Multimodal composition of the digital patient: a strategy for the knee articulation

Creating virtual bodies of real patients and using them for diagnosis and treatment planning offer the potential to further empower clinical decision making by medical experts. Virtual patient modeling allows to examine the mechanical and physiological conditions under which articulations are operating in a variety of activities without putting the patient in hazard. The continuous scientific progress has led to an increased range of musculoskeletal data and knowledge being available, covering multiple scales of the musculoskeletal system. A fuller integration of these modalities can broaden the scientific basis of virtual articulation modeling in patients, but poses challenges for data fusion and coupling of simulations. Here, we present a multimodal strategy to compose virtual models of the knee articulation based on a complementary spectrum of data that enables simulations on different scales

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35’364 ternary pools of 52’746 siRNAs targeting 17’582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrP$^{Sc}$, and its cellular counterpart PrP$^{C}$, 40 genes affected selectively PrP$^{Sc}$. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrP$^{C}$. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions

Analysis of 3d knee joint deformation using a multiscale modelling approach

The human knee joint is the largest and most complex joint of the human body. The interdependencies encountered in the musculoskeletal system are crucial in understanding musculoskeletal conditions. Patient-specific models are promising methods to unravel clinical diagnosis. However, as the range of medical and experimental data is expanding, it has become a challenge to integrate data in virtual models in a way that it is comprehensive and reliable for medical diagnosis. In this thesis, we focus on a novel modelling paradigms that encompasses mechanics with microscale and physiological data which aims at being practical for clinical investigation. We integrate microstructural and physiological data into articulation simulations, by investigating different biological organization levels such as organ, tissue, cellular, and molecular level. We consider synergies between in vitro data, medical imaging data and computational models, which can have a significant impact on the development of realistic simulation tools to answer clinical challenges

A computational approach to calculate personalized pennation angle based on MRI: effect on motion analysis

Purpose Muscles are the primary component responsible for the locomotion and change of posture of the human body. The physiologic basis of muscle force production and movement is determined by the muscle architecture (maximum muscle force, Fmo, optimal muscle fiber length, lmo, tendon slack length, lts, and pennation angle at optimal muscle fiber length, φ0). The pennation angle is related to the maximum force production and to the range of motion. The aim of this study was to investigate a computational approach to calculate subject-specific pennation angle from magnetic resonance images (MRI)-based 3D anatomical model and to determine the impact of this approach on the motion analysis with personalized musculoskeletal models. Methods A 3D method that calculates the pennation angle using MRI was developed. The fiber orientations were automatically computed, while the muscle line of action was determined using approaches based on anatomical landmarks and on centroids of image segmentation. Three healthy male volunteers were recruited for MRI scanning and motion capture acquisition. This work evaluates the effect of subject-specific pennation angle as musculoskeletal parameter in the lower limb, focusing on the quadriceps group. A comparison was made for assessing the contribution of personalized models on motion analysis. Gait and deep squat were analyzed using neuromuscular simulations (OpenSim). Results The results showed variation of the pennation angle between the generic and subject-specific models, demonstrating important interindividual differences, especially for the vastus intermedius and vastus medialis muscles. The pennation angle variation between personalized and generic musculoskeletal models generated significant variation in muscle moments and forces during dynamic motion analysis. Conclusions A MRI-based approach to define subject-specific pennation angle was proposed and evaluated in motion analysis models. The significant differences obtained for the moments and muscle forces in quadriceps muscles indicate that a personalized approach in modeling the pennation angle can provide more individual details when investigating motion behaviors in specific subjects

Automatic measurement and visualization of focal femoral cartilage thickness in stress-based regions of interest using three-dimensional knee models

Purpose Thinning of cartilage is a common manifestation of osteoarthritis. This study addresses the need of measuring the focal femoral cartilage thickness at the weight-bearing regions of the knee by developing a reproducible and automatic method from MR images. Methods 3D models derived from semiautomatic MR image segmentations were used in this study. Two different methods were examined for identifying the mechanical loading of the knee articulation. The first was based on a generic weight-bearing regions definition, derived from gait characteristics and cadaver studies. The second used a physically based simulation to identify the patient-specific stress distribution of the femoral cartilage, taking into account the forces and movements of the knee. For this purpose, four different scenarios were defined in our 3D finite element (FE) simulations. The radial method was used to calculate the cartilage thickness in stress-based regions of interest, and a study was performed to validate the accuracy and suitability of the radial thickness measurements. Results Detailed focal maps using our simulation data and regional measurements of cartilage thickness are given. We present the outcome of the different simulation scenarios and discuss how the internal/external rotations of the knee alter the overall stress distribution and affect the shape and size of the calculated weight-bearing areas. The use of FE simulations allows for a patient-specific calculation of the focal cartilage thickness. Conclusion It is important to assess the quantification of focal knee cartilage morphology to monitor the progression of joint diseases or related treatments. When this assessment is based on MR images, accurate and robust tools are required. In this paper, we presented a set of techniques and methodologies in order to accomplish this goal and move toward personalized medicine

Genome-wide transcriptomics identifies an early preclinical signature of prion infection

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets

Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies

Protective anti‐prion antibodies in human immunoglobulin repertoires

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here, we identified > 6,000 PrP‐binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage‐derived antibodies. When expressed recombinantly, these antibodies exhibited anti‐PrP reactivity. Furthermore, we surveyed 48,718 samples from 37,894 hospital patients for the presence of anti‐PrP IgGs and found 21 high‐titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti‐PrP autoimmunity is innocuous. The existence of anti‐prion antibodies in unbiased human immunological repertoires suggests that they might clear nascent prions early in life. Combined with the reported lack of such antibodies in carriers of disease‐associated PRNP mutations, this suggests a link to the low incidence of spontaneous prion diseases in human populations

Genome-wide transcriptomics identifies an early preclinical signature of prion infection

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets

Prion pathogenesis is unaltered in a mouse strain with a permeable blood-brain barrier

Transmissible spongiform encephalopathies (TSEs) are caused by the prion, which consists essentially of PrPSc, an aggregated, conformationally modified form of the cellular prion protein (PrPC). Although TSEs can be experimentally transmitted by intracerebral inoculation, most instances of infection in the field occur through extracerebral routes. The epidemics of kuru and variant Creutzfeldt-Jakob disease were caused by dietary exposure to prions, and parenteral administration of prion-contaminated hormones has caused hundreds of iatrogenic TSEs. In all these instances, the development of postexposure prophylaxis relies on understanding of how prions propagate from the site of entry to the brain. While much evidence points to lymphoreticular invasion followed by retrograde transfer through peripheral nerves, prions are present in the blood and may conceivably cross the blood-brain barrier directly. Here we have addressed the role of the blood-brain barrier (BBB) in prion disease propagation using Pdgfbret/ret mice which possess a highly permeable BBB. We found that Pdgfbret/ret mice have a similar prion disease incubation time as their littermate controls regardless of the route of prion transmission. These surprising results indicate that BBB permeability is irrelevant to the initiation of prion disease, even when prions are administered parenterally