The ARDS microenvironment enhances MSC-induced repair via VEGF in experimental acute lung inflammation.

Clinical trials investigating the potential of mesenchymal stromal cells (MSCs) for the treatment of inflammatory diseases, such as acute respiratory distress syndrome (ARDS), have been disappointing, with less than 50% of patients responding to treatment. Licensed MSCs show enhanced therapeutic efficacy in response to cytokine-mediated activation signals. There are two distinct sub-phenotypes of ARDS: hypo- and hyper-inflammatory. We hypothesized that pre-licensing MSCs in a hyper-inflammatory ARDS environment would enhance their therapeutic efficacy in acute lung inflammation (ALI). Serum samples from patients with ARDS were segregated into hypo- and hyper-inflammatory categories based on interleukin (IL)-6 levels. MSCs were licensed with pooled serum from patients with hypo- or hyper-inflammatory ARDS or healthy serum controls. Our findings show that hyper-inflammatory ARDS pre-licensed MSC conditioned medium (MSC-CMHyper) led to a significant enrichment in tight junction expression and enhanced barrier integrity in lung epithelial cells in vitro and in vivo in a vascular endothelial growth factor (VEGF)-dependent manner. Importantly, while both MSC-CMHypo and MSC-CMHyper significantly reduced IL-6 and tumor necrosis factor alpha (TNF-α) levels in the bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-induced ALI mice, only MSC-CMHyper significantly reduced lung permeability and overall clinical outcomes including weight loss and clinical score. Thus, the hypo- and hyper-inflammatory ARDS environments may differentially influence MSC cytoprotective and immunomodulatory functions.

Semiquantitative Paper-Based Microfluidic Surrogate Virus Neutralization Test for SARS-CoV-2 Neutralizing Antibodies.

Neutralizing antibodies (nAbs) produced from infection or vaccination play an important role in acquired immunity. Determining virus-specific nAb titers is a useful tool for measuring aquired immunity in an individual. The standard methods to do so rely on titrating serum samples against live virus and monitoring viral infection in cultured cells which requires high biosafety level containment. The surrogate virus neutralization test (sVNT) reduces the biohazards and it is suitable for designing rapid test device in a lateral flow assay (LFA) format. Here, we introduce the fabrication and development of a unique paper-based LFA device for determining the level of SARS-CoV-2 nAb in a sample with a semiquantitative direct colorimetric readout. A LFA-based gradient assay design was used to facilitate the sVNT, where the spike glycoprotein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) stand in as proxies for viruses and cells, respectively. The gradient assay employed multiple test dots of ACE2 spotted in increasing concentration along the sample flow path and gold nanoparticle-conjugated RBD for readout. In this way, the number of developed spots is inversely proportional to the concentration of nAbs present in the sample. The assay was tested with both standard solutions of nAb as well as human serum samples. We have demonstrated that the device can effectively provide semiquantitative test results of nAbs by direct instrument-free colorimetric detection.

Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis.

BACKGROUND AIMS: Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. METHODS: An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). RESULTS: No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen ß-chain were the most differentially expressed proteins between severity groups. CONCLUSION: Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.

Tiny Guides, Big Impact: Focus on the Opportunities and Challenges of miR-Based Treatments for ARDS.

Acute Respiratory Distress Syndrome (ARDS) is characterized by lung inflammation and increased membrane permeability, which represents the leading cause of mortality in ICUs. Mechanical ventilation strategies are at the forefront of supportive approaches for ARDS. Recently, an increasing understanding of RNA biology, function, and regulation, as well as the success of RNA vaccines, has spurred enthusiasm for the emergence of novel RNA-based therapeutics. The most common types of RNA seen in development are silencing (si)RNAs, antisense oligonucleotide therapy (ASO), and messenger (m)RNAs that collectively account for 80% of the RNA therapeutics pipeline. These three RNA platforms are the most mature, with approved products and demonstrated commercial success. Most recently, miRNAs have emerged as pivotal regulators of gene expression. Their dysregulation in various clinical conditions offers insights into ARDS pathogenesis and offers the innovative possibility of using microRNAs as targeted therapy. This review synthesizes the current state of the literature to contextualize the therapeutic potential of miRNA modulation. It considers the potential for miR-based therapeutics as a nuanced approach that incorporates the complexity of ARDS pathophysiology and the multifaceted nature of miRNA interactions.

Association of Epigenetic Age and Outcome in Critically Ill Patients.

OBJECTIVES: DNA methylation can be used to determine an individual's biological age, as opposed to chronological age, an indicator of underlying health status. This study aimed to assess epigenetic age in critically ill patients with and without sepsis to determine if higher epigenetic age is associated with admission diagnosis or mortality. DESIGN: Secondary analysis of whole blood DNA methylation data generated from a nested case-control study of critically ill septic and nonseptic patients. SETTING: Four tertiary care hospitals in Canada. INTERVENTIONS: None. PATIENTS: Critically ill patients with and without sepsis. MEASUREMENTS AND MAIN RESULTS: Epigenetic age was derived from DNA methylation data using the Hannum and PhenoAge algorithms and deviation from the patient's chronological age in years was determined. Of the 66 patients with sepsis, 34 were male (51.5%), the mean age was 65.03 years and 25 patients (37.8%) died before discharge. Of the 68 nonseptic patients, 47 were male (69.1%), the mean age was 64.92 years and 25 (36.7%) died before discharge. Epigenetic age calculated using the PhenoAge algorithm showed a significant age acceleration of 4.97 years in septic patients (p = 0.045), but no significant acceleration in nonseptic patients. Epigenetic age calculated using the Hannum algorithm showed no significant acceleration in the septic or nonseptic patients. Similarly, in the combined septic and nonseptic cohorts, nonsurvivors showed an epigenetic age acceleration of 7.62 years (p = 0.004) using the PhenoAge algorithm while survivors showed no significant age acceleration. Survivor status was not associated with age acceleration using the Hannum algorithm. CONCLUSIONS: In critically ill patients, epigenetic age acceleration, as calculated by the PhenoAge algorithm, was associated with sepsis diagnosis and mortality.

Barriers to participation in biosampling-based translational research: A cross-sectional survey of Canadian critical care researchers.

BACKGROUND AND OBJECTIVE: Collection of biosamples for translational research studies is vital for understanding biological pathways, discovering disease-related biomarkers, and identifying novel therapeutic targets. However, a lack of infrastructure for sample procurement, processing, storage, and shipping may hinder the ability of clinical research units to effectively engage in translational research. The purpose of this study was to identify the barriers to biosampling-based translational research in the critical care setting in Canada. METHODS: We administered an online survey to members of the Canadian Critical Care Trials Group (CCCTG), the Canadian Critical Care Translational Biology Group (CCCTBG), and the Canadian Critical Care Research Coordinators Group (CCCRCG). The survey focused on participants' personal experience of biosampling research, research infrastructure, motivating factors, and perceived barriers. RESULTS: We received 59 responses from 31 sites, including 6 community intensive care unit (ICU) sites. The overall response rate was 11.3%. The majority of respondents were research coordinators (44%), followed by clinician-investigators (33.8%), graduate students (10.2%), and PhD-investigators (8.5%). Although most (63.8%) respondents reported an interest in participating in translational research, they also reported that their ICUs were currently contributing to a third of the number of translational studies compared to clinical studies. For respondents with experience in participating in translational research studies, the most common barriers were lack of funding, lack of time, and insufficient research staff. For respondents without previous experience, the perceived facilitators were more interest from their research group, improved training/mentorship, increased funding, and better access to laboratory equipment. CONCLUSIONS: Our survey found that the majority of participants were interested in and recognize the value of participating in biosampling-based translational research but lacked funding, time, and research personnel trained in biosampling protocols. Our survey also identified factors that might encourage participation at new sites. Addressing these barriers will be a key step towards increasing translational research capacity across Canada.

The MSC-EV-microRNAome: A Perspective on Therapeutic Mechanisms of Action in Sepsis and ARDS.

Mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) have emerged as innovative therapeutic agents for the treatment of sepsis and acute respiratory distress syndrome (ARDS). Although their potential remains undisputed in pre-clinical models, this has yet to be translated to the clinic. In this review, we focused on the role of microRNAs contained in MSC-derived EVs, the EV microRNAome, and their potential contribution to therapeutic mechanisms of action. The evidence that miRNA transfer in MSC-derived EVs has a role in the overall therapeutic effects is compelling. However, several questions remain regarding how to reconcile the stochiometric issue of the low copy numbers of the miRNAs present in the EV particles, how different miRNAs delivered simultaneously interact with their targets within recipient cells, and the best miRNA or combination of miRNAs to use as therapy, potency markers, and biomarkers of efficacy in the clinic. Here, we offer a molecular genetics and systems biology perspective on the function of EV microRNAs, their contribution to mechanisms of action, and their therapeutic potential.

Original article: novelty of Canadian manufacture nasopharyngeal swabs for collection of samples being tested for SARS-CoV-2 in a pandemic setting.

Objectives: The COVID-19 pandemic caused a global shortage of nasopharyngeal (NP) swabs, required for RT-PCR testing. Canadian manufacturers were contacted to share NP swab innovations. The primary objective was to determine whether novel NP test swabs were comparable to commercially available swabs regarding user characteristics, ability to collect a specimen, and diagnostic performance using RT-PCR testing. Methods: Participants were randomized by swab (test/control) and nostril (left/right). A calculated positive percent agreement ≥90% was considered successful. Mean Ct values of viral genes and housekeeping gene (RNase P) were considered similar if a Ct difference ≤ 2 between control and test group was obtained. There also was a qualitative assessment of swabs usability. Results: 647 participants were enrolled from Huaycan Hospital in Lima, Peru, distributed over 8 NP swabs brands. Seven brands agreed to share their results. There were no statistically significant differences between the test swabs of these 7 brands and control swabs. Conclusion: All the seven brands are comparable to the commercially available flocked swabs used for SARS-CoV-2 regarding test results agreement, ability to collect a specimen, and user characteristics.

Efficacy and Safety of Umbilical Cord-Derived Mesenchymal Stromal Cell Therapy in Preclinical Models of Sepsis: A Systematic Review and Meta-analysis.

BACKGROUND: In preclinical studies, mesenchymal stromal cells (MSCs), including umbilical cord-derived MSCs (UC-MSCs), demonstrate the ability to modulate numerous pathophysiological processes related to sepsis; however, a systematic synthesis of the literature is needed to assess the efficacy of UC-MSCs for treating sepsis. OBJECTIVE: To examine the effects of UC-MSCs on overall mortality (primary outcome) as well as on organ dysfunction, coagulopathy, endothelial permeability, pathogen clearance, and systemic inflammation (secondary outcomes) at prespecified time intervals in preclinical models of sepsis. METHODS: A systematic search was conducted on Embase, Ovid MEDLINE, and Web of Science up to June 20, 2023. Preclinical controlled studies using in vivo sepsis models with systemic UC-MSC administration were included. Meta-analyses were conducted and expressed as odds ratios (OR) and ratios of the weighted means with 95% CI for categorical and continuous data, respectively. Risk of bias was assessed with the SYRCLE tool. RESULTS: Twenty-six studies (34 experiments, n = 1258 animals) were included in this review. Overall mortality was significantly reduced with UC-MSC treatment as compared to controls (OR: 0.26, 95% CI: 0.18-0.36). At various prespecified time intervals, UC-MSCs reduced surrogate measures of organ dysfunction related to the kidney, liver, and lung; reduced coagulopathy and endothelial permeability; and enhanced pathogen clearance from multiple sites. UC-MSCs also modulated systemic inflammatory mediators. No studies were rated as low risk across all SYCLE domains. CONCLUSIONS: These results demonstrate the efficacy of UC-MSC treatment in preclinical sepsis models and highlight their potential as a therapeutic intervention for septic shock.

Cardiac tissue model of immune-induced dysfunction reveals the role of free mitochondrial DNA and the therapeutic effects of exosomes.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.