PROTEUS Study: A Prospective Randomized Controlled Trial Evaluating the Use of Artificial Intelligence in Stress Echocardiography.

BACKGROUND: Stress echocardiography (SE) is one of the most commonly used diagnostic imaging tests for coronary artery disease (CAD) but requires clinicians to visually assess scans to identify patients who may benefit from invasive investigation and treatment. EchoGo Pro provides an automated interpretation of SE based on artificial intelligence (AI) image analysis. In reader studies, use of EchoGo Pro when making clinical decisions improves diagnostic accuracy and confidence. Prospective evaluation in real world practice is now important to understand the impact of EchoGo Pro on the patient pathway and outcome. METHODS: PROTEUS is a randomized, multicenter, 2-armed, noninferiority study aiming to recruit 2,500 participants from National Health Service (NHS) hospitals in the UK referred to SE clinics for investigation of suspected CAD. All participants will undergo a stress echocardiogram protocol as per local hospital policy. Participants will be randomized 1:1 to a control group, representing current practice, or an intervention group, in which clinicians will receive an AI image analysis report (EchoGo Pro, Ultromics Ltd, Oxford, UK) to use during image interpretation, indicating the likelihood of severe CAD. The primary outcome will be appropriateness of clinician decision to refer for coronary angiography. Secondary outcomes will assess other health impacts including appropriate use of other clinical management approaches, impact on variability in decision making, patient and clinician qualitative experience and a health economic analysis. DISCUSSION: This will be the first study to assess the impact of introducing an AI medical diagnostic aid into the standard care pathway of patients with suspected CAD being investigated with SE. TRIAL REGISTRATION: Clinicaltrials.gov registration number NCT05028179, registered on 31 August 2021; ISRCTN: ISRCTN15113915; IRAS ref: 293515; REC ref: 21/NW/0199.

Pharmacology of a Potent and Novel Inhibitor of the NOD-Like Receptor Pyrin Domain-Containing Protein 3 (NLRP3) Inflammasome that Attenuates Development of Nonalcoholic Steatohepatitis and Liver Fibrosis.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1ß production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.

Macrophages and Intervertebral Disc Degeneration.

The intervertebral disc (IVD) aids in motion and acts to absorb energy transmitted to the spine. With little inherent regenerative capacity, degeneration of the intervertebral disc results in intervertebral disc disease, which contributes to low back pain and significant disability in many individuals. Increasing evidence suggests that IVD degeneration is a disease of the whole joint that is associated with significant inflammation. Moreover, studies show elevated macrophage accumulation within the IVD with increasing levels of disease severity; however, we still need to understand the roles, be they causative or consequential, of macrophages during the degenerative process. In this narrative review, we discuss hallmarks of IVD degeneration, showcase evidence of macrophage involvement during disc degeneration, and explore burgeoning research aimed at understanding the molecular pathways regulating macrophage functions during intervertebral disc degeneration.

Three Dose Levels of a Maternal Respiratory Syncytial Virus Vaccine Candidate Are Well Tolerated and Immunogenic in a Randomized Trial in Nonpregnant Women.

BACKGROUND: Respiratory syncytial virus (RSV) causes respiratory tract infections, which may require hospitalization especially in early infancy. Transplacental transfer of RSV antibodies could confer protection to infants in their first months of life. METHODS: In this first-in-human, placebo-controlled study, 502 healthy nonpregnant women were randomized 1:1:1:1 to receive a single dose of unadjuvanted vaccine containing 30/60/120 µg of RSV fusion (F) protein stabilized in the prefusion conformation (RSVPreF3) or placebo. RESULTS: Solicited local adverse events (AEs) were more frequently reported in the RSVPreF3 groups (4%-53.2%) versus placebo (0%-15.9%); most were mild/moderate. Unsolicited AEs were comparably reported among groups. Three serious AEs were reported; none was vaccination-related. Compared with prevaccination values, anti-RSV A neutralizing antibody geometric mean titers and anti-RSVPreF3 immunoglobulin G geometric mean concentrations increased 8- to 14-fold and 12- to 21-fold at day 8 and persisted 5- to 6-fold and 6- to 8-fold higher until day 91 in the RSVPreF3 groups versus 1-fold in placebo. Comparisons at day 8 and day 31 showed that the higher dose levels were significantly more immunogenic than the lowest one. CONCLUSIONS: The RSVPreF3 vaccine was well tolerated and immunogenic. The 60 and 120 µg dose levels were selected for further investigation in pregnant women. CLINICAL TRIALS REGISTRATION: NCT03674177.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Epiphyseal cartilage canal architecture and extracellular matrix remodeling in mucopolysaccharidosis VII dogs at the onset of postnatal growth.

Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII.Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining.Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII.Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.

MicroRNA 223 3p Negatively Regulates the NLRP3 Inflammasome in Acute and Chronic Liver Injury.

The granulocyte-specific microRNA-223 (miR-223) has recently emerged as a negative regulator of NOD-like receptor 3 (NLRP3) expression, a central key player in chronic hepatic injuries such as fibrotic nonalcoholic steatohepatitis (NASH), as well as in other liver conditions including acute hepatitis. In this study, we evaluated the therapeutic effect of the synthetic miR-223 analog miR-223 3p in a murine model of lipopolysaccharide (LPS)/D-GalN-induced endotoxin acute hepatitis (EAH) or fibrotic NASH resultant of long-term feeding with a high-fat, fructose, and cholesterol (FFC) diet. miR-223 3p ameliorated the infiltration of monocytes, neutrophils, and early activated macrophages and downregulated the transcriptional expression of the pro-inflammatory cytokines Il6 and Il12 and the chemokines Ccl2, Ccl3, Cxcl1, and Cxcl2 in EAH. In fibrotic NASH, treatment with miR-223 3p led to a remarkable mitigation of fibrosis development and activation of hepatic stellate cells (HSCs). miR-223 3p disrupted the activation of the NLRP3 inflammasome by impairing the synthesis of cleaved interleukin-1ß (IL-1ß), mature IL-1ß, and NLRP3, and the activation of caspase-1 p10 in both EAH and fibrotic NASH. Our data enlightens miR-223 3p as a post-transcriptional approach to treat acute and chronic hepatitis by silencing the activation of the NLRP3 inflammasome.

7T bone perfusion imaging of the knee using arterial spin labeling MRI.

PURPOSE: To evaluate the feasibility of arterial spin labeling (ASL) imaging of epiphyseal bone marrow in the distal femoral condyle of the knee at 7T MRI. METHODS: The knees of 7 healthy volunteers were imaged with ASL using a 7T whole body MRI scanner and a 28-channel knee coil. ASL imaging used a flow-sensitive alternating inversion recovery method for labeling and a single-shot fast spin echo sequence for image readout. ASL imaging with a single oblique transverse slice was performed at 2 slice positions in the distal femoral condyle. Blood flow was measured in 2 regions of interest: the epiphyseal bone marrow and the overlying patellofemoral cartilage. To analyze perfusion SNR, 200 noise images were also acquired using the same ASL imaging protocol with RF pulses turned off. RESULTS: Knee bone marrow perfusion imaging was successfully performed with all volunteers. The overall mean of blood flow in the knee bone marrow was 32.90 ± 2.41 mL/100 g/min, and the blood flow was higher at the more distal slice position. We observed significant B0 and B1+ inhomogeneities, which need to be addressed in the future to improve the quality of ASL imaging and increase the reliability of knee bone marrow perfusion measurements. CONCLUSION: Bone marrow perfusion imaging of the distal femoral condyle is feasible using ASL at 7T. Further technical development is needed to improve the ASL method to overcome existing challenges.

Three-Dimensional GRE T1ρ mapping of the brain using tailored variable flip-angle scheduling.

PURPOSE: To introduce a new approach called tailored variable flip-angle (VFA) scheduling for SNR-efficient 3D T1ρ mapping of the brain using a magnetization-prepared gradient-echo sequence. METHODS: Simulations were used to assess the relative SNR efficiency, quantitative accuracy, and spatial blurring of tailored VFA scheduling for T1ρ mapping of brain tissue compared with magnetization-prepared angle-modulated partitioned k-space spoiled gradient-echo snapshots (MAPSS), a state-of-the-art technique for accurate 3D gradient-echo T1ρ mapping. Simulations were also used to calculate optimal imaging parameters for tailored VFA scheduling versus MAPSS, without and with nulling of CSF. Four participants were imaged at 3T MRI to demonstrate the feasibility of tailored VFA scheduling for T1ρ mapping of the brain. Using MAPSS as a reference standard, in vivo data were used to validate the relative SNR efficiency and quantitative accuracy of the new approach. RESULTS: Tailored VFA scheduling can provide a 2-fold to 4-fold gain in the SNR of the resulting T1ρ map as compared with MAPSS when using identical sequence parameters while limiting T1ρ quantification errors to 2% or less. In vivo whole-brain 3D T1ρ maps acquired with tailored VFA scheduling had superior SNR efficiency than is achievable with MAPSS, and the SNR efficiency improved with a greater number of views per segment. CONCLUSIONS: Tailored VFA scheduling is an SNR-efficient GRE technique for 3D T1ρ mapping of the brain that provides increased flexibility in choice of imaging parameters compared with MAPSS, which may benefit a variety of applications.