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Publications and source records attributed to I McConnell.
All members of the lentivirus family infect cells of the monocyte-macrophage lineage, although this may be obscured during infection in vivo by the effect of the virus on other cells, such as CD4+ lymphocytes. Macrophages are the major cell type infected by the ruminant lentivirus maedi-visna, making it a valuable model for studying the pathogenesis of lentiviruses in these cells.
Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.
Reactive changes occurring within lymph nodes draining the subcutaneous site of acute infection with maedi-visna virus (MVV) were studied, and the appearance of infected cells correlated with the immune response. Cells infected with virus were detected in the node by cocultivation from day 4 postinfection (p.i.), with maximum numbers being seen between days 7 and 14, but even then infected cells were rare, with a maximum frequency of 23 50% tissue culture infective doses (TCID50) in 10(6) lymph node cells. At later times, infected cells were still detected, but their numbers fell to 1 to 2 TCID50 per 10(6) cells. Virus-specific CD8+ cytotoxic T-cell precursors (CTLp) were isolated from infected nodes from day 10 p.i. onwards, and T-cell proliferative responses to MVV were first detected on day 7 and consistently detected after day 18. Histological analysis showed a vigorous immune response in the node. There was a marked blast reaction in the T-cell-rich zones, which was greatest at the time when the number of virally infected cells was at its height. At this stage, large numbers of plasma cells were seen in the medullary cords, indicating that extensive T-cell-dependent B-cell activation was occurring in the T-cell-rich zones. Germinal centers were prominent shortly after the onset of the T-zone response and were still present at 40 days p.i. Phenotype studies of isolated lymph node cells failed to detect major changes in the proportion or phenotype of macrophages, CD1+ interdigitating cells, and CD4+ or CD8+ T cells despite the fact that CD8+ lymphoblasts form a major population leaving the node in efferent lymph. This suggests that there is a balanced increase in the number of all cell types in response to the virus within the node and selective migration of CD8+ lymphoblasts containing virus-specific CTLp from the node. Virus-specific immune responses are therefore present within the node when infectious virus isolation is maximal, but cellular immunity may act to control the level of infection from day 18 onwards.
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Transmissions of bovine spongiform encephalopathy (BSE) from seven unrelated cattle sources have given remarkably uniform disease characteristics in mice, differing from over twenty previous and contemporary transmissions of sheep and goat scrapie. Transmissions to mice of spongiform encephalopathy from six species (including sheep and goats) which have been experimentally or naturally infected with BSE have given similar results to direct BSE transmissions from cattle. Therefore the BSE agent has retained its identity when passaged through a range of species and the 'donor' species has little specific influence on disease characteristics in mice, adding to evidence for an agent-specific informational molecule. On transmission of BSE or scrapie to mice the incubation periods are long compared with subsequent mouse-to-mouse passages (the 'species barrier'). Contributing factors include a low efficiency of infection on interspecies transmission, the apparent failure of intracerebrally injected 'foreign' inoculum to establish infection directly in mouse brain and the selection of variant strains of agent which replicate most readily in the new host species.
Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 degrees C for < or = 60 min. Bioassay in rodents showed that none of the regimens produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for < or = 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing < or = 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for < or = 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.
We have examined the types and cellular distribution of transcripts of maedi-visna virus during infection of choroid plexus fibroblasts. Early in infection (19 hours post infection) only small spliced transcripts are found in the cytoplasm. Little virus specific RNA is detected in the nucleus at this stage. Later in infection structural gene transcripts are detectable in the cytoplasm as well as the nucleus. We have measured the half life of each type of transcript in total cellular RNA late in infection by two different methods (northern blotting or S1 protection assays after alpha-amanitin treatment, or specific hybridisation after pulse chase). Both of these methods suggest half lives for unspliced viral RNAs of 8-12 hours. Although the gel based methods were unsuitable for the accurate determination of half life for the shorter transcripts, they did confirm a long half life for these transcripts: pulse chase measurements suggested that this was similar to that seen for the unspliced transcripts. These observations are consistent with the virus exerting temporal control on protein synthesis by a method analogous to that of the HIV Rev protein.
A generic, rapid and sensitive enzyme linked immunosorbent assay (ELISA) test has been developed which allows large-scale simultaneous testing of synthetic corticosteroids viz., flumethasone, dexamethasone and betamethasone. This assay can be directly applied to diluted urine samples (1 + 9) without hydrolysis of glucuronide or sulfate conjugates or any other treatment of samples. The polyclonal antibody was obtained by immunizing sheep with a flumethasone derivative linked to human serum albumin. This polyclonal antibody displayed high-reactivity with several synthetic corticosteroids whilst endogenous corticosteroids such as cortisol gave very low cross-reactivity (< 0.5%). Sensitivities obtained in this assay were 2.5, 3.1 and 12.5 ng ml-1 for flumethasone, dexamethasone and betamethasone, respectively. The ability of this assay to detect several synthetic corticosteroids was demonstrated by testing urine samples from horses to which the drugs had been administered.
Maedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.
Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis using primers complementary to sequences at the 5' and 3' ends of 60 kDa stress protein genes (encoding the '65 kDa antigens') of M. leprae and M. tuberculosis. The predicted PCR product of 1.8 kb contained the entire coding sequence of an M. paratuberculosis 60 kDa stress protein, with non-coding regions of 124 bp and 1 bp at the 5' and 3' ends, respectively. DNA encoding the entire ORF for the 60 kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia coli strain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis 60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis 60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing.
This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DR beta chains are expressed in the sheep. Two anti-beta chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DR beta chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 beta chain. Amino-terminal sequence analysis of the alpha chain associated with VPM37 beta chain shows that this alpha chain is homologous to the human DR alpha chain strongly indicating that the beta chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of post-translational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DR beta chains in the sheep.
The cell type predominantly infected by maedi-visna virus (MVV) is the macrophage, and we have looked at the ability of MVV-infected macrophages to interact with cytotoxic T lymphocytes (CTL), important effector cells against virus infections. MVV-specific CTL precursors were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in peripheral blood lymphocytes of all MVV-infected sheep. MVV-infected monocyte-derived macrophages and alveolar macrophages were able to stimulate CTL activity in vitro and were targets for these activated CTL. The major effector cell population using MVV-infected macrophage targets was CD8+ lymphocytes, although another population, lymphokine-activated killer cells, may also have been involved. There was no direct cytotoxic activity found in alveolar lymphocytes from MVV-infected sheep without lung lesions.
We have produced by gene targeting a mouse line with an inactive PrP gene. In animals heterozygous for this mutation, PrP mRNA is reduced by approximately 50% throughout the brain compared with wild type mice. The steady-state level of PrPc is also significantly reduced in heterozygotes compared to wild type mice. PrP mRNA and protein are not detected in brains of mice homozygous for the mutation. We have infected wild type mice and mice heterozygous and homozygous for the mutation with the ME7 strain of scrapie. A gene dosage effect can be seen in time of disease onset and period over which the disease symptoms develop. In heterozygotes disease onset occurs around 220 days and terminal stages are reached by 280 days. In wild type mice disease onset occurs around 130 days and the terminal stages by 160 days. The PrP-/- mice are resistant to disease up to 475 days. PrP deposition in heterozygous mice starts in the same brain area as wild type mice and can be detected as early as 50 days. The pattern of PrP deposition in the brain of heterozygotes follows an identical course to that observed in wild type mice and by terminal stages of disease the amount deposited is equivalent to wild type mice. Vacuolation is detected later than PrP deposition and distribution and degree in the terminal stages of disease is similar in wild type and heterozygous mice. These results show that signs of disease, vacuolation and PrP deposition are dependent upon PrPc in a rate dependent manner.
We have investigated several aspects of gamma delta T cells in sheep. gamma delta T cells of sheep express a unique transmembrane protein termed T19 but lack the expression of particular cell-surface molecules such as CD2, CD4 and CD8 which are typically associated with alpha beta T cells. The majority of gamma delta T cells isolated from animals of all ages examined lacked the expression of CD45RA. A faster rate of activation by gamma delta T cells compared to either CD4 or CD8 T cells was seen in the time-course of IL-2 receptor alpha chain (CD25) cell-surface expression. All gamma delta T cells expressed the CD25 protein within 8 hr of activation whereas the majority of CD4 or CD8 T cells did not express CD25 until 24 hr post-concanavalin A (Con A) stimulation. This difference in the rate of expression of activation molecules was not restricted to CD25, as a similar trend was seen with cell-surface expression of major histocompatibility complex (MHC) class II molecules. We have used the distinct phenotypic profile of ovine gamma delta T cells to purify these cells by positive selection via the T19 molecule to assess their in vitro proliferative response to various antigens. Routinely, cell populations comprising more than 93% gamma delta T cells with yields of approximately 55% were obtained. Purified gamma delta T cells were capable of responding to Mycobacterium tuberculosis antigen in a primary and secondary in vitro proliferation assay and to ovalbumin in a secondary response. Ovine gamma delta T cells showed little, if any, proliferative response to allogeneic stimulator cells.
In this study we have investigated the expression of major histocompatibility complex (MHC) class II molecules on T cells from various lymphoid compartments in the sheep. Monoclonal antibodies which react specifically with sheep MHC class II molecules homologous to the human DQ and DR molecules have been characterized. These antibodies have been used, together with the monoclonal antibodies specific for sheep CD4-, CD8- and T19-positive T cells, to quantitate DQ and DR expression on T cell subsets in adult and fetal peripheral blood, afferent lymph, lymph node and efferent lymph. The results show that expression of class II by T cells depends on the age of the animal and the physiological location of the T cell. In fetal blood there is no expression of class II on CD8+ or T19+ cells and very low expression on CD4+ T cells. In adult peripheral blood and efferent lymph a significant proportion of cells express DR but not DQ. A very different situation is found in afferent lymph and the peripheral lymph node: in afferent lymph the majority of T cells in all three subsets express both DQ and DR molecules; in the lymph node over 50% of T cells express DR and 30% are DQ+. These results suggest that within all T cell subsets there is a progression from DQ-DR- to DQ-DR+ and DQ+DR+ which correlates with physiological stages of T cell differentiation in vivo.
This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4+, CD8+ and T19+ (gamma delta T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II- or DR+DQ- but this changes to DR+DQ+ after antigenic challenge. The antigen-presenting function of these class II+ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II+ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.
The anti-CD11/CD18 monoclonal antibodies (mAbs) submitted in the Second International Workshop on Ruminant Leukocyte Differentiation Antigens, were analysed for their reactivity with the ovine homologue of CD11/CD18. Their reactivity was tested on healthy sheep tissues, and alveolar macrophages, afferent dendritic cells, peripheral blood granulocytes and monocytes. The CD11a/CD18 mAbs found positive in the sheep were reactive with all the cell populations tested. The CD11b mAbs reacted with all the cells except afferent dendritic cells, whereas CD11c were non-reactive to blood granulocytes. This is in contrast to humans and cattle where blood granulocytes express CD11c. The mAbs 72-87, F10-150, MD2B7 and MUC76A were found to be homologous to CD11a whereas BAQ30A seemed to be homologous to CD18, instead of proposed initial specificity to CD11a. CC125 and IL-A15 mAbs were found to be homologous to CD11b. OM1, which clustered with a recognized CD1 mAb in the first cluster analysis, precipitated a heterodimer of molecular weight 95,000/150,000. We propose that OM1 reacts with sheep CD11c. The mAb MF14B4 was found to react with sheep CD18.