Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

La pharmacie en contexte hospitalier : une mission à définir

<div><p>Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, <i>Trypanosoma cruzi</i>. Perturbations in host central carbon and energy metabolism have been reported in mammalian <i>T</i>. <i>cruzi</i> infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular <i>T</i>. <i>cruzi</i> amastigotes and host cellular energy metabolism. <i>T</i>. <i>cruzi</i> infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular <i>T</i>. <i>cruzi</i> amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated <i>T</i>. <i>cruzi</i> amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in ¹³C-glucose labeling and substrate utilization assays. Metabolic labeling of <i>T</i>. <i>cruzi</i>-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools <i>in situ</i>. These findings are consistent with a model in which intracellular <i>T</i>. <i>cruzi</i> amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich current views regarding available carbon sources for intracellular <i>T</i>. <i>cruzi</i> amastigotes and underscore the metabolic flexibility of this pathogen, a feature predicted to underlie successful colonization of tissues with distinct metabolic profiles in the mammalian host.</p></div

<i>T</i>. <i>cruzi</i> amastigotes incorporate exogenous glucose into multiple metabolic pathways.

<p><i>T</i>. <i>cruzi</i> amastigotes incorporate exogenous glucose into multiple metabolic pathways.</p

Intracellular <i>T</i>. <i>cruzi</i> replication is sensitive to exogenous glucose but not host mitochondrial electron transport chain activity.

<p><b>(A)</b> Proliferation of <i>T</i>. <i>cruzi</i> amastigotes in human dermal fibroblasts with ETC complex III deficiency (CIII mutant) or two independent control fibroblast lines (Normal 1 and 2) derived from flow cytometric data (as detailed in Methods). Data are normalized to represent the percentage of initial amastigotes (18 hpi) that divided the indicated number of times by 48 hpi. Mean ± SD of 2 independent experiments. Dotted lines represent average number of complete amastigote divisions achieved by 48 hpi in each condition. <b>(B)</b> Proliferation of <i>T</i>. <i>cruzi</i> amastigotes in NHDF cultured in medium with varying glucose concentrations. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. <b>(C)</b> Dose-dependent inhibition of <i>T</i>. <i>cruzi</i> growth in NHDF by 2-deoxyglucose (2-DG) in varying glucose concentrations. Relative number of <i>T</i>. <i>cruzi</i>-ß-galactosidase parasites assessed by Beta-Glo luminescence at 66 hpi shown with nonlinear fit using log(inhibitor) vs. response with variable slope. Mean ± SD of 4 biological replicates per point. <b>(D)</b> Arrest of <i>T</i>. <i>cruzi</i> amastigote proliferation in NHDF in the presence of 2 mM 2-DG under conditions of glucose depletion. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. <b>(E)</b> Fluorescence micrographs of aldehyde-fixed, DAPI-stained NHDF monolayers corresponding to conditions used in panel D, in which host cell nuclei (large) and parasite DNA (smaller dots) are readily observed. Arrows point to 2 intracellular amastigotes that persist after severe growth restriction caused by glucose withdrawal and 2-DG treatment.</p

<i>T</i>. <i>cruzi</i> infection increases host mitochondrial content and respiration.

<p><b>(A)</b> Extracellular lactate measured in culture supernatants of uninfected and <i>T</i>. <i>cruzi</i>-infected NHDF monolayers (48 hpi). Mean ± SD shown for 2 biological replicates each. Student’s t-test was applied. <b>(B)</b> Oxygen consumption rate (OCR) in uninfected and <i>T</i>. <i>cruzi</i>-infected NHDF monolayers (48hpi) before and after injection of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/A). Mean ± SD shown for 4 biological replicates. <b>(C)</b> <i>T</i>. <i>cruzi</i>-infected NHDF monolayers were treated with 1 μM ELQ300 to selectively remove amastigote respiration from the total OCR signal (Infected, <i>±</i>ELQ300). Increased host respiration during <i>T</i>. <i>cruzi</i> infection (Infected, +ELQ300). Mean ± SD shown for 4 biological replicates per condition. Two-way ANOVA with Tukey’s multiple comparisons test was applied (*p<0.05, ***p<0.001, ****p<0.0001). <b>(D)</b> Geometric mean fluorescence intensity of mitochondrial mCherry signal for each condition relative to uninfected controls in NHDF and C2C12 myoblast. Infected cells were discriminated based on <i>T</i>. <i>cruzi</i> GFP expression (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006747#ppat.1006747.s002" target="_blank">S2D Fig</a>), and the geometric mean mCherry fluorescence was determined from each subpopulation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006747#ppat.1006747.s002" target="_blank">S2E Fig</a>). Mean ± SD for 2 independent experiments. Two-way ANOVA with Dunnett’s multiple comparisons test was applied (*p< 0.05, **p< 0.01).</p

<i>T</i>. <i>cruzi</i> infection increases glucose uptake by host cells.

<p><b>(A)</b> Uptake of [³H]-2-deoxyglucose ([³H]-2-DG) into uninfected or <i>T</i>. <i>cruzi</i>-infected NHDF monolayers at 48 hours post infection (48 hpi), in which infection was established with a varying multiplicity of infection (MOI). Mean ± SD shown for 3 biological replicates per MOI. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (*p<0.05, ****p<0.0001). <b>(B)</b> Cytochalasin B (10 μM) blocks uptake of [³H]-2-DG in uninfected and infected NHDF monolayers (48 hpi). Mean ± SD shown for 3 biological replicates. Two-way ANOVA with Tukey’s multiple comparisons test was applied (****p<. 0001)<b>. (C)</b> [³H]-2-DG uptake by NHDF or <b>(D)</b> C2C12 myoblasts following a 48 h infection with <i>T</i>. <i>cruzi</i> Tulahuén, CL Brener or CL-14 strains. NHDF were infected for 2 hours with MOI 40 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. C2C12 were infected for 2 hours with MOI 80 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. Mean ± SD for 3 biological replicates. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (**p< 0.01, ***p< 0.001, ****p< 0.0001).</p

Acquisition and metabolism of glucose by intracellular <i>T</i>. <i>cruzi</i> amastigotes.

<p>Isolated <i>T</i>. <i>cruzi</i> amastigotes utilize exogenous substrates as determined by increased <b>(A)</b> oxygen consumption rate (OCR) and <b>(B)</b> extracellular acidification rate (ECAR). After establishing baseline rates, glucose (5 mM), glutamine (5 mM) or buffer were injected as substrates (subs), followed by 100 mM 2-DG to rapidly inhibit glycolysis, and 1 μM rotenone and antimycin A (R/A) to shut down mitochondrial respiration. Mean ± SD of 3 biological replicates. <b>(C)</b> Initial rate (V₀) of [³H]-2-DG uptake by isolated <i>T</i>. <i>cruzi</i> amastigotes or trypomastigotes plotted for a range of substrate concentrations. Mean ± SD of two independent experiments with biological duplicates shown for each lifecycle stage. Inset shows Lineweaver-Burk plot. <b>(D)</b> Intracellular <i>T</i>. <i>cruzi</i> amastigotes access exogenous hexose <i>in situ</i>. <i>T</i>. <i>cruzi-</i>infected monolayers were incubated with 10 μCi [³H]-2-DG in the absence or presence of cytochalasin B (15 μM) for 20 minutes prior to isolation of intracellular amastigotes for scintillation counts. Mean ± SD of two independent experiments. Student’s t-test was applied (**p< 0.01). <b>(E)</b> [³H]-2-DG is internalized by intracellular amastigotes. Following isolation from monolayers pulsed with [³H]-2-DG, treatment of amastigotes with 0.05 mg/mL alamethicin released internalized, non-bound substrate. Mean ± SD of two independent experiments. <b>(F)</b> ATP levels measured in intracellular-derived amastigotes 24 hours after incubation with the indicated carbon substrate relative to initial ATP levels of freshly isolated parasites. Mean ± SD of 3 biological replicates per condition. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the substrate deficient condition (***p< 0.001, ****p< 0.0001).</p

Microtubule plus‐end binding proteins facilitate intracellular trypanosome infection

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice