Biomedical subjects

D Werling

Publications and source records attributed to D Werling.

13 recordsLinked to original sources

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↗

PubMed2001

Morphologic and functional changes in bovine monocytes infected in vitro with the bovine leukaemia virus.

Experiments on the host cell spectrum of bovine leukaemia virus (BLV), a retrovirus closely related to the human T-cell leukaemia virus (HTLV), have yielded conflicting data. Currently, BLV is known to infect B cells, whereas its ability to infect other cell types, e.g. monocytes/macrophages, is doubtful. As monocytes/macrophages may have profound effects on the diversity of the T-cell response, we studied the possibility of in vitro infection, using bovine monocytes and SV40-transformed bovine macrophages. Proviral DNA was detected by nested polymerase chain reaction (PCR) from day 1 until the end of the experiments at either day 5 or day 80, depending on the quantity of virus used for infection. In addition, the infection was associated with morphological changes in infected cells as revealed by electron microscopy. The in vitro infection did not significantly change either the expression of surface antigens (CD11b, CD32, and major histocompatibility complex (MHC) class II) or the amounts of cytokine transcripts (interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-6 and IL-12p40) with or without lipopolysaccharide (LPS) stimulation. The data suggest that BLV can infect monocytes, but the infection does not seem to influence the function or the phenotype of these cells. Infected monocytes may, however, play a role as a viral reservoir in vivo.

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

Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva-transformed B cells.

Theileria is an intracellular parasite that causes lymphoproliferative disorders in cattle, and infection of leucocytes induces a transformed phenotype similar to tumour cells, but the mechanisms by which the parasite induces this phenotype are not understood. Here, we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase (PI3-K) activity, which appears to be necessary for proliferation, but not survival. Importantly, we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte-monocyte colony-stimulating factor (GM-CSF)-dependent autocrine loop. PI3-K induction of GM-CSF appears to be at the transcriptional level and, consistently, we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kappaB, which characterizes Theileria-infected leucocytes. Taken together, our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte.

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

Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers.

Prolonged infusions of bacterial lipopolysaccharides (LPS) are known to model gram-negative bacterial infections, but the basic mechanisms of the LPS effects on feed intake and metabolism and their potential interdependence are largely unknown. The aim of the present study was to distinguish and to better characterize the feeding suppressive and metabolic effects of LPS. Six heifers were infused intravenously for 100 min with either 1) LPS (2 microg/kg BW) with free access to feed, 2) saline with free access to feed, or 3) saline with feeding restricted to the amount of feed consumed after LPS infusion. Feed intake, body temperature, plasma concentrations of various metabolites and hormones, and the respiratory quotient and heat production were measured. The LPS reduced feed intake and induced pronounced changes in metabolic energy turnover and fat and carbohydrate metabolism that were largely independent of the concomitant feed intake reduction. Some of the metabolic changes were biphasic; the first phase resembled a stress response with increases in plasma glucose and cortisol, and the second phase reflected a beginning energy deficit with low plasma glucose and enhanced lipolysis. The coincidence of a short-term surge of plasma insulin with marked transient decreases in plasma FFA, glycerol, and beta-hydroxybutyrate as well as with the transition from hyper- to hypoglycemia indicates that insulin plays a role in some of the metabolic responses to LPS. The failure of LPS to clearly increase energy expenditure despite the increase in body temperature suggests that anaerobic mechanisms of heat production and, perhaps, a reduced peripheral blood flow contributed to the fever. Many of the initial metabolic responses occurred before and, therefore, independent of, an increase in circulating tumor necrosis factor-alpha.

Adipose Tissue↗

PubMed1998

Analysis of the phenotype and phagocytic activity of monocytes/macrophages from cattle infected with the bovine leukaemia virus.

The bovine leukaemia virus (BLV) is a retrovirus that infects mainly B lymphocytes of cattle, but proviral DNA can also be isolated from monocytes/macrophages. This study investigated the effect of BLV infection on surface antigens on freshly isolated peripheral blood monocytes and cultured monocyte-derived macrophages, with and without lipopolysaccharide (LPS) stimulation. The effect of BLV infection on phagocytic activity of CD14+ monocytes was also assessed. The percentage of monocytes expressing the surface antigens CD11b, CD32 (FcgammaRII), MHC class II and the surface antigen recognised by mAb DH59B were increased in BLV-positive cattle. In contrast, expression intensity of all markers was low in samples from BLV-positive cattle. CD14+ monocytes from BLV-positive cattle showed less Fcgamma-receptor-mediated phagocytosis compared to monocytes from BLV-negative cattle. After 7 days in culture, there was evidence for shedding/downregulation of surface antigens on monocyte-derived macrophages, in particular on cells from BLV-positive cattle. LPS stimulation decreased the percentage of cells expressing the measured markers in monocyte-derived macrophages taken from BLV-negative cattle, but not in cultures derived from BLV-positive cattle. The results provide further evidence for an altered function of monocytes and macrophages in BLV-infected cattle.

Animals↗

PubMed1998

Gammadelta T cells present antigen to CD4+ alphabeta T cells.

Gammadelta and alphabeta TCR+ T cells share many properties and their interactions are likely to be coordinated and regulated. We provide evidence that cattle gammadelta T cells are able to present antigen to CD4+ T cells. To help elucidate their function gammadelta T cell lines were propagated for extensive characterization. Cells expressed high levels of MHC class II and production of co-stimulatory molecules as evidenced by the binding of a CTLA-4 fusion protein and synthesis of CD80 transcripts. These properties and the presence of a well-developed endosomal compartment indicated the cells might function as antigen-presenting cells. Resting CD4+ T cells from calves immunized with ovalbumin or respiratory syncytial virus-antigen proliferated in response to gammadelta T cells pulsed with antigen that appeared to be endocytosed via clathrin-coated pits or specific receptors.

Abatacept↗

PubMed1996

Characterisation of the acute phase response of heifers to a prolonged low dose infusion of lipopolysaccharide.

The effect of a prolonged low dose infusion of bacterial lipopolysaccharide (LPS) on acute phase-like reactions was examined in heifers. LPS (2 micrograms kg-1 dissolved in 100 ml water), or saline was infused (at 1 ml min-1) intravenously for 100 minutes and blood samples were taken at various times before, during and after the infusion. The serum concentrations of tumour necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and serum amyloid A (SAA) and the rectal temperature increased in response to the LPS infusion. Serum TNF alpha increased before the increases in IL-1 beta and IL-6 and remained high from 20 minutes after the onset of the infusion until the end of the sampling period (six hours). The LPS-induced increases in serum IL-1 beta and IL-6 were biphasic. Plasma cortisol and lactate concentrations also increased, and plasma glucose and beta-hydroxybutyrate concentrations decreased in response to the LPS infusion. The similarity of these reactions to changes observed in response to bacterial infections shows that the prolonged infusion of low doses of LPS is a good model for studying the acute phase response to Gram-negative bacterial infection in heifers.

Acute-Phase Proteins↗

PubMed1995

Effect of bovine leukemia virus infection on bovine peripheral blood monocyte responsiveness to lipopolysaccharide stimulation in vitro.

The effects of bovine leukemia virus (BLV) infection on cytokine activity of bovine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared to supernatants of LPS-stimulated monocytes from BLV-negative cows, supernatants from BLV-positive cows contained about four times more interleukin-1 beta (IL-1 beta) (as measured by an enzyme-linked immunosorbent assay (ELISA) for bovine IL-1 beta). Despite their higher IL-1 beta concentration, supernatants from BLV-positive cows stimulated proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide) assay similar to supernatants from BLV-negative cows, but showed about 30% less IL-1 activity than supernatants from BLV-negative cows on the IL-1-dependent cell line LBRM-33 1 A-5, and about five times more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results demonstrate that BLV infection changes the cytokine response of bovine monocytes to LPS stimulation in vitro. The results are consistent with the assumption that BLV infection leads to the production and secretion of a soluble IL-1 inhibitor by LPS-stimulated peripheral blood monocytes.

Animals↗

PubMed1994

Caprine arthritis encephalitis virus infection changes caprine blood monocyte responsiveness to lipopolysaccharide stimulation in vitro.

The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results indicate that CAEV infection changes caprine monocyte cytokine responsivity.

Animals↗