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Biomedical subjects

J E Beesley

Publications and source records attributed to J E Beesley.

At least 37 records · Page 2Linked to original sources
PubMed1992

The human squamous cervical carcinoma cell line, HOG-1, is responsive to steroid hormones.

Growth of the human squamous cervical carcinoma cell line, HOG-I, was stimulated in response to oestradiol in serum-containing and chemically defined medium. The oestradiol-stimulated growth could be inhibited by 4-OH tamoxifen, progesterone and medroxyprogesterone acetate; the last 2 compounds also inhibited basal cell growth in serum-containing and chemically defined media. The data are consistent with the sensitivity of human squamous cervical cancer to sex-steroid hormones and suggest that endocrine therapies may be of benefit in this disease.

Adult↗
PubMed1992

Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.

Animals↗
PubMed1992

Adjuvanticity and ISCOM formation by structurally diverse saponins.

Adjuvant activity and immunostimulating complex (ISCOM) formation by a series of saponins and glycoalkaloids differing in the structures of their aglycones and sugar chains were examined. The only two saponins apart from Quillaia that were adjuvant-active were Gypsophila and Saponaria, which resemble Quillaia in that they contain saponins with branched sugar chains attached to positions 3 and 28 of the aglycone. Glycoalkaloids with a branched sugar chain lacked adjuvant activity. Saponaria saponins formed irregular ISCOM-like structures, and Gypsophila produced a sheet of joined pore-like structures. The alfalfa hederagenin saponin and Quinoa also formed pore-sheets, despite lacking adjuvanticity.

Adjuvants, Immunologic↗
PubMed1992

Microbiochemical analysis of changes in proteoglycan and collagen in joint tissues during the development of antigen-induced arthritis in the rabbit.

Microbiochemical assays of the proteoglycan and collagen content of articular cartilage and synovial lining have been performed on tissue sections taken from rabbits with antigen-induced arthritis. This experimental arthritis is a close analogue of the natural disease-rheumatoid arthritis. Animals were killed at intervals during the first 21 days following induction of arthritis to assess changes in the composition of the extracellular matrices of the synovial lining and articular cartilage during the early development of this experimental lesion. In confirmation of earlier studies these microbiochemical assays revealed a rapid and significant loss of proteoglycan from the articular cartilage. This loss was, however, not uniform but was restricted to the intermediate zone of the cartilage. Over the period studied, there was only a slight loss of proteoglycan from the superficial zone of the cartilage facing the joint cavity. These findings demonstrate that, at least in this model, cartilage proteoglycan loss is not due to the action of proteases present in the synovial fluid. Moreover it suggests that the chondrocytes in the mid-zone of the cartilage are responsive to those signals stimulating proteoglycan breakdown. There was no significant loss of collagen from the cartilage over the time period of this study. The synovial lining from arthritic joints, in contrast, showed a progressive increase in both proteoglycan and collagen.

Animals↗
PubMed1992

Scanning X-ray microanalysis of microcarrier cultured endothelial cells: elemental changes during the transition to confluency and the effect of ionophore A23187.

Porcine endothelial cells were grown on microcarrier beads and examined by scanning electron microscopy (SEM) at various times after initiation of culture. Total cell coverage on the bead surface varied from mean values of approximately 7% (3h) to 80% (96h). Beam penetration into the subcellular matrix presents a major problem with SEM X-ray microanalysis of microcarrier cultured cells and necessitates the use of an accelerating voltage not exceeding 10kV. At this voltage and below, X-ray contribution from elements present in the microcarrier bead has minimal effect on the determination of cell elemental levels. Washing the cells with 0.15M sucrose was the least perturbing of the rinsing techniques investigated, removing surface culture medium but not internal diffusible ions. X-ray microanalysis revealed detectable levels of Na, P, S, Cl, K and Ca in the cells, with well-marked changes from initial attachment to confluency. The level of K decreased from approximately 1.0% at 3h to 0.4% at 24h, with a corresponding decrease in the K/Na ratio. This unexpectedly low level of K was invariably observed after 24h, and is a genuine feature of established microcarrier culture. The effect of ionophore A23187 was determined at the 3h culture stage, and resulted in significant increases in the concentration of divalent cations (Mg2+, Ca2+), monovalent ions (Na+, Cl-) and a decrease in the level of K+.

Animals↗
PubMed1991

CD11/CD18 cell surface adhesion glycoproteins: discordance of monocyte function and expression in response to stimulation.

The adherence of blood leukocytes to vascular endothelium precedes their diapedesis into the extravascular space. These processes require the expression of adherence glycoproteins on the cell surface of the leukocyte. The relative importance of these adherence molecules is so far poorly understood. However, there is evidence to suggest that a disparity exists between the surface receptor expression of these glycoproteins and leukocyte adherence to vascular endothelial cells in culture. We have investigated the importance of each of the adhesion glycoproteins CD11a, CD11b, and CD11c in mediating the adherence of human monocytes to endothelial cells in culture. We have also investigated the chronological relationship between changes in monocyte adherence to endothelial cells and the surface expression of CD11a, CD11b, and CD11c following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The increase in adherence occurred within 1 minute, but declined if monocytes were preincubated with fMLP for up to 30 minutes. The surface expression of adherence molecules demonstrated a significant increase in CD11a and CD11b in the presence of fMLP after 10 min and was maintained while monocyte adherence to endothelium declined. These changes in surface receptor expression were quantitated using an immunolabeling technique. It is suggested that fMLP stimulation of monocyte adherence is unlikely to be solely dependent on increased surface receptor expression of adhesion molecules.

Adult↗
PubMed1991

Mitochondria of mammalian Plasmodium spp.

Highly purified mitochondrial fractions have been isolated from the intraerythrocytic stages of two mammalian Plasmodium spp., Plasmodium yoelii of rodents and Plasmodium falciparum of man. Mitochondria of the former parasite are cristate whereas those of the latter are essentially acristate. Isolated mitochondria from both parasite species were heterogeneous with respect to size, shape, density of matrix staining and extent of internal structure. Respiratory assay, by reduction of exogenous cytochrome c, showed NADH, alpha-glycerophosphate and succinate to be the substrates with the greatest potential for metabolism. Additionally, proline, dihydroorotate and glutamate (P. falciparum only) were oxidized at low rates. A number of NAD(+)-linked substrates were not utilized. The NADH-dependent reduction of cytochrome c was insensitive to rotenone and antimycin A. Fumarate inhibited the NADH-dependent reduction of cytochrome c and stimulated the oxidation of NADH, suggestive of an NADH-fumarate reductase pathway. Oxidation of either alpha-glycerophosphate or succinate was fully inhibited by standard mitochondrial electron transport inhibitors, including a number of Complex III inhibitors, although the concentrations required of such inhibitors (notably myxothiazol) were relatively high compared to mammalian mitochondria. Dithionite-reduced minus oxidized difference spectra indicated the presence of cytochromes aa3, b, c and c1 in mitochondria of both parasite species, but at a higher cytochrome to protein ratio in P. yoelii. Freshly isolated mitochondria from either species exhibited only low respiratory control ratios with alpha-glycerophosphate or succinate as substrates. The apparent absence of a respiratory chain 'Site I' in such mitochondria may mean that NADH-fumarate reductase serves to reoxidize mitochondrial NADH.

Animals↗
PubMed1990

Ultrastructural localization of interleukin 1 in human peripheral blood monocytes; evidence for IL-1 beta in mitochondria.

The pathway of interleukin 1 (IL-1) secretion from the cell remains unclear. IL-1 beta is the major form produced by human monocytes, and is synthesized as a precursor of 35 kDa which is processed to the extracellular biologically active 17 kDa form. We have examined the intracellular localization of IL-1 beta in lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes, by immunocytochemistry and immunoprecipitation of subcellular fractions. LPS treatment slightly damaged the cells. Unstimulated cells showed very little immunolabelling. In contrast, there was heavy immunolabelling on LPS stimulated cells. Immunolabelling occurred within the cytoplasm, nucleus and mitochondria. There was no immunolabelling on the membranous secretory organelles and the plasma membrane. Blebs of cytoplasm budding from the cell surface were immunolabeled, suggesting an alternative route of secretion of IL-1 beta from the cell. Immunoprecipitation studies confirmed these results.

Humans↗
PubMed1990

Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties.

Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

Amino Acid Sequence↗
PubMed1990

Identification of mRNA for PDGF B-chain in human megakaryocytes isolated using a novel immunomagnetic separation method.

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.

Antibodies, Monoclonal↗
PubMed1989

Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation.

A new rabbit model of atherosclerosis is described in which several of the features seen in early human atherosclerosis are generated within a period of 7 days. The positioning of a hollow silastic collar around the carotid artery of a cholesterol-fed rabbit results in macrophage and smooth muscle cell infiltration into the arterial subendothelium, foam cell formation and the deposition of extracellular lipid. A time-dependent accumulation of extracellular cholesteryl ester occurs within the arterial wall. Each of these changes occurs in the presence of a morphologically intact endothelium as assessed using light microscopy, scanning and transmission electron microscopy. A high cholesterol diet did not affect the extent of proliferation but exacerbated cholesteryl ester accumulation. It is proposed that the changes induced by the collar may be mediated by obstruction of the adventitial vasa vasorum with the creation of a localised ischaemic region.

Animals↗
PubMed1989

Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold.

The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions.

Antibodies, Monoclonal↗
PubMed1989

Microbiological immunocytochemistry: a review of current trends and applications.

It has been considered worthwhile to update previous reviews of microbiological immunocytochemistry in order to identify areas of current importance in this continually expanding area of research. Publications in virology, bacteriology and protozoology indicate a continued interest in immunocytochemistry. Deployment of colloidal gold techniques is almost universal in these applications. The post-embedding technique was the most widely applied technique although a few studies employed the immunonegative stain, pre-embedding and immunoreplica techniques, thereby reflecting the use of colloidal gold in all other areas of the biological sciences.

Animals↗
PubMed1988

An attempt to produce Ostertagia circumcincta infections in Mongolian gerbils.

Adult infections of Ostertagia circumcincta were successfully established in gerbils, although worm growth was retarded compared with infections in sheep. Chronic administration of beta-methasone to the host enhanced development of the parasite reproductive system but did not increase infection rate or prevent worm rejection. Infection rates achieved with single infections were not increased by using daily or weekly trickle infection regimes with or without prior exsheathment of larvae. Examination of stomachs from infected gerbils demonstrated pathological changes in the pyloric region which was the predilection site for O. circumcincta. This is in contrast to infections in sheep in which the fundic region of the abomasum is the preferred site.

Animals↗
PubMed1988

Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes.

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha-granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.

Animals↗
PubMed1986

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica serotype A7 organisms.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A7 organisms was purified and characterized by chemical analysis and by 1H and 13C NMR spectroscopy using one- and two-dimensional methods. The polymer has the repeating unit----3)-beta-2-acetamido-2-deoxygalactopyranose-(1----3)-alpha- 2-acetamido- 2-deoxy-6-O-acetyl-glucopyranose-(1-phosphate----. It was immunogenic (capable of eliciting antibodies) for sheep. Chemical removal of O-acetyl groups destroyed both the ability of the polymer to adhere to sheep erythrocytes at neutral pH and the ability to form immune precipitates with specific antisera. Studies using the protein A-gold technique in the electron microscope showed the polysaccharide to be peripherally localized on the bacterial surface.

Amino Acids↗
PubMed1986

The use of gold markers in immunocytochemical studies of microbiological organisms: a review.

Colloidal gold probes have revolutionized electron immunocytochemistry and are now used extensively for microbiological studies. The gold probes can be applied in one of several methods to gain valuable information from the sample. The pre-embedding technique, the post-embedding technique, the immunonegative stain technique, the immunoreplica technique and the immunofreeze-fracture technique, are each described and their applications in microbiology, for diagnosis, for the detection of the site of antigen synthesis in host cells, for vaccine production and for genetic engineering are reported.

Bacteria↗