The Efficacy of Tetracyclines in Peripheral and Intracerebral Prion Infection

We have previously shown that tetracyclines interact with and reverse the protease resistance of pathological prion protein extracted from scrapie-infected animals and patients with all forms of Creutzfeldt-Jakob disease, lowering the prion titre and prolonging survival of cerebrally infected animals. To investigate the effectiveness of these drugs as anti-prion agents Syrian hamsters were inoculated intramuscularly or subcutaneously with 263K scrapie strain at a 10−4 dilution. Tetracyclines were injected intramuscularly or intraperitoneally at the dose of 10 mg/kg. A single intramuscular dose of doxycycline one hour after infection in the same site of inoculation prolonged median survival by 64%. Intraperitoneal doses of tetracyclines every two days for 40 or 44 days increased survival time by 25% (doxycycline), 32% (tetracycline); and 81% (minocycline) after intramuscular infection, and 35% (doxycycline) after subcutaneous infection. To extend the therapeutic potential of tetracyclines, we investigated the efficacy of direct infusion of tetracyclines in advanced infection. Since intracerebroventricular infusion of tetracycline solutions can cause overt acute toxicity in animals, we entrapped the drugs in liposomes. Animals were inoculated intracerebrally with a 10−4 dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 µg/ 20 µl of doxycycline or minocycline entrapped in liposomes was administered 60 days after inoculation, when 50% of animals showed initial symptoms of the disease. Median survival increased of 8.1% with doxycycline and 10% with minocycline. These data suggest that tetracyclines might have therapeutic potential for humans

Molecular Interactions between Prions as Seeds and Recombinant Prion Proteins as Substrates Resemble the Biological Interspecies Barrier In Vitro

Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrPC) into the pathogenic isoform (PrPSc). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble α-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrPSc as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrPSc from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers

Comparative expression pattern of Matrix-Metalloproteinases in human glioblastoma cell-lines and primary cultures

<p>Abstract</p> <p>Background</p> <p>Glioblastomas (GBM), the most frequent malignant brain tumors in adults, are characterized by an aggressive local growth pattern and highly invasive tumor cells. This invasion is facilitated by expression of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twenty-three family members are known. Elevated levels of several of them have been reported in GBM. GBM cell-lines are used for <it>in vitro </it>studies of cell migration and invasion. Therefore, it is essential to know their MMP expression patterns. Only limited data for some of the cell-lines are published, yet. To fill the gaps in our knowledge would help to choose suitable model systems for analysis of regulation and function of MMPs during GBM tumorigenesis, cell migration and invasion.</p> <p>Findings</p> <p>We analysed MMP-1, -8, -9, -10, -11, -13, -17, -19, -20, -21, -23, -24, -26, -27, and MMP-28 expression in seven GBM cell-lines (SNB-19, GaMG, U251, U87, U373, U343, U138) and in four primary cell cultures by semiquantitative RT-PCR, followed changes in the MMP expression pattern with increasing passages of cell culture and examined the influence of TNF-α and TGF-β1 stimulation on the expression of selected MMPs in U251 and U373 cells.</p> <p>MMP-13, -17, -19 and -24 were expressed by all analyzed cell-lines, whereas MMP-20 and MMP-21 were not expressed by any of them. The other MMPs showed variable expression, which was dependent on passage number. Primary cells displayed a similar MMP-expression pattern as the cell-lines. In U251 and U373 cells expression of MMP-9 and MMP-19 was stimulated by TNF-α. MMP-1 mRNA expression was significantly increased in U373 cells, but not in U251 cells by this cytokine. Whereas TGF-β1 had no impact on MMP expression in U251 cells, it significantly induced MMP-11 and MMP-24 expression in U373 cells.</p> <p>Conclusions</p> <p>Literature-data and our own results suggest that the expression pattern of MMPs is highly variable, dependent on the cell-line and the cell-culture conditions used and that also regulation of MMP expression by cytokines is cell-line dependent. This is of high impact for the transfer of cell-culture experiments to clinical implementation.</p

Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics

Molecular pathology of human prion disease

Human prion diseases are associated with a range of clinical presentations and are classified by both clinicopathological syndrome and aetiology with sub-classification according to molecular criteria. Considerable experimental evidence suggests that phenotypic diversity in human prion disease relates in significant part to the existence of distinct human prion strains encoded by abnormal PrP isoforms with differing physicochemical properties. To date, however, the conformational repertoire of pathological isoforms of wild-type human PrP and the various forms of mutant human PrP has not been fully defined. Efforts to produce a unified international classification of human prion disease are still ongoing. The ability of genetic background to influence prion strain selection together with knowledge of numerous other factors that may influence clinical and neuropathological presentation strongly emphasises the requirement to identify distinct human prion strains in appropriate transgenic models, where host genetic variability and other modifiers of phenotype are removed. Defining how many human prion strains exist allied with transgenic modelling of potentially zoonotic prion strains will inform on how many human infections may have an animal origin. Understanding these relationships will have direct translation to protecting public health

Methods for Molecular Diagnosis of Human Prion Disease.

Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe updated procedures that are currently used within the MRC Prion Unit at UCL to determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in the brain or peripheral tissues