Biomedical subjects

Peter Staeheli

Publications and source records attributed to Peter Staeheli.

At least 19 recordsLinked to original sources

PubMed2007

Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken.

The chicken represents the best-characterized animal model for B cell development in the so-called gut-associated lymphoid tissue (GALT) and the molecular processes leading to B cell receptor diversification in this species are well investigated. However, the mechanisms regulating B cell development and homeostasis in GALT species are largely unknown. Here we investigate the role played by the avian homologue of B cell-activating factor of the tumor necrosis factor family (BAFF). Flow cytometric analysis showed that the receptor for chicken B cell-activating factor of the tumor necrosis factor family (chBAFF) is expressed by mature and immature B cells. Unlike murine and human BAFF, chBAFF is primarily produced by B cells both in peripheral lymphoid organs and in the bursa of Fabricius, the chicken's unique primary lymphoid organ. In vitro and in vivo studies revealed that chBAFF is required for mature B cell survival. In addition, in vivo neutralization with a decoy receptor led to a reduction of the size and number of B cell follicles in the bursa, demonstrating that, in contrast to humans and mice, in chickens BAFF is also required for the development of immature B cells. Collectively, we show that chBAFF has phylogenetically conserved functions in mature B cell homeostasis but displays unique and thus far unknown properties in the regulation of B cell development in birds.

Animals↗

PubMed2006

Protective role of beta interferon in host defense against influenza A virus.

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.

Animals↗

PubMed2006

Serological evidence for Borna disease virus infection in humans, wild rodents and other vertebrates in Finland.

BACKGROUND: Borna disease virus (BDV) can infect many vertebrate species, including humans. BDV infection may lead to meningoencephalomyelitis in animals. An association with human neuropsychiatric diseases has been reported, but the causal relationship between BDV and human disease remains unclear. OBJECTIVES AND STUDY DESIGN: To find out whether BDV is present in Finland and to look for a potential reservoir, we examined a large panel of blood samples from different vertebrate species with immunofluorescence assay. Samples from horses, cats, dogs, sheep, cattle, large predators, grouse, wild rodents and humans were included. Most positive results were confirmed by other specific methods and in other laboratories. RESULTS AND CONCLUSIONS: BDV-specific antibodies were detected in 10 horses, 2 cats, as well as 2 horses and 1 dog from farms housing a previously detected seropositive horse. Interestingly, BDV-specific antibodies were further detected in three wild rodents. In humans, BDV-specific antibodies were detected in a veterinarian and in two patients suspected to have a Puumala hantavirus infection. Our serological analysis suggests that BDV infects various vertebrates in Finland, including humans. Furthermore, our data indicate for the first time that BDV infects also wild rodents.

Animals↗

PubMed2006

Neurons produce type I interferon during viral encephalitis.

Type I interferons, also referred to as IFN-alpha/beta, form the first line of defense against viral infections. Major IFN-alpha/beta producers in the periphery are the plasmacytoid dendritic cells (pDCs). Constitutive expression of the IFN regulatory factor (IRF)-7 enables pDCs to rapidly synthesize large amounts of IFN-alpha/beta after viral infection. In the central nervous system (CNS), pDCs are considered to be absent from the parenchyma, and little is known about the cells producing IFN-alpha/beta. The study presented here aimed to identify the cells producing IFN-alpha/beta in the CNS in vivo after infection by neurotropic viruses such as Theiler's virus and La Crosse virus. No cells with high constitutive expression of IRF-7 were detected in the CNS of uninfected mice, suggesting the absence of cells equivalent to pDCs. Upon viral infection, IFN-beta and some subtypes of IFN-alpha, but not IFN-epsilon or IFN-kappa, were transcriptionally up-regulated. IFN-alpha/beta was predominantly produced by scattered parenchymal cells and much less by cells of inflammatory foci. Interestingly, in addition to some macrophages and ependymal cells, neurons turned out to be important producers of both IFN-alpha and IFN-beta. However, only 3% of the infected neurons produced IFN-alpha/beta, suggesting that some restriction to IFN-alpha/beta production existed in these cells. All CNS cell types analyzed, including neurons, were able to respond to type I IFN by producing Mx or IRF-7. Our data show that, in vivo, neurons take an active part to the antiviral defense by being both IFN-alpha/beta producers and responders.

Animals↗

PubMed2006

RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication.

De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5'-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5' termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

Borna disease virus↗

PubMed2006

Innate immune responses in NF-kappaB-repressing factor-deficient mice.

NF-kappaB-repressing factor (NRF) is a transcriptional silencer protein that specifically counteracts the basal activity of several NF-kappaB-dependent promoters by direct binding to specific neighboring DNA sequences. In cell culture experiments, the reduction of NRF mRNA leads to a derepression of beta interferon, interleukin-8, and inducible nitric oxide synthase transcription. The X chromosome-located single-copy NRF gene is ubiquitously expressed and encodes a protein of 690 amino acids. The N-terminal part contains a nuclear localization signal, the DNA-binding domain, and the NF-kappaB-repressing domain, while the C-terminal part is responsible for double-stranded RNA binding and nucleolar localization. To study the function of NRF in a systemic context, transgenic mice lacking the NRF gene were created. Against predictions from in vitro experiments, mice with a deletion of the NRF gene are viable and have a phenotype that is indistinguishable from wild-type mice, even after challenge with different pathogens. The data hint towards an unexpected functional redundancy of NRF.

Animals↗

PubMed2005

Genome trimming: a unique strategy for replication control employed by Borna disease virus.

Genome and antigenome synthesis of negative-strand RNA viruses is initiated at promoters located in inverted terminal repeats (ITR). The ITR of Borna disease virus (BDV), a persisting neurotropic virus with a nuclear replication phase, are exceptional in that they appear to be noncomplete. Our analysis showed that the vast majority of genomic and antigenomic RNA molecules of BDV lack four 5'-terminal nucleotides required for perfect complementarity with the 3' ITR. By using a previously undescribed reverse genetics system, we investigated whether the structure of the ITR would affect virus propagation. BDV rescued from cDNA encoding complete ITR (rBDVc) showed wild-type virulence, whereas virus rescued from cDNA encoding a viral genome with noncomplete ITR (rBDVnc) was strongly attenuated. Both recombinant viruses expressed similar RNA and protein levels in persistently infected cells. However, rBDVnc particles were less infectious, indicating that complete ITR are required for high viral replicase but not transcriptase activity. Interestingly, genomic RNA from purified rBDVc particles lacked 5'-terminal nucleotides like authentic BDV, strongly suggesting programmed genome truncation. By specifically trimming its genome at the 5' terminus, BDV seems to limit viral genome amplification, which may favor noncytolytic viral persistence.

Base Sequence↗

PubMed2005

The functional avidity of virus-specific CD8+ T cells is down-modulated in Borna disease virus-induced immunopathology of the central nervous system.

Borna disease virus (BDV) infection of the central nervous system (CNS) leads to severe neurological symptoms in susceptible MRL mice. The disease is mainly mediated by CD8+ T cells specific for the immunodominant epitope TELEISSI in the BDV nucleoprotein. In this study, TELEISSI/MHC class I tetramers were used to directly visualize antigen-specific CD8+ T cells. We found that on average approximately 30% of the ex vivo analyzed CD8+ T cells in the CNS of diseased mice were specific for TELEISSI. Unexpectedly, the frequency of tetramer-reactive brain-derived CD8+ T cells doubled following overnight culture in the absence of antigen. The majority of CD8+ T cells showed enhanced tetramer binding without up-regulation of T cell receptor surface expression. The frequency of IFN-gamma-secreting CD8+ T cells after antigen-specific stimulation was higher in overnight cultures than in freshly isolated BDV-specific brain lymphocytes, and enhanced tetramer binding correlated with elevated sensitivity to lower levels of peptide antigen in cytotoxicity assays. These results indicate that the functional avidity of virus-specific CD8+ T cells was down-modulated in vivo. Thus, quantification of tissue-infiltrating CD8+ T cells by the tetramer technique must be interpreted with caution as it may underestimate the real frequency of antigen-specific CD8+ T cells.

Animals↗

PubMed2005

Vaccine-induced protection against Borna disease in wild-type and perforin-deficient mice.

Borna disease virus (BDV) can persistently infect the central nervous system and induce CD8+ T-cell-mediated neurological disease in MRL mice. To determine whether specific immune priming would prevent disease, a prime-boost immunization protocol was established in which intramuscular injection of a recombinant parapoxvirus expressing BDV nucleoprotein (BDV-N) was followed by intraperitoneal infection with vaccinia virus expressing BDV-N. Immunized wild-type and perforin-deficient mice remained healthy after intracerebral infection with BDV and contained almost no virus in the brain at 5 weeks post-challenge. Immunization failed to induce resistance against BDV in mice lacking mature CD8+ T cells. Immunization of perforin-deficient mice with a poxvirus vector expressing mutant BDV-N lacking the known CD8+ T-cell epitope did not efficiently block multiplication of BDV in the brain and did not prevent neurological disease, indicating that vaccine-induced immunity to BDV in wild-type and perforin-deficient mice resulted from the action of CD8+ T cells.

Animals↗

PubMed2005

CD8 T cells require gamma interferon to clear borna disease virus from the brain and prevent immune system-mediated neuronal damage.

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.

Age Factors↗

PubMed2004

Overlap of interaction domains indicates a central role of the P protein in assembly and regulation of the Borna disease virus polymerase complex.

The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.

Binding, Competitive↗

PubMed2004

The interferon system of non-mammalian vertebrates.

Genetic analysis in mice and other mammals showed that the interferon (IFN) system is important for resistance to viral and bacterial infections. IFN is thought to play a similar role in non-mammalian species, although knowledge is lagging behind. Molecular cloning revealed low but significant conservation of IFN genes in birds and fish. Whereas chickens and other birds have multiple genes for IFN-alpha and single genes for IFN-beta and IFN-gamma like most mammals, fish appear to have fewer IFN genes. Nonetheless, regulation of IFN-alpha and IFN-beta genes in response to viral and non-viral activators is well conserved in birds and fish, and similar sets of genes are induced in response to IFN in mammalian and non-mammalian vertebrates. IFN-like activities were also observed in reptiles and amphibians, but genes encoding these activities have not yet been cloned. Insights into the IFN system of non-mammalian species may lead to improved prophylactic and therapeutic approaches against virus infections in poultry and fish.

Amphibians↗

PubMed2004

Cerebral expression of interleukin-12 induces neurological disease via differential pathways and recruits antigen-specific T cells in virus-infected mice.

Transgenic expression of interleukin-12 (IL-12) in astrocytes causes a spontaneous inflammatory central nervous system disorder in aged mice. Here we show that spontaneous disorder developed only when both mature lymphocytes and interferon (IFN)-gamma were present. Infection with noncytolytic Borna disease virus (BDV) did not affect wild-type mice but accelerated disease of IL-12 transgenic mice. Infection of transgenic mice lacking lymphocytes did not result in neurological symptoms. In contrast, BDV infection of transgenic mice lacking IFN-gamma induced neurological disease with delayed onset of symptoms that resembled those in infected transgenic mice with a functional IFN-gamma gene. In BDV-infected transgenic mice devoid of IFN-gamma no cerebellar calcification was observed, and multiplication of BDV was not inhibited. To determine the antigen specificity of lymphocytes in brains of diseased animals, the IL-12 transgene was introduced into an H-2k genetic background. Infection of IL-12 transgenic H-2k mice resulted in extensive lymphocytic infiltration into the cerebellum but not into other brain regions that also contained viral antigen but expressed the transgene at lower levels. Tetramer analysis revealed that most CD8 T cells in the cerebellum of such mice were BDV-specific. Our results thus demonstrate that IFN-gamma secreting lymphocytes are responsible for disease of IL-12 transgenic mice. They further suggest that expression of IL-12 in the central nervous system may lead to localized recruitment of T cells that recognize antigens expressed in the brain.

Animals↗

PubMed2004

Neurological disorder after Borna disease virus infection in the absence of either interferon-gamma, Fas, inducible NO synthase, or chemokine receptor CXCR3.

Borna disease virus (BDV) can induce severe neurological disorder in Lewis rats and MRL mice. Antiviral CD8 T cells have been shown to be the mediators of disease in these animals. To define molecules involved in the disease process, we performed infection studies in MRL mice lacking either interferon-gamma, a functional Fas/FasL system, chemokine receptor CXCR3, or inducible NO synthase. We further used transgenic MRL mice expressing interferon-gamma-inducible, T cell-attracting chemokine CXCL10 in brain astrocytes. After intracerebral infection with BDV, wild-type and mutant mice developed CD8 T cell responses and neurological disease at similar frequency and with similar kinetics, suggesting that these factors are not required for initiation and maintenance of the immunopathological process. Similarly, the course of disease could not be altered by treating infected MRL mice or Lewis rats with the drug L-N(6)-(1-iminoethyl)-lysine (L-NIL) that specifically blocks the activity of the inducible NO synthase. We therefore have excluded a number of important factors that have been demonstrated to be crucial in the pathogenesis of a broad number of pathologic conditions. Thus, BDV-induced disease may not result from the action of a single dominant T cell-dependent effector molecule. Disease rather reflects a combined influence of several as yet undefined factors from CD8 T cells.

Animals↗

PubMed2004

Synthesis of IFN-beta by virus-infected chicken embryo cells demonstrated with specific antisera and a new bioassay.

Transcripts of interferon-alpha (IFN-alpha) and IFN-beta genes are present in virus-infected chicken cells, but because of a lack of appropriate assays and reagents, it was unclear if biologically active IFN-beta is secreted. We have established a nonviral bioassay for the sensitive detection of chicken IFN (ChIFN). This assay is based on a quail cell line that carries a luciferase gene that is controlled by the IFN-responsive chicken Mx promoter. Luciferase activity was strongly stimulated when the indicator cells were incubated with ChIFN-alpha, ChIFN-beta, or ChIFN-gamma but not with chicken interleukin-1beta (ChIL-1beta). Unlike the classic antiviral assay that preferentially detects ChIFN-alpha, the Mx-luciferase assay detected ChIFN-alpha and ChIFN-beta with similar sensitivity. With the help of this novel assay and with rabbit antisera specific for either IFN-alpha or IFN-beta, we analyzed the composition of IFN in supernatants of virus-infected chicken embryo cells. Virtually all IFN produced in response to Newcastle disease virus (NDV) was IFN-alpha. However, IFN produced in response to influenza A or vaccinia virus (VV) was a mixture of usually more than 80% IFN-alpha and up to 20% IFN-beta. Thus, IFN-alpha and IFN-beta both contribute to the cytokine activity in supernatants of virus-infected chicken cells. Furthermore, the infecting virus appears to determine the IFN subtype composition.

Animals↗

PubMed2004

Chicken BAFF--a highly conserved cytokine that mediates B cell survival.

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells.

Amino Acid Sequence↗

PubMed2004

The X protein of Borna disease virus regulates viral polymerase activity through interaction with the P protein.

Borna disease virus polymerase activity is negatively regulated by the viral X protein. Using a virus minireplicon system it was found that all X mutants that no longer interacted with the viral P protein failed to exhibit significant inhibitory activity. The action of X could further be neutralized by expression of a P fragment that contained the X interaction domain but lacked all domains known to mediate interaction with other viral proteins. X thus appears to regulate the activity of the Borna disease virus polymerase by targeting the polymerase cofactor P.

Borna disease virus↗

PubMed2004

Immunization with dendritic cells can break immunological ignorance toward a persisting virus in the central nervous system and induce partial protection against intracerebral viral challenge.

Dendritic cells (DCs) have been used successfully to induce CD8 T cells that control virus infections and growth of tumours. The efficacy of DC-mediated immunization for the control of neurotropic Borna disease virus (BDV) in mice was evaluated. Certain strains of mice only rarely develop spontaneous neurological disease, despite massive BDV replication in the brain. Resistance to disease is due to immunological ignorance toward BDV antigen in the central nervous system. Ignorance in mice can be broken by immunization with DCs coated with TELEISSI, a peptide derived from the N protein of BDV, which represents the immunodominant cytotoxic T lymphocyte epitope in H-2(k) mice. Immunization with TELEISSI-coated DCs further induced solid protective immunity against intravenous challenge with a recombinant vaccinia virus expressing BDV-N. Interestingly, however, this immunization scheme induced only moderate protection against intracerebral challenge with BDV, suggesting that immune memory raised against a shared antigen may be sufficient to control a peripherally replicating virus, but not a highly neurotropic virus that is able to avoid activation of T cells. This difference might be due to the lack of BDV-specific CD4 T cells and/or inefficient reactivation of DC-primed, BDV-specific CD8 T cells by the locally restricted BDV infection. Thus, a successful vaccine against persistent viruses with strong neurotropism should probably induce antiviral CD8 (as well as CD4) T-cell responses and should favour the accumulation of virus-specific memory T cells in cervical lymph nodes.

Animals↗