Thermal Inactivation of Carcasses of Mice and Rabbits Infected with Pathogens of Risk Groups Two to Four.

Pathogenesis of viruses or other agents that are infectious to humans is frequently studied in vivo using natural or genetically modified animals. Depending on the risk group of the pathogen, the majority of such experimental studies are performed at least under biosafety level 2 (BSL-2) conditions. Biosafety considerations are therefore critical at all steps of research involving potentially infectious pathogens. Inactivation of pathogens studied using in vitro experiments is usually performed using moist heat sterilization. However, few standardized and validated protocols are currently available for the thermal inactivation of carcasses from laboratory animals infected with such human pathogens. To comply with laboratory biologic safety rules and requirements imposed by regulatory authorities, documentation of appropriate inactivation conditions or use of a validated procedure according to national or international standards is critical. In the current study, we evaluated inactivation protocols in a standard laboratory autoclave for carcasses of either frozen mice or recently terminated rabbits, which were placed inside autoclave bags with bedding material in stainless steel containers. Temperature sensors were placed into different tissues of the carcasses to continuously record temperature in situ and in real-time, and a reference sensor was placed in the autoclave. To achieve pathogen inactivation, autoclaving protocols had to be optimized for both species. Frozen mice required 2 different fractionated prevacuum stages, whereas recently terminated rabbits required 3 different fractionated prevacuum stages. This study provides a template for an evaluation procedure to safely and effectively inactivate mice and rabbits infected with risk group 2 to 4 pathogens.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

Quantitative Real-Time Quaking-Induced Conversion Allows Monitoring of Disease-Modifying Therapy in the Urine of Prion-Infected Mice.

Prion diseases are fatal neurodegenerative diseases characterized by accumulation of the pathogenic prion protein PrP in the brain. We established quantitative real-time quaking-induced conversion for the measurement of minute amounts of PrP in body fluids such as urine. Using this approach, we monitored the efficacy of antiprion therapy by quantifying the seeding activity of PrP from the brain and urine of mice after prion infection. We found that the aggregation inhibitor anle138b decreased the levels of PrP in the brain and urine. Importantly, variations of PrP levels in the urine closely corresponded to those in the brain. Our findings indicate that quantification of urinary PrP enables measurement of prion disease progression in body fluids and can substitute for immunodetection in brain tissue. We expect PrP quantification biologic fluids (such as urine and cerebrospinal fluid) with quantitative real-time quaking-induced conversion to emerge as a valuable noninvasive diagnostic tool for monitoring disease progression and the efficacy of therapeutic approaches in animal studies and human clinical trials of prion diseases. Moreover, highly sensitive methods for quantifying pathologic aggregate seeds might provide novel molecular biomarkers for other neurodegenerative diseases that may involve prion-like mechanisms (protein aggregation and spreading), such as Alzheimer disease and Parkinson disease.

Establishing quantitative real-time quaking-induced conversion (qRT-QuIC) for highly sensitive detection and quantification of PrPSc in prion-infected tissues.

BACKGROUND: PrPSc, the only known constituent of prions, the infectious agents causing prion diseases, can be detected by real-time quaking-induced conversion (RT-QuIC). However, there is no efficient method to quantify the amount of PrPSc by RT-QuIC. RESULTS: Here we introduce quantitative RT-QuIC (qRT-QuIC) to quantify with high accuracy minute amounts of PrPSc in the brain and various peripheral tissues at levels far below detection by in vivo transmission. PrPSc is relatively resistant to treatment with proteinase K (PK). However, as there can also be a fraction of pathological PrP that is digested by PK, we use the term PrP27-30 to denote to the amount of PrPSc that can be detected by immunoblot after PK treatment. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. By seeding known amounts of PrP27-30 quantified by immunoblot into qRT-QuIC a standard calibration curve can be obtained. Based on this calibration curve, seeded undetermined amounts of PrP27-30 can be directly calculated. qRT-QuIC allowed to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units obtained by in vivo i.c. transmission studies. We find that PrP27-30 concentration increases steadily in the brain after inoculation and can be detected at various time points during the incubation period in peripheral organs (spleen, heart, muscle, liver, kidney) in two experimental scrapie strains (RML, ME7) in the mouse. CONCLUSIONS: We suggest that an automatic quantitative system to measure disease progression as well as prion contamination of organs, blood and food product is feasible. Moreover, the concept of qRT-QuIC should be applicable to measure other disease-associated proteins rich in ß-pleated structures (amyloid) that bind ThT and that show seeded aggregation.

Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease.

In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrP(Sc)) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood-brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.