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Biomedical subjects

J C Hope

Publications and source records attributed to J C Hope.

At least 19 recordsLinked to original sources
PubMed2003

Differential effects of bovine viral diarrhoea virus on monocytes and dendritic cells.

Various pathogens have been shown to infect antigen-presenting cells and affect their capacity to interact with and stimulate T-cell responses. We have used an antigenically identical pair of non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea virus (BVDV) isolates to determine how the two biotypes affect monocyte and dendritic cell (DC) function. We have shown that monocytes and DCs are both susceptible to infection with ncp BVDV and cp BVDV in vitro. In addition, monocytes infected with ncp BVDV were compromised in their ability to stimulate allogeneic and memory CD4(+) T cell responses, but DCs were not affected. This was not due to down-regulation of a number of recognized co-stimulatory molecules including CD80, CD86 and CD40. Striking differences in the response of the two cell types to infection with cytopathic virus were seen. Dendritic cells were not susceptible to the cytopathic effect caused by cp BVDV, whereas monocytes were killed. Analysis of interferon (IFN)-alpha/beta production showed similar levels in monocytes and DCs exposed to cp BVDV, but none was detected in cells exposed to ncp BVDV. We conclude that the prevention of cell death in DCs is not associated with enhanced production of IFN-alpha/beta, as proposed for influenza virus, but is by a distinct mechanism.

Animals↗
PubMed2002

Co-stimulation and modulation of the ensuing immune response.

As a consequence of the central role of dendritic cells (DC) in stimulating primary immune responses any bias in the response introduced by the DC has the potential for having a long-term effect on immunity. Examination and analysis of ruminant afferent lymph dendritic cells derived by cannulation allows studies on the properties of ex vivo DC that is not possible in humans and rodents and information can be derived from ruminants that has implications of generic relevance. Previous studies have identified two major populations of DC in afferent lymph draining the skin of cattle that differ in their capacity to stimulate CD4 and CD8 T cells. Differences in expression of cytokine transcripts have now been shown for the two types of DC. The CD11a(+)/SIRPalpha(-) population synthesised more IL-12, whilst the CD11a(-)/SIRPalpha(+) population produced more IL-10. This is likely to affect the bias of the immune response following presentation of antigen to T cells by one DC sub-population or the other. An inability to synthesise IL-1alpha was the reason for the failure of the CD11a(+)/SIRPalpha(-) DC to stimulate CD8 T cells. This property would potentially affect the induction of CD8 responses. Expression of the co-stimulatory molecules CD80, CD86 and CD40 appeared similar for both DC populations and not to relate to differences in function. A further examination of the SIRPalpha molecule on DC indicated that on cross-linking it was tyrosine phosphorylated and that it recruited the SHP-2 protein phosphatase. Associated with this was a blocking of TNFalpha secretion on exposure to LPS. The interaction of SIRPalpha with its ligand CD47 on T cells appeared to be an early event in the stimulation of T cells as binding of the ligand was reduced on activated T cells. CD26 was identified as another molecule expressed by the SIRPalpha(-) DC sub-population. This is reported to have an enzymatic activity on certain chemokines that could result in the promotion of a Th1 bias.A model is proposed that takes these observations into account in which SIRPalpha(-) DC would be expected to promote a Th1 biased response and the SIRPalpha(+) DC a more balanced one.

Animals↗
PubMed2002

Role of bovine chemokines produced by dendritic cells in respiratory syncytial virus-induced T cell proliferation.

Respiratory syncytial virus (RSV) has been reported to induce the production of chemokines in the airway epithelia. Dendritic cells (DC) are the most potent antigen-presenting cells. They are located throughout the body and release chemokines in response to inflammation and infection. We have investigated the chemokine profile of bovine DC in response to exposure to bovine RSV (BRSV). Transcripts for several chemokines were detected by RT-PCR, subsequently cloned and expressed, and the products analysed by western blotting. To test the effect of the recombinant chemokines on RSV-induced T cell proliferation, DC were pulsed with BRSV, irradiated, and added to purified bovine CD4(+) T cells from RSV-immune cattle in combination with various concentrations of recombinant chemokines, and the proliferative response of the T cells assessed. Eotaxin was the only chemokine, of those investigated, that specifically enhanced the T cell response to BRSV-pulsed DC. Addition of MIP-1alpha to control wells or to wells containing BRSV-pulsed DC had similar effects, suggesting non-specific stimulation of T cells. RANTES and MIP-3alpha did not seem to influence the proliferative response of T cells co-cultured with BRSV-pulsed DC. Thus, although BRSV induced the production of several chemokines by DC, only eotaxin promoted a BRSV specific CD4(+) T cell proliferative response.

Animals↗
PubMed2002

Development of detection methods for ruminant interleukin (IL)-12.

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Animals↗
PubMed2002

Development of an ELISA for bovine IL-10.

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFN gamma by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFN gamma synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.

Animals↗
PubMed2002

Exposure to Mycobacterium avium primes the immune system of calves for vaccination with Mycobacterium bovis BCG.

The objective of the investigation was to provide data on how a prior exposure of cattle to Mycobacterium avium, used here as a model of exposure to an environmental mycobacterium, affected the cellular immune response that follows vaccination with Mycobacterium bovis BCG. The assessment of cellular immune responses included lymphocyte proliferation assays, the delayed hypersensitivity skin test and IFN-gamma synthesis in whole blood cultures. One group of calves was inoculated subcutaneously with M. avium followed 12 weeks later by M. bovis-BCG. The other group was vaccinated subcutaneously with BCG alone. Calves previously exposed to M. avium responded more rapidly, as assessed in the in vitro assays, to purified protein derivative (PPD) from M. avium (PPD-A) or M. bovis (PPD-B) than did calves inoculated with BCG only, indicating that the exposure to M. avium had primed the immune response in these calves. Following inoculation of BCG the intensity of the in vitro responses and the delayed hypersensitivity skin test to PPD-A was higher for the M. avium-primed animals while the responses to PPD-B were similar in the M. avium-primed and BCG-only groups. The results are consistent with a model in which prior exposure to environmental mycobacteria does not necessarily inhibit the immune response to the vaccine strain, BCG. They suggest that M. avium infection primes the immune system of calves and that the detection of an immune response specific for M. bovis BCG is masked by reactivity to antigens also present in M. avium.

Adjuvants, Immunologic↗
PubMed2002

Alpha/beta and gamma interferons are induced by infection with noncytopathic bovine viral diarrhea virus in vivo.

In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor beta (TGF-beta) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-beta.

Acute Disease↗
PubMed2001

Masking of two in vitro immunological assays for Mycobacterium bovis (BCG) in calves acutely infected with non-cytopathic bovine viral diarrhoea virus.

Acute infection of calves, previously vaccinated with bacille Calmette-Guerin (BCG), with non-cytopathic viral diarrhoea virus (BVDV) resulted in the temporary suppression of two in vitro assays used to monitor Mycobacterium bovis infection. Lymphocyte proliferation and interferon-gamma production by whole blood cultures containing purified protein derivatives prepared from Mycobacterium avium (PPD-A) and M bovis (PPD-B) were markedly suppressed. The implication is that acute infections of cattle with non-cytopathic BVDV may temporarily compromise diagnostic tests for M. bovis infections and result in a failure to identify cattle with tuberculosis.

Acute Disease↗
PubMed2001

Differences in the induction of CD8+ T cell responses by subpopulations of dendritic cells from afferent lymph are related to IL-1 alpha secretion.

The major subset of dendritic cells (DC) from bovine afferent lymph expresses the SIRP alpha MyD-1 antigen, but not CD11a or the antigen recognized by mAb CC81, and potently stimulates CD4+ and CD8+ T lymphocyte proliferation. The minor subpopulation, that is CD11a+ CC81+ MyD-1-, effectively stimulates CD4+ but not CD8+ T lymphocyte proliferation. CD11a+ CC81+ MyD-1- DC did not induce anergy or death or secrete an inhibitory factor. However, supernatant from cultures of CD8+ T cells with CD11a- CC81- MyD-1+ DC significantly enhanced proliferation of CD8+ T cells in response to CD11a+ CC81+ MyD-1- DC, an effect that was blocked by interleukin (IL)-1alpha, but not IL-1beta, specific mAb. The proliferation of CD8+ T cells with CD11a+ CC81+ MyD-1- DC was also enhanced by adding IL-1alpha. IL-1beta slightly enhanced proliferation, whereas IL-2, IL-6, IL-12, and IL-15 had no effect. We conclude that the failure to stimulate CD8+ T cell proliferation results from the lack of IL-1alpha synthesis by this population, which may have important consequences in vivo.

Adjuvants, Immunologic↗
PubMed2000

Dendritic cells, implications on function from studies of the afferent lymph veiled cell.

Studies of afferent lymph veiled cells (ALVC) show that the full biological function of dendritic cells in peripheral tissue is not explained by a simple model in which immature dendritic cells at the body surface take up antigen, migrate via the afferent lymph ducts, mature and then effectively present antigens to T-cells in the draining lymph node. Furthermore, it is evident from various investigations that the dendritic cells in afferent lymph draining from the body surfaces are not a homogeneous population of cells. They comprise a mixture of cell phenotypes defined by staining with monoclonal antibodies, and the different sub-populations have distinct biological functions and roles in vivo. The molecular basis for differences between the function of afferent lymph dendritic cell subsets is only now being explored and defined but some progress has been made in understanding the role of co-stimulatory molecules. It should be possible to exploit knowledge of the functions of these cells and aid future vaccination strategies in domesticated animals thereby improving animal health and reducing economic loss, and, as a consequence, improving human health. By deliberately targeting functionally distinct subsets of either precursor or mature dendritic cells in vivo, it should become feasible to achieve an appropriately biased immune response.

Animals↗
PubMed2000

Deficiency of IL-2 or IL-6 reduces lymphocyte proliferation, but only IL-6 deficiency decreases the contact hypersensitivity response.

We evaluated the importance of IL-2 and IL-6 in primary antigen-induced proliferation of lymph node cells (LNC) and the induction of contact hypersensitivity (CH). These responses were examined in cytokine-deficient mice following application of the contact sensitizer, oxazolone (OX). Proliferation and induction of IL-6 by LNC from IL-2-deficient (IL-2(-/-)) mice were reduced by approximately 95 %, relative to the proliferation of LNC from IL-2(+/+) mice, although induction and elicitation of CH responses was not significantly affected. In contrast, the proliferation of LNC from sensitized IL-6(- /-) mice was reduced by approximately 50% and the CH response was significantly reduced, relative to responses of IL-6(+/+) mice. Although nonspecific inflammatory responses induced by croton oil were similar in IL-6(+/+) and IL-6(-/-) mice, both the acute inflammatory response to OX and the second phase of the inflammatory response were significantly reduced. Thus IL-2 and IL-6 play a significant role in the total proliferative response of LNC following primary contact sensitization. However, the proliferation they promote is not critical for priming the antigen-specific effector cells responsible for eliciting CH responses and IL-6 appears to be more important for expression of the later phases of acute inflammation and the CH induced by OX.

Animals↗
PubMed2000

Flt-3 ligand, in combination with bovine granulocyte-macrophage colony-stimulating factor and interleukin-4, promotes the growth of bovine bone marrow derived dendritic cells.

Culture of bone marrow precursor cells with cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and the tyrosine kinase receptor binding proteins Flt-3 ligand (Flt-3L) and stem-cell factor (SCF), has previously been shown, in both mouse and human, to result in the generation of large numbers of dendritic cells. We extend these findings to bovine dendritic cells. Culture of bovine bone marrow cells with GM-CSF, IL-4 and either Flt-3L or SCF enhanced the generation of low buoyant-density dendritic cells. However, only the addition of Flt-3L to cells cultured with GM-CSF and IL-4 was shown to increase the number of dendritic cells and induce the differentiation of dendritic cells with potent capacity to stimulate allogeneic T cells and resting CD4+ memory T cells. The effective ability to stimulate T cells was associated with the expression of major histocompatibility complex (MHC) class II molecules and CD80/86 by dendritic cells. Bovine bone marrow derived dendritic cells appeared to be exclusively of myeloid origin because they expressed the myeloid-related antigens CD14, MyD-1 and CD11b.

Animals↗
PubMed2000

Dendritic cells induce CD4+ and CD8+ T-cell responses to Mycobacterium bovis and M. avium antigens in Bacille Calmette Guérin vaccinated and nonvaccinated cattle.

Few data are available regarding the induction of memory T-lymphocyte responses in cattle following Bacille Calmette Guérin (BCG) vaccination. Studies of the immune response induced by BCG vaccination provide an insight into the basis of antimycobacterial immunity that could be exploited for the development of more effective vaccination strategies. We used autologous dendritic cells (DC) infected with Mycobacterium bovis Bacille Calmette Guérin (BCG) or pulsed with purified protein derivative from M. bovis (PPD-B) or M. avium (PPD-A) to assess responses of CD4+, CD8+ and WC1+ gammadelta TCR+ lymphocytes from BCG vaccinated and nonvaccinated cattle. Mycobacteria-specific CD4+ and CD8+, but not WC1+ gammadelta TCR+, memory T lymphocytes were demonstrated in BCG-vaccinated cattle. CD4+ and CD8+ lymphocytes proliferated and produced interferon (IFN)-gamma in response to BCG-infected or PPD-B-pulsed DC. Proliferative responses were greater for CD4+ than CD8+ lymphocytes, although secretion of IFN-gamma was higher from the CD8+ T cells. Responses to PPD-A-pulsed DC were lower, with no CD8+ response. Lymphocytes from nonvaccinated calves were also stimulated to proliferate by BCG-infected DC, although the magnitude of proliferation was lower. The findings suggest that immunity to M. bovis induced by BCG vaccination in cattle may involve CD8+ memory T cells which produce IFN-gamma, as well as CD4+ memory T cells.

Animals↗
PubMed1999

Dendritic cells in cattle: phenotype and function.

Dendritic cells are professional antigen presenting cells derived from the bone marrow and distributed throughout body tissues where they are located in sites that are suitable for antigen uptake. They are central to the induction of immune responses in naive animals and thus have become targets in strategies that are aimed at modulating resistance to infection. Studies in cattle have shown that the dendritic cells are phenotypically heterogeneous and that the different phenotypes have different biological properties. The molecular basis for this variation has begun to be investigated and has led to the identification of a member of the SIRPalpha family of signal regulatory proteins (MyD1) on a subset of dendritic cells in afferent lymph. Uptake of antigen by cattle dendritic cells is by a number of mechanisms that can involve endocytosis via clathrin coated pits or via caveolae as well as macropinocytosis.

Animals↗
PubMed1999

The top3(+) gene is essential in Schizosaccharomyces pombe and the lethality associated with its loss is caused by Rad12 helicase activity.

The topoisomerase III gene ( top3 (+)) from Schizosaccharomyces pombe was isolated and a targeted gene disruption ( top3 :: kan (R)) was used to make a diploid strain heterozygous for top3 (+). The diploid was sporulated and the top3 :: kan (R)spores went through four to eight cell divisions before arresting as elongated, predominantly binucleated cells with incompletely segregated chromosomes. This demonstrates that top3 (+)is essential for vegetative growth in fission yeast. The aberrant chromosomal segregation seen in top3 :: kan (R)cells is unlike the 'cut' phenotype seen in mitosis-defective mutants and so we refer to this phenotype as 'torn'. A deletion mutant, rad12-hd ( rad12 is a homolog of Saccharomyces cerevisiae SGS1), partially suppressed the lethality of top3 mutants. A point mutant, rad12-K547I, which presumably eliminates helicase activity, also suppresses the lethality of top3 mutants, demonstrating that the lethality seen in top3 (-)cells is most likely caused by the helicase activity of Rad12. This double mutant grows very slowly and has much lower viability compared to rad12-hd top3 :: kan (R)cells, implying that the helicase activity of Rad12 is not the only cause of top3 (-)lethality. The low viability of rad12 (-) top3 (-)mutants compared with rad12 single mutants suggests that Top3 also functions independently of Rad12.

Amino Acid Sequence↗
PubMed1999

Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells.

The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established.

Amiloride↗
PubMed1996

Antigen-induced unresponsiveness in contact sensitivity: association of depressed T lymphocyte proliferative responses with decreased interleukin 6 secretion.

Topical exposure of mice to the contact allergen oxazolone induces both 4 persistent antigen-specific down-regulation of subsequent lymph node cell (LNC) proliferative responses stimulated by the same chemical and a more transient depression of LNC proliferative responses provoked by exposure to unrelated chemical sensitizers: the latter being associated with antigenic competition in contact sensitivity. In this paper a relationship between reduced LNC proliferative activity and the secretion of interleukin 6 (IL-6) is described. Pretreatment of mice with oxazolone caused a persistent, dose-dependent inhibition of LNC proliferative activity and a parallel reduction of IL-6 secretion when mice were re-exposed, at a different site, to the same chemical. Consistent with dendritic cells (DC) being the major source of IL-6 within allergen-activated lymph nodes, depletion of Thy-lt T lymphocytes did not compromise production of this cytokine. Although in mice pretreated with oxazolone IL-6 secretion by cultured LNC was impaired markedly, the initial IL-6 content of freshly isolated LNC was apparently normal. These data suggest that the down-regulation of lymphocyte proliferative responses induced by exposure of mice to oxazolone, and the consequential impaired responsiveness, is associated with, and possibly secondary to, the reduced secretion by lymph node DC of IL-6, a cytokine that is a costimulator of T lymphocyte activation and the production of which correlates closely with the vigour of LNC proliferative activity.

Adjuvants, Immunologic↗