Search PubMed⌕ Search

Biomedical subjects

P Sopp

Publications and source records attributed to P Sopp.

At least 19 recordsLinked to original sources
PubMed2006

Flow cytometric detection of gamma interferon can effectively discriminate Mycobacterium bovis BCG-vaccinated cattle from M. bovis-infected cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-gamma) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-gamma-producing lymphocytes by flow cytometric analysis of intracellular IFN-gamma expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-gamma-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+ T lymphocytes expressing IFN-gamma, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle.

Animals↗
PubMed2005

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Animals↗
PubMed2004

The role of dendritic cells in shaping the immune response.

Dendritic cells are central to the initiation of primary immune responses. They are the only antigen-presenting cell capable of stimulating naive T cells, and hence they are pivotal in the generation of adaptive immunity. Dendritic cells also interact with and influence the response of cells of the innate immune system. The manner in which dendritic cells influence the responses in cells of both the innate and adaptive immune systems has consequences for the bias of the adaptive response that mediates immunity to infection after vaccination or infection. It also provides an opportunity to intervene and to influence the response, allowing ways of developing appropriate vaccination strategies. Mouse and human studies have identified myeloid, lymphoid and plasmacytoid dendritic cells. Studies in domesticated animals with agents of specific infectious diseases have confirmed the applicability of certain of the generic models developed from mice or from in vitro studies on human cells. In vivo and ex vivo studies in cattle have demonstrated the existence of a number of subpopulations of myeloid dendritic cells. These cells differ in their ability to stimulate T cells and in the cytokines that they produce, observations clearly having important implications for the bias of the T-cell response. Dendritic cells also interact with the innate immune system, inducing responses that potentially bias the subsequent adaptive 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↗
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 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

Phenotypic analysis of local cellular responses in calves infected with bovine respiratory syncytial virus.

Changes in lymphocyte subsets in the trachea, pulmonary tissue, bronchoalveolar lavage (BAL), peripheral blood and bronchial lymph node (BLN) of gnotobiotic calves infected with bovine respiratory syncytial virus (BRSV) were analysed by flow cytometry. Following BRSV infection, virus titres in the nasopharynx reached a peak between days 5 and 7 and infection was resolving from day 10. Although calves did not develop signs of clinical respiratory disease, there was evidence of gross pneumonia and histological changes typical of BRSV bronchiolitis, which were most extensive from day 710 of infection. Following BRSV infection there was a recruitment of CD8+ T cells into the trachea and lung, which peaked on day 10 after infection. Thus, there were approximately equal numbers of CD8+ and CD4+ T cells in the lung and trachea of uninfected calves, whereas by day 10 of infection, CD8+ cells outnumbered CD4+ cells by 3:1 in the lungs and 6:1 in the trachea of the infected calves. Although the increase in CD4+ T cells into the lungs was less marked than that of CD8+ T cells, changes in expression of CD45R, CD45RO, L-selectin and interleukin-2 receptors all suggested that CD4+ T cells were activated during BRSV infection. Changes in gamma delta T cells were not observed in BRSV-infected calves. There was a marked increase in B cells in the BLN after infection and BLN CD4+ T cells changed from the majority expressing L-selectin and CD45R in uninfected calves to a predominance of L-selectin- CD45R- CD45RO+ phenotype, 10 days after infection. In conclusion, CD8+ T cells constitute the major lymphocyte subpopulation in the respiratory tract of calves recovering from BRSV infection.

Animals↗
PubMed1997

Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells.

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.

Animals↗
PubMed1997

Cross-reactivity of monoclonal antibodies to defined human leucocyte differentiation antigens with bovine cells.

Thirty-seven subpanels of monoclonal antibodies (mAbs) included within the Vth International Workshop on Human Leucocyte Differentiation Antigens (Vth Workshop) were assayed for reactivity with bovine peripheral blood leucocytes. Sixty-five of the 772 mAbs (8.4%) stained bovine cells. mAbs from each of the 27 different CD groups that contained a mAb reacting with cattle were further investigated to compare the cellular expression of the antigen in cattle with that reported for the different CD antigens in humans. Two-colour immunofluorescence staining of the Vth Workshop mAbs against characterized bovine leucocyte subpopulation markers that identified monocytes, B cells, CD4, CD8 and WC1 +T cells were used for these analyses. Eighteen of the mAbs to different human CD antigens (CD11a, CD14, CD18, CD21, CD27, CD29, CD49a, CD49b, CD49d, CD49e, CD51, CD61, CD62L, CD62P, CD63, CDw78, CD98, CD100) stained bovine antigens with an almost identical cellular distribution to that reported in humans. This implies that these mAb react with the homologous cattle molecules. Nine mAbs (CD35, CD37, CD49c, CD50, CD54, CD66, CD81, CD88, CD102) stained bovine cells but the cellular distribution of the bovine antigen was different to that reported in humans implying either a different cellular distribution for these antigens in cattle or a reaction with a different molecule. The investigation has allowed the identification of several bovine homologues of human CD antigens that have not been previously defined in cattle and the cross-reacting mAbs will be valuable reagents for future investigations of bovine immunology.

Animals↗
PubMed1996

Cluster analysis.

Cluster analysis was performed on flow cytometry data generated from the reactivities of the 302 workshop monoclonal antibodies with 36 target cell preparations. The antibodies were assigned to 42 preliminary clusters that were subjected to further examination in subsequent stages of the workshop.

Animals↗