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I McConnell

Publications and source records attributed to I McConnell.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1990

Afferent lymph dendritic cells: a model for antigen capture and presentation in vivo.

We review the phenotypic and functional properties of sheep afferent lymph dendritic cells. These dendritic cells bear surface immunoglobulin and can acquire antigen/antibody complexes, both in vitro and in vivo. Our data suggest a role for Fc receptors in the capture of antigen by these cells. Dendritic cells collected after in vivo antigen pulsing are capable of stimulating T cell proliferation in an antigen-specific manner. Afferent dendritic cells express all the known groups of presentational molecules involved in activation of T cells, namely MHC class I and class II, and CD1. These results suggest a role for afferent dendritic cells in the activation of alpha beta and gamma delta T cells.

Animals↗
PubMed1990

IL-2-like activity in lymph fluid following in vivo antigen challenge.

The majority of studies that characterize lymphokines utilize in vitro activation of lymphocytes. In an attempt to identify and characterize lymphokines released from tissue sites, we have cannulated sheep lymphatic vessels and collected lymph that drains a site of in vivo antigen challenge. Lymph draining directly from a site of intradermal antigen challenge (afferent lymph) and lymph draining an antigen-stimulated lymph node (efferent lymph) were assayed for lymphokine activity by the ability of cell-free lymph fluid to stimulate the proliferation of sheep Con A-blasts. Afferent and efferent lymph, both collected at 24 and 48 hr following in vivo antigen challenge, with either ovalbumin or PPD in primed animals, stimulates the proliferation of sheep Con A-blast cells. This in vivo-derived lymphokine activity and in vitro-generated sheep Con A supernatant has an active component with properties similar to interleukin-2 (IL-2) that has been described in several other species. The IL-2-like material is precipitated by 40-80% ammonium sulphate saturation, has a molecular weight (MW) of 20,000 MW as judged by gel filtration chromatography, and is eluted from an anion-exchange HPLC column with 125 mM NaCl. HPLC ion-exchange fractionation of the 20,000 MW material from lymph fluid shows differences between afferent and efferent lymph material. The fractionation of afferent material is similar to that of in vitro generated Con A supernatant material with a single peak of activity eluted by 125 mM NaCl. In contrast, the 20,000 MW material from efferent lymph elutes with peaks of activity at 125 and 300 mM NaCl.

Animals↗
PubMed1990

Characterization of monoclonal antibodies specific for alpha- and beta-chains of sheep MHC class II.

In order to identify and characterize the sheep major histocompatibility complex (MHC) class II molecules, a panel of 19 monoclonal antibodies (mAb) has been raised following immunization of mice with a variety of class II antigen preparations. Antibodies were selected by ELISA using immunopurified sheep class II as antigen and further screened for the ability to react on immunoblots. Nine mAb reacted with the beta-chain and four reacted with the alpha-chain. The chain specificity of the remainder could not be determined as they did not blot. The anti-alpha and anti-beta mAb all reacted with deglycosylated class II. With the exception of one anti-alpha and one anti-beta antibody, the antibodies reacted with both efferent lymph cells and peripheral blood mononuclear cells (PBMC). Three of the anti-sheep class II mAb reacted with human PBMC.

Animals↗
PubMed1989

Characterization of sheep afferent lymph dendritic cells and their role in antigen carriage.

We have ablated peripheral lymph nodes in sheep and subsequently cannulated the pseudo-afferent lymphatic vessel that arises as a consequence of afferent lymphatic vessels reanastomosing with the former efferent duct. This technique allows the collection of lymph with a cellular composition that resembles true afferent fluid, and in particular, containing 1-10% dendritic cells. A 16-h collection of this lymph may contain between 10(6) and 10(7) dendritic cells. This dendritic cell population may be enriched to greater than 75% by a single-density gradient centrifugation step. We have generated a mAb that recognizes sheep CD1. This monoclonal not only reacts with afferent dendritic cells, but with dendritic cells in the skin and paracortical T cell areas of lymph nodes. The expression of CD1 suggests afferent dendritic cells are related to skin Langerhans' cells and other dendritic cells that act as accessory cells for T cell responses. Consistent with this is the high level of expression by dendritic cells of molecules involved in antigen recognition by T cells, including MHC class I and class II. Afferent dendritic cells express high levels of the cellular adhesion molecule LFA-3, and at the same time express a ligand for this molecule, namely CD2. The accessory functions of afferent dendritic cells resemble those displayed by mature Langerhans' cells and by lymph node interdigitating cells. These include clustering with resting T cells and stimulating their proliferation in a primary response to antigen. Afferent dendritic cells are capable of acquiring soluble protein antigen in vivo or in vitro and presenting the material directly to autologous T cells in an antigen-specific manner. We conclude that afferent dendritic cells represent a lymph-borne Langerhans' cell involved in antigen carriage to the lymph node.

Animals↗
PubMed1989

In vivo modulation of CD1 and MHC class II expression by sheep afferent lymph dendritic cells. Comparison of primary and secondary immune responses.

The experiments described in this article characterize the phenotypic and functional changes in afferent lymph cell populations that occur as a result of in vivo immune stimulation. During the primary immune response (in antigen-naive sheep) there are very transient increases in level of CD1 expression by subpopulations of dendritic cells (DC) but no alterations in cell kinetics or MHC class II expression. In contrast, secondary antigenic challenge (in primed sheep) into the drainage area of an afferent lymphatic causes profound changes in the cell output, characterized by a greater than threefold drop in total cell output on days 1-3 followed by an approximate fivefold rise on day 5. There is also a substantial increase in both the proportion of MHC class II-positive T lymphocytes (from 28 to 54%) and in the quantitative expression of class II by both DC and lymphocytes. Class II expression by DC increases five- to sixfold by day 5, while the level of expression of class II on lymphocytes approximately doubles. The increase in CD1 expression during the secondary response is more prolonged than during the primary response, being detectable between days 2 and 6 after challenge. The rise in class II affects the whole DC population, in contrast to CD1 where the increase affects only a subpopulation of cells. In terms of functional properties, afferent lymph DC isolated during a primary response show no alteration of their activity, whereas DC taken 4-5 d after secondary challenge are up to fivefold more active in their ability to present soluble antigen to primed autologous T cells and to antigen-specific cell lines as well as to stimulate in the MLR. The relative expression of class II correlates temporally with an increased capacity of DC to present antigen. Monoclonal anti-class II antibodies totally inhibit the in vitro assays but anti-CD1 antibodies have no effect. The previous paper has demonstrated that afferent DC can associate with antigen in vivo and can present that antigen to antigen-specific T cells. This article extends our knowledge of DC biology and demonstrates that DC, activated during secondary in vivo immune responses, have an enhanced ability to present an antigen, unrelated to that used for challenge, to specific T cell lines. This enhancement correlates directly with quantitative variation of expressed class II and not CD1 and suggests that this variation in class II expression plays a physiological role in in vivo immune regulation.

Animals↗
PubMed1989

Non-random migration of CD4 and CD8 T cells: changes in the CD4: CD8 ratio and interleukin 2 responsiveness of efferent lymph cells following in vivo antigen challenge.

In this investigation we have examined some of the cellular and molecular changes in efferent lymph that drains from an antigen-stimulated peripheral lymph node. Resting efferent lymph is characterized by a higher percentage of CD4+ cells and consequently, a higher CD4/CD8 ratio than peripheral blood. Following antigen stimulation of a cannulated peripheral lymph node in antigen-primed sheep, the percentage of CD4+ cells in efferent lymph increases above the resting level during days 1, 2 and 3 post antigen stimulation. This is followed on days, 3, 4, and 5 after antigen stimulation by an increase in the percentage of CD8+ cells above the resting level which occurs as the percentage of CD4+ cells returns to the resting level. These changes cause the CD4/CD8 ratio to first increase above the resting value during the CD4 phase and then decrease below the resting value during the CD8 phase. During the CD4 phase a lymphokine activity is present in cell-free lymph fluid. Lymph fluid collected at this time supports the proliferation of activated T cells. Supernatants generated from efferent cells collected at a similar time and cultured in vivo for 24 h without any further stimulation are capable of releasing this material. During the CD8 phase cells expressing functional interleukin(IL)2 receptors appear in lymph fluid. The data suggests a sequential exit of T cell subsets from an antigen-stimulated lymph node and that the appearance of IL2-like activity and IL2-responsive cells in efferent lymph fluid are temporally distinct events.

Animals↗
PubMed1989

Antigen presentation in the sheep: generation of antigen-specific T-cell lines.

Antigen-specific sheep T-cell lines have been generated in vitro from peripheral blood mononuclear cells (PBMC). PBMC prepared from antigen-primed animals were cultured in the presence of ovalbumin (OVA) or purified protein derivative (PPD). After 5 days of culture, activated antigen-specific cells were expanded by further culture in the presence of recombinant human interleukin-2 (IL-2). Cell lines generated after two cycles of antigen stimulation followed by expansion with IL-2 show a proliferative response to antigen only in the presence of autologous antigen-presenting cells (APC) and recognize only the antigen used in the original stimulation. An OVA-specific cell line was found to be capable of recognizing a synthetic peptide corresponding to amino acid residues 323-338 of OVA. The cell lines also responded by proliferation in an allogeneic mixed leucocyte reaction (MLR). Cell-surface phenotyping shows that the cell lines comprise both CD4- and CD8-positive cells.

Animals↗
PubMed1989

The scrapie disease process is unaffected by ionising radiation.

The incubation period of scrapie, its degenerative neuropathology and the replication of its causal unconventional virus are all tightly controlled parameters of the experimental disease in mice. Each parameter can vary depending on the strain and dose of virus, on the route of infection, and on the host genotype. Exposure to whole-body gamma-irradiation from Caesium 137 has no effect on the progress or development of the disease, based on the three independent indices of incubation period, neuropathology, or infectibility by high or low doses of virus. These results are based on an extensive series of experiments in many mouse strains and are consistent using different strains (ME7, 22A, 79A, 87V) and doses of virus, routes of infection, timing and dose of radiation (3-15 Gy) administered as single or fractionated exposures with or without bone-marrow (b.m.) replacement therapy. Levels of infection in the spleen are unaltered after lethal whole-body irradiation of the scrapie-infected host, despite several-fold reductions in tissue mass due to the loss of proliferating myeloid and lymphoid precursor cells and their progeny. Contrary to our earlier suggestion, scrapie infection with the 22A virus does not reduce the effectiveness of post-exposure bone-marrow replacements to recolonise an infected host after repeated ionising radiation totalling 15Gy. This work narrows the search for the candidate cells and biosynthetic systems which replicate the virus in the lymphoreticular and central nervous systems. Many programmed cellular events are radiation sensitive but protein synthesis is extremely radioresistant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Lymphocyte subpopulations in the blood of sheep persistently infected with border disease virus.

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.

Aging↗
PubMed1987

Linkage of the scrapie-associated fibril protein (PrP) gene and Sinc using congenic mice and restriction fragment length polymorphism analysis.

Sinc, with two alleles p7 and s7, is the major gene determining the incubation period of all strains of scrapie in mice. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and we have used a cDNA copy of the hamster PrP mRNA to carry out restriction fragment length polymorphism (RFLP) analysis of different inbred mouse strains including VM(Sincp7) and VM(Sincs7) congenic mice. In VM(Sincp7) mice, a 5.5 kb XbaI fragment hybridized to the PrP cDNA sequence whereas VM(Sincs7) congenic mice had a 3.8 kb XbaI fragment. The VM X VM(Sincs7) congenic F1 mice had both the 5.5 kb and the 3.8 kb fragments. The Sincs7 donor mouse strain, C57BL, had the 3.8 kb fragment suggesting that the Sinc gene and the gene coding for PrP are linked, and could even be the same gene. Other Sincp7 inbred mice (IM and MB) had the 5.5 kb fragment but so too did some Sincs7 strains (RIII and VL), implying that the XbaI site polymorphism is not functionally involved in the difference between the two Sinc alleles. We have mapped the polymorphic XbaI site to the 3' flanking region of the PrP gene. TaqI and HhaI were also found to show polymorphisms in the inbred mouse strains studied. The apparent RFLP with HhaI may be a result of differences in methylation rather than in sequence.

Animals↗
PubMed1987

Antibody-independent activation of the classical pathway of complement by Epstein-Barr virus.

A purified preparation of Epstein-Barr virus (EBV) has been shown to activate the classical complement pathway by direct interaction with the first component of complement, C1, without the intervention of antibody. No evidence was found for activation of the alternative pathway. Following classical pathway activation the specific affinity of EBV for B cells can be presumed to be lost since the virus will become opsonized for clearance by phagocytic cells bearing complement receptors, CR1 and CR3. This activation is further evidence that complement plays a role in defence mechanisms independently of antibody activity.

Antigens, Viral↗
PubMed1986

An improved procedure for obtaining germ-free laboratory mice.

The economy of time and effort accruing from the use of laminar air-flow cabinets to obtain germ-free mice has already been established. However, a further modification of the technique, utilizing small intermediate isolators, has made it possible to transfer to the large, recipient isolators only those litters which are viable and demonstrably free from contamination.

Animal Husbandry↗