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Biomedical subjects

G Entrican

Publications and source records attributed to G Entrican.

At least 19 recordsLinked to original sources
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↗
PubMed2005

Immunopathology of Chlamydophila abortus infection in sheep and mice.

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.

Abortion, Veterinary↗
PubMed2004

Variability in cytokine production and cell proliferation by mitogen-activated ovine peripheral blood mononuclear cells: modulation by interleukin (IL)-10 and IL-12.

T-cell reactivity is typically measured by cell proliferation and/or production of cytokines in response to antigenic/mitogenic stimulation. The choice of assays is more limited in ruminants than rodents, and complicated by the variability inherent in outbred populations. We have measured proliferation and production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC) from 24 sheep, and compared the responses between sheep, within sheep over several sample points, and also drawn comparisons between the two assays. PBMC derived from different sheep varied by as much as ten-fold in both proliferation and IFN-gamma production, though not necessarily at the same sample time. Thus, there was a poor correlation between the two assays and also considerable variation in the responses from the same animal at different time points. Both parameters could be modulated by exogenous recombinant ovine interleukin (IL)-10 and IL-12, but we were unable to correlate IFN-gamma production with endogenous cytokine production in the assays. These data highlight the importance of assay selection for the measurement of immune responsiveness and also demonstrate the variation that can be expected between sheep and over time.

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↗
PubMed2001

A role for tryptophan in immune control of chlamydial abortion in sheep.

Tryptophan (Trp) catabolism appears to be an important mechanism for regulation of inflammatory responses, resulting in T-cell tolerance and survival of semi-allogeneic concepti during pregnancy. Trp catabolism can be induced by IFN-gamma, and is therefore an important host defence mechanism against intracellular pathogens. Chlamydophila abortus is a bacterial pathogen that can cause persistent infection in non-pregnant sheep, but invades the placenta and causes abortion in late pregnancy. IFN-gamma was found to control the growth of Chlamydophila abortus in ovine cells in a highly dose-dependent manner. Addition of 200U/ml IFN-gamma eradicated all traces of infection from the cultures, whereas concentrations less than 50U/ml failed to control the growth of the organism, resulting in cell lysis. However, concentrations in the range of 50-100U/ml were found to restrict growth to an extent that a persistent infection was established, allowing survival of the organism in tissue culture for several months. Removal of IFN-gamma resulted in the re-appearance of infectious organisms. Addition of exogenous Trp to the cells treated with 50-100U/ml IFN-gamma prevented the establishment of persistence. These effects in tissue culture are analogous to the persistent infection observed in pregnant sheep prior to abortion. These data suggest that control of C. abortus growth in the periphery is linked to the balance of pro-inflammatory cytokine production and availability of Trp during pregnancy.

Abortion, Veterinary↗
PubMed1999

Cytokine release by ovine macrophages following infection with Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen that causes abortion in both sheep and humans. The disease in sheep (but not humans) is characterized by a long-term persistent phase that appears to be under the control of interferon-gamma. However, nothing is known about cytokine induction that precedes the persistent phase in sheep. Primary alveolar lavage cells recovered from normal adult sheep were used to study cytokine production in the first 72 h of infection with C. psittaci. These cells were phenotypically characteristic of macrophages, being adherent, phagocytic, CD14+ and staining positive for non-specific esterase. In vitro infection of the macrophages with C. psittaci resulted in the release of IL-1beta, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as measured by ovine-specific ELISAs. Heat-treated chlamydiae (1 h at 65 degrees C) did not induce the release of IL-1beta, but the release of IL-8 was similar to that induced by untreated organisms. The cells from different sheep varied most notably in their patterns of GM-CSF release in response to heat-treated and untreated organisms.

Animals↗
PubMed1998

Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants.

The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.

Adjuvants, Immunologic↗
PubMed1998

Cytokines and the protective host immune response to Chlamydia psittaci.

The immunobiology of enzootic abortion of ewes (EAE) is incompletely understood. The causative agent is Chlamydia psittaci, which infects many ruminant species and has zoonotic potential. The organism can survive in the ovine host for many months without causing clinical symptoms but does not generate a sterile immunity during this time. It has been postulated that the organism persists in the host entering at a latent phase, possibly mediated by host cytokine production. The effects of cytokines on chlamydial multiplication vary between host species, between different cell types within those species and also vary between chlamydial species and strains. The multiplication of the EAE strain of C. psittaci in ovine ST-6 cells can be restricted by interferon-gamma (IFN-gamma) but not with comparable concentrations of IFN-alpha. Altering the nutrient composition of the cultures by addition of tryptophan partially reverses the antichlamydial effects of the IFN-gamma. This offers a potential mechanism by which C. psittaci can persist in sheep. The implications of these observations for the pathogenesis of EAE are discussed.

Abortion, Veterinary↗
PubMed1997

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

Animals↗
PubMed1996

Development of a sandwich ELISA for ovine granulocyte/macrophage colony-stimulating factor.

Recombinant ovine granulocyte/macrophage colony-stimulating factor (rOv GM-CSF) has been expressed in Chinese hamster ovary cells. A stable, cloned line of these cells has been established which secretes high levels (40 mu g ml(-1)) of rOv GM-CSF. Three murine monoclonal antibodies (mAbs) were produced which reacted with rOv GM-CSF on Western blots. These mAbs also neutralised the activity of both recombinant and native Ov GM-CSF in a bone marrow haemopoietic progenitor cell assay. Two of the mAbs, which recognise mutually exclusive epitopes, were selected for the development of a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GM-CSF in biological samples of ovine origin.

Animals↗
PubMed1996

The activation status of ovine CD45R+ and CD45R- efferent lymph T cells after orf virus reinfection.

The dynamics and activation status of CD4+ and CD8+ T-cells differentially expressing the CD45R (220 kDa) antigen were studied in prefemoral efferent lymph draining the site of cutaneous reinfection with orf virus. CD4+, CD45R+ lymphoblasts preceded CD4+, CD45R- lymphoblasts during the first 48 h after reinfection. Thereafter, the output of both total and blast-transformed CD4+, CD45R- T-cells increased in proportion to the CD4+, CD45R+ cells for the duration of the virus reinfection. Output of CD8+, CD45R+ T-cells exceeded that of the CD8+, CD45R+ cells both before and after reinfection. However, within the lymphoblast population, CD8+, CD45R+ and CD8+, CD45R- T-cells increased and decreased in parallel. CD4+, CD45R- and CD8+, CD45R- T-cells produced interleukin-2, interferon-gamma and granulocyte-macrophage colony-stimulating factor after culture for 24 h without exogenous restimulation, whereas CD4+, CD45R+ T-cells produced only interleukin-2. The results show that although both CD45R+ and CD45R- alpha beta receptor+ T-cell subsets are activated as a consequence of virus reinfection in vivo, it is the CD45R- subset that predominates in the later stages of reinfection and is the principal cellular source of lymphokines in the efferent lymph.

Animals↗
PubMed1996

Interferon-gamma mediates long-term persistent Chlamydia psittaci infection in vitro.

The long-term anti-chlamydial effects of recombinant ovine interferon gamma (rOvIFN-gamma) were studied in ovine ST-6 fibroblasts infected with the S26/3 strain of Chlamydia psittaci. Chlamydial multiplication was assessed by enzyme-linked immunosorbent assay analysis of supernate lipopolysaccharide, titration of inclusion-forming units in culture supernates, and enumeration of inclusion bodies in cultured cells at 7-day intervals. Concentrations of 250 and 1000 U/ml of rOvIFN-gamma resulted in a microbistatic inhibition of C. psittaci growth, which appeared to become microbicidal when rOvIFN-gamma was maintained in the cultures for 14 days or more. There were no signs of C. psittaci multiplication when cultures were maintained in 25 or 100 U/ml of rOvIFN-gamma. However, subsequent removal of rOvIFN-gamma from these cultures resulted in a re-emergence of viable, infectious chlamydiae, which eventually killed all the fibroblasts. This re-emergence was more rapid in cultures initially treated with 25 U/ml of rOvIFN-gamma than in those treated with 100 U/ml.

Abortion, Veterinary↗
PubMed1996

Cloning and biologic activities of a bovine interferon-alpha isolated from the epithelium of a rotavirus-infected calf.

A cDNA encoding a distinct bovine (Bo) interferon (IFN) alpha, designated BoIFN-alpha E, was generated from gut epithelial cells isolated from a rotavirus-infected calf. The BoIFN-alpha E cDNA sequence shared a greater than 90% identity with the other BoIFN-alpha subtypes. The cDNA encoding BoIFN-alpha E has been expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Insect cells infected with recombinant virus secreted a protein with a relative molecular mass of 19,500 into the culture medium not observed in cells infected with wild-type AcMNPV. Supernatants harvested from cultures of insect cells infected with the recombinant AcMNPV encoding IFN-alpha E inhibited the replication of Semliki Forest virus in a bovine cell line and typically showed 10(6) dilution units/ml of antiviral activity. However, differences were observed between the activities of recombinant BoIFN-alpha E and BoIFN-alpha 1 1 on the proliferation of WC1+ gamma/delta T cells. Purified ( > 99%) WC1+ gamma/delta T cells failed to proliferate to IFN-alpha 1 1 or concanavalin A and IFN-alpha E acted as a weak proliferative signal to these cells, demonstrating a functional difference between two closely related BoIFN-alpha subtypes.

Amino Acid Sequence↗
PubMed1995

Production and characterisation of ovine GM-CSF expressed in mammalian and bacterial cells.

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) was isolated and two forms of recombinant ovine GM-CSF were produced. A glycosylated form was produced in mammalian cells infected with a recombinant vaccinia virus encoding ovine GM-CSF. Recombinant ovine GM-CSF was also produced in Escherichia coli and purified by affinity chromatography. Both forms of the protein were detected by ovine GM-CSF-specific monoclonal antibodies, and exhibited activity on ovine bone marrow haemopoetic progenitor cells.

Animals↗
PubMed1995

A double monoclonal antibody ELISA for detecting pestivirus antigen in the blood of viraemic cattle and sheep.

A panel of monoclonal antibodies (mAbs) has been produced to the p125/p80 non-structural polypeptide of border disease virus (BDV) and bovine virus diarrhoea virus (BVDV). This polypeptide appears to be highly conserved among BDV and BVDV isolates and consequently the mAbs directed against it have a broad cross-reactivity with pestivirus isolates. The epitope specificities of these mAbs were determined by competitive binding and four of the mAbs with mutually exclusive epitope specificities were selected for the development of a diagnostic ELISA. Two mAbs were used to capture virus antigen prepared from the blood of infected cattle and sheep, then two different mAbs used to detect the captured antigen. This double mAb ELISA was compared to existing ELISAs which rely on polyclonal antibodies (pAbs) for detecting captured antigen. The mAb detection ELISA was more sensitive than the pAb detection ELISAs for both cattle and sheep and resulted in higher optical densities for positive samples without an increase in background readings of negative controls.

Animals↗
PubMed1995

Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells.

The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.

Animals↗
PubMed1994

The importance of interferon-gamma in an early infection of Chlamydia psittaci in mice.

Athymic mice (nu/nu) and their hairy littermates (nu/+) were infected experimentally with Chlamydia psittaci and the role of endogenous interferon-gamma (IFN-gamma) on the resolution of the infection was studied. The pathological changes produced in the spleen, liver and lung were exacerbated by administration of a monoclonal antibody (mAb) to IFN-gamma and an increased number of viable chlamydiae were recovered from the tissues of both nu/+ and nu/nu mice treated in this way.

Animals↗