Background
This large-scale analysis pools individual data about the Clinical Frailty Scale (CFS) to predict outcome in the intensive care unit (ICU).
Methods
A systematic search identified all ...clinical trials that used the CFS in the ICU (PubMed searched until 24th June 2020). All patients who were electively admitted were excluded. The primary outcome was ICU mortality. Regression models were estimated on the complete data set, and for missing data, multiple imputations were utilised. Cox models were adjusted for age, sex, and illness acuity score (SOFA, SAPS II or APACHE II).
Results
12 studies from 30 countries with anonymised individualised patient data were included (n = 23,989 patients). In the univariate analysis for all patients, being frail (CFS ≥ 5) was associated with an increased risk of ICU mortality, but not after adjustment. In older patients (≥ 65 years) there was an independent association with ICU mortality both in the complete case analysis (HR 1.34 (95% CI 1.25–1.44), p < 0.0001) and in the multiple imputation analysis (HR 1.35 (95% CI 1.26–1.45), p < 0.0001, adjusted for SOFA). In older patients, being vulnerable (CFS 4) alone did not significantly differ from being frail. After adjustment, a CFS of 4–5, 6, and ≥ 7 was associated with a significantly worse outcome compared to CFS of 1–3.
Conclusions
Being frail is associated with a significantly increased risk for ICU mortality in older patients, while being vulnerable alone did not significantly differ. New Frailty categories might reflect its “continuum” better and predict ICU outcome more accurately.
Trial registration:
Open Science Framework (OSF:
https://osf.io/8buwk/
).
Graphical Abstract
Abstract Background aims The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving ...product yield and driving manufacturing consistency. Methods Bone marrow–derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate ( P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation ( R2 = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.
The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, ...lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25–5.0 μg/mL) and/or carbendazim (0.8–1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the “DeepFlow” neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for ‘mononucleates’, ‘binucleates’, ‘mononucleates with MN’ and ‘binucleates with MN’, respectively. Successful classifications of ‘trinucleates’ (90%) and ‘tetranucleates’ (88%) in addition to ‘other or unscorable’ phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.
The purpose of this study was to develop a bioartificial liver (BAL) to treat patients with severe liver failure until they can be either transplanted or recover spontaneously.
Severe acute liver ...failure is associated with high mortality. Liver transplantation has emerged as an effective therapy for patients who did not respond to standard management. However, because of the donor organ shortage and urgent need for transplantation, many patients die before they can be transplanted and others do not survive after transplantation, primarily because of intracranial hypertension.
Three groups of patients with severe acute liver failure were treated with the BAL. In group 1 (n = 18) were patients with fulminant hepatic failure (FHF), in group 2 (n = 3) were patients with primary nonfunction (PNF) of a transplanted liver, and in group 3 (n = 10) were patients with acute exacerbation of chronic liver disease. Patients in groups 1 and 2 were candidates for transplantation at the time they entered the study, whereas patients in group 3 were not.
In group 1, 16 patients were "bridged" successfully to transplantation, 1 patient was bridged to recovery without a transplant, and 1 patient died because of concomitant severe pancreatitis. In group 2, all patients were bridged successfully to retransplantation. In group 3, two patients were supported to recovery and successful transplants at later dates; the other eight patients, although supported temporarily with the BAL, later died because they were not candidates for transplantation.
The authors' clinical experience with the BAL has yielded encouraging results. A randomized, controlled, prospective trial (phase II-III) is being initiated to determine the efficacy of the system.
Dialysis-related amyloidosis (DRA) is a complication of hemodialysis where β2-microglobulin (β2m) forms plaques mainly in cartilaginous tissues. The tissue-specific deposition, along with a known ...intransigence of pure β2m to form fibrils in vitro at neutral pH in the absence of preformed fibrillar seeds, suggests a role for factors within cartilage in enhancing amyloid formation from this protein. To identify these factors, we determined the ability of a derivative lacking the N-terminal six amino acids found in ex vivo β2m amyloid deposits to form amyloid fibrils at pH 7.4 in the absence of fibrillar seeds. We show that the addition of the glycosaminoglycans (GAGs) chrondroitin-4 or 6-sulfate to fibril growth assays results in the spontaneous generation of amyloid-like fibrils. By contrast, no fibrils are observed over the same time course in the presence of hyaluronic acid, a nonsulfated GAG that is abundant in cartilaginous joints. Based on the observation that hyaluronic acid has no effect on fibril stability, while chrondroitin-6-sulfate decreases the rate of fibril disassembly, we propose that the latter GAG enhances amyloid formation by stabilizing the rare fibrils that form spontaneously. This leads to the accumulation of β2m in fibrillar deposits. Our data rationalize the joint-specific deposition of β2m amyloid in DRA, suggesting mechanisms by which amyloid formation may be promoted.
Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, in situ segmentation of all cells imaged. Currently, achieving this is complex and requires ...exogenous fluorescent labels that occupy significant spectral bandwidth, increasing the duration and complexity of imaging experiments while limiting the number of channels remaining to address the study’s objectives. We demonstrate that the excitation light reflected during routine confocal microscopy contains sufficient information to achieve accurate, label-free cell segmentation in 2D and 3D. This is achieved using a simple convolutional neural network trained to predict the probability that reflected light pixels belong to either nucleus, cytoskeleton, or background classifications. We demonstrate the approach across diverse lymphoid tissues and provide video tutorials demonstrating deployment in Python and MATLAB or via standalone software for Windows.
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•Cell segmentation of tissues can be achieved from reflected laser excitation light•Single-cell information is freely established for many tissue microscopy studies•Windows software is provided alongside extensive video tutorials and data
Across the biomedical sciences, there is an urgent need to move beyond qualitative imaging to quantitative, cell-based reporting of tissue microscopy data. Typically, cell segmentation requires fluorescent labeling of nucleus and cytoplasm, which limits the spectral bandwidth available for other reporter molecules. However, recent advances in deep-learning algorithms have transformed automated image classification, and this raises the possibility of proceeding with reduced image information. Here, we show that 2D and 3D cell segmentation of lymphoid tissues can be freely established from the reflected laser excitation light always present during routine confocal microscopy using entirely standard equipment.
Cell segmentation is an essential step in quantitative tissue microscopy. Wills et al. show this can be achieved simply using the reflected laser light always present during routine imaging by confocal microscopy. This frees up microscope channels and establishes single-cell information as an attainable start point for many tissue microscopy experiments.
Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly ...defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiffness was found in diabetic GK compared with Wistar control rats. This correlated with impaired structural organization of collagen fibers and a reduced expression of collagen I and III in the injured tendons of the diabetic GK compared with Wistar control. Moreover, MMP-3 gene expression was downregulated in the injured diabetic GK tendons compared with injured Wistar controls. Our results indicate that in a rat model of diabetes tendon healing is impaired mainly due to altered expression of collagen and MMPs reflecting decreased degradation of matrix proteins and impaired tissue remodeling. Further our data suggest that therapeutic modulation of collagens or MMPs might be targets for new regenerative approaches in operated, injured, or maybe also degenerative tendon diseases in diabetes.
Mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) cause autosomal dominant forms of Alzheimer disease (ADAD). More than 280 pathogenic mutations have been ...reported in APP, PSEN1, and PSEN2. However, understanding of the basic biological mechanisms that drive the disease are limited. The Dominantly Inherited Alzheimer Network (DIAN) is an international observational study of APP, PSEN1, and PSEN2 mutation carriers with the goal of determining the sequence of changes in presymptomatic mutation carriers who are destined to develop Alzheimer disease.
We generated a library of 98 dermal fibroblast lines from 42 ADAD families enrolled in DIAN. We have reprogrammed a subset of the DIAN fibroblast lines into patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized for pluripotency markers.
This library represents a comprehensive resource that can be used for disease modeling and the development of novel therapeutics.
In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli ...fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case. However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7.
Background: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal ...rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. Methods and results: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. Conclusion: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.
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