Biomedical subjects

C J Howard

Publications and source records attributed to C J Howard.

At least 37 records · Page 2Linked 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↗

PubMed2003

Modulation of the bovine delayed-type hypersensitivity responses to defined mycobacterial antigens by a synthetic bacterial lipopeptide.

The use of defined protein and peptide antigens can overcome specificity limitations of purified protein derivatives in the detection of bovine tuberculosis when the antigens are used in blood-based tests. Since the use of these specific antigens as skin test reagents could have practical advantages, we investigated the potential of Mycobacterium bovis-specific antigens to stimulate delayed-type hypersensitivity (DTH) responses in cattle experimentally infected with M. bovis. A cocktail of the recombinant antigens ESAT-6, MPB83, and MPB64 failed to stimulate in vivo DTH in cattle that had been experimentally infected with M. bovis despite the fact that the antigens were recognized in vitro by the same animals. However, it was possible to stimulate antigen-specific bovine DTH responses by using ESAT-6 in combination with a synthetic bacterial lipopeptide. This lipopeptide stimulated the release of the proinflammatory cytokine tumor necrosis factor alpha from monocyte-derived bovine dendritic cells in vitro, thereby providing a possible mechanism for its DTH-enhancing properties.

Animals↗

PubMed2003

Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8(+) T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-gamma) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-gamma and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-gamma response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.

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

Infection and transformation of dendritic cells from bovine afferent lymph by Theileria annulata.

Following incubation with sporozoites of the protozoan parasite Theileria annulata, dendritic cells (DC), extracted from bovine afferent lymph, became infected and transformed into large, rounded, continuously proliferating cell lines. Phenotypic analysis of the transformed cells by immunostaining and flow cytometry revealed that they expressed MHC class I and II antigens, the myeloid marker MyD (SIRP alpha) and the bovine WC6 (workshop cluster 6) molecule. Transformed DC cell lines differed from those produced from infection of macrophages and B cells in that some lines expressed CD21 and a proportion of cells continued to express the antigen stained by the mAb CC81, a marker which defines a subpopulation of DC in afferent lymph. Both of the main populations of DC that have been identified in bovine afferent lymph appeared to be equally permissive for infection and transformation with T. annulata. These findings raise the possibility that the transformed proliferating cells characteristic of in vivo infections could be derived from DC as well as macrophages. This could have consequences for understanding the pathogenesis of the disease and for developing methods to manipulate immune responses to eliminate the parasite.

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

Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2.

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.

Amino Acid Sequence↗

PubMed2001

IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood.

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.

Animals↗

PubMed2001

Cross-reactivity with bovine cells of monoclonal antibodies submitted to the 6th International Workshop on Human Leukocyte Differentiation Antigens.

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.

Animals↗

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

Cattle immune responses to tetanus toxoid elicited by recombinant S. typhimurium vaccines or tetanus toxoid in alum or Freund's adjuvant.

Cattle were immunised orally, nasally or subcutaneously with either S. typhimurium 4/74 aroA(-) aroD(-) or S. typhimurium 4/74 htrA-based live vaccines expressing Fragment C (TetC) of tetanus toxin from plasmid pTetnir15. Oral inoculation with S. typhimurium 4/74 aroA(-) aroD(-)- (pTetnir15) elicited mucosal anti-TetC IgA but no measurable systemic humoral responses to TetC. Subcutaneous inoculation with the same strain elicited both mucosal IgA and systemic anti-TetC IgG1 responses. Nasal inoculation did not elicit any detectable anti-TetC responses. Oral delivery of S. typhimurium htrA(-) proved fatal in inoculated animals. None of the animals inoculated with either mutant S. typhimurium developed detectable T cell proliferative responses to the guest antigen. Cattle were also inoculated with tetanus toxoid adsorbed in alum or emulsified in Freund's complete adjuvant. Animals inoculated subcutaneously with Ttox emulsified in FCA developed systemic IgG1 and IgG2 antibody, while animals inoculated with Ttox adsorbed in alum developed systemic IgG1 but little IgG2 to Ttox. Both of these groups of animals developed measurable TetC-specific proliferative T cell responses that were associated with the production of IFNgamma.

Alum Compounds↗

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↗