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Biomedical subjects

D C Cottell

Publications and source records attributed to D C Cottell.

17 recordsLinked to original sources
PubMed2004

Ionizing radiation induces a stress response in primary cultures of rainbow trout skin.

Fish skin is very vulnerable to damage from physical and chemical pollutants because it is in direct contact with the aquatic environment. In this study, the effect of gamma radiation on primary cultures of rainbow trout skin was investigated. Primary cultures containing two cell types, epidermal cells and goblet mucous cells, were exposed to doses ranging from 0.5-15 Gy 60Co gamma radiation. Expression of PCNA, c-myc and BCL2 was investigated as well as growth and levels of apoptosis and necrosis. Morphological and functional changes were also studied. The irradiated cultures showed evidence of a dose-dependent increase in necrosis and enhanced proliferation as well as morphological damage. In addition, mucous cell area was found to decrease significantly after irradiation. The study shows the value of these primary cultures as in vitro models for studying radiation effects. They provide an effective alternative to whole-animal exposures for radiation risk assessment.

Animals↗
PubMed2001

Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production.

The objective of this study was to describe the ultrastructure of blastocysts derived by in vivo and in vitro methods and to investigate how the morphology is affected by exposure to cryoprotectant (10% glycerol) or cryopreservation by conventional slow freezing. In vivo derived blastocysts were characterized by a narrow perivitelline space (PvS), a continuous cover of numerous stacked microvilli (MV) on the plasma membrane, a well-defined system of cell-to-cell coupling and a large population of round or elongated mitochondria with numerous transverse cristae. Exposure of these blastocysts to cryoprotectant was manifested by shrinkage of the blastocysts and swelling of the mitochondria. Cryopreservation resulted in further shrinkage, damage to the MV, and accumulation of cellular debris. In comparison, the in vitro matured (IVM)/in vitro fertilized (IVF) in vivo cultured blastocysts displayed a wider PvS; they appeared to possess less MV and all blastocysts displayed some cellular debris in their PvS. There was also a decrease in the number of junctional contacts between the trophoblastic cells. The reaction of these blastocysts to exposure to cryoprotectant was similar to that of the in vivo derived blastocysts. However, they appeared to be more susceptible to cryopreservation. The totally in vitro produced (IVP) blastocysts displayed a wider PvS, no stacking of the MV, increased numbers of lipid droplets and a further reduction in the junctional contacts between trophoblastic cells. The IVP blastocysts sustained breakage of the zona pellucida on exposure to cryoprotectant and were extremely sensitive to cryopreservation, losing all cell structure and organization. The findings of the present study indicate that in vivo derived blastocysts possess certain structural characteristics that confer a greater tolerance on them to exposure to cryoprotectant and cryopreservation.

Animals↗
PubMed1999

Human endothelial cells cultured on microporous filters used for leukocyte transmigration studies form monolayers on both sides of the filter.

A growing number of studies on the mechanism of leukocyte transendothelial migration use endothelial cells grown on microporous filters as an in vitro model of endothelium. Ultrastructural examination of such a model system previously demonstrated that human pulmonary artery endothelial cells (HPAEC) formed confluent monolayers on both sides of the 3-microm-pore filter (Mackarel et al., 1999). To determine whether this was a characteristic specific to pulmonary artery endothelial cells, the growth characteristics of a human pulmonary microvascular endothelial cell type (HMVEC-L) and the widely used human umbilical vein endothelial cells (HUVEC) on 3-microm microporous filters were examined by transmission electron microscopy (TEM). Similar to HPAEC, HMVEC-L and HUVEC were also found to grow on both sides of the filter. All three endothelial cell types were capable of migrating through the 3 microm pores of the filter to form a monolayer on the filter underside. The endothelial cells on the underside were orientated in an inverted position with the luminal surface facing away from the filter. Such 'bilayer' formation was observed at a range of seeding densities and in different culture media. Despite the presence of a bilayer of endothelial cells, TEM demonstrated that neutrophils migrated successfully across the cell-filter-cell system. Previous transmigration reports in which an in vitro model similar to ours was used have often assumed only one layer of endothelial cells. The observations reported here indicate that while endothelial cells on microporous filters are useful models for examining leukocyte-endothelial interactions, they are not appropriate for studies examining endothelial cell 'sidedness.'

Cell Culture Techniques↗
PubMed1999

Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors.

It has long been speculated that neutrophils deploy proteases to digest subendothelial matrix as they migrate from the bloodstream. Direct evidence for the involvement of proteases in neutrophil transendothelial migration is, however, lacking. To address this issue we used transmission electron microscopy to verify the presence of continuous basal lamina beneath pulmonary endothelial cells grown on microporous filters, and then examined the effects of protease inhibitors on neutrophil migration through the endothelial cells and their associated subcellular matrix. Inhibitors of the two major matrix-degrading protease groups present in neutrophils, the matrix metalloproteinases (MMPs) and serine proteases, were assessed for their ability to modulate neutrophil transendothelial migration in response to the chemoattractant n-formylmethionyl leucylphenylalanine (FMLP). Neither the naturally occurring MMP inhibitor, tissue inhibitor of metalloproteinase-1, nor the hydroxamic acid-based inhibitors GM-6001, BB-3103, or Ro 31-9790 had any significant effect on FMLP-stimulated neutrophil migration across endothelial cells and associated basal lamina, with >/= 80% of neutrophils migrating through the system, even in the presence of inhibitors, at concentrations that totally inhibited all the gelatinase B (MMP-9) released upon stimulation with FMLP. Similarly, with serine protease inhibitors no significant inhibition of neutrophil migration was observed with a naturally occurring inhibitor, secretory leukocyte protease inhibitor, or a low molecular-weight synthetic inhibitor, Pefabloc SC. These results indicate that neither MMP nor serine protease digestion of sub-endothelial matrix is required for successful neutrophil transendothelial migration.

Cell Movement↗
PubMed1996

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells.

The phenomenon of delayed heritable lethal damage (often referred to as "lethal mutations") in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny.

Animals↗
PubMed1996

Effect of a tobacco-related nitrosamine on intercellular communication in human urothelial cells: a possible factor in smoking-related bladder carcinogenesis.

Bladder cancer is associated with smoking. Among the tobacco-derived carcinogens suspected of being involved in initiating the disease are nitrosamines found in urine. In this study a nitrosamine found in the urine of smokers was tested using a tissue culture model of normal human urothelium. Explant cultures were established from ureters and exposed to 5 ng/ml of the derivative. This level had been demonstrated previously to induce a variety of changes associated with initiation of carcinogenesis. Proliferation of the cultures was increased following exposure to the carcinogen, and the gap junction intercellular communication was reversibly inhibited. Examination of the connexin 43 protein and message status showed that the mRNA was unaffected, but the protein was not detectable using anti-connexin 43 antibody. The expression of the protein recovered within 24 h of removal of the carcinogen, indicating that the continued presence of the agent was necessary. Given the roles of cell proliferation and cell communication in carcinogenesis, the results may suggest a mechanism involving pre- or post-initiation deregulation of cell communication systems. Whether the enhanced growth is a separate effect or a consequence of reduced communication is an intriguing question.

Carcinogens↗
PubMed1994

Three-dimensional structure of lung elastin demonstrated by scanning electron microscopy/stereo-pair images.

The aim of this study was to expose the inflated 3-D structure of lung elastin. Formic acid digestion followed by freeze-drying unveiled the lamellar framework. The 3-D structure of elastin was well preserved within the alveolar septa and ducts, as demonstrated by scanning electron microscopy/stereo-pair photography. Elastin fibers are seen in the alveolar septa, which are continuous with the lamellae. The removal of collagen fibers and cells by formic acid was visualised as a function of time: The optimum was 48 hours. Transverse sections still retained some collagen fibrils and partially digested cells in addition to elastin as shown by transmission electron microscopy (TEM). Formic acid digestion followed by critical point drying caused damage to the lamellar structures and they appeared to collapse. Sodium hydroxide digestion combined with freeze-drying did not preserve the 3-D lamellar structure of elastin, but converted it into flat ribbonlike bands. The main structures remaining following alkali treatment were identified by TEM as collagen fibrils well preserved in their original locations.

Animals↗
PubMed1991

Development of the lung in mice with bromodeoxyuridine-induced cleft palate.

Clinical and laboratory observations show that denial of free communication between the amniotic fluid and lung fluid results in pulmonary hypoplasia. Thus, cleft palate resulting from tongue obstruction to palatal shelf elevation might be associated with disturbed lung development. This association exists in the Pena-Shokeir phenotype. The goal of these experiments was to see what effect bromodeoxyuridine (BUdR)-induced cleft palate had on lung development. LACA mice were injected with 500 mg/kg BUdR on E11 or E11 and E12 of gestation, a treatment known to produce a 25% and 50% incidence of cleft palate, respectively. BUdR had a direct retarding effect on lung growth but, when cleft palate occurred as well, the lungs were more severely affected. Morphometry showed that lungs from fetuses with cleft palate had only one-half the saccular volume of controls or of treated fetuses with normal palates. Although hypoplastic, lungs associated with cleft palate had type I and type II pneumocytes, and the latter were shown by electron microscopy to be capable of producing surfactant. Hence, cellular differentiation had not been affected by the treatment. Fetuses with cleft palate had less amniotic fluid than controls but significantly more than those with normal palates after treatment. Thus, the pattern of abnormalities in this animal model bears some resemblance to that of the human Pena-Shokeir phenotype.

Animals↗
PubMed1990

Study of the mechanisms of BUdR-induced cleft palate in the mouse.

This study was designed to examine the pathogenesis of bromodeoxyuridine (BUdR)-induced clefts of the secondary palate in the LACA mouse. Intraperitoneal injections of BUdR (500 mg/kg body weight) were given at various days and combinations of days between E11 and E15 (plug day = E1). Treatment on E11 alone resulted in approximately 22% of fetuses with cleft palate when the latter were examined either on E16 or E19. Treatment on E11 and E12 approximately doubled the above incidence, and treatment on E11, 12 and 13 raised it to 100%. However, no treatment, either single or multiple, caused cleft palate when given later than E11. This suggests that the cellular changes caused by BUdR that lead to cleft palate must be inflicted during E11 and that such damage can be repaired in about 80% of embryos. All fetuses with cleft palate had severe micrognathia on E16 and E19, which skeletal staining showed to be the result of a bilateral sigmoid buckling of Meckel's cartilage. Studies with the scanning electron microscope (SEM) on E15, 16, and 19 suggested strongly that the micrognathia caused a relative macroglossia and hence mechanical interference with palatal shelf reorientation. Histological studies with the light microscope showed that BUdR caused cellular necrosis in many embryonic tissues during the 24 hours after its administration. This necrosis was strikingly more severe in the mandibular rudiment of the first branchial arch than in the maxillary. The latter observation accords well with findings by other workers that cell proliferation is more rapid in the mandibular blastema than in the maxillary. Transmission electron microscope (TEM) studies of the buckled region of Meckel's cartilage failed to reveal any ultrastructural differences from control Meckel's cartilage. Hence BUdR had only interfered with the shape of the cartilage but not with its histiogenesis. We conclude that BUdR, by its cytotoxicity or antidifferentiative effects, interfered with the formation of the anterior end of Meckel's cartilage, initiating a chain of events leading through micrognathia and relative macroglossia to failure of palatal shelf reorientation and cleft palate.

Animals↗
PubMed1989

Immunohistochemical staining of bromodeoxyuridine-labelled cells in the mouse embryo.

The aim of this study was to compare two immunohistochemical methods, avidin-biotin peroxidase and immunogold silver staining (IGSS), in the detection of 5-bromodeoxyuridine incorporation in mouse embryo tissues. In addition, two fixation schedules, formal-saline and a mixture of formaldehyde and glutaraldehyde (4FIG), and two embedding procedures, paraffin wax and the acrylic resin L.R. White, were also compared. Pregnant mice were injected with 600 mg per kilogram body weight on days 10 or 15 (plug day = day 1) of gestation and the embryos recovered 2 h after treatment and fixed in formal-saline or 4FIG. Fixed material was then processed into wax blocks or L. R. White resin. After sectioning, the antigen-antibody reaction was visualized using either the avidin-biotin peroxidase or IGSS methods. All methods tested gave a well-contrasted, highly specific reaction, but IGSS in combination with 4FIG and L. R. White gave a clearer and more sensitive signal than other combinations.

Animals↗
PubMed1985

Some limitations of the use of tannic acid as a marker of damaged skeletal muscle fibres.

Mouse biceps brachii muscles containing single transverse incisions were treated with low molecular weight tannic acid. In the absence of fibre degeneration, intracellular spread of tannic acid was confined to a short distance along the fibres from the damaged portions of the cell membranes. Beyond this, damaged and undamaged fibres could not be distinguished from one another. Tannic acid is not a reliable marker of localized trauma to skeletal muscle fibres.

Animals↗
PubMed1983

A method for the detection of amyloid in human tissue embedded in epon using a combination of polarizing light microscopy and electron microscopy.

This paper describes a method for demonstrating amyloid in human tissue embedded in epon using a combination of congo red staining and polarizing microscopy. The method involves the removal of the embedding medium from semi-thin sections prior to staining with congo red and examination in the polarizing microscope. Typical apple green birefringence was detected in areas of amyloid deposition; these areas were mapped for selective trimming and ultra-thin sections taken serially were examined in the electron microscope. The method was found to be reliable, easy to use and particularly of value as a sampling procedure prior to investigation in the electron microscope.

Amyloid↗
PubMed1977

Alkaline phosphatase in mitochondria.

In electron microscope cytochemical studies alkaline phosphatase activity was present in the mitochondria of all liver cells and associated with the plasma membrane of the cells of bile canaliculi. The mitochondrial activity was partially inhibited by L-phenylalanine and Levamisole but the plasma membrane associated activity was completely inhibited by Levamisole. Biochemical assays have shown that a significant amount of the total mouse liver alkaline phosphatase activity was present in the mitochondria fraction. Starch gel electrophoresis showed that this mitochondrial alkaline phosphatase had a characteristic isoenzyme pattern, consisting of 3 distinct bands which were not retarded by neuraminidase treatment. The enzyme in the mitochondria-free supernatant showed one wide band which was retarded by neuraminidase.

Alkaline Phosphatase↗
PubMed1977

Alkaline phosphatase activity in human bladder tumor cell lines.

The cellular localization and isoenzyme pattern of alkaline phosphatase in five cell lines derived from human bladder carcinomas (T24, RT4, RT112, J82, EJ) shown not to be HeLa cells has been established. RT112 cells had a high level of alkaline phosphatase. RT4 had a moderate amount of alkaline phosphatase but in the other three lines, levels were extremely low. Prednisolone caused a small (2 to 3-fold) increase in total alkaline phosphatase in T24 and RT112 lines only. Electrophoretic separation of isoenzymes showed that RT112 and RT4 cells (derived from more highly differentiated tumor types) had three heat stable bands equivalent to placental alkaline phosphatase and three slower bands of a modified placental type. Prednisolone increased only the former. In T24 cells the enzyme resembled the liver-type alkaline phosphatase in electrophoretic mobility and sensitivity to heat denaturation. Cytochemical studies confirmed the presence of cell surface-associated extramembraneous placental type enzyme in RT112 cells. All five cell lines had small deposits of intramembraneous alkaline phosphatase in the plasma membrane and deposits associated tith the mitochondrial membranes and the endoplasmic reticulum that were not completely inhibited by phenylalanine or Levamisole.

Alkaline Phosphatase↗