Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity

Validation of Messenger Ribonucleic Acid Markers Differentiating Among Human Acute Respiratory Distress Syndrome Subgroups in an Ovine Model of Acute Respiratory Distress Syndrome Phenotypes

BACKGROUND: The discovery of biological subphenotypes in acute respiratory distress syndrome (ARDS) might offer a new approach to ARDS in general and possibly targeted treatment, but little is known about the underlying biology yet. To validate our recently described ovine ARDS phenotypes model, we compared a subset of messenger ribonucleic acid (mRNA) markers in leukocytes as reported before to display differential expression between human ARDS subphenotypes to the expression in lung tissue in our ovine ARDS phenotypes model (phenotype 1 (Ph1): hypoinflammatory; phenotype 2 (Ph2): hyperinflammatory). METHODS: We studied 23 anesthetized sheep on mechanical ventilation with observation times between 6 and 24 h. They were randomly allocated to the two phenotypes (n = 14 to Ph1 and n = 9 to Ph2). At study end, lung tissue was harvested and preserved in RNAlater. After tissue homogenization in TRIzol, total RNA was extracted and custom capture and reporter probes designed by NanoString Technologies were used to measure the expression of 14 genes of interest and the 6 housekeeping genes on a nCounter SPRINT profiler. RESULTS: Among the 14 mRNA markers, in all animals over all time points, 13 markers showed the same trend in ovine Ph2/Ph1 as previously reported in the MARS cohort: matrix metalloproteinase 8, olfactomedin 4, resistin, G protein-coupled receptor 84, lipocalin 2, ankyrin repeat domain 22, CD177 molecule, and transcobalamin 1 expression was higher in Ph2 and membrane metalloendopeptidase, adhesion G protein-coupled receptor E3, transforming growth factor beta induced, histidine ammonia-lyase, and sulfatase 2 expression was higher in Ph1. These expression patterns could be found when different sources of mRNA – such as blood leukocytes and lung tissue – were compared. CONCLUSION: In human and ovine ARDS subgroups, similar activated pathways might be involved (e.g., oxidative phosphorylation, NF-κB pathway) that result in specific phenotypes

Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity

Neurological manifestations of COVID-19 in adults and children

Different neurological manifestations of coronavirus disease 2019 (COVID-19) in adults and children and their impact have not been well characterized. We aimed to determine the prevalence of neurological manifestations and in-hospital complications among hospitalized COVID-19 patients and ascertain differences between adults and children. We conducted a prospective multicentre observational study using the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) cohort across 1507 sites worldwide from 30 January 2020 to 25 May 2021. Analyses of neurological manifestations and neurological complications considered unadjusted prevalence estimates for predefined patient subgroups, and adjusted estimates as a function of patient age and time of hospitalization using generalized linear models. Overall, 161 239 patients (158 267 adults; 2972 children) hospitalized with COVID-19 and assessed for neurological manifestations and complications were included. In adults and children, the most frequent neurological manifestations at admission were fatigue (adults: 37.4%; children: 20.4%), altered consciousness (20.9%; 6.8%), myalgia (16.9%; 7.6%), dysgeusia (7.4%; 1.9%), anosmia (6.0%; 2.2%) and seizure (1.1%; 5.2%). In adults, the most frequent in-hospital neurological complications were stroke (1.5%), seizure (1%) and CNS infection (0.2%). Each occurred more frequently in intensive care unit (ICU) than in non-ICU patients. In children, seizure was the only neurological complication to occur more frequently in ICU versus non-ICU (7.1% versus 2.3%, P &lt; 0.001). Stroke prevalence increased with increasing age, while CNS infection and seizure steadily decreased with age. There was a dramatic decrease in stroke over time during the pandemic. Hypertension, chronic neurological disease and the use of extracorporeal membrane oxygenation were associated with increased risk of stroke. Altered consciousness was associated with CNS infection, seizure and stroke. All in-hospital neurological complications were associated with increased odds of death. The likelihood of death rose with increasing age, especially after 25 years of age. In conclusion, adults and children have different neurological manifestations and in-hospital complications associated with COVID-19. Stroke risk increased with increasing age, while CNS infection and seizure risk decreased with age.</p

Characterizing preclinical sub-phenotypic models of acute respiratory distress syndrome:An experimental ovine study

Abstract The acute respiratory distress syndrome (ARDS) describes a heterogenous population of patients with acute severe respiratory failure. However, contemporary advances have begun to identify distinct sub‐phenotypes that exist within its broader envelope. These sub‐phenotypes have varied outcomes and respond differently to several previously studied interventions. A more precise understanding of their pathobiology and an ability to prospectively identify them, may allow for the development of precision therapies in ARDS. Historically, animal models have played a key role in translational research, although few studies have so far assessed either the ability of animal models to replicate these sub‐phenotypes or investigated the presence of sub‐phenotypes within animal models. Here, in three ovine models of ARDS, using combinations of oleic acid and intravenous, or intratracheal lipopolysaccharide, we investigated the presence of sub‐phenotypes which qualitatively resemble those found in clinical cohorts. Principal Component Analysis and partitional clustering identified two clusters, differentiated by markers of shock, inflammation, and lung injury. This study provides a first exploration of ARDS phenotypes in preclinical models and suggests a methodology for investigating this phenomenon in future studies

Studying the endothelial glycocalyx in vitro: what is missing?

All human cells are coated by a surface layer of proteoglycans, glycosaminoglycans (GAGs) and plasma proteins, called the glycocalyx. The glycocalyx transmits shear stress to the cytoskeleton of endothelial cells, maintains a selective permeability barrier, and modulates adhesion of blood leukocytes and platelets. Major components of the glycocalyx, including syndecans, heparan sulfate, and hyaluronan, are shed from the endothelial surface layer during conditions including ischaemia and hypoxia, sepsis, atherosclerosis, diabetes, renal disease, and some viral infections. Studying mechanisms of glycocalyx damage in vivo can be challenging due to the complexity of immuno-inflammatory responses which are inextricably involved. Previously, both static as well as perfused in vitro models have studied the glycocalyx, and have reported either imaging data, assessment of barrier function, or interactions of blood components with the endothelial monolayer. To date, no model has simultaneously incorporated all these features at once, however such a model would arguably enhance the study of vasculopathic processes. This review compiles a series of current in vitro models described in the literature that have targeted the glycocalyx layer, their limitations, and potential opportunities for further developments in this field

Combined Mesenchymal Stromal Cell Therapy and ECMO in ARDS:A Controlled Experimental Study in Sheep

Rationale: Mesenchymal stromal cell (MSC) therapy is a promising intervention for acute respiratory distress syndrome (ARDS), although trials to date have not investigated its use alongside extracorporeal membrane oxygenation (ECMO). Recent preclinical studies have suggested that combining these interventions may attenuate the efficacy of ECMO. Objectives: To determine the safety and efficacy of MSC therapy in a model of ARDS and ECMO. Methods: ARDS was induced in 14 sheep, after which they were established on venovenous ECMO. Subsequently, they received either endobronchial induced pluripotent stem cell-derived human MSCs (hMSCs) (n = 7) or cell-free carrier vehicle (vehicle control; n = 7). During ECMO, a low VT ventilation strategy was employed in addition to protocolized hemodynamic support. Animals were monitored and supported for 24 hours. Lung tissue, bronchoalveolar fluid, and plasma were analyzed, in addition to continuous respiratory and hemodynamic monitoring. Measurements and Main Results: The administration of hMSCs did not improve oxygenation (PaO2/FIO2 mean difference =2146mmHg; P= 0.076) or pulmonary function.However, histological evidence of lung injury(lung injuryscoremeandifference=20.07;P=0.04) and BALIL-8 were reduced. In addition, hMSC-treated animals had a significantly lower cumulative requirement for vasopressor. Despite endobronchial administration, animals treated with hMSCs had a significant elevation in transmembrane oxygenator pressure gradients. Thiswas accompanied by more pulmonary artery thromboses and adherent hMSCs found on explanted oxygenator fibers. Conclusions: Endobronchial hMSC therapy in an ovine model of ARDS and ECMO can impair membrane oxygenator function and does not improve oxygenation. These data do not recommend the safe use of hMSCs during venovenous ECMO. </p

Administration of mesenchymal stem cells during ECMO results in a rapid decline in oxygenator performance

Mesenchymal stem cells (MSCs) have attracted attention as a potential therapy for Acute Respiratory Distress Syndrome (ARDS). At the same time, the use of extracorporeal membrane oxygenation (ECMO) has increased among patients with severe ARDS. To date, early clinical trials of MSCs in ARDS have excluded patients supported by ECMO. Here we provide evidence from an ex-vivo model of ECMO to suggest that the intravascular administration of MSCs during ECMO may adversely impact the function of a membrane oxygenator. The addition of clinical grade MSCs resulted in a reduction of flow through the circuit in comparison to controls (0.6 ±0.35 L min -1 vs 4.12 ± 0.03 L min -1 , at 240 minutes) and an increase in the transoygenator pressure gradient (101±9 mmHg vs 21±4 mmHg, at 240 minutes). Subsequent immunohistochemistry analysis demonstrated quantities of MSCs highly adherent to membrane oxygenator fibres. This study highlights the potential harm associated with MSC therapy during ECMO and suggests further areas of research required to advance the translation of cell therapy in this population. </p