Search PubMed⌕ Search

Biomedical subjects

I McConnell

Publications and source records attributed to I McConnell.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inactivation of the 22A strain of scrapie agent by autoclaving in sodium hydroxide.

Samples of macerated mouse-brain infected with the 22A strain of scrapie agent were subjected to gravity-displacement autoclaving at 121 degrees C for 30 minutes in the presence of 2 M sodium hydroxide. No infectivity was detectable by mouse bioassay in samples which were either held for an hour at room temperature before autoclaving, or autoclaved immediately after adding the hydroxide. In contrast, all of the mice injected with a control sample, held for an hour in distilled water before autoclaving, developed scrapie. If adopted as a routine decontamination procedure it is recommended that hydroxide-treated waste should be autoclaved in sealed heat-resistant containers which can withstand the pressures involved. This would make the procedure safer for the operator, and avoid autoclave damage through exposure to sodium hydroxide.

Animals↗
PubMed1997

The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue.

Brain-tissue infected with scrapie-like agents remains infectious after histological fixation, and represents a source of occupational exposure. Infectivity titres in formol or paraformaldehyde-lysine-periodate (PLP)-fixed mouse-brains infected with the 301V strain of BSE agent were reduced by around six logs following treatment with formic acid. After PLP fixation and formic acid treatment, no infectivity was detectable in mouse-brain infected with the 87V strain of scrapie agent. Similarly-treated mouse-brain infected with the ME7 strain of scrapie agent showed a titre loss of approximately 5 logs. No infectivity was detectable in PLP-fixed, BSE-infected bovine brain after formic acid treatment, but this was an unreliable result. With unfixed brain homogenates, formic acid reduced the titre of ME7 by > or = 5.0 logs; technical problems limited the measured loss of BSE infectivity to > or = 1.0 logs. These studies confirm that formic acid treatment during fixation of brain-tissue significantly reduces the infectivity titres of scrapie-like agents, thus reducing the level of any occupational exposure.

Animals↗
PubMed1996

Pharmacokinetics and toxicokinetics of an orally active tripeptide, IRI-695, in animals.

Pharmacokinetics and toxicokinetics of IRI-695, a tripeptide, were investigated in the rat, rabbit, dog, and monkey. Tissue distribution and excretion of [14C]IRI-695 were determined in the rat. Following a single intravenous (IV) injection, the elimination half-life (t1/2) of IRI-695 in the rabbit, dog, and monkey was similar (about 65 min) and approximately four times that in the rat (15 min). This difference in t1/2 can be attributed to about four times higher clearance of the drug in rats (11.2 mL min-1 kg-1). The volume of distribution (Vss) in these four species, 132-234 mL kg-1, suggested negligible preferential distribution of IRI-695 to body tissue. After a 5 mg kg-1 oral dose, the absolute bioavailability of IRI-695 was 2.0% in rats and 3.1% in dogs. However, systemic drug exposure in the dog was about five to 10 times that in the rat, which is related to the slower clearance of the peptide in the dog. Toxicokinetic studies in the rat and dog indicated linear kinetics and systemic exposure of IRI-695 up to 300 mg kg-1 d-1 oral doses throughout the 28 d toxicity study. Accumulation of the drug after the repeated oral dosing was negligible. After a single 0.10 mg kg-1 [14C]IRI-695 IV injection in rats, almost all of the radioactivity administered was excreted in urine within 24 h postdose.

Amino Acid Sequence↗
PubMed1996

The phenotype and phagocytic activity of macrophages during maedi-visna virus infection.

Macrophages from maedi-visna virus (MVV) infected sheep have been shown to have an activated phenotype from sites of lesions in vivo. Here we have looked at the direct effect of virus infection on macrophage phenotype and activity in vitro by flow cytometry. There was no significant difference in the expression of several surface markers (CD4, CD8, MHC Class I, MHC Class II, lymphocyte function associated antigen(LFA)-1 and LFA-3) on monocyte-derived macrophages (MDM) by 5 days post MVV infection. In contrast the phagocytic activity of MVV-infected MDM for the yeast Candida utilis and erythrocytes was decreased by 5 days p.i. although the surface binding of erythrocytes was not affected. Interestingly, an activated phenotype was seen on alveolar macrophages (AM) from sheep with maedi (surface expression of MHC Class I, Class II and LFA-1 was increased), but there was no difference in the binding and phagocytosis of erythrocytes by these cells. However the binding and phagocytosis of the bacterium, Pasteurella hemolytica was increased with AM from MVV-infected sheep without lesions. Similarly there was no significant difference in the phagocytic and erythrocyte rosetting activity between fresh monocytes from MVV-infected and uninfected control sheep. Therefore the phenotype of macrophages taken from sites of lesions caused by MVV does not correspond to a direct effect by the virus on these cells or to particular activities of the macrophages.

Animals↗
PubMed1996

Identification of the sheep homologue of the monocyte cell surface molecule--CD14.

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.

Animals↗
PubMed1996

Electron microscope studies of the replication of a British isolate of maedi visna virus in macrophages and skin cell lines.

The replication of EV1, a British isolate of maedi visna virus (MVV), in macrophages has not previously been studied. We therefore used transmission and scanning electron microscopy (TEM and SEM respectively) to compare the replication of EV1 in macrophages versus skin cell lines. Monocyte-derived macrophages (MDM), alveolar macrophages (AM) and skin cell lines were all permissive for replication by EV1. Virus grew rapidly and to high titers in skin cell lines and mature MDM. However replication was slower in less mature MDM or AM. Virion budding occurred through (i) cytoplasmic membranes only (skin cells), (ii) cytoplasmic and vesicular membranes (MDM) or (iii) vesicular membranes only (AM). This meant that virions accumulated in vacuoles within macrophages. Retroviral intracytoplasmic type A particles were seen in the cytoplasm of AM and MDM infected with strain EV1, but not MDM infected with strain 1514 (an Icelandic MVV strain) and were shown to contain MVV gag antigen by immunogold staining.

Animals↗
PubMed1996

The effect of dry heat on the ME7 strain of mouse-passaged scrapie agent.

Partial survival of lyophilized scrapie agent has been reported previously following exposure to dry heat at 360 degrees C for 1 h, and led to speculation that scrapie-like agents might not be completely inactivated by incineration. However, it is known that dried infectivity is more difficult to inactivate by heat than that in hydrated samples. In this present study it was shown that the infectivity in macerates of mouse-brain infected with the ME7 strain of scrapie agent was not completely inactivated by exposure to dry heat at temperatures up to 180 degrees C for 1 h but the titre of surviving infectivity reduced progressively as the temperature was increased. No infectivity was recovered after a 1 h exposure at 200 degrees C. These data suggest that scrapie-like agents are unlikely to survive incineration.

Animals↗
PubMed1996

Scrapie infection can be established readily through skin scarification in immunocompetent but not immunodeficient mice.

Scarification of the skin is a possible route of entry for scrapie infectivity in sheep, and for Creutzfeldt-Jakob disease agent in humans within the context of occupational exposure to infected brain in the autopsy room or laboratory. The effectiveness of skin scarification routes as portals of entry for infectivity had not previously been tested experimentally but this study has shown that these are efficient routes for establishing infection in mice using the 139A and ME7 strains of scrapie agent. Scarification had much the same efficiency as inoculation by the intraperitoneal, intravenous or perivenous routes but was not effective in immunocompromised (SCID) mice. It was concluded that replication of infectivity within the lymphoreticular system, which is precluded in SCID mice, is a necessary prerequisite for the development of infection in the central nervous system following inoculation via scarification.

Animals↗
PubMed1996

Replication of scrapie in spleens of SCID mice follows reconstitution with wild-type mouse bone marrow.

SCID mice are resistant to intraperitoneal infection with 10(3) and 10(4) intracerebral ID50 units of ME7 scrapie agent whereas they develop disease after intracerebral challenge. However, higher doses introduced, by intraperitoneal or subcutaneous routes, produce disease. Immunocompetent mice of the same strain (CB20) developed scrapie following either intracerebral or intraperitoneal infection. Bioassay of spleens from SCID mice infected with 10(-1) dilutions of ME7 scrapie by intraperitoneal, intracerebral or abdominal subcutaneous injection showed traces or low levels of infectivity in spleen. However, subcutaneous injection beneath the skin of the neck failed to infect the spleen. CB20 bone marrow reconstitution of SCID mice resulted in the regeneration of a normal lymphoid architecture in the spleen. Spleens from these reconstituted mice, infected intracerebrally with a 10(-1) dilution of ME7 contained high levels of infectivity. These results suggest that the ability to replicate scrapie agent in spleen or lymphoid tissue depends on the restoration of normal lymphoid structure and in particular the presence of differentiated follicular dendritic cells. The possibility that SCID mice can select minor strains of scrapie which are normally unrecognized in cloned ME7 is discussed.

Animals↗
PubMed1995

Sequence and repeat structure variants in the long terminal repeat of maedi-visna virus EV1.

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i.) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.

Animals↗
PubMed1995

Molecular cloning and expression of DNA encoding ovine interleukin 2.

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Amino Acid Sequence↗
PubMed1995

Analysis of the expression and secretion of isotypes of sheep B cell immunoglobulins with a panel of isotype-specific monoclonal antibodies.

Monoclonal antibodies to sheep light chain, IgM and IgG were produced and used to assess total immunoglobulin (Ig) synthesis by sheep B cells in culture and antibodies to specific antigens. By using these antibodies in a dual fluorescence-activated cell sorting analysis of sheep efferent lymph B lymphocytes the percentage change in surface Ig isotype of B lymphoblasts from IgM to IgG after the antigenic stimulation of the local lymph node was measured. An extension of this analysis to paired blood and afferent or efferent lymph B cells made it possible to investigate the recirculation characteristics of B cells expressing different Ig isotypes.

Animals↗
PubMed1995

Variable expression of major histocompatibility complex class II in the domestic cat.

This paper describes the characterisation of six independently produced monoclonal antibodies (mAbs) specific for non-polymorphic determinants of feline major histocompatibility complex (MHC) class II. One mAb is an anti-sheep class II which cross-reacts with the cat and five have been produced in response to immunisation with purified feline immunodeficiency virus. Despite their independent source all the mAbs have identical reactivities, immunoprecipitating two complex groups of polypeptides of M(r) 33 to 36.000 (MHC class II alpha chains) and M(r) 28 to 31,000 (MHC class II beta chains). Immunoblot analysis showed them to be beta chain-specific. One and two-dimensional electrophoresis revealed the complexity of feline class II mass and charge and implied the expression of multiple class II loci in the cat. Furthermore, it was demonstrated that distinct cell populations expressed a distinct range of class II variants. This suggesting either the differential expression or the distinct post-translational modification of lymphocytes from different sites. The mAbs have also been used for the detailed examination of the cellular distribution and tissue localisation of MHC class II in the cat.

Animals↗
PubMed1995

Identification of CD45 (leucocyte common antigen) in the domestic cat.

Feline CD45 (leucocyte common antigen, LCA) was characterised by using the mouse monoclonal antibody, WC45a. Its specificity was established on the basis of its reactivity with different lymphoid tissues, as judged by flow cytometry and immunohistochemistry, and of the molecular weight of its antigen in different tissues. It reacted with all the leucocyte populations tested including T and B lymphocytes, granulocytes and monocytes. By immunohistology it reacted only with leucocytes and not with endothelia, epithelia or connective tissue. It precipitated cell surface polypeptides, of M(r) 180,000 to 220,000 from lymph node cells, and therefore apparently recognised an epitope represented on most of the isoforms of the CD45 family.

Animals↗
PubMed1995

Biochemical and phenotypic characterization of the ovine beta 2 (leucocyte) integrins.

This paper is concerned with the relationship of the three distinct members of the ovine beta 2-integrin family of leucocyte adhesion molecules that play an important role in cell-cell and cell-matrix interactions. A panel of monoclonal antibodies (mAbs) specific for sheep and cattle macrophages was characterized by flow cytometry, immunohistology, and immunoprecipitation for reactivity to the beta 2 integrins. Immunoprecipitation analysis of sheep antigens showed that these monoclonal antibodies could be divided into four distinct groups. All precipitated an M(r) 95,000 beta chain but they differed in the size or number (or both) of the alpha chains recognized. Group 1 precipitated alpha chains of M(r) 180,000; group 2 had M(r) 170,000 alpha chains; the group 3 alpha chain was of M(r) 150,000 and group 4 mAbs precipitated alpha chains of all three sizes. The relationship between these antibodies was demonstrated by sequential immunoprecipitation, which showed that the reactivities of antibodies in groups 1, 2 and 3 were mutually exclusive but that group 4 antibodies shared a common specificity with the other three groups. By analogy with the human and murine beta 2 integrin families, group 1 antibodies seemed to be specific for CD11a (LFA-1); group 2 were CD11b (CR3 or Mac1); group 3 were CD11c (CR4 or p150/95) and group 4 were CD18. In addition to different molecular weights, these antibodies had different cellular and tissue distributions. CD11a and CD18 were distributed identically. The antigens recognized by both were present on all the leucocyte populations. The mAbs recognizing CD11b reacted with a sub-population of peripheral blood B lymphocytes and all myeloid cells (alveolar macrophages, peripheral blood monocytes and granulocytes) except afferent lymph dendritic cells (ADC). Anti-CD11c (p 150/95 or CR4) antibodies reacted strongly with macrophages and ADC but were weakly reactive on monocytes and negative on neutrophils. CD11c was also present on a sub-population of peripheral blood B cells.

Animals↗