Biomedical subjects

M A Hofmann

Publications and source records attributed to M A Hofmann.

At least 19 recordsLinked to original sources

PubMed2002

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease.

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.

Alzheimer Disease↗

PubMed2002

RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response.

The receptor for advanced glycation end products (RAGE) and its proinflammatory S100/calgranulin ligands are enriched in joints of subjects with rheumatoid arthritis (RA) and amplify the immune/inflammatory response. In a model of inflammatory arthritis, blockade of RAGE in mice immunized and challenged with bovine type II collagen suppressed clinical and histologic evidence of arthritis, in parallel with diminished levels of TNF-alpha, IL-6, and matrix metalloproteinases (MMP) 3, 9 and 13 in affected tissues. Allelic variation within key domains of RAGE may influence these proinflammatory mechanisms, thereby predisposing individuals to heightened inflammatory responses. A polymorphism of the RAGE gene within the ligand-binding domain of the receptor has been identified, consisting of a glycine to serine change at position 82. Cells bearing the RAGE 82S allele displayed enhanced binding and cytokine/MMP generation following ligation by a prototypic S100/calgranulin compared with cells expressing the RAGE 82G allele. In human subjects, a case-control study demonstrated an increased prevalence of the 82S allele in patients with RA compared with control subjects. These data suggest that RAGE 82S upregulates the inflammatory response upon engagement of S100/calgranulins, and, thereby, may contribute to enhanced proinflammatory mechanisms in immune/inflammatory diseases.

Alleles↗

PubMed2001

Quantitative TaqMan RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus.

Since two different types of porcine reproductive and respiratory syndrome virus (PRRSV), the European (EU) and the North American (US) strain, occur or coexist in European swine herds, their rapid and reliable detection and differentiation is essential for disease surveillance. A quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) is described for PRRSV detection and strain differentiation. Sensitivity and specificity were compared with a conventional PRRSV RT-PCR and to the detection of both PRRSV types in cell cultures and both were found to be equal or superior to the reference methods. Reproducibility was tested and proved that the assay was very reliable. Standard dilutions included in each test allowed absolute quantitation of the amount of viral RNA. The TaqMan assay described below is time-saving, easy to handle, exhibits a decreased risk of cross-contamination and is highly sensitive and specific. It is, therefore, considered to be a powerful tool for the rapid detection and differentiation of PRRSV.

Animals↗

PubMed2001

A recombinant classical swine fever virus with a marker insertion in the internal ribosome entry site.

Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.

5' Untranslated Regions↗

PubMed2001

Receptor for advanced glycation end products mediates inflammation and enhanced expression of tissue factor in vasculature of diabetic apolipoprotein E-null mice.

Advanced glycation end products (AGEs) and their cell surface receptor, RAGE, have been implicated in the pathogenesis of diabetic complications. Here, we studied the role of RAGE and expression of its proinflammatory ligands, EN-RAGEs (S100/calgranulins), in inflammatory events mediating cellular activation in diabetic tissue. Apolipoprotein E-null mice were rendered diabetic with streptozotocin at 6 weeks of age. Compared with nondiabetic aortas and kidneys, diabetic aortas and kidneys displayed increased expression of RAGE, EN-RAGEs, and 2 key markers of vascular inflammation, vascular cell adhesion molecule (VCAM)-1 and tissue factor. Administration of soluble RAGE, the extracellular domain of the receptor, or vehicle to diabetic mice for 6 weeks suppressed levels of VCAM-1 and tissue factor in the aorta, in parallel with decreased expression of RAGE and EN-RAGEs. Diabetic kidney demonstrated increased numbers of EN-RAGE-expressing inflammatory cells infiltrating the glomerulus and enhanced mRNA for transforming growth factor-beta, fibronectin, and alpha(1) (IV) collagen. In mice treated with soluble RAGE, the numbers of infiltrating inflammatory cells and mRNA levels for these glomerular cytokines and components of extracellular matrix were decreased. These data suggest that activation of RAGE primes cells targeted for perturbation in diabetic tissues by the induction of proinflammatory mediators.

Animals↗

PubMed2001

Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model.

Although hyperhomocysteinemia (HHcy) is a well-known risk factor for the development of cardiovascular disease, the underlying molecular mechanisms are not fully elucidated. Here we show that induction of HHcy in apoE-null mice by a diet enriched in methionine but depleted in folate and vitamins B6 and B12 increased atherosclerotic lesion area and complexity, and enhanced expression of receptor for advanced glycation end products (RAGE), VCAM-1, tissue factor, and MMP-9 in the vasculature. These homocysteine-mediated (HC-mediated) effects were significantly suppressed, in parallel with decreased levels of plasma HC, upon dietary supplementation with folate and vitamins B6/B12. These findings implicate HHcy in atherosclerotic plaque progression and stability, and they suggest that dietary enrichment in vitamins essential for the metabolism of HC may impart protective effects in the vasculature.

Animals↗

PubMed2000

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

Animals↗

PubMed2000

Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases.

The receptor for advanced glycation end products (RAGE), a multi-ligand member of the immunoglobulin superfamily of cell surface molecules, interacts with distinct molecules implicated in homeostasis, development and inflammation, and certain diseases such as diabetes and Alzheimer's disease. Engagement of RAGE by a ligand triggers activation of key cell signalling pathways, such as p21ras, MAP kinases, NF-kappaB and cdc42/rac, thereby reprogramming cellular properties. RAGE is a central cell surface receptor for amphoterin, a polypeptide linked to outgrowth of cultured cortical neurons derived from developing brain. Indeed, the co-localization of RAGE and amphoterin at the leading edge of advancing neurites indicated their potential contribution to cellular migration, and in pathologies such as tumour invasion. Here we demonstrate that blockade of RAGE-amphoterin decreased growth and metastases of both implanted tumours and tumours developing spontaneously in susceptible mice. Inhibition of the RAGE-amphoterin interaction suppressed activation of p44/p42, p38 and SAP/JNK MAP kinases; molecular effector mechanisms importantly linked to tumour proliferation, invasion and expression of matrix metalloproteinases.

Animals↗

PubMed2000

Classical swine fever virus: a ring test to evaluate RT-PCR detection methods.

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Two sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 34 samples of random primed cDNA. These had been synthesised from viral RNA representative of seven different genetic subtypes of CSFV. The other set comprised 40 clinical samples containing tonsil, spleen, whole blood or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the clinical samples were also compared with those obtained by virus isolation and antigen ELISA.ELISA. Both RT-PCR and RT-nested PCR appeared to give some false positive results. Several of the PCR tests appear suitable in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratories, and to show that improved control procedures can eliminate problems due to false positive reactions.A limited comparison of extraction and reverse transcription procedures showed similar results in each of three participating laboratories, even though the methods were not standardised.

Classical Swine Fever Virus↗

PubMed2000

Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol.

A method for storing samples containing classical swine fever virus (CSFV) or foot-and-mouth disease virus (FMDV), respectively, was developed, which abolishes the infectivity of both plus strand RNA viruses, and allows storage of samples above 0 degrees C for an extended time, yet preserves the viral RNA in a state which allows its detection by reverse transcription-polymerase chain reaction (RT-PCR), and even rescue of infectious virus after transfection of the extracted RNA into susceptible cells. Supernatants from infected cell cultures as well as organs from diseased animals were stored in Trizol(R) for 1-4 weeks at -20 degrees C, 4 degrees C, room temperature, or 37 degrees C. RNA was then extracted and used subsequently for RT-PCR, as well as transfection into susceptible cells to initiate the replication of progeny virus. Formaldehyde-fixed samples were also included in this study. Storage up to 4 weeks at 37 degrees C in Trizol(R) still yielded positive RT-PCR results and rescue of infectious virus upon RNA transfection. In contrast, formaldehyde fixation reduced drastically the detectability of viral RNA. This method represents a safe and inexpensive alternative to -70 degrees C (dry ice) storage or transport of samples, and abolishes the biosafety risks involved in shipping deep-frozen infectious materials.

Animals↗

PubMed2000

Weekly oxaliplatin, high-dose infusional 5-fluorouracil and folinic acid as palliative third-line therapy of advanced colorectal carcinoma.

The efficacy of oxaliplatin combined with high-dose 5-fluorouracil (5-FU) and folinic acid (FA) as an outpatient salvage treatment for patients with metastasized colorectal cancer was retrospectively analyzed in one center. Tumor progression had occurred for the majority of patients during two regimens (n = 11) otherwise during one (n = 1) regimen of prior 5-FU-based chemotherapy, which had been applied in a standardized sequential fashion. As third-line therapy oxaliplatin was infused intravenously over 2 h at a dose of 60 mg/m2 prior to a 2-h infusion of FA (500 mg/m2). 5-FU (2,600 mg/m2) was subsequently given over 24 h. A favorable response was observed in 9/12 (75%) of the heavily pretreated patients, including partial remissions in 3/12, minor responses in 2/12 and stable disease in 4/12 patients. The median progression free time was 23 weeks (interquartile range i. r. 0-28) for all patients, the median survival time from start of third-line therapy 55 weeks (i. r. 40-86). The median survival time from the beginning of first-line palliative chemotherapy was 34 months (i. r. 25-45 months). The highest toxicity was WHO grade III and was observed in six patients: Nausea (2), diarrhea (3), vomiting (2) and peripheral neuropathy (1). The quality of life was not adversely affected by the oxaliplatin/5-FU/FA-regimen as assessed by the EORTC QLQ-C30 questionnaire. Thus, the results show the efficiency and low toxicity of oxaliplatin/high-dose 5-FU/FA as palliative third-line therapy of patients with metastasized colorectal cancer and emphasize that sequential palliative chemotherapy may lead to extended survival of these patients.

Adult↗

PubMed1999

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

Amino Acid Sequence↗

PubMed1999

A survey of Newcastle disease in Swiss laying-hen flocks using serological testing and simulation modelling.

Newcastle disease (ND) is a highly contagious viral disease of birds particularly domestic poultry. Switzerland is currently declared free from ND; since vaccination is prohibited, the detection of antibodies against ND virus (NDV) results in the destruction of the respective flock (stamping-out policy). However, in 1995 and 1996, antibody-positive flocks were detected and sporadic ND outbreaks even occurred in Switzerland. Therefore, a serosurvey was done to look for evidence of NDV infections in Swiss laying-hen flocks. The survey was designed to provide 95% confidence of detecting at least one seropositive flock if the flock prevalence were 1%. Thirty blood samples from each of 260 commercial laying-hen flocks were collected during 1996 in a central poultry slaughterhouse. Sera were screened for NDV antibodies with a commercial blocking enzyme-linked immunosorbent assay (ELISA). Samples with a questionable or positive test result were retested with the same ELISA. A stochastic computer model was applied to define a cut-off number of test-positive samples to help to differentiate between true- and false-positive flocks and to estimate the true flock prevalence of infection. Four flocks were identified as NDV-seropositive and the NDV true seroprevalence among commercial laying-hen flocks in Switzerland was most likely between 1.35 and 1.55%. This indicates that Swiss laying-hen and parental flocks with more than 150 animals have been in contact with strains of NDV that cause subclinical infection in chicken, because no clinical symptoms have been observed. In this context, computer simulation was a useful technique to interpret survey results.

Animals↗

PubMed1999

[Non-enzymatic glycation and oxidative stress in chronic illnesses and diabetes mellitus].

UNLABELLED: New approaches in biochemistry and molecular biology have increased the knowledge on the pathophysiology of chronic diseases as late diabetic complications, Alzheimer's disease, arteriosclerosis and vascular disease by defining the concept of "AGE-formation and oxidative stress." Nonenzymatic glycation, in which reducing sugars are covalently bound to free aminogroups of macromolecules, results in the formation of Advanced Glycation End products (AGEs) which accumulate during aging and at accelerated rate during the course of diabetes. Glycation accompanying oxidation processes support AGE-formation. AGE-formation changes the physicochemical properties of proteins, lipids and nucleic acids. In addition, binding of AGEs to specific surface receptors induces cellular signalling and cell activation. Interaction of AGEs with one of the receptors, RAGE, generates intracellular oxidative stress, which results in activation of the transcription factor NF-kappa B and subsequent gene expression, which might be relevant in late diabetic complications. CONCLUSION: Knowledge of the basis molecular mechanisms allows to understand the interplay of different inducers such as redicals, cytokines, AGE-proteins and amyloid-beta-peptids and to define oxidative stress as a "common endpoint" of cell dysfunction. With respect to therapeutic options it is now possible not only to optimize blood glycemic control, but also to design drugs such as AGE-inhibitors and AGE-"cross-link" breakers. In addition patients with chronic disease associated with increased oxidative stress ay benefit from an antioxidant rich (and AGE protein poor?) nutrition.

Alzheimer Disease↗

PubMed1999

Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB.

Increased oxidative stress and subsequent activation of the transcription factor NF-kappaB has been linked to the development of late diabetic complications. To determine whether oxidative stress dependent NF-kappaB activation is evident in patients with diabetic nephropathy we used an Electrophoretic Mobility Shift Assay based semiquantitative detection system which enabled us to determine NF-kappaB activation in ex vivo isolated peripheral blood mononuclear cells. We examined 33 patients with diabetes mellitus (Type I and Type II). Patients with diabetic nephropathy showed higher NF-kappaB binding activity in Electrophoretic Mobility Shift Assays and stronger immunohistological staining for activated NF-kappaBp65 than patients without renal complications. NF-kappaB binding activity correlated with the degree of albuminuria (r = 0.316) and with thrombomodulin plasma concentrations (r = 0.33), indicative for albuminuria associated endothelial dysfunction. In a 3 day intervention study in which 600 mg of the antioxidant thioctic acid (alpha-lipoic acid) per day were given to nine patients with diabetic nephropathy oxidative stress in plasma samples was decreased by 48% and NF-kappaB binding activity in ex vivo isolated peripheral blood mononuclear cells by 38%. In conclusion, activation of the transcription factor NF-kappaB in ex vivo isolated peripheral blood mononuclear cells of patients with diabetes mellitus correlates with the degree of diabetic nephropathy. NF-kappaB activation is at least in part dependent on oxidative stress since thioctic acid (alpha-lipoic acid) reduced NF-kappaB binding activity.

Adult↗

PubMed1999

Classical swine fever virus is genetically stable in vitro and in vivo.

Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene'93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5' NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies.

Animals↗

PubMed1999

Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus.

To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N(pro), C, E(rns), E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.

Animals↗

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↗