Biomedical subjects

Thomas W Jungi

Publications and source records attributed to Thomas W Jungi.

10 recordsLinked to original sources

PubMed2006

Human monocytoid cells as a model to study Toll-like receptor-mediated activation.

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

Antibodies, Monoclonal↗

PubMed2005

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages.

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Animals↗

PubMed2005

Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines.

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

Animals↗

PubMed2004

Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists.

Toll-like receptors (TLR) have been described as partially sharing signalling pathways but showing unique ligand specificity and tissue distribution. Here, the response of bovine macrophages (Mphi) and dendritic cells (DC), both derived from monocytes, was compared by exposing them to the TLR-specific ligands lipopolysaccharide, poly(I:C)-double-stranded RNA, and CpG-DNA, as well as inactivated Gram-negative and Gram-positive bacteria, shown to bind to TLR. The production of NO, superoxide anion, interleukin-10 (IL-10), IL-12 and tumour necrosis factor (TNF) was determined. Compared to monocytes, Mphi expressed more TLR2 and similar levels of TLR4 mRNA transcripts, as analysed by quantitative polymerase chain reaction, whereas DC expressed reduced amounts. Although both DC and Mphi recognized the TLR ligands, dramatic differences were seen in their reaction pattern to them. Both cell types responded with the production of TNF, but DC produced more IL-12, whereas Mphi produced more IL-10, regardless of the TLR agonist used. Co-stimulation with interferon-gamma influenced the amount of cytokine production, but did not alter the cell type-specific response pattern. Compared to Mphi, DC produced > 10 times less NO upon triggering with TLR ligands. In addition, DC produced superoxide anion to opsonized and non-opsonized zymosan, but not to phorbol 12-myristate 13-acetate, a response pattern confirmed for human Mphi and DC, respectively. Different protein kinase C isoforms and extracellular signal-regulated kinase patterns were detected in cell lysates of resting and stimulated Mphi and DC. Collectively, our results point to profound differences in pathogen-derived signal-response coupling occurring commensurate with distinct functions carried out by Mphi or DC.

Animals↗

PubMed2003

Exploration of stress-induced immunosuppression in chickens reveals both stress-resistant and stress-susceptible antigen responses.

In the present study, depriving chickens of foraging material was shown to induce stress. The impact of this type of stress on the immune response was compared with feeding of corticosterone (1.5 mg per bird per day), a hormone known to be immunosuppressive and to be the major stress hormone of chickens. Corticosterone feeding induced stress as revealed by higher heterophil/lymphocyte (H/L) ratios, longer tonic immobility (TI) reaction, reduced body weight gain and reduced egg production. Blood corticosterone levels were increased. Corticosterone feeding decreased the antibody response to tetanus toxoid and SRBC, DTH to PPD from Mycobacterium tuberculosis and the inflammatory response to PHA. Housing chickens on slats also induced chronic stress, as evidenced by increased H/L ratios, prolonged TI duration and decreased egg production. Corticosterone levels were slightly but not significantly enhanced. This novel form of chronic stress strongly suppressed humoral and cellular immune responses as evidenced by lower antibody titers to sheep red blood cells (SRBC) and tetanus toxoid (TT) decreased DTH reaction to PPD and inflammatory reaction to PHA in the skin. In contrast, the antibody response to human serum albumin (HSA) was neither influenced by corticosterone feeding nor by keeping the birds on slats. Even the combination of corticosterone feeding and housing the birds on slats did not significantly impair antibody responses to HSA. In conclusion, the present study showed that chronic stress induced by depriving the birds of foraging material led to a similar impairment of humoral and cell-mediated immunity as did feeding with corticosterone. More importantly, it showed for the first time that depending on the antigen tested, there are stress-resistant and stress-susceptible antigen responses.

Animals↗

PubMed2003

TOLL-like receptors linking innate and adaptive immune response.

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, which is mediated by B and T lymphocytes, recognises pathogens by rearranged high affinity receptors. However, the establishment of adaptive immunity is often not rapid enough to eradicate microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defense mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors (PRR). Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns (PAMP) to TLR induces the production of reactive oxygen and nitrogen intermediates (ROI and RNI), pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this review, we will summarize the discovery and the critical roles of the TLR family in host defense, briefly allude to signaling mechanisms mediating the response to TLR ligands, and will provide an update on current knowledge regarding the ligand specificity of these receptors and their role in immunity of domestic animals, particularly cattle.

Animals↗

PubMed2003

Toll-like receptor-4 is involved in eliciting an LPS-induced oxidative burst in neutrophils.

The lipopolysaccharide (LPS) receptor complex of mononuclear phagocytes is composed of Toll-like receptor-4 (TLR4), MD-2 and CD14. Other phagocyte populations may express similar LPS receptors. The transmembrane glycoprotein TLR4 was shown to induce or upregulate a variety of gene products, which collectively are the mediators of an LPS effect. In this study, an involvement of TLR4 in mediation of an oxidative burst was determined using murine peritoneal exsudate neutrophils and lucigenin-enhanced chemiluminescence (CL). The CL response was dependent on the LPS dose and the presence of serum, putatively a source of lipopolysaccharide-binding protein (LBP). In the absence of serum, a CL signal was elicited by 4 microg/ml LPS in peritoneal exsudate cells (PEC) from TLR4-sufficient (C3H/HeN) but not TLR4 deficient (C3H/HeJ) mice. The signal obtained in PEC from TLR4-sufficient mice was completely abrogated by superoxide dismutase (SOD), which indicated that the response depended on the formation of superoxide anion, and was also seen in purified neutrophils but not purified macrophages (Mphi). In the presence of serum, lower LPS concentrations (e.g. 40 ng/ml) elicited a strong CL response in PEC from TLR4-sufficient, and a weak signal in cells from TLR-4-deficient mice. This suggests that TLR4 engagement is involved in promoting an oxidative burst in murine neutrophils.

Acute-Phase Proteins↗

PubMed2003

BVDV and innate immunity.

Infection with bovine viral diarrhea virus (BVDV) is prevalent in the cattle population worldwide. The virus exists in two biotypes, cytopathic and non-cytopathic, depending on the effect of the viruses on cultured cells. BVDV may cause transient and persistent infections which differ fundamentally in the host's antiviral immune response. Transient infection may be due to both cytopathic and non-cytopathic biotypes of BVDV and leads to a specific immune response. In contrast, only non-cytopathic BVD viruses can establish persistent infection as a result of infection of the embryo early in its development. Persistent infection is characterized by immunotolerance specific for the infecting viral strain. In this paper we discuss the role of innate immune responses in the two types of infection. In general, both transient and persistent infections are associated with an increased frequency of secondary infections. Associated with the increased risk of such infections are, among others, impaired bacteria killing and decreased chemotaxis. Interestingly, non-cytopathic BVDV fails to induce interferon type I in cultured bovine macrophages whereas cytopathic biotypes readily trigger this response. Cells infected with non-cytopathic BVDV are also resistant to induction of interferon by double stranded RNA, a potent interferon inducer signalling the presence of viral replication in the cell. Thus, non-cytopathic BVDV may dispose of a mechanism suppressing a key element of the antiviral defence of the innate immune system. Since interferon is also important in the activation of the adaptive immune response, suppression of this signal may be essential for the establishment of persistent infection and immunotolerance.

Animals↗

PubMed2002

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions.

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Animals↗

PubMedDate not supplied

[The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes].

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants.

Animals↗