3D multicellular systems in disease modelling: From organoids to organ-on-chip.

Cell-cell interactions underlay organ formation and function during homeostasis. Changes in communication between cells and their surrounding microenvironment are a feature of numerous human diseases, including metabolic disease and neurological disorders. In the past decade, cross-disciplinary research has been conducted to engineer novel synthetic multicellular organ systems in 3D, including organoids, assembloids, and organ-on-chip models. These model systems, composed of distinct cell types, satisfy the need for a better understanding of complex biological interactions and mechanisms underpinning diseases. In this review, we discuss the emerging field of building 3D multicellular systems and their application for modelling the cellular interactions at play in diseases. We report recent experimental and computational approaches for capturing cell-cell interactions as well as progress in bioengineering approaches for recapitulating these complexities ex vivo. Finally, we explore the value of developing such multicellular systems for modelling metabolic, intestinal, and neurological disorders as major examples of multisystemic diseases, we discuss the advantages and disadvantages of the different approaches and provide some recommendations for further advancing the field.

Prospective Assessment of the Diagnostic Accuracy of Multi-site Photoplethysmography Pulse Measurements for Diagnosis of Peripheral Artery Disease in Primary Care.

Peripheral arterial disease (PAD) is associated with cerebral and coronary artery disease. Symptomatic PAD affects about 5% of people over 55 years; many more have asymptomatic PAD. Early detection enables modification of arterial disease risk factors. Diagnostically, assessment of symptoms or signs can be unreliable; ankle brachial pressure index (ABPI) testing is time-consuming and few healthcare professionals are properly trained. This study assessed the diagnostic accuracy of multi-site photoplethysmography (MPPG), an alternative non-invasive test for PAD, in primary care. PAD patients identified from general practice registers were age- and sex-matched with controls. Participants were assessed using MPPG, ABPI and duplex ultrasound (DUS). Outcome measures were sensitivity and specificity of MPPG and ABPI (relative to DUS) and concordance. MPPG test results were available in 249 of 298 eligible participants from 16 practices between May 2015 and November 2016. DUS detected PAD in 101/249 (40.6%). MPPG sensitivity was 79.8% (95% confidence interval [CI] 69.9-87.6%), with specificity 71.9% (95% CI 63.7-79.2%). ABPI sensitivity was 80.2% (95% CI 70.8-87.6%), with specificity 88.6% (95% CI 82-93.5%). With comparable sensitivity to ABPI, MPPG is quick, automated and simpler to do than ABPI; it offers the potential for rapid and accessible PAD assessments in primary care.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation.

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Reliability of the Edinburgh Claudication Questionnaire for Identifying Symptomatic PAD in General Practice.

The Edinburgh Claudication Questionnaire (ECQ) was developed to help identify peripheral arterial disease (PAD) in the general population but has not been validated against diagnostic arterial imaging methods such as Duplex Vascular Ultrasound Scanning (DUS). In the present study, we assessed the accuracy of the ECQ for diagnosis using DUS. As part of a National Institute of Health Research funded project looking at novel diagnostic methods, 250 patients were studied from 15 general practices across North East England from May 2015 and November 2016. Practices identified those with a PAD diagnosis from their registers as well as age- and sex-matched controls. All the ECQs were recorded by a vascular specialist nurse. Duplex vascular ultrasound scanning was used as a reference standard for the diagnosis of occlusive PAD. The ECQ had a sensitivity of 52.5% (95% CI: 42.3%-62.5%), specificity of 87.1% (95% CI: 80.6%-92.0%), positive likelihood ratio of 4.06 (95% CI: 2.57-6.42), and negative likelihood ratio of 0.55 (95% CI: 0.44-0.68) compared with reference standard DUS. The ECQ has relatively poor overall diagnostic test accuracy in isolation. It may be helpful in ruling out PAD or as a supplementary test to improve diagnosis of symptomatic disease in General Practice.

Accuracy of Peripheral Arterial Disease Registers in UK General Practice: Case-Control Study.

BACKGROUND: Approximately 20% of the UK population aged 55 to 75 years have evidence of peripheral arterial disease (PAD). PAD affects quality of life and life expectancy if not appropriately diagnosed and managed. At risk patients require accurate diagnosis to ensure optimal treatment to slow disease progression and minimize adverse outcomes. AIM: To assess the accuracy of general practice (GP) registration of the diagnosis of peripheral arterial disease (PAD). DESIGN AND SETTING: An observational analytic case-control study. As part of a National Institute for Health Research-funded (ISRCTN13301188) project assessing novel diagnostic methods set in GP practice. METHODS: A total of 125 patients registered as having PAD and 125 age- and sex-matched controls were recruited from 15 general practices across North East England. The register was then assessed for accuracy of diagnosis. Duplex vascular ultrasound scanning (DUS) undertaken by vascular scientists was used as the gold standard reference for PAD. RESULTS: The PAD register had a sensitivity of 86% (95% CI 77%-92%) and specificity of 74% (95% CI 67%-81%) when compared with DUS. The positive predictive value, however, was 69.6% (95% CI 63%-75%) and negative predictive value 88.8% (95% CI 82%-92%). The overall diagnostic effectiveness of the PAD register was 79.2% (95% CI 73%-84%). CONCLUSION: This analysis indicates that while PAD is detected with reasonable sensitivity in primary care, many patients registered with a diagnosis of PAD lacked DUS-proven disease. Improved approaches to the objective diagnosis of PAD may improve diagnosis and management of PAD in primary care.