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Biomedical subjects

R A Collins

Publications and source records attributed to R A Collins.

At least 37 records · Page 2Linked to original sources
PubMed2000

Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.

Animals↗
PubMed2000

Genome rearrangement by replication-directed translocation.

Gene order in bacteria is poorly conserved during evolution. For example, although many homologous genes are shared by the proteobacteria Escherichia coli, Haemophilus influenzae and Helicobacter pylori, their relative positions are very different in each genome, except local functional clusters such as operons. The complete sequences of the more closely related bacterial genomes, such as pairs of Chlamydia, H. pylori and Mycobacterium species, now allow identification of the processes and mechanisms involved in genome evolution. Here we provide evidence that a substantial proportion of rearrangements in gene order results from recombination sites that are determined by the positions of the replication forks. Our observations suggest that replication has a major role in directing genome evolution.

Biological Evolution↗
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↗
PubMed2000

Development and use of monoclonal antibodies to chicken fibronectin to show that the chicken hepatocellular carcinoma cell line, LMH, constitutively expresses fibronectin.

Fibronectin (Fn) is a high molecular weight glycoprotein and acute phase reactant that contributes to a variety of cellular activities including proliferation and wound healing. Production of Fn is influenced by cytokines such as IL-1alpha, IL-6 and TNF -alpha, and in serum Fn levels can function as an indicator of sepsis and reticulo-endothelial function. Here we describe the production of a panel of mAb to chicken Fn and give evidence that a chicken hepatocellular carcinoma cell line, LMH, constitutively expresses Fn. A capture ELISA to measure chicken Fn was developed using an IgG1 mAb (AV62) as the capture Ab, and biotinylated AV63 (IgG2b) as the detecting Ab. This study identified a single commercially available mAb directed against human Fn that also recognised chicken Fn. By contrast, the anti-chicken Fn mAbs did not cross-react with either human or bovine Fn.

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

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↗
PubMed1999

Bovine interleukin-12 and modulation of IFNgamma production.

The effects of IL-12 on the responses of cattle peripheral blood mononuclear cells (PBMC) to bovine respiratory syncytial virus (BRSV) antigen and ovalbumin (OVA) were tested, in vitro. IL-12 did not affect the proliferative responses of PBMC to these antigens but markedly accelerated and augmented the level of IFNgamma secreted. When tested on lymphoblasts rather than resting T-cells IL-12 also enhanced proliferation. In contrast IL-4 and, to greater extent, IL-10 inhibited the response. The effect of IL-12 on IFNgamma synthesis was confirmed at the level of IFNgamma. mRNA expression using Taqman PCR. CD4 and CD8 T-cell populations produced IFNgamma, however, CD4 T-cells comprised the largest contributors to the IFNgamma production. Gamma/delta T-cells did not contribute markedly. A comparison of the species cross-reactivity showed bovine IL-12 was also active in the human system. This study shows that antigen-driven responses in cattle can be significantly influenced by exogenous cytokines and suggests the IL-12/IL-10 balance is crucial for regulation of IFNgamma.

Animals↗
PubMed1999

The nutritional supplement chromium(III) tris(picolinate) cleaves DNA.

Chromium(III) tris(picolinate) [Cr(pic)3] is currently a very popular nutritional supplement; however, its safety has recently been questioned, especially with regard to its ability to act as a clastogen. At physiologically relevant concentrations, Cr(pic)3 is reduced by biological reductants, including ascorbate and thiols, to Cr(II)-containing species. These species are susceptible to air oxidation, resulting in the catalytic generation of the potent DNA-damaging agent hydroxyl radical. In the absence of reductants, H2O2 can interact with Cr(pic)3 to produce hydroxyl radicals by a second, less efficient mechanism. Cr(pic)3 is extremely stable, which allows the complex to be readily absorbed but also to potentially be incorporated into cells intact. In this form, Cr(pic)3 is primed by its redox potential to enter into the generation of hydroxyl radicals. This study suggests that investigation of the long-term effects of supplementation of the diet with Cr(pic)3 are needed to assess the safety of this material.

Animals↗
PubMed1999

Phenotypic analysis of peripheral leukocytes in piglets infected with classical swine fever virus.

The phenotypic changes in circulating leukocytes in swine fever influenced by classical swine fever virus (CSFV) infection with different strain virulence was studied in piglets. The phenotypic differences were measured by monoclonal antibodies specific for porcine differentiation antigens. The pattern of phenotypic change varied with the virulence of CSFV. Infection with virulent, but not the attenuated strain of CSFV resulted in the dramatic early loss of CD8-bearing T lymphocytes from the circulation. A similar trend was also seen in the gammadelta T-cell compartment following infection with the highly virulent strain, Washington. The loss of circulating B-lymphocytes was consistent with the failure to generate neutralising antibody. These observations contrasted the finding that the number of leukocytes expressing the CD4 surface antigen increased in piglets infected with CSFV. These data provide preliminary information on the potential range of leukocyte changes produced in piglets following infection with CSFV.

Animals↗
PubMed1999

Production of interleukin-12 as a self-processing 2A polypeptide.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked subunits (p40 and p35) encoded by separate genes. We used the apparent autocleavage property of a 2A peptide from the foot-and-mouth disease virus (FMDV) to express bovine (Bo) IL-12 as a self-processing polypeptide (p402Ap35). We demonstrate that 2A will mediate the cleavage of p402Ap35 into two separate subunits in a manner similar to that observed during the processing of the FMDV polypeptide. Furthermore, this 2A polypeptide encoded a functional heterodimer, which elicited activities associated with IL-12 in other species. We propose that this strategy of self-processing polypeptides may be used in many applications where the coordinated and stoichiometric expression of complex proteins is required.

Animals↗
PubMed1999

Differential cytokine responses of CD4+ and CD8+ T cells in response to bovine viral diarrhoea virus in cattle.

Virus-specific T cell responses were measured in cattle seropositive for bovine viral diarrhoea virus (BVDV) and compared with those from BVDV-seronegative animals. CD4+ and CD8+ T cells were purified and co-cultured in vitro with autologous, BVDV-infected monocytes over a time-course to assess the kinetics of the proliferative response. Supernatants from parallel T cell cultures were harvested and the presence of the cytokines interleukin (IL)-2, IL-4 and interferon-gamma (IFN-gamma) were measured by ELISA (IFN-gamma) or by a bioassay (IL-2 and IL-4). CD4+ and CD8+ T cells from all seropositive, but not seronegative, cattle proliferated specifically in response to BVDV-infected monocytes. Measurement of cytokines in the supernatants from proliferating T cell cultures showed that the CD4+ T cell response was type 2-like, with extremely high levels of B cell growth factor and IL-4 activity together with comparatively low levels of IL-2 activity and IFN-gamma protein. The CD8+ T cell response, although more variable, appeared to be type 1-like, with increased IL-2 and IFN-gamma but no IL-4 or B cell stimulatory activity.

Animals↗
PubMed1999

Immune cells and cytokine production in the bovine corpus luteum throughout the oestrous cycle and after induced luteolysis.

Immune cells and their cytokine products have powerful local effects within body tissues. There has been great interest in the potential role of these cells, not only during destruction of the corpus luteum but also during its functional lifespan. In this study, lymphocytes, macrophages and major histocompatibility complex class II molecules were quantified using immunohistochemistry and the reverse transcription-polymerase chain reaction was used to detect mRNA for tumour necrosis factor alpha and interferon gamma within corpora lutea from three groups of cows: (1) corpora lutea collected at an abattoir and assessed visually into four stages (stage I (days 1-5), stage II (days 6-12), stage III (days 13-18) and stage IV (days 19-21) of the oestrous cycle); (2) corpora lutea collected around natural luteolysis (days 14-20); and (3) corpora lutea collected 6, 12 and 24 h after prostaglandin F 2 alpha-induced luteolysis. The numbers of T lymphocytes (CD5+ and CD8+) were significantly higher (P < 0.05) at stage IV and from day 16 onwards, before functional luteolysis. There were significantly higher numbers (P < 0.01) of macrophages at stages I, III and IV compared with stage II in visually staged tissue. Major histocompatibility complex class II molecules were increased (P < 0.05) at stages I and IV compared to stage II and at all times after induced luteolysis. Using reverse transcription-polymerase chain reaction, mRNA encoding tumour necrosis factor alpha and interferon gamma was detected in all luteal tissue collected around natural luteolysis and after induced luteolysis. These findings, particularly the increase in T lymphocytes before functional luteolysis, provide further evidence of a significant role for the immune system in affecting reproductive function in cows.

Animals↗
PubMed1998

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4.

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Animals↗
PubMed1998

Phenotype, growth regulation and cytokine transcription in Ovine Herpesvirus-2 (OHV-2)-infected bovine T-cell lines.

The causal agent of sheep-associated malignant catarrhal fever (MCF), Ovine Herpesvirus-2 (OHV-2), can be propagated in IL-2-dependent lymphoblastoid cell lines derived from diseased cattle and deer providing a useful model for the investigation of the pathogenesis of MCF. In this study, five interleukin-2 (IL-2)-dependent cell lines were established from affected cattle to examine their growth regulation and cytokine transcription. All cell lines expressed CD2, CD5 and CD25. Three of the cell lines were CD4+ and one CD8+, whereas one cell was of mixed CD4 and CD8 phenotye. The growth of these cell lines was reduced when cultured with antibody against CD25, the IL-2 receptor alpha subunit. All cell lines showed a lack of response to Con A and their cell growth was inhibited by Cyclosporin A which is known to inhibit cytokine promoters. It was decided therefore, to examine the cell lines for the presence of mRNA of different cytokines. The results showed that the cell lines transcribed message for IFNgamma, TNFalpha, IL-4 and IL-10 whereas no mRNA for IL-2 or IL-1beta was detected. In conclusion, the OHV-2-immortalised cell lines resemble anergic T-cells which may be activated giving rise to the characteristic lesions of MCF.

Animals↗
PubMed1998

Identification of phosphate groups involved in metal binding and tertiary interactions in the core of the Neurospora VS ribozyme.

We have used ethylation protection experiments and modification interference using phosphorothioate nucleosides to identify phosphate groups involved in the magnesium-dependent tertiary structure and function of the VS ribozyme, a small, self-cleaving RNA. Phosphorothioate interference-rescue experiments in the presence of the thiophilic manganese ion implicate four phosphate groups in direct metal ion binding. Phosphorothioate substitution also creates a new manganese binding site that increases the cis cleavage rate of the ribozyme, possibly by disrupting an inhibitory structure. Interpreting these data in the context of a recently developed structural model shows that almost all of the important phosphate groups are located in the central core of the ribozyme. The model suggests roles for certain phosphate groups in particular steps of RNA folding and identifies a candidate region for the active site of the ribozyme.

Base Sequence↗
PubMed1998

Influence of IL-12 on interferon-gamma production by bovine leucocyte subsets in response to bovine respiratory syncytial virus.

The cytokine IL-12 is a key molecule in the regulation of CD4+ T cell development and specifically potentiates the development of T helper 1 responses in mouse and man. However the biological effects mediated by bovine IL-12 have not been defined in cattle. To produce the expression of the two mature proteins a polyprotein approach was used. This system is employed by positive strand viruses and encodes both products from a single open reading frame (ORF). The 2A region of foot-and-mouth disease virus (FMDV) encodes a site that appears to undergo auto-cleavage. Here the 2A was flanked by sequences encoding the p35 and p40 polypeptides of the heterodimeric cytokine to mediate their cleavage. Formation of the correct heterodimeric structure is an absolute requirement for IL-12 biologic activity. Using bovine respiratory syncytial virus (BRSV) and ovalbumin (OVA) we studied the effects of IL-12 on the responses of peripheral blood mononuclear cells (PBMC) to these antigens, in vitro. The presence of IL-12 markedly influenced the level of IFNg secreted by these cells, and although IL-12 induced IFNg production in the absence of antigenic stimulation, IFNg production was accelerated and augmented in response to IL-12 and antigen. Analysis of the T cell subsets by flow cytometry showed that CD4+ T cells comprised the largest contributors to IFNg production. The WC1 + gd T cells did not appear to contribute to the production of IFNg.

Animals↗