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Biomedical subjects

I McConnell

Publications and source records attributed to I McConnell.

At least 19 recordsLinked to original sources
PubMed1992

Clinicopathological investigation of primary, uncomplicated maedi-visna virus infection.

Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.

Animals↗
PubMed1992

Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25.

A 1.3-kb cDNA that encodes the entire 825-bp coding region of ovine CD25, the interleukin-2 receptor 55-kDa protein, has been isolated. Comparison of the deduced amino acid sequence with CD25 proteins from other species shows the ovine sequence to have the greatest homology with that of the bovine species.

Amino Acid Sequence↗
PubMed1992

Lysosomes as key organelles in the pathogenesis of prion encephalopathies.

The causation, structural origin, and mechanism of formation of spongiform lesions in transmissible encephalopathies are unknown. We have used immunogold electron microscopy to locate ubiquitin conjugates, hsp 70, and beta-glucuronidase (markers of the lysosomal compartment) and prion protein (PrP) in both control and scrapie-infected mouse brain. In scrapie-infected brain, lysosomes and lysosome-related structures (multivesicular and tubulovesicular dense bodies) are present in abnormally high numbers in neuronal cell processes. These structures contain PrP, together with the lysosomal markers ubiquitin conjugates, hsp 70, and beta-glucuronidase, which could also be identified spilling from tubulovesicular dense bodies into areas of early rarefaction in neuronal processes; we suggest that these areas of rarefaction are the precursor lesions of spongiform change. We advance the hypothesis that spongiform change is brought about by cytoskeletal disruption in neuronal processes caused by liberation of hydrolytic enzymes from lysosomes overloaded with the abnormal isoform of PrP (PrPsc). We suggest that the lysosomal system is probably acting as the bioreactor for processing of normal PrP to the abnormal isoform. The continuous production of increasing quantities of abnormal PrPsc in lysosome-related bodies will eventually cause disruption of the lysosomal membrane with destruction of the neuronal cytoskeleton and the initiation of vacuolation. Later, death of the cell will be associated with release of the PrPsc isoform into the extracellular environment. Repeated rounds of phagocytosis, lysosomal biogenesis of PrPsc, lysosomal membrane rupture, hydrolytic enzyme release, and neuronal lysis will lead to an exponential increase in cell damage and cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characteristics of the T cell-mediated immune response to maedi-visna virus.

Virus-specific T cell-mediated immunity was investigated in healthy sheep persistently infected with the ruminant lentivirus maedi-visna. Visna-specific lymphocyte proliferation was demonstrated in response to both purified virions and recombinant p25, the major core protein of maedi-visna virus. The responding T cell population in this assay was mainly of the CD4+ phenotype, although in some individuals CD8+ T cells were also shown to respond to visna antigen in this system.

Animals↗
PubMed1992

Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay.

The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.

Animals↗
PubMed1992

Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.

The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.

Animals↗
PubMed1992

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

Acetone↗
PubMed1992

Are Sinc and the PrP gene congruent? Evidence from PrP gene analysis in Sinc congenic mice.

Congenic mouse strains VM/Dk and VM-Sincs7/Dk differ at the Sinc gene, which controls the incubation period of scrapie in mice; VM/Dk mice are Sincp7p7 and VM-Sincs7/Dk mice are Sincs7s7. Restriction fragment length polymorphism and DNA sequencing analysis demonstrated that the PrP genes also differ in these strains, confirming the close genetic linkage of Sinc and PrP. Using the restriction enzyme HhaI, we have shown that at least 100 kb of DNA flanking the PrP gene differs between the two strains, and therefore the congruence of the Sinc and PrP genes is still not certain.

Animals↗
PubMed1992

Transmission of bovine spongiform encephalopathy and scrapie to mice.

Transmission from four cases of bovine spongiform encephalopathy (BSE) to mice resulted in neurological disease in 100% of recipient animals, after incubation periods of between 265 and 700 days post-injection. The results from the four cases were very similar to one another. There were major differences in the incubation period between the four inbred strains of mice tested, and even between strains of the same Sinc genotype, and the incubation periods of Sinc heterozygote mice were much longer than those for any of the inbred strains. Transmission from a case of natural scrapie differed in two important respects: there were no differences in the incubation period between mouse strains of the same Sinc genotype, and that of the heterozygotes was between those of the Sinc homozygotic parental strains. The distribution of vacuolar degeneration in the brains of mice infected with scrapie also differed from those infected with the BSE isolates. Transmission was also achieved from formol-fixed BSE brain. These results show that the same strain of agent caused disease in the BSE cases, and that the relationship of BSE to scrapie in sheep is unclear.

Animals↗
PubMed1992

Cell phenotypes in the efferent lymph of sheep persistently infected with Border disease virus.

The prefemoral efferent lymphatics of sheep persistently infected (PI) with Border disease virus (BDV) were cannulated in order to study the effects of the virus on cells of the immune system. Efferent lymphocytes recovered from PI sheep were phenotyped using a panel of monoclonal antibodies (MoAb) specific for ovine cell-surface markers and compared to lymphocytes recovered from normal, healthy controls. PI sheep had an increased percentage of cells expressing the T cell-associated molecules CD5, CD4, CD8 and T19, also an increase in cells expressing CD1 and a population of cells expressing low levels of the T19 molecule which was not present in control sheep. The lymphocytes were examined for the presence of BDV using virus-specific MoAb. On average 8.5% of the efferent lymphocytes from PI sheep carried virus antigen. BDV antigen was also found in the mononuclear cells and connective tissue of lymph nodes indicating widespread virus dissemination within the lymphoid system of PI sheep.

Animals↗
PubMed1992

Phenotypic analysis of lymphocyte populations in the lungs and regional lymphoid tissue of sheep naturally infected with maedi visna virus.

We have analysed the phenotype of lymphocytes in lung and regional lymph node of symptomatic and asymptomatic sheep infected with the ovine lentivirus, maedi visna virus (MVV). Compared to equivalent tissues from age-matched, non-infected controls, MVV-infected sheep show increased numbers of lymphocytes in the lung, both in the bronchus-associated lymphoid tissue (BALT) and in the alveolar septae. Both CD8+ and CD4+ T lymphocyte numbers in alveolar septae were increased, particularly in animals with clinical respiratory disease. The ratio of CD8+ to CD4+ lymphocytes was similar to that in normal lung. In both MVV-infected and uninfected animals a high proportion of pulmonary lymphocytes, particularly in the alveolar septae, did not express the CD5 antigen, suggesting that they were activated. The number of activated cells was higher in infected sheep. Variable numbers of alveolar macrophages containing MVV-core protein were present in alveolar lumina, the majority of positive cells showing morphological evidence of activation. In regional lymphoid tissue there were increased numbers of CD8+ and gamma delta expressing T cells in lymphoid follicles and germinal centres of infected animals. The specificity of these cells is unknown and we could find no evidence for the presence of cells productively infected with the virus in these structures. This study shows that activated T lymphocytes, particularly of the CD8 subset, play a major part in the pathogenesis of MVV-induced pulmonary and regional lymph node lesions.

Animals↗
PubMed1992

Vasopressin antagonist inhibition of clotting factor release in the rhesus monkey (Macaca mulatta).

The antidiuretic (V2) agonist, dDAVP, stimulates release of the clotting factors von Willebrand factor (vWF) and factor VIIIc (FVIIIc) in humans. The objective of these studies was to identify and characterize dDAVP stimulation of clotting factor release in pentobarbital-anesthetized rhesus monkeys using V2 receptor agonists (dDAVP, SK&F 101926, SK&F 104146, SK&F 104244) and a V2 receptor antagonist (SK&F 105494) given i.v. dDAVP (3.0 micrograms/kg) stimulated release of vWF, FVIIIc and renin and was associated with tachycardia and blood pressure-lowering activity. The V2 receptor antagonist, SK&F 105494 (30 micrograms/kg, i.v.), had no effect on clotting factor release, heart rate or blood pressure, but prevented dDAVP stimulation of clotting factor release and tachycardia. The V2 agonists SK&F 101926, SK&F 104146 and SK&F 104244 did not stimulate clotting factor release, blood pressure, heart rate or plasma renin activity in this species at the doses tested. Thus, the rhesus monkey is a nonhuman primate species in which dDAVP stimulation of FVIIIc and vWF occurs. The results support the hypothesis that dDAVP stimulation of clotting factor release is mediated by a low-affinity, V2-like receptor mechanism. The tachycardia and renin release associated with dDAVP administration are most likely secondary to vasodilation, also mediated by a V2-like mechanism.

Animals↗
PubMed1991

In vivo detection of metabolic changes in a mouse model of scrapie using nuclear magnetic resonance spectroscopy.

In vivo proton nuclear magnetic resonance (NMR) spectroscopy studies of scrapie in a mouse model have shown the appearance of an abnormal peak in the brain early in the incubation period. This abnormal peak was detected weeks before the detection of a protease-resistant form of a membrane protein and vacuolar histopathology in vitro, and several months before clinical signs, and the signal increased in intensity as the disease progressed. In the chronic stage of the disease, a reduction in N-acetyl aspartate levels was observed using in vivo and in vitro proton NMR spectroscopy.

Animals↗
PubMed1991

The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis.

Mouse lines which are congenic for Sinc, the major gene controlling scrapie incubation period, have been produced by selective breeding from the inbred C57BL(Sincs7) and VM(Sincp7) strains; the s7 allele of Sinc has been introduced into a VM background by 18 serial backcrosses, at each generation selecting on the basis of the incubation period with the ME7 scrapie strain. The characteristics of the disease produced by seven scrapie strains have been compared in Sincs7 and Sincp7 congenic mice and in the F1 cross between them. As previously found in non-congenic mice, each scrapie strain has a characteristic, precisely reproducible incubation period pattern in the three Sinc genotypes. The Sinc gene controls the incubation period for all scrapie strains tested but the direction of allelic action and the apparent dominance pattern differs between scrapie strains. Comparison with non-congenic mice shows that other genes also have a minor effect on incubation period. The distribution of vacuolar degeneration in the brain depends mainly on the scrapie strain but is also influenced by Sinc and other unspecified mouse genes. Restriction fragment length polymorphism analysis has already shown that the close linkage between Sinc and the gene encoding PrP has been maintained in the Sinc congenic lines, strengthening the possibility that PrP is the Sinc gene product. The present study confirms that scrapie strains carry information which is independent of the host but nevertheless suggests that host PrP protein interacts with this information to regulate the progression of the disease.

Alleles↗
PubMed1991

Nucleotide sequence of EV1, a British isolate of maedi-visna virus.

We have isolated a maedi-visna-like virus from the peripheral blood mononuclear cells of a British sheep displaying symptoms of arthritis and pneumonia. After brief passage in fibroblasts this virus (designated EV1) was used to infect choroid plexus cells. cDNA clones of the virus were prepared from these cells and sequenced. Gaps between non-overlapping clones were filled using gene amplification by the polymerase chain reaction. The genome structure is similar to that described for visna virus strain 1514, and differs from that described for visna virus strain SA-OMVV in not having a W reading frame. Overall the genome differs by about 20% between each of these strains, but there is fivefold variation in the amount of divergence of derived amino acid sequences of different open reading frames. Two sequenced EV1 clones each contain only one copy of the 43 bp repeat, with paired AP-1 sites, which is a feature of other ruminant lentiviral long terminal repeats (LTRs). However, analysis of viral DNA in infected cells by gene amplification shows that LTRs with two repeats do occur, albeit at a relatively low frequency.

Amino Acid Sequence↗
PubMed1991

Identification of a putative cellular receptor for the lentivirus visna virus.

One mechanism by which viral tropism may be controlled is by the expression of a specific virus receptor on the cell surface. This paper reports the identification of a putative cellular receptor for visna virus, the prototype virus of the family Lentiviridae. Using a virus overlay protein blot assay we identified a group of polypeptides of apparent Mr 30K to 33K which interacts with visna virus and is present on permissive but not non-permissive cells. A rat polyclonal anti-ovine major histocompatibility complex (MHC) class II antigen (Ag) serum raised to immunopurified MHC class II Ag, but not preimmune serum, blocked the interaction of visna virus with these polypeptides. In an ELISA, immunopurified MHC class II Ag bound to visna virus but not to bovine parainfluenza 3 virus. Preincubation of visna virus with immunopurified soluble MHC class II Ag resulted in a marked decrease in virus-induced syncytium formation, i.e. preincubation with class II Ag inhibited infection with visna virus, but we have been unable to inhibit infection using class II Ag-specific antisera. These results suggest that ovine MHC class II Ag acts as a component of a cellular receptor for visna virus. This is of particular interest owing to the close similarities between visna virus and human immunodeficiency virus (HIV), and the relationship between MHC class II and CD4, the cellular receptor for HIV. It is also of relevance to recent reports that a growing number of viruses utilize polypeptides of the Ig supergene family as receptors.

Animals↗
PubMed1990

Uptake of antigen by afferent lymph dendritic cells mediated by antibody.

Dendritic cells isolated from sheep afferent lymph were examined for their ability to bind soluble protein and peptide antigens labeled with fluorescein both in in vitro assays and following intradermal injection of antigen in vivo. Analysis of dendritic cells by flow cytometry revealed weak direct binding of proteins and peptide antigens. However, the degree of uptake was greatly enhanced in the presence of specific antibody in vitro or if antigen was injected intradermally into antigen-primed sheep. About 60% of dendritic cells possessed the ability to take up antigen in both the in vitro and in vivo experiments. The uptake of antigen occurred very rapidly, reaching maximum values in terms of cell numbers and fluorescence intensity in less than 5 min in vitro and 20-40 min following in vivo challenge. Both sheep IgG subclasses could mediate this effect, but F(ab')2 fragments were ineffective. Procedures adopted to remove complement components from the in vitro test mixtures did not result in any reduction in the binding of antigen by dendritic cells. Two-color flow cytometry analysis of the dendritic cell population further showed that 43% of cells taking up the antigen/antibody complexes were CD1+, suggesting a relationship between these cells and Langerhans' cells or other dendritic cells in skin. The results, thus, indicate that approximately two thirds of sheep afferent lymph dendritic cells bind antigen/antibody complexes via an Fc receptor, a mechanism which could be important in the accentuated accessory function of these cells known to occur following secondary antigen challenge.

Animals↗