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Biomedical subjects

J Greenwood

Publications and source records attributed to J Greenwood.

At least 19 recordsLinked to original sources
PubMed1995

Antigen presentation by rat brain and retinal endothelial cells.

Brain and retinal endothelial cells (EC) form the blood-brain and vascular blood-retinal barriers, respectively, and are believed to play a role in mediating T cell responses in the central nervous system. In this study, Lewis rat retinal and brain EC grown in vitro were capable of expressing MHC class II I-A but not I-E molecules following treatment with interferon-gamma. In the presence of their antigen, CD4+ antigen-specific T cells were able to mediate lysis of retinal EC monolayers to a similar extent as brain EC. T cell proliferation was poorly supported by confluent retinal or brain EC monolayers, but subconfluent EC monolayers supported proliferation in a MHC class II (I-A)-restricted manner (P < 0.001). Exposure of T cells to confluent retinal EC monolayers resulted in them becoming less responsive to subsequent antigen presentation by thymocytes. Conversely, pre-exposure with subconfluent EC had no such effect. These results suggest that a non-proliferating EC monolayer is able to downregulate T cell responsiveness which may have important implications during lymphocyte traffic across the blood-tissue barriers of the central nervous system.

Animals↗
PubMed1995

Two strategies for promoting clinical competence in pre-registration nursing students.

A 'cluster' model of clinical teaching was successfully introduced in 1989 to improve the quality of the clinical component of one pre-registration nursing programme in South Australia. A cognitive model of skills acquisition will be introduced in 1995 to further enhance the effectiveness of 'cluster' teaching. Both models are described together with their implementation processes.

Clinical Competence↗
PubMed1995

Engineering a peptide epitope display system on filamentous bacteriophage.

The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein-protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines.

Amino Acid Sequence↗
PubMed1994

Engineering multiple-domain forms of the therapeutic antibody CAMPATH-1H: effects on complement lysis.

Antibody-mediated lysis of cells involves a complex interaction between the cell, the target antigen, the antibody and host effector mechanisms. One such mechanism, complement-mediated cell lysis, requires the interaction of C1q with the antibody heavy chain constant regions, and in particular the CH2 domain. Here we investigate the potential benefit of multiple-domain forms of the therapeutic monoclonal antibody CAMPATH-1H. This antibody is directed against the CDw52 antigen expressed by human lymphocytes and has proven lytic abilities both in vitro and in vivo. Using target cells with either high or low antigen density, engineered antibodies that contained additional domains in tandem (CH2, hinge-CH2 or Fc intramolecular repeats) showed no improvement in complement-mediated lysis when compared with controls. However, a homodimeric form of the antibody that was engineered by mutation of a serine residue to cysteine near the carboxy-terminal of the CH3 domain, exhibited markedly improved lysis using target cells expressing antigen at low density. Interestingly, no improvement was seen using cells expressing antigen at high density. These results suggest that dimeric forms of antibodies could be useful for converting cells with low density antigens into useful targets for therapy.

Alemtuzumab↗
PubMed1992

Therapy with monoclonal antibodies. An in vivo model for the assessment of therapeutic potential.

A set of rat-human and rat-rat chimeric mAb has been created, all possessing V regions identical in their specificity for the mouse CD8 Ag. In vitro all antibodies were able to block cell-mediated lysis but varied greatly in their capacity to utilize rabbit complement. We examined the ability of these chimeric antibodies to deplete in vivo and established a clear hierarchy. Of the human IgG subclasses, only IgG1, 2, and 3 could fix complement in vitro, yet all (IgG1-4) were remarkably potent at depleting CD8+ PBL in vivo. In contrast, human IgA2 and IgE were ineffective at clearing CD8+ PBL. The vector system used to create these antibodies together with the small doses of antibodies required to deplete in vivo make this a simple and rapid system for testing the effects of different antibody isotypes and mutants. We have shown that a mutant of human IgG1, which is incapable of fixing complement, depletes perfectly well in vivo, whereas an aglycosyl IgG1 mutant is rendered inactive. Our model provides a unique opportunity to study effector functions and motifs that are used by mAb in vivo and will help in the design of improved antibodies for human therapy.

Animals↗
PubMed1992

Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro.

Retinal vascular endothelia form one aspect of the blood-retinal barrier and, like the blood-brain barrier, control the passage of molecules and cells into the parenchyma. To facilitate comparative in vitro studies, rat retinal endothelial cells have been cultured and characterised. Using immunocytochemical techniques, retinal endothelium was positive for von Willebrand's factor, tight junction-associated polypeptide (ZO-1) and the transferrin receptor. The cells also expressed high-affinity uptake of acetylated low-density lipoprotein. Using the monoclonal antibodies JSB-1 and C219, the product of the multidrug resistance gene, P-glycoprotein, was found to be expressed on primary cultures of both brain and retinal endothelium.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Persuading general practitioners to prescribe--good husbandry or a recipe for chaos?

Edinburgh's Community Drug Problem Service was set up three years ago in response to the high prevalence of HIV infection amongst local injecting drug users and the lack of existing statutory drug services in Lothian. From the outset, a policy of shared care and substitute prescribing by the druguser's general practitioner (GP) was adopted. Over 800 referrals later, this discussion paper looks at the methods used to persuade GPs to cooperate in longterm drug management, and examines the advantages, disadvantages and outcome of such a shared care approach.

Drug Prescriptions↗
PubMed1992

Effect of lymphocytic infiltration on the blood-retinal barrier in experimental autoimmune uveoretinitis.

Using an experimental model of autoimmune uveoretinitis, we have examined the relationship of T cell infiltration in the retina to blood-retinal barrier (BRB) breakdown. Sensitive quantitative in vivo techniques were used to examine BRB permeability to sucrose, a low mol. wt non-transported solute. Electron microscopy was also used to localize extravasated horseradish peroxidase, a macromolecular visual tracer, from the retinal vasculature and to identify the route by which any leakage was occurring. No increase in BRB permeability was found prior to lymphocytic infiltration. By day 10 of the disease inflammatory cells could be seen within the structurally intact retina, which was shortly followed by an increase in the permeability of the BRB to sucrose. Only later in the disease process, when damage to the photoreceptor layer became apparent, did extravasation of the macromolecule HRP occur. At no stage of the disease process was there any detectable damage to inter-endothelial tight junctions. The size-dependancy of tracer extravasation in the initial stages of the disease is indicative of a paracellular route being responsible for the increase in BRB permeability. In later stages of the disease some evidence of horseradish peroxidase filled 'vesicle-like' profiles was observed. We suggest that the devastating complication of BRB breakdown in ocular inflammation is a direct consequence of lymphocytic infiltration.

Animals↗
PubMed1992

Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier.

Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like cell:cell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VIII-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells. Contaminants can be kept to < ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca(2+)- and Mg(2+)-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.

Animals↗
PubMed1992

The blood-retinal barrier in experimental autoimmune uveoretinitis (EAU): a review.

The blood-retinal barrier (BRB) is believed to play an important part in the pathogenesis of experimental autoimmune uveoretinitis (EAU). Central to the disease process is the recruitment of inflammatory cells from the circulation, a mechanism that is controlled in part by the BRB. As the disease progresses the BRB becomes disrupted first to small and then to large molecular weight tracers. In these two respects EAU shares many similarities with experimental autoimmune encephalomyelitis (EAE) in which there is dysfunction of the blood-brain barrier (BBB). In EAU, however, the differential roles played by the two barrier sites that comprise the BRB are not clear although some evidence would suggest that it is the retinal endothelium that is initially involved. BRB breakdown in EAU has been found to occur concomitantly with lymphocyte infiltration by mechanisms that remain to be elucidated.

Animals↗
PubMed1991

Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens.

We describe here two systems for encoding foreign amino acid sequences in the exposed N-terminal segment of the major coat protein of bacteriophage fd. Small peptides can be encoded directly; larger peptides are encoded in hybrid bacteriophage particles, in which the capsid is formed from a mixture of wild-type and modified coat proteins. In both cases, the peptides are present in multiple copies per phage particle. Peptides that represent the circumsporozoite protein, the major surface antigen of the sporozoites of the malaria parasite, Plasmodium falciparum, were inserted in this way and found to be highly immunogenic. These systems should prove to be valuable in displaying specific or random peptides as antigens, and could lead to cheap and effective vaccines. They will also allow rapid screening of peptides as potential agents of other biological effects, with important applications in biomolecular design.

Amino Acid Sequence↗
PubMed1991

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA.

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.

Amino Acid Sequence↗
PubMed1991

Mechanisms of blood-brain barrier breakdown.

The functional status of the blood-brain barrier (BBB) must be taken into account when designing and interpreting brain imaging techniques. The integrity of the BBB is affected in many diseases of the brain, with the potential involvement of a number of different but poorly understood cellular mechanisms. Factors known to disrupt the BBB experimentally include arachidonic acid and the eicosanoids, bradykinin, histamine and free radicals. These active compounds, released in pathological tissue, may alter cytosolic calcium levels and induce second messenger systems leading to an alteration in BBB permeability. Extravasation of plasma proteins may occur via disrupted tight junctions, stimulation of fluid-phase vesicular transport or the formation of transcellular pores or channels.

Animals↗
PubMed1991

The effect of bile salts on the permeability and ultrastructure of the perfused, energy-depleted, rat blood-brain barrier.

The action of bile salts upon the rat blood-brain barrier (BBB) was assessed in the absence of energy-yielding metabolism. Brains were perfused in situ with a Ringer solution for 5 min followed by a 1 min perfusion containing either sodium deoxycholate (DOC), taurochenodeoxycholate (TCDC), or Ringer/DNP. The integrity of the BBB was then determined by perfusing with the radiotracer [14C]mannitol for 2.5 min. Alternatively, the brains were perfusion fixed for ultrastructural assessment. At 0.2 mM DOC, the BBB remained intact and the cerebral ultrastructure was similar to the controls. At 1 mM and above, disruption of the BBB became evident. At 2 mM, the cerebral cortex became severely vacuolated, with damaged endothelium and collapsed capillaries. With TCDC, BBB disruption occurred at 0.2 mM without any apparent ultrastructural damage to the microvasculature. Following 2 mM TCDC, similar, but less widespread, structural changes to the 2 mM DOC-perfused animals was apparent. Opening of the BBB occurred at a concentration lower than that required to cause lysis of either red blood cells or cultured cerebral endothelial cells. It is proposed that the effect of bile salts at concentrations of 1.5 mM and above is largely due to their lytic action as strong detergents on endothelial cell membranes, but that at lower concentrations a more subtle modification of the BBB occurs.

Animals↗