Biomedical subjects

Andreas Zurbriggen

Publications and source records attributed to Andreas Zurbriggen.

At least 19 recordsLinked to original sources

PubMed2006

Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins.

The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.

Adaptation, Biological↗

PubMed2006

Detection and distribution of apoptotic cell death in normal and diseased canine cranial cruciate ligaments.

One of the possible initiating factors in canine cranial cruciate ligament (CCL) rupture could be an abnormal pattern of ligament cell death. This study compared apoptotic cell death in sections of ruptured CCLs and normal controls, and examined nitric oxide (NO) production in joint tissues and correlated this to apoptosis. CCLs and cartilage from the lateral femoral condyle were harvested from 10 healthy dogs and 15 dogs with CCL rupture and ligaments were further processed to detect cleaved caspase-3 and to determine supernatant NO production in explant cultures. Apoptotic activity was greater in ruptured ligaments compared to controls. NO in ligaments showed a moderate but significant positive correlation with caspase-positive cells. The results suggest that increased apoptosis has a role in CCL rupture and that apoptosis may be influenced by local NO production.

Animals↗

PubMed2006

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.

Animals↗

PubMed2005

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Amino Acids↗

PubMed2005

Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis.

Canine distemper virus (CDV), a negative stranded RNA morbillivirus, causes a multisystemic disease in dogs, which is associated with a severe immune suppression. The aim of the study was to examine the influence of early CDV infection on leukocyte depletion, lymphopenia and virus-induced cell death in dogs infected with a virulent CDV strain. From 10 infected dogs, peripheral blood leukocytes were harvested periodically, phenotyped and analyzed for CDV antigen content and apoptosis using Annexin V-FITC and propidium iodide labeling. CDV infection induced a severe CD3+ T cell and CD21+ B cell depletion in all animals at 3 days post-infection (d.p.i.). For dogs with severe distemper, developing virus persistence in the lymphoid tissue and central nervous system, this lymphopenia lasted until the end of the experiment. Increased levels of lymphocyte apoptosis were found at 3 d.p.i., and monocyte apoptosis at 6 d.p.i. This was more prominent in the group of animals with severe distemper. At 3 d.p.i. no leukocyte infection was detectable indicating that the early lymphocyte depletion and apoptosis was not a direct consequence of virus infection. Taken together, our results demonstrate that CDV-induced lymphopenia is an early event and that the degree of lymphocyte depletion correlates with the severity of disease and virus persistence in the lymphoid tissue and central nervous system.

Animals↗

PubMed2005

Demyelination in canine distemper virus infection: a review.

Canine distemper virus (CDV) causes severe immunosuppression and neurological disease in dogs, associated with demyelination, and is a model for multiple sclerosis in man. In the early stage of the infection, demyelination is associated with viral replication in the white matter. In acute demyelinating lesions there is massive down-regulation of myelin transcription and metabolic impairment of the myelin-producing cells, but there is no evidence that these cells are undergoing apoptosis or necrosis. Oligodendroglial change is related to restricted infection of these cells (transcription but no translation) and marked activation of microglial cells in acute lesions. Concomitant with immunological recovery during the further course of the disease, inflammation occurs in the demyelinating plaques with progression of the lesions in some animals. A series of experiments in vitro suggests that chronic inflammatory demyelination is due to a bystander mechanism resulting from interactions between macrophages and antiviral antibodies. Autoimmune reactions are also observed, but do not correlate with the course of the disease. The progressive or relapsing course of the disease is associated with viral persistence in the nervous system. Persistence of CDV in the brain appears to be favored by non-cytolytic selective spread of the virus and restricted infection, in this way escaping immune surveillance in the CNS. The CDV Fusion protein appears to play an important role in CDV persistence. Similarities between canine distemper and rodent models of virus-induced demyelination are discussed.

Animals↗

PubMed2004

Age-period-cohort analysis of the Bovine Spongiform Encephalopathy (BSE) epidemic in Switzerland.

Cattle born after animal-feed control measures were implemented in 1990 have become BSE cases in Switzerland, indicating sub-optimal effectiveness of these measures. To evaluate these measures, the incidence of BSE cases in Switzerland recorded through clinical case reporting from January 1991 to June 2000 (categorized into age groups and birth cohorts of 6-month duration) was analyzed by Poisson log-linear regression using an age-period-cohort model. The incidence was maximum in the cattle cohort born from October 1989 to March 1990, and dropped to zero in the cohort born from April to September 1991. A second peak was observed in a cohort born from April to September 1994. The first drop of incidence is interpreted as a result of initial implementation of the feed ban in 1990. The second peak might be related to exposure of cattle to feed intended for pigs and poultry.

Animal Feed↗

PubMed2004

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

Animals↗

PubMed2004

Microglial cell activation in demyelinating canine distemper lesions.

Microglia cells are the principal immune effector elements of the brain responding to any pathological event. To elucidate the possible role of microglia in initial non-inflammatory demyelination in canine distemper virus (CDV) infection, microglia from experimentally CDV infected dogs were isolated ex vivo by density gradient centrifugation and characterized immunophenotypically and functionally using flow cytometry. Results from dogs with demyelinating lesions were compared to results from recovered dogs and two healthy controls. CDV antigen could be detected in microglia of dogs with histopathologically confirmed demyelination. Microglia of these dogs showed marked upregulation of the surface molecules CD18, CD11b, CD11c, CD1c, MHC class I and MHC class II and a tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), B7-2 (CD86), whereas no increased expression was found for CD44 and CD45. Functionally, microglia exhibited distinctly enhanced phagocytosis and generation of reactive oxygen species (ROS). It was concluded that in CDV infection, there is a clear association between microglial activation and demyelination. This strongly suggests that microglia contribute to acute myelin destruction in distemper.

Animals↗

PubMed2004

Characterization of canine microglial cells isolated ex vivo.

Microglia are the principal immune effector elements of the brain sharing immunophenotypic and functional characteristics of macrophages as well as of antigen presenting cells (APCs). The purpose of this study was to isolate canine microglial cells and make them available for ex vivo characterizations of their functions and immunophenotype. After isolation, carried out by density gradient centrifugation, microglial cells accumulated on distinct interfaces of 1.077 and 1.066 g/ml of a Percoll gradient. Identification of microglial cells in other species is realized by their specific immunophenotype of CD11b/c+ and CD45low. Our results indicate, that expression of CD45 is very low or even absent in canine microglial cells. In addition, they expressed CD18 and CD11b/c+, as determined by flow cytometry and immunohistochemistry. Fourteen additional monoclonal antibodies (mAbs) were used to characterize and compare canine microglial cells with monocytes. Microglia and monocytes can be clearly distinguished by their differential expression intensity of surface antigens (CD45, CD44, CD14). Functional characterization was assessed by a reactive oxygen species (ROS)-generation test and phagocytosis assay using flow cytometry. In conclusion, ex vivo examination of microglia is possible in dogs and most probably reflects the conditions in vivo. The measurement of tissue culture artifacts can be largely avoided using this method.

Animals↗

PubMed2004

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Adaptation, Physiological↗

PubMed2004

Canine distemper virus infection of canine footpad epidermis.

Infection of the footpad epidermis can occur in natural canine distemper virus (CDV) infection of dogs. Footpads from 19 dogs experimentally inoculated with virulent distemper strain A75/17 and from two nonexposed dogs were examined histopathologically and assessed for the presence of viral antigen and nucleoprotein mRNA, as well as number of inflammatory and apoptotic cells. Dogs were divided into four groups based on inoculation status and postmortem examination: inoculated dogs with severe distemper (group 1, n = 7); inoculated dogs with mild distemper (group 2, n = 4); inoculated dogs without distemper (group 3, n = 8); and noninoculated dogs (group 4, n = 2). Footpads from dogs of all groups had a comparably thick epidermis. Eosinophilic viral inclusions and syncytial cells were present in footpad epidermis of one dog of group 1. Footpads of group 1 dogs contained viral antigen and mRNA in the epidermis with strongest staining in a subcorneal location. Additionally, in these dogs footpad dermal structures including eccrine glands and vascular walls were positive for virus particles. No CDV antigen or mRNA was present in the footpad epidermis and dermis of any other dog. Group 1 dogs had more CD3-positive cells and apoptotic cells within the basal layer of the epidermis when compared to the other groups. These findings demonstrate that in experimental infection CDV antigen and mRNA were colocalized in all layers of the infected canine footpad epidermis. The scarcity of overt pathological reactions with absence of keratinocyte degeneration indicates a noncytocidal persisting infection of footpad keratinocytes by CDV.

Animals↗

PubMed2004

Up-regulation of cytokeratin expression in canine distemper virus-infected canine footpad epidermis.

Cytokeratin expression was assessed in footpad epidermis from dogs using immunohistochemistry. Four groups of dogs were studied: dogs with experimentally induced distemper and with canine distemper virus (CDV) in footpad epidermis (group 1, n = 7); dogs with experimentally induced distemper and without CDV in footpad epidermis (group 2, n = 4); inoculated dogs without distemper and without CDV in the footpad epidermis (group 3, n = 8), and noninoculated dogs without distemper (group 4, n = 2). No increase in thickness of the footpad epidermis was present in any of these groups. Sections of metacarpal or metatarsal pads were stained for cytokeratin (CK)14 (proliferation-associated), CK10 (correlated with early differentiation), and for involucrin (associated with terminal differentiation). CK14 was present in basal keratinocytes of all groups, but staining intensity decreased towards the corneal layer in groups 2-4, but not in group 1. CK10 was present in the spinous and granular layer of all groups, but staining of the granular layer was much stronger in group 1. Involucrin was present in the granular layer of footpads of group 1 and only in the upper part of this layer in groups 2-4. The results demonstrate increased staining intensity and/or wider distribution within the footpad epidermis in group 1 dogs when compared to the other groups. This was interpreted as up-regulation in expression of these proteins. These findings suggest that presence of CDV antigen and mRNA in footpad epidermis was associated with an increase in expression of CK14, CK10 and involucrin. The potential role of this up-regulation in cytokeratin expression in the development of CDV-induced digital hyperkeratosis remains speculative at the moment and requires further studies.

Animals↗

PubMed2004

Two functionally redundant isoforms of Drosophila melanogaster eukaryotic initiation factor 4B are involved in cap-dependent translation, cell survival, and proliferation.

Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome. DrosophilaeIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs. Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle. The proteins are cytoplasmic with polarized distribution. The two isoforms bind RNA with the same affinity. eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system. RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed. Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.

Amino Acid Sequence↗

PubMed2003

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.

Animals↗

PubMed2003

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease.

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.

Animals↗

PubMed2003

Canine distemper, a re-emerging morbillivirus with complex neuropathogenic mechanisms.

Paramyxoviruses are responsible for a wide variety of diseases both in humans and in animals. Common to many paramyxoviruses is the fact that they can cause neurological symptoms in their final host. Newly discovered paramyxoviruses, such as the Hendra and Nipah viruses, show the same pattern of pathogenesis as that of the paramyxoviruses already known. Canine distemper virus (CDV) is a well-studied member of the genus Morbillivirus. Study of the neuropathogenesis of CDV might give insight into disease mechanisms and suggest approaches for the prevention of other recently discovered paramyxovirus infections.

Animals↗

PubMed2003

A new paraclinical CSF marker for hypoxia-like tissue damage in multiple sclerosis lesions.

Recent studies on the immunopathology of multiple sclerosis revealed a heterogeneity in the patterns of demyelination, suggesting interindividual differences in the mechanism responsible for myelin destruction. One of these patterns of demyelination, characterized by oligodendrocyte dystrophy and apoptosis, closely mimics myelin destruction in acute white matter ischaemia. In the course of a systematic screening for virus antigen expression in multiple sclerosis brains, we identified a monoclonal antibody against canine distemper virus, which detects a cross-reactive endogenous brain epitope, highly expressed in this specific subtype of actively demyelinating multiple sclerosis lesions with little or no immunoreactivity in other active multiple sclerosis cases. The respective epitope, which is a phosphorylation-dependent sequence of one or more proteins of 50, 70 and 115 kDa, is also expressed in a subset of active lesions of different virus-induced inflammatory brain diseases, but is present most prominently and consistently in acute lesions of white matter ischaemia. Its presence is significantly associated with nuclear expression of hypoxia-inducible factor-1 alpha within the lesions of both inflammatory and ischaemic brain diseases. The respective epitope is liberated into the CSF and, thus, may become a useful diagnostic tool to identify clinically a defined multiple sclerosis subtype.

Acute Disease↗