Search PubMed⌕ Search

Biomedical subjects

G R Newman

Publications and source records attributed to G R Newman.

At least 19 recordsLinked to original sources

Scanning electron microscopic examination of bacterial immobilisation in a carboxymethyl cellulose (AQUACEL) and alginate dressings.

Dressings have been applied to open wounds for centuries. Traditionally they have been absorbent, permeable materials, i.e. gauze that could adhere to desiccated wound surfaces, inducing trauma on removal. With the advent of modern wound care products many dressings are now capable of absorbing large volumes of exudate whilst still continuing to provide a moist wound healing environment. Equally important is their ability to lock exudate in the dressing (i.e. bacterial retention within the dressing matrix) such that upon removal from a wound surface bacterial dispersion is minimised. In these studies detailed scanning electron microscopy techniques have demonstrated the fluid controlling properties of alginate wound dressings and a carboxymethylated cellulose wound dressing (AQUACEL) Hydrofiber) dressing (CMCH)). It was demonstrated that following hydration of the latter wound dressing, the subsequent formation of a cohesive gel was effective in encapsulating large populations of potentially pathogenic bacteria such as Psuedomonas aeruginosa and Staphylococcus aureus under the gelled surface, as well as being immobilised within the swollen fibres. In contrast, hydrated alginate wound dressings did not form a uniform, cohesive gel structure, with the result that fewer bacteria were immobilised within the gel matrix. Many bacteria were trapped on individual, non-hydrated fibres. The unique absorbent gelling properties of the CMCH dressing appears to provide an ideal environment for immobilising bacteria.

Alginates↗

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Androstadienes↗

A cytochemical method for visualising blood vessels selectively in semithin LR white sections.

Traditional cytochemical techniques for illustrating blood vessels are usually unsuitable for use in high resolution, 0.35 microm-thick LR White sections due to the thinness of the tissue and, if collagenous, high background staining. We report that a modification of the periodic acid-thiocarbohydrazide-silver proteinate-silver enhancement method (PATCH-SP-SE) selectively visualises, with low background, even the smallest blood vessels in semithin LR White sections of highly collagenous tissues such as human parietal peritoneum.

Acrylic Resins↗

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.

Animals↗

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function.

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.

Animals↗

Morphologic analysis of a strand recovered from a prosthetic mitral valve: No evidence of fibrin.

We report the first morphologic analysis of a linear mobile structure (strand) detected by transesophageal echocardiography on a bioprosthetic mitral valve and then recovered at surgery. Electron microscopy showed it to consist of a sparsely cellular component, with extracellular amorphous or fibrillary areas. Collagen was largely responsible for the fibrillary appearance.

Echocardiography, Transesophageal↗

Resins for combined light and electron microscopy: a half century of development.

The last fifty years have seen enormous improvements in the way biological specimens are prepared for microscopy. The Fifties produced the essential groundwork upon which many of our current methodologies are based. Acrylic resin embedding was introduced in 1949, with subsequent publications seeking improvements to resin formulations, embedding protocols, and modes of polymerisation. Procedures for progressive lowering of temperature processing, cryosubstitution, freeze-drying and polymerisation by ultra-violet light at low temperatures, all had their genesis in this decade of great innovation. The Sixties marked the period when the acrylics were eclipsed by the more stable and reliable epoxy resins, and much of our present-day understanding of ultrastructure was elucidated. The Seventies carried on this work with advances in technical developments concerned mainly with freezing methodologies. The beginning of the Eighties saw a resurrection of the acrylic resins, with new formulations of these resins giving reliable and stable embeddings. The low temperature and freezing methodologies pioneered in the Fifties, backed up by recent improvements to low temperature technologies, were used to further our understanding of ultrastructure and breathe new life into the science of immunocytochemistry. The remainder of the Eighties and Nineties has seen the ever increasing application of these various microscopical techniques to a wide range of biological studies. The flexibility offered by the acrylic resins in choosing between different processing, embedding and polymerisation methods has provided the impetus for detailed studies to bring to the attention of microscopists the underlying trends governing specimen preparation. Therefore, looking forward to the new Millennium, this has allowed for a more reasoned choice in organising a strategy to deal with a variety of microscopical requirements and for planning an appropriate protocol.

Catalysis↗

Silver development in microscopy and bioanalysis: past and present.

With the experience accumulated from more than a century of silver applications in biology and medicine, physical development has become a powerful bioanalytical tool for marker amplification in blotting procedures, in situ hybridization, immunocytochemistry, histochemistry, and cytochemistry. Early, empirical techniques of silver impregnation followed by development in a reducing solution (chemical developer), or a solution which contained both silver reducers and silver salts (physical developer) were often capricious and suffered from unwanted silver precipitation caused by light and self-nucleation. To accommodate the modern demand for accurate physical development, various strategies have been devised to counter these problems. One approach has been to introduce organic colloids into the developer to keep the silver ions and reducer molecules apart, whilst excluding light by using a dark-room or by covering the solution. Albumen, gelatin, and complex polysaccharides have all been tested, but gum arabic is preferred. In addition, further control can be achieved by slowing down the rate of development with low pH and by changing from silver nitrate to silver lactate, which dissociates more slowly. Effective colloid protection in a physical developer is also provided by the inclusion of tungsten salts which can delay light-catalysed silver reduction and keep the developer clear for many minutes. The same result has been achieved by complexing the silver salt in the physical developer with very large organic molecules, restricting ionization. 'Light insensitive' commercial designer products have resulted. Probably no single formulation can satisfy all conditions of use, but with increased understanding of the mechanisms of physical developers a more flexible, user-friendly approach is anticipated.

3,3'-Diaminobenzidine↗

Ultrastructural analysis of ruptured tendon from anabolic steroid users.

Tendon rupture has been linked with anabolic steroid abuse on the basis of a small number of published case reports. Although experimental data from animal models suggest steroids alter the biomechanical properties of tendon, ultrastructural evidence to support this theory is lacking. Indeed, microscopic analysis of human tendon from steroid users has not previously been reported. In this study, specimens of ruptured human tendon from four patients were biopsied during surgical repair. Two of the subjects were anabolic steroid users, and two subjects were used as nonsteroid-user controls. Ruptured tendon from both groups was examined using electron microscopy. No differences in collagen fibril ultrastructure were seen. We conclude that anabolic steroids did not induce ultrastructural collagen changes that might predispose to tendon rupture in humans.

Adult↗

Silver development in microscopy and bioanalysis: a new versatile formulation for modern needs.

Potentially, silver development could unify most modern demands for clean, accurately localized marker amplification in microscopy and bioanalysis. However, the existing technology leaves room for improvement in developer design. A new formulation has been devised which, by using principles of silver chelation, avoids problems of self-nucleation and catalysis by light. It is made, just before use, by mixing together equal amounts of stock solutions containing high molarity, Tris-buffered silver nitrate and alcoholic, buffered pyrogallol. The two stocks are easily prepared and have very long shelf-lives. The developer is light insensitive for up to an hour at room temperature, so that development can proceed under ambient light conditions and at the neutral pH most suited to biological systems. The powerful reducer in the suggested formulation should allow the detection of low concentrations of marker signal in a wide range of applications.

Humans↗

A biochemical and immuno-electron microscopical analysis of chondroitin sulphate-rich proteoglycans in human alveolar bone.

This study used biochemical and immunohistochemical methods to characterize the chondroitin sulphate-rich proteoglycans from human alveolar bone obtained from an oral source. Proteoglycans were extracted from bone by a sequential 4 M guanidine HCl extraction process, and purified by DEAE-ion exchange chromatography. SDS-PAGE and Western blot analysis, using CS-56 monoclonal antibody, demonstrated one major proteoglycan species with a core protein of 58 kDa, glycosaminoglycan chains of 45-66 kDa and a mean molecular weight of 205 kDa. This work confirmed the biochemistry of chondroitin sulphate-rich proteoglycans from a novel source of adult human alveolar bone, and pointed towards a proteoglycan with a high glutamate, glycine, aspartate, alanine, serine and leucine content. Sections of alveolar bone were embedded in LR White resin, labelled with CS-56 antibody and examined with the light and electron microscopes. At the light microscope level, labelling was restricted to the osteocyte lacunae and canaliculi. Ultrastructural observations showed that the labelling was localized to fine filamentous material in the walls of the osteocytes and canaliculi. Sparse labelling was associated with the collagen fibres immediately subjacent to the lamina limitans, but no labelling of the mineralized matrix was observed. These findings also indicated subtle differences in the distribution of chondroitin sulphate compared with previously reported work, which may indicate species or age differences in the samples used in this study. Ultrastructural analysis confirmed and extended observations of glycosaminoglycan localization at the osteocyte cell membrane of mature human alveolar bone.

Adult↗

Role of complement in the aetiology of Pick's disease?

Complement in the postmortem brains of 15 cases of Pick's disease has been widely analyzed immunohistochemically and, in 2 cases, by immunoelectron microscopy. Astrocytes and the Pick bodies and cytoplasm of ballooned neurons were immunoreactive with antibodies to classical pathway components C1, C1q, C4, C2 and C3 and the terminal complex components C5, C6 and C8. In almost all cases, no immunostaining was obtained with antibodies against C9 and neoepitopes in the membrane attack complex (MAC), the complement complex responsible for cytotoxicity. However, unequivocal staining with antibodies to two soluble complement regulatory proteins, S-protein and clusterin, and to the membrane complement inhibitor CD59 was found, although three other membrane inhibitors, CR1(CD35), DAF (CD55), and MCP (CD46), were not detected. The complement immunoreactivity of astrocytes and neurons could be the result of complement biosynthesis or attack. Complement attack will be restricted by the expressed regulatory proteins. However, neurons may be the victims of attack since they show pathological change. The internalization of complement-attacked membrane, perhaps involving the genesis of Pick bodies and ballooning, may explain the intracellular immunolocalization of complement in damaged neurons. Immunoglobulins, as a possible source of complement activation, were observed in only two cases, leaving unresolved the trigger for complement activation in the other cases.

Aged↗

Immunocytochemically detectable metallothionein is expressed by astrocytes in the ischaemic human brain.

The metallothioneins in vitro are both effective free radical and transitional metal ion scavengers. For this reason we investigated the expression of this protein in the normal CNS and after ischaemia using a monoclonal anti-metallothionein antibody to metallothionein isoforms I and II. Using immunohistochemistry and confocal laser scanning microscopy, we found anti-GFAP and anti-metallothionein were co-localized in astrocytes at the edges of infarcts. Energy dispersive X-ray microanalysis demonstrated high levels of copper and iron in the metallothionein positive reactive astrocytes. On the basis of the in vitro properties of these proteins, the functional importance of metallothionein in reactive astrocytes could be as a free radical scavenger and metal ion chelator.

Aged↗

Immunomicroscopy: resin techniques and on-section labelling with immunocolloidal gold or immunoperoxidase--planning a protocol.

On-section immunocytochemistry is divided into two parts: (i) processing of biological tissue for section microscopy and (ii) immunolabelling of sections. Many of the more successful microscopical methods employ delicate aldehyde fixation of biological tissue followed by "sympathetic" processing into an acrylic resin. Processing regimens do not have to be complicated. Simple and cost effective room temperature protocols utilising partial dehydration have been devised and they can be as effective as the more complex low temperature techniques in preserving both ultrastructure and antigenic reactivity. The embedded material can be investigated by either light or electron microscopy. Frozen sections can be cut and immunolabelled but only if the tissue is chemically fixed first, as in resin embedding. Fixation with low concentrations of aldehyde will normally better preserve tissue immunoreactivity but this may be at the expense of good ultrastructure with these protocols. If so, low temperature resin embedding methods or rapid freezing and cryosubstitution can be tried. The choice of processing protocol will determine which acrylic resin to use, as will the preference for subsequent immunolabelling with either colloidal gold or peroxidase/diaminobenzidine (DAB). Both types of labelling system offer advantages to localisation studies and can be used in combination for double or even triple labelling. Silver enhancement of the colloidal gold or DAB allows for improved observation by light microscopy.

Animals↗

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy.

To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.

Actinin↗

A functional role for corpora amylacea based on evidence from complement studies.

Few theories have been advanced for the production of corpora amylacea (CA) by the normal ageing brain and by the CNS under various neurological conditions. Proteins derived from neurons and oligodendrocytes are found in CA and to understand their origins brain tissue from patients with Alzheimer's disease (AD), multiple sclerosis (MS) and Pick's disease (PD) were tested for complement activity. All CA were immunopositive for antisera to classical pathway-specific components, the activation products C3d and the terminal complement complex (TCC), the C3 convertase regulator membrane cofactor protein (MCP) and the fluid phase regulators S-protein and clusterin. CA were immunonegative for the alternative complement pathway proteins and the complement regulators, decay accelerating factor (DAF) and CD59. Western immunoblotting of isolated solubilized CA from the same tissues demonstrated a weak band for MCP but TCC was more easily shown by immunoprecipitation. A filamentous fringe around CA, probably of astrocytic origin, was also immunopositive for complement factors. CA consist of an inert mucopolysaccharide matrix encasing ubiquitinated proteins, resulting from death of and damage to neurons, myelin and oligodendrocytes. A function of CA, therefore, could be to prevent the recognition of these immunogenic proteins by lymphocytes and microglia and thus protect the CNS from further injury.

Aging↗

New immunocytochemical evidence for a neuronal/oligodendroglial origin for corpora amylacea.

Studies employing a bank of antisera applied to sections of LR White embedded AD and normal ageing brain tissue, may throw new light on the derivation of CA. Conspicuous levels of immunoreactivity were found in the CA of both tissues with markers for oligodendrocytic proteins such as antisera against myelin basic proteolipid protein, galactocerebroside and myelin/oligodendrocyte glycoprotein. CA were unreactive with MRC OX-42, a marker for microglia and macrophages. In a previous publication we demonstrated that the much more abundant CA in the brains of Alzheimer's disease (AD) sufferers, although slightly more varied in their immunoreactivity than those found in normally ageing controls, were universally immunoreactive with anti-tau, a neuronally derived protein and often also contained amyloid. The cores of CA were not immunoreactive with anti-GFAP, suggesting a lack of involvement with astrocytes. Our results now show that in addition to amyloid and neuronal proteins, a significant proportion of the content of CA is derived from oligodendrocytes and/or myelin. The substantial Fe peak previously reported following X-ray microanalysis of CA was probably due to ferritin. However, immunostaining with antisera to ferritin showed that high ferritin immunoreactivity was common to both micro- and macroglia as well as CA. More significantly, the immunoreactivity of CA with anti-ubiquitin suggests that degeneration of neuronal/oligodendrocytic elements may precede CA formation.

Aged↗