Biomedical subjects

M A Hofmann

Publications and source records attributed to M A Hofmann.

At least 37 records · Page 2Linked to original sources

PubMed1999

[Classical swine fever in wild boars in Switzerland].

In May 1998, wild boars with classical swine fever (CSF) symptoms were detected in the southern part (Canton Ticino) of Switzerland. CSF virus was isolated from the submitted samples and RT-PCR followed by direct nucleotide sequencing of the 5' non-translated region showed that this virus was identical to the isolate previously recognized in wild boars from the area of Varese (Italy). In most animals, antibodies to CSF virus were detected as well. An immediate measurement was taken by limiting the movement of pigs and identifying both risk and surveillance zones. In order not to disturb potentially infected wild boars within their habitat a complete hunting prohibition for 2 months was enforced. The different possibilities of the control of CSF outbreaks in wild boars are discussed.

Animals↗

PubMed1998

Low density blood granulocytic cells induced during classical swine fever are targets for virus infection.

Classical swine fever virus infection of pigs causes a severe leukopenia and immunosuppression. In the present study, the kinetics of virus infection, and identification of target cells for the virus in peripheral blood were analysed. Virus infection was often not detectable before 5-7 days p.i. A minority of animals yielded detectable infected cells at 3 days p.i., but < 5% PBMC. It was not until 10 days p.i. that this figure increased-to 35-70% PBMC depending on the animal. Detailed analysis of Ficoll-Hypaque-purified PBMC identified the major population to be SWC3+SWC8+CD14+MHCII- granulocytic cells. Microscopic observations determined that these low density granulocytic cells in the PBMC from CSFV infected animals were indeed immature cells. Both the low density granulocytic cells and monocytes were major targets for CSFV infection in the peripheral blood. This is the first demonstration that low density granulocytic cells dominate the blood leukocyte population during CSF, and that such cells are targets for virus infection. The present work also demonstrates that the leukocyte population changes, such as B lymphocyte depletion and the relative dominance of myeloid cells in the blood during CSF, occur before virus infection of the affected cells. Thus, the pathological mechanism therein is not a direct consequence of virus infection.

Animals↗

PubMed1998

AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept.

OBJECTIVE: This is the first part of a bipartite review that summarizes the rising knowledge on the molecular mechanisms underlying the action of advanced glycation endproducts (AGEs) and their contribution to diabetic complications and vascular disease. While the first part presented here focusses on AGE formation, the second part will describe the AGE-protein/receptor interactions and their role in mediating AGE-dependent intracellular signalling. RESULTS: Nonenzymatic glycation, in which reducing sugars are covalently attached to free amino groups and ultimately form AGEs, has been found to occur during normal aging and at accelerated rate in diabetes mellitus. Oxidation, accompanying glycation in vivo, further supports chemical modifications. AGE formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signalling, activation of transcription factors and subsequent gene expression in vitro and in vivo. CONCLUSIONS: AGEs elicit a wide range of cell-mediated responses that might contribute to the pathogenesis of diabetic complications, vascular and renal disease and Alzheimer's disease. Substances that inhibit AGE formation, reduce oxidative stress or destroy already formed crosslinks may limit the progression of disease and may offer new tools for therapeutic interventions in the therapy of AGEs mediated disease.

Diabetes Mellitus↗

PubMed1998

Comparative detection of classical swine fever virus in striated muscle from experimentally infected pigs by reverse transcription polymerase chain reaction, cell culture isolation and immunohistochemistry.

Classical swine fever (CSF) is a highly contagious viral disease, which can be transmitted by CSFV-contaminated swill. In 1993, four CSF outbreaks in Switzerland were caused presumably by feeding pigs with improperly heated swill. The aim of the investigations was to find a suitable method for CSFV detection in striated muscle samples of infected pigs in order to allow routine testing of meat for virus contamination. The sensitivity of virus detection in striated muscle was compared with the detection in target organs. Using reverse transcription polymerase chain reaction (RT-PCR), cell culture isolation and immunohistochemistry on samples from 14 experimentally infected pigs, CSFV was detected in target organs of ten, and in striated muscle of six pigs, respectively. Overall, only 58% of muscle samples from CSFV-positive animals were positive by RT-PCR and 40% by virus isolation in cell culture, whereas the virus was detected in target organs of these pigs. Virus detection from striated muscle was primarily successful in severely diseased animals infected with highly virulent CSFV strains. It is concluded that striated muscle is not suitable for sensitive CSFV detection, and additional organs have to be examined for reliable diagnosis.

Animals↗

PubMed1998

Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect.

Cytopathogenicity of classical swine fever virus (CSFV) depends on the presence of defective particles containing a subgenomic (sg) RNA with a defined deletion. In a previous report we described the spontaneous generation of this sg RNA and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the CPE and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK-6 cells that had survived the CPE (CPE(surv)cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a CPE, CPE(surv) cells were protected from the CPE when superinfected with cp CSFV or with cp bovine viral diarrhoea virus. Similarly, cells which were rescued and further propagated after acute infection with cp CSFV also proved to be protected from the CSFV-induced CPE. When either virus obtained from CPE(surv) cells that had spontaneously lost the sg RNA or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the CPE after superinfection with cp CSFV. These findings suggest that the virus contained in CPE(surv) cells confers on the host cell the ability to resist the CSFV-induced CPE. However, when naive cells were infected with supernatants from CPE(surv)cells that contained defective virus particles, the CPE reappeared within three to five virus passages, indicating that the sg RNA retained its cytopathogenic potential.

Animals↗

PubMed1998

A recombinant classical swine fever virus stably expresses a marker gene.

The gene coding for bacterial chloramphenicol acetyltransferase (CAT) was inserted in frame into the viral Npro gene of the full-length cDNA clone pA187-1 of the classical swine fever virus (CSFV) strain Alfort/187. RNA transcribed in vitro from the resulting plasmid was transfected into SK-6 porcine kidney cells. Infectious progeny virus vA187-CAT recovered from transfected cells had growth characteristics indistinguishable from those of parental virus vA187-1. In cells infected with vA187-CAT the predicted fusion protein, CAT-Npro, was detected, and it retained the enzymatic activities of both CAT and Npro. The CAT gene remained stably inserted in the viral genome after 10 virus passages. Thus, marker virus vA187-CAT represents a useful tool for quantitative analysis of viral replication and gene expression.

Animals↗

PubMed1998

Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture.

The sequence encoding the viral leader proteinase Npro was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187. The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus. At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA. This RNA lacked the same viral genes as the subgenomic RNA which has been found in all cytopathogenic CSFV strains analyzed so far, but it maintained the ubiquitin sequence.

Animals↗

PubMed1998

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: Considering that elevated blood levels of homocyst(e)ine represent a known independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular complications. RESEARCH DESIGN AND METHODS: Homocyst(e)ine and thrombomodulin plasma levels, a marker of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with stable, well-controlled IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload homocyst(e)ine plasma levels than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had homocyst(e)ine plasma levels above the normal range (defined as mean +2 SD of values obtained in the control group). The IDDM patients with hyperhomocyst(e)inemia had higher thrombomodulin plasma levels (62.2 vs. 38.2 ng/ml; P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l; P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells show an increased release of thrombomodulin into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications of patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Adult↗

PubMed1998

Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes.

OBJECTIVE: The redox-sensitive transcription factor nuclear factor-kappa B (NF-kappa B) is believed to contribute to late diabetic complications. It is unknown whether NF-kappa B is influenced by glycemic control. RESEARCH DESIGN AND METHODS: To determine whether NF-kappa B is activated in patients with insufficient glycemic control (HbA1c > 10%), we developed a tissue culture-independent electrophoretic mobility shift assay (EMSA)-based semiquantitative detection system that allowed us to determine NF-kappa B activation in ex vivo-isolated peripheral blood mononuclear cells (PBMCs). We included 43 patients with type 1 diabetes in this cross-sectional study. 10 of those received the antioxidant thioctic acid (600 mg/day p.o.) for 2 weeks. RESULTS: Monocytes of patients with HbA1c levels > 10% demonstrated significantly higher NF-kappa B binding activity in an EMSA and a stronger NF-kappa B staining in immunohistochemistry than monocytes of patients with HbA1c levels of 6-8%. The increase in NF-kappa B activation correlated with an increase in plasmatic markers of lipid peroxidation. Treatment with the antioxidant thioctic acid decreased NF-kappa B binding activity. CONCLUSIONS: Hyperglycemia induces activation of the transcription factor NF-kappa B in ex vivo-isolated PBMCs of patients with type 1 diabetes. NF-kappa B activation is at least partially dependent on oxidative stress, since the antioxidant thioctic acid significantly lowered the extent of NF-kappa B binding activity.

Adult↗

PubMed1997

An indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen.

An indirect enzyme-linked immunosorbent assay termed rnPRRS ELISA using baculovirus-expressed and affinity-purified viral nucleocapsid protein (rNC) antigen was developed for detecting antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) in swine sera. Sera (1395) originating from different European countries were used for the validation of this assay. The rnPRRS ELISA was capable of detecting antibodies in all sera known to contain anti-PRRSV antibodies, resulting in 100% sensitivity. The specificity was 95.8%. The rnPRRS ELISA was more sensitive compared to the most widely used tests for the diagnosis of porcine reproductive and respiratory syndrome (PRRS) (i) a commercially available ELISA; (ii) the indirect immunofluorescence assay (IIFA); and (iii) the immunoperoxidase monolayer assay (IPMA). The main advantage of the rnPRRS ELISA compared to an ELISA using whole virus antigen is the use of a single immunogenic protein instead of infectious PRRSV. The rnPRRS ELISA is suitable for routine diagnosis of PRRS and also for epidemiological surveys since it allows highly reliable testing of a large number of sera within a short period of time.

Animals↗

PubMed1997

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines.

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Animals↗

PubMed1997

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: While elevated blood levels of homocyst(e)ine represent an independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular diseases. RESEARCH DESIGN AND METHODS: Plasma levels of homocyst(e)ine and thrombomodulin (TM), markers of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload plasma levels of homocyst(e)ine than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had plasma homocyst(e)ine levels above the normal range (means +/- 2 SD of values obtained in the control group). These IDDM patients with hyperhomocyst(e)inemia had higher plasma TM levels (62.2 vs. 38.2 ng/ml, P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l, P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; and macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells showed an increased release of TM into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications in patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Administration, Oral↗

PubMed1996

Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2.

Recombinant envelope protein E2 (gp55) of classical swine fever virus (CSFV) strain Alfort/187 was evaluated as an alternative to whole virus as ELISA antigen for the detection of antibodies against CSFV. A glycosylated and a non-glycosylated form of E2 was expressed in the baculovirus system. Six histidine residues added at the carboxy terminus of each of the recombinant proteins allowed purification by nickel-chelate affinity chromatography. Comparison of the antigenic properties of the two proteins in indirect and blocking ELISAs revealed that the glycosylated form resulted in both higher sensitivity and specificity. The indirect ELISA, using glycosylated E2, either derived from crude cell extract or affinity-purified, was validated by testing a total of 2719 porcine sera. Its final version proved to be as sensitive (98.3%) as the virus neutralization test when sera from infected pig herds were examined, and highly specific (99.6%) when applied to test negative sera. It is therefore suitable for large scale monitoring of classical swine fever.

Animals↗

PubMed1996

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter. Cleavage at an SrfI restriction site introduced at the exact 3' end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription. This RNA proved to be infectious after transfection into porcine kidney cells. Infectivity was not increased after capping of the synthetic RNA. Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody. RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells. A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells. An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP. The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis.

Animals↗

PubMed1996

Detection of extraneous agents in vaccines using the polymerase chain reaction of Newcastle disease virus in poultry biologicals.

Reverse transcription-polymerase chain reaction (RT-PCR) in poultry vaccine was applied to the detection of Newcastle disease virus (NDV), using two primer pairs spanning the cleavage site of the FO fusion protein coding sequence. Amplification of a specific cDNA segment was possible from live and inactivated, oil-adjuvanted NDV vaccines without previous treatment. The RT-PCR was able to detect between 5 x 10(2) EID50 (in live vaccine preparations) and 10(5) EID(50) or 0.056 haemagglutinating units of NDV (in inactivated vaccine preparations). In addition, live vaccine preparations were inactivated with beta-propiolactone (beta-PL). Amplified cDNA was obtained after treatment with 0.1% beta-PL, whereas at a concentration of 1% or 10% no specific bands were visible in the agarose gel. These results demonstrate the applicability of the method for the control of poultry vaccines by ensuring the absence of extraneous agents.

Animal Testing Alternatives↗

PubMed1995

Detection of Newcastle disease virus in poultry vaccines using the polymerase chain reaction and direct sequencing of amplified cDNA.

In order to develop an in vitro method for the control of poultry vaccines for identity and the absence of extraneous agents, reverse transcription-polymerase chain reaction (RT-PCR) was applied for the detection of Newcastle disease virus (NDV). NDV vaccines were employed for the establishment of the method, using two primer pairs spanning the cleavage site of the F0 fusion protein coding sequence. Amplification of a specific cDNA segment was possible from live and inactivated, oil-adjuvanted NDV vaccines without prior treatment. The cDNA was characterized by restriction endonuclease digestion as well as by direct nucleotide sequencing. The RT-PCR was able to detect between 5 x 10(2) EID50 (in live vaccine preparations) and 10(5) EID50 or 0.056 haemagglutinating units of NDV (in inactivated vaccine preparations). In addition, live vaccine preparations were inactivated with beta-propiolactone (beta-PL). Amplified cDNA was obtained after treatment with 0.1% beta-PL, whereas at a concentration of 1% or 10% no specific bands were visible in the agarose gel. These results demonstrate the applicability of the method for the control of poultry vaccines for identity and for the absence of extraneous agents, and additionally allow a rapid characterization of the respective NDV strain.

Animals↗

PubMed1995

[Diagnosis of contagious diseases in animals using PCR].

The PCR is used for diagnostic purposes as it allows to detect infections agents within a much shorter time than by cultural isolation. In addition, it can detect non-infectious viruses and bacteria in clinical samples. These advantages are important factors in the diagnosis of highly contagious animal diseases (mainly caused by viruses) since a rapid laboratory diagnosis will allow to take immediate disease control actions. PCR is routinely used at the Institute of African and classical swine fever virus, foot and mouth disease virus, Aujeszky's disease virus, porcine reproductive and respiratory syndrome virus, as well as Newcastle disease virus. The isolate can be further characterized by direct nucleotide sequencing of the amplified DNA. Since reliability of the results as well as as prevention of contaminations are vital to PCR, this method should be carried out by appropriately trained personnel. In addition, it requires a high level of technical infrastructure.

Animals↗

PubMed1994

Role of subgenomic minus-strand RNA in coronavirus replication.

Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgenomic mRNAs) probably function in mRNA amplification by serving as templates for transcription from internal (intergenic) promoters, rather than by faithful (full-length) mRNA replication.

Base Sequence↗