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Biomedical subjects

A G Murray

Publications and source records attributed to A G Murray.

At least 19 recordsLinked to original sources
PubMed2005

Modelling salmon lice dispersal in Loch Torridon, Scotland.

A particle transport model is described that is being used to simulate the dispersal of salmon lice (Lepeophtheirus salmonis) larvae in the waters of Loch Torridon. A hydrodynamic model, forced by tides and winds, drives the transport model. Particle movements are strongly influenced by winds, which can lead to formation of lice concentrations in coastal areas several kilometres from the source. Idealised constant wind simulations have been used to locate areas that larval lice may potentially reach from given source locations. Detailed analysis of simulations forced with real wind data is required to assess areas that larval lice from these sources are likely to reach. Further field and experimental work on the viability of lice is required to assess infection risk.

Animals↗
PubMed2004

Assessment of different transfection parameters in efficiency optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 +/- 2.28%; p = 0.001) followed by M199 (24 +/- 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 +/- 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 +/- 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 +/- 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 +/- 1.54% to 27 +/- 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

Cells, Cultured↗
PubMed2003

In vitro senescence occurring in normal human endothelial cells can be rescued by ectopic telomerase activity.

Telomerase activation is a means to delay in vitro replicative senescence in human cells via telomere maintainence; however, this enzymatic activity is virtually absent in almost all normal somatic cells. As a result, cell senesce, leading to an eventual loss of graft function. Aging allografts, either due to cell injury related to transplantation and/or the use of organs from older donors, pose a threat to the long-term survival of a graft as constitutive cells of an aging organ have a much reduced ability to thrive after transplantation. In our study, human endothelial cells were found to undergo replicative senescence in culture with an increase in the percentage of senescent cells (beta-gal staining at pH 6) and a decrease in both the fraction of S-phase cycling cells and the proliferative index measured using CFDA-SE dye. Aging endothelial cells also demonstrated slow rates of proliferation and migration compared to younger cells. Unlike control cells that were transfected with an irrelevant gene vector, telomerase-transfected endothelial cells recovered rapidly after media replacement in cultures that had been serum starved for 2 weeks. Telomerase-transfected cells also retained a high proliferative index comparable to young cells as opposed to untransfected control cells. This young phenotype provided by telomerase expression through restoration of the telomeres may help to increase the longevity of organ transplants.

Cell Cycle↗
PubMed2003

An evaluation of current diagnostic tests for the detection of infectious salmon anaemia virus (ISAV) following experimental water-borne infection of Atlantic salmon, Salmo salar L.

Four commonly used diagnostic tests [reverse transcription polymerase chain reaction (RT-PCR), indirect fluorescent antibody test (IFAT), virus culture and light microscopy] were evaluated for their ability to detect infectious salmon anaemia virus (ISAV) or tissue pathology following experimental infection of Atlantic salmon. Fish were infected with ISAV by water-borne exposure which mimics the route of natural infection. Forty-five per cent of pre-clinical fish tested yielded positive results by RT-PCR for at least one of the organs tested (kidney, heart, gill, liver, blood). No significant difference was detected between organs in the number or time of first occurrence of positive result. Virus culture identified a total of 14% of pre-clinical fish as ISAV-infected. The presence of ISAV in heart tissue was particularly notable (13% of fish sampled) as was the inability to culture virus from spleen tissue. In the case of IFAT, 15% of fish sampled were positive, although tissue other than kidney proved unsuitable for use in this method. Only limited ISAV-specific pathology was detectable by histological examination of fish prior to the onset of clinical disease. These findings reveal important information regarding the optimal choice of both tissue sample and diagnostic test for the routine diagnosis of ISAV.

Anemia↗
PubMed2002

A tractable deterministic model with realistic latent period for an epidemic in a linear habitat.

This paper describes a model for the spatial spread of an epidemic in which the latent period has a Gamma distribution with arbitrary mean and variance while the infected and uninfected may have different diffusion coefficients. Using a variant of the "linear chain trick" we derive a simple algorithm for the propagation speed of the infection front. By way of verification, we show that, with suitably matched parameters, the algorithm agrees closely with speeds obtained numerically from the corresponding model in which the latent period is a constant.

Algorithms↗
PubMed2002

Gene therapy: a lipofection approach for gene transfer into primary endothelial cells.

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37 degrees C, 24 degrees C, and 6 degrees C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 microg; Fugene 6, 0.5 microg; DOTAP, 2.5 microg) and lipids (Effectene, 10 microl; Fugene 6, 6 microl; DOTAP, 15 microl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 +/- 1.3% (mean +/- SEM), 33 +/- 1.4%, and 18 +/- 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37 degrees C. Transfection results were lower at 24 degrees C with mean efficiencies of 26 +/- 2.4% for Effectene, 14 +/- 2.9% for Fugene 6, and 15 +/- 3.2% for DOTAP. Furthermore, mean efficiencies at 6 degrees C were 6 +/- 0.5%, 8 +/- 1.5%, and 6 +/- 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37 degrees C vs. 24 degrees C: p < 0.0001; 37 degrees C vs. 6 degrees C: p < 0.0001; 24 degrees C vs. 6 degrees C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

Analysis of Variance↗
PubMed2001

Infectious salmon anaemia virus in wild fish from Scotland.

Following the outbreak of infectious salmon anaemia (ISA) at salmon farms in Scotland, UK, a survey was established to determine the extent of infection in wild fish. All fish tested were free from the clinical symptoms of ISA. Isolations of ISAV were made from 5 sea trout within areas where ISA affected salmon farms were located. Evidence for ISAV in other sea trout was provided by ISA RT-PCR diagnostic tests. Results from ISA RT-PCR tests reveal evidence for ISAV being present in salmon parr, adult salmon and juvenile brown trout in rivers distant from salmon farms and indicate that, at the time of the survey (1998-1999), ISAV may have been widely distributed. Nucleotide sequence analysis of segments 2 and 8 showed that for most sequences from wild fish there was 100% homology with ISAV isolated from clinically affected farmed fish although evidence is presented which indicates variability in ISAV sequences from wild fish. Modelling the RT-PCR findings indicates that ISAV among salmonid fish was spatially non-random. Brown trout, sea trout and salmon (adult and parr) show a pattern of occasionally large numbers of positive samples against a background of very low numbers.

Animals↗
PubMed2001

Simple models of massive epidemics of herpesvirus in Australian (and New Zealand) pilchards.

In March 1995, a mass mortality of pilchard started to occur in South Australia. This spread very rapidly throughout the Australian pilchard's range, later reaching New Zealand. In November 1998, a similar mass mortality broke out in South Australia and also spread, at a slower rate, throughout the Australian range. The mortality appeared to be caused by a herpesvirus. The mortality spread as a classical epidemic front, but its speed of progress and the brief duration of mortalities at a given location are extreme. We apply simple epidemic modelling techniques, SIR and SEIR modelling, to examine the factors behind the spread of this mortality and the differences between the 1995 and 1998/9 epidemics. We discuss biological factors influencing the critical processes of long-distance (D) and local (beta) transmission of infection.

Animals↗
PubMed2001

Modelling treated waste disposal in Port Phillip Bay and Bass Strait biogeochemical and physical removal.

Large cities, such as Melbourne, generate substantial quantities of sewage, which, after treatment, must be disposed. Melbourne's sewage is disposed via two routes, that treated at the Western Treatment Plant (WTP) is discharged in enclosed Port Phillip Bay, while the Boags Rock outfall empties into the exposed Bass Strait. In Port Phillip Bay biogeochemical processes control the fate of waste, while in the Bass Strait physical mixing rapidly disperses the waste. These different processes require very different ecosystem models. Port Phillip Bay requires detailed modelling of water-column and in-sediment processes, in particular detailed models of recycling processes, and also modelling of benthos-water-column interactions. Interaction of these components gives the model a nonlinear response to change in load. The Bass Strait ecosystem model is simple with no modelling of the sediment and limited modelling of water-column recycling. This model's behaviour is largely controlled by the physical environment.

Animals↗
PubMed2001

Noncytolytic human lymphocytes injure dermal microvessels in the huPBL-SCID skin graft model.

Recent transplantation experiments using perforin-deficient mice as allograft recipients have challenged the concept that allograft rejection is mediated exclusively by CTL. We sought to determine if human noncytolytic lymphocytes could mediate rejection of allogeneic human skin grafts in the huPBL-SCID mouse model of rejection. We generated short term lines of human lymphocytes from peripheral blood mononuclear cells using PHA as a mitogen. The first group was stimulated with PHA alone, the second with PHA plus IL-4 and neutralizing antibody to IL-12, and in the third group PBL were depleted of B cells and monocytes before stimulation as in group 2. After two passages, lines were tested for cytolytic ability and IFN-gamma production. Each line was injected i.p. to mice bearing allogeneic skin grafts. The grafts were harvested between day 16 and 21 after PBL injection, then the histology was scored by a blinded observer for degree of infiltration, microvessel injury, induction of epidermal MHC class II, and perforin expression. In vitro we found that PBL in groups 2 and 3 were unable to lyse cultured endothelial cells in a lectin-directed 111In release assay. In vivo 80% of the IL-4/anti-IL-12 groups maintained the IFN-gamma-low phenotype, and no perforin was detected in these grafts. Nevertheless, human microvessel injury was similar between the two groups. This was not antibody-dependent since the B-cell-depleted group showed similar injury. Moreover adjacent murine vessels were intact. We interpret these observations to show (1) these human PBL lines maintained their phenotype following in vivo restimulation, and (2) noncytolytic graft-infiltrating lymphocytes specifically promote injury of allogeneic human microvessels.

Animals↗
PubMed2000

Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment.

Plasmodium falciparum-infected erythrocytes roll on and/or adhere to CD36, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and P-selectin under shear conditions in vitro. However, the lack of an adequate animal model has made it difficult to determine whether infected erythrocytes do indeed interact in vivo in microvessels. Therefore, we made use of an established model of human skin grafted onto severe combined immunodeficient (SCID) mice to directly visualize the human microvasculature by epifluorescence intravital microscopy. In all grafts examined, infected erythrocytes were observed to roll and/or adhere in not just postcapillary venules but also in arterioles. In contrast, occlusion of capillaries by infected erythrocytes was noted only in approximately half of the experiments. Administration of an anti-CD36 antibody resulted in a rapid reduction of rolling and adhesion. More importantly, already adherent cells quickly detached. The residual rolling after anti-CD36 treatment was largely inhibited by an anti-ICAM-1 antibody. Anti-ICAM-1 alone reduced the ability of infected erythrocytes to sustain rolling and subsequent adhesion. These findings provide conclusive evidence that infected erythrocytes interact within the human microvasculature in vivo by a multistep adhesive cascade that mimics the process of leukocyte recruitment.

Animals↗
PubMed1999

Activated lymphocytes promote endothelial cell detachment from matrix: a role for modulation of endothelial cell beta 1 integrin affinity.

In vivo, MHC class I-restricted injury of allogeneic tissue or cells infected by intracellular pathogens occurs in the absence of classical cytolytic effector mechanisms and Ab. Modulation of the target cell adhesion to matrix may be an additional mechanism used to injure vascular or epithelial cells in inflammation. We studied the mechanisms of human umbilical vein endothelial cell (EC) detachment from matrix-coated plastic following contact by concanamycin A-treated lymphocytes as an in vitro model of perforin-independent modulation of EC basement membrane adhesion. Human PBL were depleted of monocytes, stimulated, then added to an EC monolayer plated on either fibronectin or type I collagen matrices. Activated, but not resting, PBL induced progressive EC detachment from the underlying matrix. Injury of the EC monolayer required direct cell contact with the activated lymphocytes because no detachment was seen when the PBL were placed above a Transwell membrane. Moreover plasma membranes prepared from activated but not resting PBL induced EC detachment. Adherent EC stimulated with activated PBL did not show evidence of apoptosis using TUNEL and annexin V staining at time points before EC detachment was observed. Finally, neither the matrix metalloproteinase inhibitors o-phenanthroline and BB-94 nor aprotinin blocked EC detachment. However, activation of EC beta1 integrin using mAb TS2/16 or Mg2+ decreased EC detachment. These data indicate that cell-cell contact between activated PBL and EC reduces adhesion of EC to the underlying matrix, at least in part by inducing changes in the affinity of the endothelial beta 1 integrin.

Adjuvants, Immunologic↗
PubMed1999

Neonatal porcine islet cells induce human CD4+, but not CD8+, lymphocyte proliferation and resist cell-mediated cytolytic injury in vitro.

Xenotransplantation of porcine tissue to human recipients promises to alleviate the organ shortage. Human antibody-mediated and cell-mediated immune responses against porcine grafts, however, represent barriers to successful xenotransplantation. We compared neonatal porcine islet cells (NPICs) and neonatal porcine splenocytes for the ability to stimulate proliferation of human peripheral blood lymphocytes (PBLs), and for their susceptibility to human natural killer (NK) and cytotoxic T-lymphocyte (CTL)-mediated lysis. Human peripheral blood CD4+ lymphocytes showed strong proliferation in response to NPICs, likely because of occasional swine leukocyte antigen (SLA) class II+ cells in the NPIC preparations. In contrast, human peripheral blood CD8+ lymphocytes did not proliferate in response to NPICs, although they showed clear responses to both porcine splenocytes and endothelial cells. Both human CTL-raised-against-porcine splenocytes and endogenous NK cells lysed porcine splenocytes, but the same cells showed little or no lytic activity against NPICs. Lysis of porcine splenocyte targets was completely abrogated by pretreatment of the human NK or CTL populations with concana-mycin A, suggesting a perforin-dependent effector mechanism. Pretreatment of the NPIC targets with proinflammatory porcine cytokines to upregulate SLA class I expression failed to enhance human CTL-mediated lysis. However, lysis of NPICs by human CTLs could be elicited when a lectin was added to form stable effector:target cell conjugates. It appears that NPICs do not express sufficiently high levels of co-stimulatory and/or adhesion molecules to either activate human CD8+ T-cells or to be effective targets for activated human CTLs. These data suggest that NPICs may not be destroyed by NK- or CTL-mediated lytic mechanisms after transplantation into humans.

Animals↗
PubMed1998

Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin.

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.

Animals↗
PubMed1997

Pig but not human interferon-gamma initiates human cell-mediated rejection of pig tissue in vivo.

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-gamma (IFN-gamma) but not human IFN-gamma or tumor necrosis factor. Grafts injected with pig IFN-gamma also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-gamma (but not human IFN-gamma or heat-inactivated pig IFN-gamma) induced human CD4(+) and CD8(+) T cells and macrophages to more extensively infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

Animals↗
PubMed1995

Human vascular smooth muscle cells poorly co-stimulate and actively inhibit allogeneic CD4+ T cell proliferation in vitro.

We compared immunologic functions of human vascular smooth muscle cells (VSMC) with those of endothelial cells (EC) cultured from saphenous vein. Both cell types can express comparable levels of MHC class II molecules. However, class II-positive VSMCs, unlike ECs, do not stimulate resting CD4+ T cell proliferation. Limiting dilution analyses revealed IL-2-producing cells alloreactive to class II-positive ECs but not VSMCs. Class II molecules on VSMCs are functional, inducing CD25 expression on resting CD4+ T cells and stimulating proliferation of CD4+ T cells that have been pre-activated by ECs. VSMC expression of the co-stimulator molecules CD44, CD54, CD58, and CD59 is comparable to EC expression, and neither VCAM-1 nor B7 are expressed on either cell. However, VSMCs are less efficient than ECs at co-stimulating IL-2 production by PHA-stimulated PBL. VSMCs but not ECs cultured across a Transwell inhibit CD4+ T cell proliferation to allogeneic ECs and, to a lesser extent, IL-2 production in the same assay. Inhibition of proliferation cannot be transferred by VSMC-conditioned media, nor reversed by inhibitors of prostaglandin synthesis, TGF-beta 1, or nitric oxide synthesis. CD4+ T cells cocultured with class II-positive VSMCs proliferate to a subsequent challenge with ECs from the same donor as well as freshly isolated T cells. We conclude that VSMCs express functional MHC class II molecules and stimulate pre-activated T cells. However, VSMCs lack adequate costimulators to fully stimulate resting T cells, and VSMCs inhibit T cell proliferation.

Antigen-Presenting Cells↗