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A Zurbriggen

Publications and source records attributed to A Zurbriggen.

At least 73 records · Page 4Linked to original sources
PubMed1996

[RT-PCR: a tool for the generation of clones for research on canine distemper].

In the present work an adapted method of the reverse transcriptase polymerase chain reaction (RT-PCR) is described. This procedure was an important tool for cloning a variety of gene sequences derived from oligodendrocytes and canine distemper virus (CDV). Among other applications, the obtained clones were used to produce probes for in situ hybridization and to sequence the inserted DNAs. Due to these molecularbiological techniques new insights were gained into the pathogenesis of CDV-induced demyelination and CDV-persistence in the central nervous system.

Animals↗
PubMed1995

Restricted infection with canine distemper virus leads to down-regulation of myelin gene transcription in cultured oligodendrocytes.

Canine distemper virus (CDV) induces oligodendroglial degeneration and multifocal demyelination in the central nervous system. The mechanism of oligodendrocyte degeneration is not understood but it has been shown that there is a restricted infection of these cells without viral protein production. Using a combination of immunocytochemistry and in situ hybridization we were able to demonstrate the transcription of the entire virus genome throughout the whole observation period (7-35 days after infection) in oligodendrocytes in CDV-infected brain cell cultures. Therefore, the lack of viral protein and particle production can not be explained on the basis of a defective viral transcription. The present study also shows that a restricted infection of oligodendrocytes with CDV down-regulates the transcription of the major myelin genes coding for proteolipid protein, myelin basic protein (MBP) and myelin-associated glycoprotein in a very similar way. Using densitometry for in situ hybridization products of MBP in populations of normal and infected oligodendrocytes, an effect could be observed long before morphological changes were detectable. The present results strongly suggest that demyelination in distemper is induced by a restricted CDV infection of oligodendrocytes which down-regulates the expression of a variety of cellular genes, in particular those coding for myelin proteins. Consequently, the infected cells are no longer able to synthesize all the membrane compounds which are necessary for maintaining their structural integrity.

Animals↗
PubMed1995

Studies on canine distemper virus persistence in the central nervous system.

Chronic progressive demyelination in canine distemper virus (CDV) infection is associated with persistence of the virus in the nervous system. We studied persistence by examining expression of CDV mRNA corresponding to all genes of the virus as well as genomic CDV RNA in brain sections of dogs with acute and chronic demyelinating disease. All virus mRNAs were expressed in acute demyelinating lesions in a way similar to that seen in lymphoid tissues, the primary replication site of CDV. Their distribution corresponded very well with immunohistochemical detection of virus protein. In contrast, much more CDV mRNA than virus protein was found in gray matter areas suggesting that translation of CDV can be impaired in nervous distemper. Virus protein and RNA were cleared from chronic inflammatory demyelinating lesions. mRNA corresponding to the distal genes (F; H; L) of CDV disappeared first in inflammatory lesions for technical reasons associated with the particular mode of transcription of morbilliviruses. CDV RNA and protein persisted in chronically ill dogs in other areas of the CNS in which inflammation had not occurred. Our results suggest that persistence of CDV is favored by non-cytolytic spread of the virus and restricted infection of certain cells with reduced viral protein expression. Both tend to delay immune recognition of the virus.

Animals↗
PubMed1995

Neuroimmunological studies in steroid-responsive meningitis-arteritis in dogs.

The clinical and pathological expression of steroid-responsive meningitis-arteritis (SRMA) is predominantly neurological and the present study was therefore mostly concerned with the intrathecal humoral immune responses of 13 dogs suffering from the disease. All the dogs synthesised IgG intrathecally, indicating that the immune response in SRMA is in part specifically directed towards the central nervous system (CNS). Half of the dogs also had high levels of IgM only in the cerebrospinal fluid (CSF) and nearly all of them had high levels of IgA in the CSF as well as in the serum. Six of the dogs had circulating immune complexes in the serum, but not in the CSF. Neither IgM nor IgA rheumatoid factors were found. A chemotaxis assay revealed enhanced migration of neutrophils into the CSF in three cases. All the dogs had marked meningeal inflammation and arterial lesions of the meningeal blood vessels. Only one dog had arterial involvement outside the CNS. The acute vascular lesions consisted mostly of degenerative changes of the media and periarterial inflammation, and there was no evidence of immune complex deposition. Chronic lesions were mostly characterised by stenosis, adventitial thickening and periarteritis. Focal deposits of IgA were found in the vascular wall of one chronic case. It was concluded that the meningeal lesions in SRMA are a primary event, rather than the result of a generalised immune complex disease. These lesions are associated with an intrathecal humoral immune response, in which IgA appears to play a central role.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1995

Nucleotide and deduced amino acid sequences of the nucleocapsid protein of the virulent A75/17-CDV strain of canine distemper virus.

Virus persistence is essential in the chronic inflammatory canine distemper virus (CDV)-induced demyelinating disease. In the case of CDV there is a close association between persistence and virulence. Virulent CDV isolated from dogs with distemper shows immediate persistence in primary dog brain cell cultures (DBCC) and in different cell lines. We have evidence that the nucleocapsid (NP) protein plays an important role in the development of persistence. The NP-protein, the most abundant structural virus protein, also influences virus assembly and has some regulatory functions in virus transcription and replication. In this study we compared the nucleotide and deduced amino acid sequence of a virulent CDV strain (A75/17-CDV) to a culture-attenuated non-virulent strain (OP-CDV). Viral RNA was extracted from DBCC infected with virulent CDV. Virulent CDV retains its in vivo properties, such as virulence and ability to cause demyelination, when propagated in these DBCC. The viral RNA was reverse transcribed and the resulting cDNA amplified by polymerase chain reaction for subsequent cloning. The nucleotide sequences of these clones were determined by the dideoxy chain termination method. The number of nucleotides and the putative NP-protein of the virulent strain matched the attenuated CDV strain. We observed a total of 105 nucleotide differences. Three were localised within the 3' and five within the 5' non-coding region of the NP-gene. The 97 nucleotide changes within the coding region resulted in 22 amino acid differences. 10 of these amino acid (AA) modifications were within the N-terminal region (AA 1 to 159) and 12 within the C-terminal area (AA 351 to 523).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The neurobiology of canine distemper virus infection.

Canine distemper virus (CDV) invades the nervous system and replicates in neurons and glial cell of the white matter during a period of severe viral induced immunosuppression. Demyelination occurs in infected white matter areas in the absence of inflammation. The mechanism of demyelination is not apparent because there is no ultrastructural evidence of viral replication in the oligodendrocytes, the myelin producing cells. However, brain tissue culture studies have shown that oligodendrocytes support transcription of all CDV genes and later on degenerate, although no viral proteins can be found in these cells. It remains to be shown how such a restricted infection leads to demyelination. Concomitant with immunologic recovery during the further course of the disease, inflammation occurs in the demyelinating lesions with progression of the lesions in some animals. A series of experiments in vitro suggested that chronic demyelination is due to a bystander mechanism associated with the virus-induced immune response in which antibody dependent cell-mediated reactions play an important role. The progressive, or even relapsing, course of the disease is associated with viral persistence in the nervous system. Persistence of CDV in the brain appears to be due to non-cytolytic selective spread of the virus with very limited budding. In this way CDV escaped immune surveillance.

Acute Disease↗
PubMed1995

Selective spread and reduced virus release leads to canine distemper virus persistence in the nervous system.

In primary dog brain cell cultures (DBCC) the attenuated canine distemper virus (CDV) is cytolytic, whereas virulent CDV is not. Thus, the question why cytolysis does or does not occur appears to be intimately associated with the mechanism of persistence. Persistence is most likely related to the way these viruses replicate. In the present study we used morphological and immunocytochemical approaches to compare several aspects of virus replication between a cytolytic and a virulent CDV strain using DBCC to study both strains. Quantitative measurements did not detect a difference in the rate of virus protein synthesis at the level of the single cell, between the two types of infection. Electron microscopical results and virus titration experiments showed marked differences in virus spread and virus release. Immunocytochemical studies showed differences in the distribution of the nucleocapsid and matrix proteins between the two infections. Budding and cytolysis are strongly limited in the virulent A75/17-CDV infection as compared to attenuated viruses. This is probably due to structural changes in the virus proteins leading to modifications of virus assembly. Thus the present study supports a mechanism of CDV persistence through a type of virus maturation and spread by which very little virus is released outside of the cell.

Animals↗
PubMed1995

In situ hybridization and immunohistochemistry for prion protein (PrP) in bovine spongiform encephalopathy (BSE).

In about 5% of the cows showing clinical signs of bovine spongiform encephalopathy (BSE) the histopathological examination is not conclusive. In order to rule out BSE in these cases, additional methods are necessary. For that reason, non-radioactive in situ hybridization (ISH) was performed using a riboprobe against the messenger RNA coding for the prion protein (PrP). In addition, a polyclonal antibody for immunohistochemistry (IHC) was generated against a synthetic peptide derived from bovine PrP. ISH and IHC were used to analyse brain sections of cattle suffering from BSE and various neurological diseases including four cows with clinically suspect but histologically unconfirmed BSE. ISH revealed no differences in localization, distribution and neuronal levels of PrP mRNA between BSE positive and negative cattle. However, there was a BSE-specific staining pattern in IHC allowing to exclude BSE the four suspected cases. Additionally, IHC for PrP is an elegant alternative to search for scrapie associated fibrils by electron microscopy.

Animals↗
PubMed1995

Canine distemper virus persistence in the nervous system is associated with noncytolytic selective virus spread.

Canine distemper virus (CDV), a negative-strand RNA morbillivirus, causes a progressive demyelinating disease in which virus persistence plays an essential role. The antiviral immune response leads to virus clearance in the inflammatory lesions. However, CDV can replicate and persist outside these inflammatory lesions within the brain. How CDV is capable of persisting in the presence of an effective antiviral immune response is poorly understood. In the present investigation, we studied several aspects of virus replication in primary dog brain cell cultures (DBCC), comparing an attenuated CDV strain and a virulent CDV strain. Confluent DBCC were infected with either virulent A75/17-CDV or attenuated Onderstepoort-CDV and monitored for 60 days. Persistence was not associated with defective virus production, because all mRNAs and corresponding proteins were continuously expressed in the noncytolytic infection. Quantitative measurements did not detect a difference between the two types of infection in the rate of virus transcription and protein synthesis at the level of the single cell. However, electron microscopy and virus titration experiments showed that in the persistent CDV infection virus budding is strongly limited compared with that of the attenuated virus. Morphometry and immunocytochemistry showed profound differences in the way the two viruses spread in the culture. The attenuated CDV spread randomly to immediately adjacent cells, whereas persistent CDV spread selectively to more-distant cells by way of cell processes. In conclusion, the present study supports a mechanism of CDV persistence through selective spread by way of cell processes, enabling virulent CDV to invade the central nervous system without the need of releasing much virus into the extracellular space.

Animals↗
PubMed1994

Intrathecal synthesis of major immunoglobulin classes in inflammatory diseases of the canine CNS.

The IgG index, IgM and IgA contents in cerebrospinal fluid and serum were examined retrospectively using an enzyme-linked immunosorbent assay (ELISA) in 69 dogs with inflammatory central nervous system (CNS) diseases of various aetiologies. Fifteen normal dogs were used as controls. After measuring IgG and albumin contents in serum and cerebrospinal fluid (CSF), the IgG index was calculated according to the formula of Link and Tibbling to demonstrate intrathecal immunoglobulin synthesis. A surprisingly high number of animals with encephalitis, including dogs with protracted diseases such as chronic distemper encephalitis and granulomatous meningoencephalomyelitis, showed in addition to an elevated IgG production evidence of intrathecal IgM and IgA production. The highest values of intrathecal as well as systemic IgA levels were found in dogs suffering from steroid responsive meningitis-arteritis. It was concluded that the control of the humoral immune response in the brain differs from that in other tissues. Because of striking similarities between dogs and humans in respect to humoral neuroimmunological reactions, the dog can be considered to be a useful animal model for the study of the intrathecal humoral immune response.

Animals↗
PubMed1994

A chemiluminescence assay to detect antibodies to brain surface antigens in human sera.

A chemiluminescence assay was developed based on the interaction between antibodies binding to the surface of living brain cells in culture and macrophages. Such interaction leads to production of reactive oxygen radicals which can be measured by a chemiluminescence assay. This assay was used to detect anti brain antibodies in serum samples from humans with various neurological diseases. Such antibody activity was found in a high proportion of these patients. Subsequent experiments with purified IgG fractions and corresponding F(ab')2 fragments showed that the observed reactions were highly specific. It was concluded that the chemiluminescence assay is a sensitive and useful technique to detect autoantibodies in neurological diseases.

Animals↗
PubMed1993

Distribution of glycosylphosphatidylinositol-specific phospholipase D mRNA in bovine tissue sections.

It has been reported that mammalian serum, and to a lower extent mammalian liver, brain, pancreas, udder, and milk, contain glycosylphosphatidylinositol-specific phospholipase D activity. However, the sites of synthesis have not been determined. In order to study in which cell(s) of the organism synthesis of glycosylphosphatidylinositol-specific phospholipase D takes place, we undertook a systematic screening of 12 different bovine tissues. In situ hybridization experiments with a specific anti-sense RNA probe, derived from a bovine liver cDNA, revealed that glycosylphosphatidylinositol-specific phospholipase D mRNA is present in mast cells of the adrenal gland, lung, and liver. On the other hand, our specific probe detected no mRNA in bovine pancreas, brain, and udder, although enzyme activity has been reported in these tissues. Northern blot analysis of total bovine liver RNA demonstrated two distinct glycosylphosphatidylinositol-specific phospholipase D mRNAs of approximately 3.3 kb and 4 kb length suggesting that two forms of the enzyme may exist.

Adrenal Glands↗
PubMed1993

Saponin treatment for in situ hybridization maintains good morphological preservation.

The commonly used procedures for in situ hybridization require treatment of the tissue with non-ionic detergents and proteolytic enzymes, resulting in considerable loss of morphological detail. In this study proteinase pre-treatment of the tissue was replaced by saponin, a highly surface-active plant glycoside. This saponin treatment allowed good preservation of tissue morphology, as determined by differential interference and contrast enhanced video microscopy. Saponin pre-treatment resulted in an equal or even better hybridization sensitivity with probes recognizing viral (canine distemper virus) and cellular (myelin) nucleic acid sequences in tissue cultures as well as in paraffin sections. Probable mechanisms of how saponin allows probe penetration while maintaining the morphological details are discussed.

Animals↗
PubMed1993

In situ hybridization of virulent canine distemper virus in brain tissue, using digoxigenin-labeled probes.

Only a few hybridization experiments have been performed for detection of canine distemper virus (CDV) nucleic acid sequences in tissue cultures and in various tissues. Those published studies used probes derived from tissue culture-adapted CDV, and hybridization signals were not obtained in the CNS tissue, although infective CDV and viral antigen were detectable in this tissue. We developed probes complementary to virulent CDV and were able to detect viral RNA not only in primary brain cell cultures, but also in brain tissues, by use of in situ hybridization. Sensitivity of the test at least equaled that of immunohistochemistry. We applied digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding viral nucleic acid sequence. Our results indicate that to detect CDV nucleic acid sequences in brain tissues, it is essential to use probes derived from the virulent virus.

Animals↗
PubMed1993

Restricted canine distemper virus infection of oligodendrocytes.

BACKGROUND: Canine distemper virus, a morbillivirus induces multifocal demyelination in the central nervous system. The acute demyelination correlates with virus replication in brain cells, especially astrocytes. Observations in vivo and in vitro demonstrated degeneration of oligodendrocytes, the myelin producing cells. However, the mechanism of oligodendroglial degeneration in distemper remained unexplained. Infection of the myelin producing cells, the most obvious explanation for the phenomenon of demyelination, could not be supported by extensive searches for viral particles or antigens in these cells neither in vivo nor in vitro. EXPERIMENTAL DESIGN: In the present study, we combined in situ hybridization to visualize viral nucleic acid sequences with immunofluorescence for oligodendroglial antigens. RESULTS: The nonradioactive in situ hybridization technique in combination with contrast enhanced video microscopy allowed us to unequivocally demonstrate the presence of canine distemper virus nucleic acid sequences in cultured oligodendrocytes. Many oligodendrocytes close to infected foci in the brain cell cultures were found to contain viral nucleic acid sequences. Only 1% of the viral nucleic acid sequences containing oligodendrocytes also contained viral antigen. Canine distemper virus replication in these cells is clearly restricted. CONCLUSIONS: Different possibilities why oligodendrocytes do not support a productive virus infection and mechanisms by which such a restricted infection leads to oligodendroglial degeneration and ensuing demyelination are discussed. While our results have advanced our understanding of the pathogenesis of acute demyelination in distemper, they may also offer a possible explanation for the chronic progressive or even relapsing course of the disease. A restricted infection of the oligodendrocytes may be the mechanism by which canine distemper virus persists in the central nervous system. Virus persistence is probably a key event in many chronic viral induced inflammatory demyelinating diseases.

Animals↗
PubMed1992

[Spongiform encephalopathies with special reference to bovine spongiform encephalopathy].

In Switzerland bovine spongiform encephalopathy (BSE) was detected for the first time in November 1990. It is a transmissible disease of the central nervous system similar to Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and kuru in man, and, in animals, scrapie in sheep and goats, chronic wasting disease (CWD) in captive mule deer and elk of North America and transmissible mink encephalopathy (TME) of farm reared mink. The infectious agent of the spongiform encephalopathies (SEs) is extremely small (greater than 100 microns) and very resistant against physical and chemical disinfectants. The infectiousness is closely linked to the so-called prion proteins, the biological and genetic properties of which are not yet fully understood. The first outbreak of BSE occurred in England, and the disease spread rapidly all over Great Britain. The source of infection proved to be bone and meat meal obtained from scrapie-infected slaughter wastes. So far we have diagnosed 17 cases of BSE in our country. The risk of humans contracting CJD by ingestion of meat, milk and their products contaminated with BSE is probably minimal.

Animals↗