by using a collagen gel system in which leukocyte movement is less dependent on adherence. Unfractionated human milk leukocytes (HML) or fractionated peripheral blood leukocytes were placed on collagen gels in microwells and the leading edge of migration was determined by inverted phase microscopy. The mean rates of invasion of HML, blood neutrophils, and mononuclear blood leukocytes were 14, 240, and less than 1 mu/h, respectively (p less than 0.01). We then examined the identity of motile HML by immunoperoxidase techniques using antibodies to selected cell markers. Motile HML were positive for a specific macrophage marker (cathepsin B) and a neutrophil and monocyte marker (Mac-1) but were negative for specific neutrophil (cathepsin G) or lymphocyte markers (CD3 and CD5). The directed motility of these cells was not enhanced by exposure to the chemoattractant, N-formyl-L-methionyl-L-phenylalanine, but was inhibited by a T cell lectin, phytohemagglutinin. The movement was actin dependent but was not dependent on calcium or Mac-1 surface glycoproteins. Thus, the diminished motility of milk neutrophils does not appear to be due to decreased adherence per se, and those HML that are motile are macrophages. This suggests a dichotomy for the function of HML. Neutrophils may be relegated to the lumen of th alimentary tract, whereas macrophages may penetrate into mucosal sites for host defense.

Cell Migration Inhibition↗

PubMed2006

Hyperoxia and apoptosis in developing mouse lung mesenchyme.

Hyperoxia contributes to the development of bronchopulmonary dysplasia in former premature infants. Injurious environmental factors such as hyperoxia may disrupt distal airway branching and alveolar septation, as these critical stages in lung development occur following birth in extremely premature infants. To test if hyperoxia directly inhibited distal airway branching, we cultured E16 fetal mouse lung explants in either 20% (control) or 95% oxygen (hyperoxia). Hyperoxia reduced the number of distal airways to less than 50% of controls. Explants cultured in 95% oxygen also had fewer complex distal airways compared with controls. Mesenchymal cells adjacent to distal airways in hyperoxic explants appeared apoptotic by phase microscopy. Consistent with increased apoptosis, explants cultured in hyperoxia had increased caspase 3/7 activity compared with controls. Hyperoxia also increased mesenchymal caspase 3 expression and annexin V binding within cultured explants as visualized by fluorescence microscopy. We measured increased annexin V binding in isolated primary fetal lung mesenchymal cells cultured in 95% oxygen suggesting a direct effect on cells within the mesenchyme. Hyperoxia can lead to NF-kappaB activation, which mediates inflammatory cascades and may protect cells from apoptosis. We detected NF-kappaB activation and nuclear p65 localization in explants exposed to 48 h of hyperoxia. Inhibition of NF-kappaB prevented the hyperoxia-induced activation of caspase 3. NF-kappaB activation may therefore contribute to apoptosis in the developing fetal mouse lung following hyperoxia exposure. Our data suggest hyperoxia inhibits distal airway branching and directly induces apoptosis of the fetal mouse lung mesenchyme.

Animals↗

PubMed2006

Mucoadhesive, thermosensitive, prolonged-release vaginal gel for clotrimazole:beta-cyclodextrin complex.

The purpose of this study was to achieve a better therapeutic efficacy and patient compliance in the treatment for vaginitis. Clotrimazole (1%) has been formulated in a vaginal gel using the thermosensitive polymer Pluronic F127 (20%) together with mucoadhesive polymers such as Carbopol 934 and hydroxypropylmethylcellulose (0.2% for both). To increase its aqueous solubility, clotrimazole was incorporated as its inclusion complex with 1:1 molar ratio with beta-cyclodextrin. The inclusion complex was thoroughly characterized using various techniques, including 1H NMR spectroscopy, FT IR spectrophotometry, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and determination of stability constant (k(1:1)). The gelation temperature and rheological behavior of different formulations at varying temperatures were measured. In vitro release profiles of the gels were determined in pH 5.5 citrate buffer. It was observed that complexation with cyclodextrin slowed down the release of clotrimazole considerably. Carbopol 934, on the other hand, was found to interact with beta-cyclodextrin, inducing precipitation. As far as rheological properties are concerned, thermosensitive in situ gelling was obtained with formulations containing drug:cyclodextrin complex rather than with free drug. Thus, the optimum formulation for a controlled-release thermosensitive and mucoadhesive vaginal gel was determined to be clotrimazole:beta-cyclodextrin 1% with 0.2% hydroxypropylmethylcellulose in Pluronic F127 gel (20%) providing continuous and prolonged release of active material above MIC values.

Antifungal Agents↗

PubMed1983

Muscle plasma membrane abnormalities in infants with Duchenne muscular dystrophy.

Muscle biopsies from one preclinical case, one early case, four symptomatic cases of Duchenne muscular dystrophy, and six controls were investigated for the presence of delta lesions by phase optics and conventional electronmicroscopy. By phase microscopy the frequency of delta lesions was 4.9% in the preclinical and early cases and 4.0% in the four symptomatic cases. The incidence of delta lesions in the controls was 0.4%. Electronmicroscopic studies of delta lesions revealed similar findings in the preclinical, early, and symptomatic cases. The common denominator of the delta lesions was disruption of the muscle plasma membrane. Plasma membrane lesions occur early in Duchenne muscular dystrophy.

Cell Membrane↗

PubMed1980

Angiotensin-converting enzyme: II. Pulmonary endothelial cells in culture.

Direct studies of the function of a given cell type often require that the cell type be obtained in pure culture. A number of different specific metabolic activities have been attributed to pulmonary endothelial cells, yet with few exceptions the conclusions were based on indirect evidence. Thus, to improve our ability to examine directly for specific metabolic activities, we began a program to obtain pulmonary endothelial cells in culture. Two methods have been developed: (1) cells can be obtained from pulmonary artery and vein of large animals (cow, pig), and (2) cells can be obtained from the microvasculature of small animals (rat, guinea pig, and rabbit). The latter technique can also be used to obtain cells from a lobe of lung from large animals and may be adaptable for use with human tissue. In the first technique, pulmonary arteries, free of blood, are filled with collagenase (0.25%, 500 units) in Puck's saline for 25 min. The collagenase mixture containing cells is removed and centrifuged. The pellet is resuspended and seeded into culture flasks. In the second method, lungs are perfused (artery to vein) with Krebs-Henseleit solution until the effluent is blood-free. Collagenase (0.25%, 500 units) is introduced, and the lungs are then perfused in the opposite direction (vein to artery) until the flow stops spontaneously (ca. 15 min). The detached cells are collected and seeded as before. The endothelial cells attach as small clumps (10-50 cells). Those flasks which contain more than 95% endothelial cells (by phase microscopy) are retained for culture and the lines are purified usng differential adherence procedures. The cells grow as monolayers with a cobblestone appearance. They contain Weibel-Palade bodies. They possess converting enzyme activity and are reactive with antibodies to converting enzyme, Factor VIII and alpha 2-macroglobulin. The cells synthesize prostaglandins and related substances. In addition, they possess ADPase and synthesize angiotensin-converting enzyme.

Adenosine Diphosphate↗

PubMedDate not supplied

[The prevalence of Demodex folliculorum and Demodex brevis in a population sample from Botucatu, São Paulo, Brazil].

A survey was conducted to determine the prevalence of D. folliculorum and D. brevis for the first time in Brazil. In this study, association between the presence of mites and host factors (age, sex and race) were analysed. Samples were obtained from 100 individuals submitted to a facial cleaning in an aesthetic clinic in Botucatu city. All the samples were mounted in Berlese's medium and examined by dark-field phase microscopy. From 100 studied individuals, 72 were positive, among the positive cases, 51% showed D. folliculorum, 2% showed D. brevis and 19% both species. The parasite distribution in relation to sex was not taken in account because the sex ratio favoured females (90%). According to age, prevalence was high in all age groups. The factors influencing this distribution could be due. 1. the examination of extensive skin areas, 2. the group examined composed of individuals in treatment in an anaesthetic clinic could be more infested than a normal population, 3. the fact that in tropics, the prevalence is often high in all ages.

Adolescent↗

PubMed2005

Normalization of high-flow or removal of flow cannot stop high-flow induced endothelial proliferation.

Endothelial cells (ECs) are activated in response to high-flow. Our previous studies using arteriovenous fistula (AVF) model have demonstrated that high-flow in blood vessels induces an early and rapid proliferation of ECs before arterial dilatation. Here, we investigated the proliferation of ECs, which had once been stimulated by high-flow loading, in a situation without the influence of high-flow. First, we induced high-flow in the rabbit common carotid artery by using AVF. Then, we removed the influence of high-flow by normalization of high-flow with the closure of AVF or by removal of flow itself with tissue isolation and organ culture or with cell culture of ECs, at the timing considered that ECs began to proliferate. Kinetics of ECs was investigated by a laser scanning confocal microscopy, phase-contrast microscopy and light microscopy using bromodeoxyuridine labeling method. We found that ECs, which had once been stimulated by high-flow, transiently proliferated even after normalization of high-flow or removal of flow. We assume that proliferation of ECs is promised when these cells start to proliferate after high-flow loading.

Animals↗

PubMed1986

The reversible inhibition of myoblast proliferation by gamma-hexachlorocyclohexane.

The effects of treating non-fusing myoblast variants, fu-1 and M3A, with two levels (1 X 10(-4) M and 2 X 10(-4) M) of gamma-hexachlorocyclohexane, an inhibitor of phosphatidylinositol synthesis, on myoblast proliferation were evaluated by measuring myoblast proliferation (counting cells) and visual inspection via phase microscopy. In the presence of gamma-hexachlorocyclohexane, these cells were arrested, presumably in G1. The inability of these cells to replicate did not appear to be due to a toxic effect of gamma-hexachlorocyclohexane, because these cells were capable of resuming proliferation once they were transferred to media lacking gamma-hexachlorocyclohexane. Cells were grown in media containing myo-[2-3H]inositol and the radioactive content of water-soluble metabolites, the end product of phosphatidylinositides hydrolysis, was quantitated. Cells were grown in the presence of gamma-hexachlorocyclohexane, in addition to the loss of proliferative ability, also contained significantly less water-soluble metabolites. Therefore, it appears that there is a direct relationship between phosphatidylinositol metabolism and cell proliferation in the cell lines studied.

Animals↗

PubMed1998

Replication and pathogenesis of white sturgeon iridovirus (WSIV) in experimentally infected white sturgeon Acipenser transmontanus juveniles and sturgeon cell lines.

Characteristics of the in vitro propagation of the white sturgeon iridovirus (WSIV) were examined in 6 sturgeon cell lines. One new cell line originating from gonadal tissues (WSGO) produced up to 12-fold more WSIV [approximately 22 TCID50 (50% tissue culture infective dose) cell-1], than that of a previously established reference spleen cell line (WSS-2). Infected WSGO cell cultures were examined using phase microscopy, viral infectivity assay and transmission electron microscopy (TEM). At 15 degrees C, both mature virions and infectious virus were first detected after 7 d post-infection. Capsids acquired envelopes in the cytoplasm and virions remained primarily cell-associated during the 35 d replication cycle. Cellular changes including hyper-refractility and cytoplasmic swelling with dense cytoplasmic inclusions correlated to extensive proliferation of cytoplasmic vesicles and viral assembly sites. These cytological characteristics corresponded to changes in target cells of WSIV-infected juvenile white sturgeon following bath challenge. Microscopic changes in stained tissue sections of the host epithelium were detected 4 d post-challenge, approximately 8 d prior to the onset of clinical signs. Hypertrophied Malpighian cells surrounded by a prominent pericellular cisternum characterized epithelial lesions in the skin. Similar changes to epithelial cells of the barbels, olfactory organs and esophagus were also observed. Destruction of the sensory epithelium is suggested as a cause for cessation of feeding which occurs early in the infection of white sturgeon juveniles with WSIV.

Animals↗