Endothelial cell-derived extracellular vesicles modulate the therapeutic efficacy of mesenchymal stem cells through IDH2/TET pathway in ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and fatal disease. Although mesenchymal stem cell (MSC)-based therapy has shown remarkable efficacy in treating ARDS in animal experiments, clinical outcomes have been unsatisfactory, which may be attributed to the influence of the lung microenvironment during MSC administration. Extracellular vesicles (EVs) derived from endothelial cells (EC-EVs) are important components of the lung microenvironment and play a crucial role in ARDS. However, the effect of EC-EVs on MSC therapy is still unclear. In this study, we established lipopolysaccharide (LPS) - induced acute lung injury model to evaluate the impact of EC-EVs on the reparative effects of bone marrow-derived MSC (BM-MSC) transplantation on lung injury and to unravel the underlying mechanisms. METHODS: EVs were isolated from bronchoalveolar lavage fluid of mice with LPS - induced acute lung injury and patients with ARDS using ultracentrifugation. and the changes of EC-EVs were analysed using nanoflow cytometry analysis. In vitro assays were performed to establish the impact of EC-EVs on MSC functions, including cell viability and migration, while in vivo studies were performed to validate the therapeutic effect of EC-EVs on MSCs. RNA-Seq analysis, small interfering RNA (siRNA), and a recombinant lentivirus were used to investigate the underlying mechanisms. RESULTS: Compared with that in non-ARDS patients, the quantity of EC-EVs in the lung microenvironment was significantly greater in patients with ARDS. EVs derived from lipopolysaccharide-stimulated endothelial cells (LPS-EVs) significantly decreased the viability and migration of BM-MSCs. Furthermore, engrafting BM-MSCs pretreated with LPS-EVs promoted the release of inflammatory cytokines and increased pulmonary microvascular permeability, aggravating lung injury. Mechanistically, LPS-EVs reduced the expression level of isocitrate dehydrogenase 2 (IDH2), which catalyses the formation of α-ketoglutarate (α-KG), an intermediate product of the tricarboxylic acid (TCA) cycle, in BM-MSCs. α-KG is a cofactor for ten-eleven translocation (TET) enzymes, which catalyse DNA hydroxymethylation in BM-MSCs. CONCLUSIONS: This study revealed that EC-EVs in the lung microenvironment during ARDS can affect the therapeutic efficacy of BM-MSCs through the IDH2/TET pathway, providing potential strategies for improving the therapeutic efficacy of MSC-based therapy in the clinic.

Endothelial cell-derived extracellular vesicles expressing surface VCAM1 promote sepsis-related acute lung injury by targeting and reprogramming monocytes.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life-threatening syndrome with no effective pharmacotherapy. Sepsis-related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell-derived EVs (EC-EVs) play a crucial role in ALI/ARDS development, but the mechanism remains largely unknown. Here, we demonstrated that the number of circulating EC-EVs was increased in sepsis, exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1), overexpressed on EC-EVs during sepsis, activated the NF-κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface, rather than the tissue resident macrophage surface, thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC-EVs or blocking ITGA4 on monocytes. Furthermore, the number of VCAM1+ EC-EVs was significantly increased in patients with sepsis-related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis-related ALI/ARDS, but also provide potential novel targets and strategies for its precise treatment.

Activation of Wnt/ß­Catenin­p130/E2F4 promotes the differentiation of bone marrow­derived mesenchymal stem cells into type II alveolar epithelial cells through cell cycle arrest.

The results of our previous study demonstrated that activation of the Wnt/ß-catenin pathway increased the differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells; however, the specific mechanisms remain unclear. The present study aimed to evaluate the role of Wnt/ß-catenin-p130/E2F transcription factor 4 (E2F4) in regulating the differentiation of mouse MSCs (mMSCs) into AT II cells, and to determine the specific mechanisms. mMSCs with p130 or E2F4 overexpression were constructed using lentiviral vectors. Differentiation of mMSCs into AT II cells was promoted using a modified coculture system with murine lung epithelial-12 cells incubated in small airway growth medium for 7-14 days. The differentiation efficiency was detected using immunofluorescence, western blot analysis and transmission electron microscopy. To detect the association between the canonical Wnt pathway and p130/E2F4, 4 mmol/l lithium chloride (LiCl) or 200 ng/ml Dickkopf-related protein 1 (DKK-1) was also added to the coculture system. Following differentiation, the cell cycle of mMSCs was evaluated using flow cytometry. The results of the present study demonstrated that surfactant protein C (SP-C) protein expression was higher in the p130 overexpression (MSC-p130) and E2F4 overexpression (MSC-E2F4) groups compared with the normal control mMSCs group following differentiation into AT II cells. Similar results for SP-C protein expression and lamellar body-like structures were also observed using immunofluorescence analysis and electron microscopy. Following the addition of LiCl into the coculture system for activation of the Wnt/ß-catenin signaling pathway, phosphorylated (p)-p130/p130 was slightly decreased at 7 days and E2F4 was increased both at 7 and 14 days in mMSCs. Furthermore, the p-p130/p130 ratio was significantly increased at 14 days and E2F4 was decreased both at 7 and 14 days following DKK-1-mediated inhibition of the Wnt pathway. The results of the present study demonstrated that the numbers of cells in G1 and S phases were increased following activation of the Wnt pathway and decreased following Wnt pathway inhibition. However, the number of cells in G1 phase was increased following the differentiation of mMSCs overexpressing p130 or E2F4. Therefore, the results of the present study revealed that the canonical Wnt signaling pathway may affect the differentiation of MSCs into AT II cells via regulation of downstream p130/E2F4. The specific mechanisms may be associated with G1 phase extension in the cell cycle of MSCs.

Effect of EIT-guided PEEP titration on prognosis of patients with moderate to severe ARDS: study protocol for a multicenter randomized controlled trial.

BACKGROUND: Acute respiratory syndrome distress (ARDS) is a clinical common syndrome with high mortality. Electrical impedance tomography (EIT)-guided positive end-expiratory pressure (PEEP) titration can achieve the compromise between lung overdistension and collapse which may minimize ventilator-induced lung injury in these patients. However, the effect of EIT-guided PEEP titration on the clinical outcomes remains unknown. The objective of this trial is to investigate the effects of EIT-guided PEEP titration on the clinical outcomes for moderate or severe ARDS, compared to the low fraction of inspired oxygen (FiO2)-PEEP table. METHODS: This is a prospective, multicenter, single-blind, parallel-group, adaptive designed, randomized controlled trial (RCT) with intention-to-treat analysis. Adult patients with moderate to severe ARDS less than 72 h after diagnosis will be included in this study. Participants in the intervention group will receive PEEP titrated by EIT with a stepwise decrease PEEP trial, whereas participants in the control group will select PEEP based on the low FiO2-PEEP table. Other ventilator parameters will be set according to the ARDSNet strategy. Participants will be followed up until 28 days after enrollment. Three hundred seventy-six participants will be recruited based on a 15% decrease of 28-day mortality in the intervention group, with an interim analysis for sample size re-estimation and futility assessment being undertaken once 188 participants have been recruited. The primary outcome is 28-day mortality. The secondary outcomes include ventilator-free days and shock-free days at day 28, length of ICU and hospital stay, the rate of successful weaning, proportion requiring rescue therapies, compilations, respiratory variables, and Sequential Organ Failure Assessment (SOFA). DISCUSSION: As a heterogeneous syndrome, ARDS has different responses to treatment and further results in different clinical outcomes. PEEP selection will depend on the properties of patients and can be individually achieved by EIT. This study will be the largest randomized trial to investigate thoroughly the effect of individual PEEP titrated by EIT in moderate to severe ARDS patients to date. TRIAL REGISTRATION: ClinicalTrial.gov NCT05207202. First published on January 26, 2022.

Identifying high-risk phenotypes and associated harms of delayed time-to-antibiotics in patients with ICU onset sepsis: A retrospective cohort study.

PURPOSE: To identify phenotypes of Intensive Care Unit (ICU) onset sepsis and its associated harms of delayed time-to-antibiotics. MATERIALS AND METHODS: The Medical Information Mart for Intensive Care IV (MIMIC-IV) database was employed to identify patients with ICU onset sepsis. The primary exposure was time-to-antibiotics, as measured from sepsis recognition to first antibiotic administered. Latent profile analysis (LPA) was used to identify phenotypes of sepsis based on individual organ failure score derived from Sequential Organ Failure Assessment (SOFA). Interactions between phenotypes and time-to-antibiotics on 28-day mortality were explored. RESULTS: 6246 patients were enrolled in final analysis. The overall 28-day mortality was 12.7%. Delayed time-to-antibiotics was associated with increased 28-day mortality in patients with ICU onset sepsis (HR 1.12, 95% CI 1.08-1.18). Four phenotypes of sepsis were identified: phenotype 1 was characterized by respiratory dysfunction, phenotype 2 was characterized by cardiovascular dysfunction, phenotype 3 was characterized by multiple organ dysfunction, and phenotype 4 was characterized by neurological dysfunction. The adjusted HR of 28-day mortality was 1.16 (95% CI 1.08-1.25) in phenotype 1, and 1.06 (95% CI 1.00-1.13) in phenotype 2, while no significant interaction was observed. CONCLUSIONS: Septic patients with respiratory or cardiovascular dysfunction were associated with harms of delayed time-to-antibiotics.

Pharmacokinetic/Pharmacodynamic Parameters of Linezolid in the Epithelial Lining Fluid of Patients With Sepsis.

The aim of this study was to investigate the pharmacokinetic/pharmacodynamic parameters of linezolid in both the plasma and epithelial lining fluid (ELF) of patients with pneumonia-induced sepsis. Blood specimens and bronchoalveolar lavage samples were collected at defined time points after administration of linezolid. The concentration in the ELF was calculated by urea dilution method. PK parameters were calculated, and probability of target attainment was evaluated by Monte Carlo simulations. Twenty-three patients were enrolled, 8 of whom had septic shock. The maximum concentration of linezolid was higher in the ELF than in the plasma (36.02 ± 13.17 vs 19.51±4.83 mg/L, P < .001) in all of the patients. In patients with septic shock, the maximum concentration in the ELF was significantly higher than that in the non-septic shock group (45.25 ± 11.70 vs 31.10 ± 11.38 mg/L, P = .01), while there was no significant difference in the plasma. The corresponding probability of target attainment values were 90.5% and 65.1% in ELF and plasma, respectively, with a minimum inhibitory concentration of 2 mg/L, which were 99.9% in the ELF in the patients with septic shock. Linezolid possesses an efficient penetration into the ELF of patients with pneumonia-induced sepsis with mechanical ventilation. When minimum inhibitory concentration ≤ 2 mg/L, 600 mg of linezolid every 12 hours could achieve the optimal therapeutic targets in the ELF rather than in the plasma of patients with pneumonia-induced sepsis.

Therapeutic effects of high molecular weight hyaluronic acid in severe Pseudomonas aeruginosa pneumonia in ex vivo perfused human lungs.

We previously reported that extracellular vesicles (EVs) released during Escherichia coli (E. coli) bacterial pneumonia were inflammatory, and administration of high molecular weight hyaluronic acid (HMW HA) suppressed several indices of acute lung injury (ALI) from E. coli pneumonia by binding to these inflammatory EVs. The current study was undertaken to study the therapeutic effects of HMW HA in ex vivo perfused human lungs injured with Pseudomonas aeruginosa (PA)103 bacterial pneumonia. For lungs with baseline alveolar fluid clearance (AFC) <10%/h, HMW HA 1 or 2 mg was injected intravenously after 1 h (n = 4-9), and EVs released during PA pneumonia were collected from the perfusate over 6 h. For lungs with baseline AFC > 10%/h, HMW HA 2 mg was injected intravenously after 1 h (n = 6). In vitro experiments were conducted to evaluate the effects of HA on inflammation and bacterial phagocytosis. For lungs with AFC < 10%/h, administration of HMW HA intravenously significantly restored AFC and numerically decreased protein permeability and alveolar inflammation from PA103 pneumonia but had no effect on bacterial counts at 6 h. However, HMW HA improved bacterial phagocytosis by human monocytes and neutrophils and suppressed the inflammatory properties of EVs released during pneumonia on monocytes. For lungs with AFC > 10%/h, administration of HMW HA intravenously improved AFC from PA103 pneumonia but had no significant effects on protein permeability, inflammation, or bacterial counts. In the presence of impaired alveolar epithelial transport capacity, administration of HMW HA improved the resolution of pulmonary edema from Pseudomonas PA103 bacterial pneumonia.

Synbiotic Therapy Prevents Nosocomial Infection in Critically Ill Adult Patients: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials Based on a Bayesian Framework.

Background: The efficacy of synbiotics, probiotics, prebiotics, enteral nutrition or adjuvant peripheral parenteral nutrition (EPN) and total parenteral nutrition (TPN) in preventing nosocomial infection (NI) in critically ill adults has been questioned. We conducted a systematic review and network meta-analysis (NMA) of randomized controlled trials (RCTs) to evaluate and rank the effectiveness of these therapies on NI amongst critically ill adults. Methods: Four electronic databases were systematically searched up to June 30, 2019 for RCTs comparing the administration of probiotics, prebiotics, synbiotics, EPN and TPN in critically ill adults. The primary outcome was NI. The relative efficacy of all outcomes was determined by a Bayesian framework with random effects NMA. We estimated the odds ratio (OR) and mean difference (MD) and ranked the comparative effects of all regimens with the surface under the cumulative ranking probabilities. The study has been registered on PROSPERO (CRD42019147032). Results: Fifty-five RCTs (7,119 patients) were identified. Primary outcome showed that synbiotics had the best effect in preventing NI than EPN (OR 0.37; 95% CrI 0.22-0.61), probiotics followed (OR 0.52; 95% CrI 0.34-0.77), whereas TPN significantly increased NI (OR 2.29; 95% CrI 1.48-3.67). Subgroup analysis showed that TPN significantly increased NI in intensive care unit (ICU) patients (OR 1.57; 95% CrI 1.01-2.56) and severe acute pancreatitis (SAP) patients (OR 3.93; 95% CrI 1.74-9.15). Secondary outcomes showed that synbiotics were more effective in preventing hospital-acquired pneumonia (HAP) (OR 0.34; 95% CrI 0.11-0.85), catheter-related bloodstream infection (OR 0.08; 95% CrI 0.01-0.80), urinary tract infection (OR 0.27; 95% CrI 0.08-0.71) and sepsis (OR 0.34; 95% CrI 0.16-0.70) than EPN. Amongst the treatments, probiotics were most effective for shortening the mechanical ventilation duration (MD -3.93; 95% CrI -7.98 to -0.02), prebiotics were most effective for preventing diarrhea (OR 0.24; 95% CrI 0.05-0.94) and TPN was the least effective in shortening hospital length of stay (MD 4.23; 95% CrI 0.97-7.33). Conclusions: Amongst the five therapies, synbiotics not only prevented NI in critically ill adults but also demonstrated the best treatment results. By contrast, TPN did not prevent NI and ranked last, especially in ICU and SAP patients. Take-Home Message: Nosocomial infection is a leading cause of mortality in critically ill patients in the ICU. However, the efficacy of synbiotics, probiotics, prebiotics, enteral nutrition or adjuvant peripheral parenteral nutrition and total parenteral nutrition in preventing nosocomial infection in critically ill adults has been questioned. The network meta-analysis provides evidence that amongst the five therapies, synbiotics not only prevented NI in critically ill adults but also demonstrated the best treatment results. By contrast, TPN did not prevent NI and ranked last, especially in ICU and SAP patients. The results of this study will provide a new scientific basis and a new idea for the debate on the efficacy of synbiotics and other treatments in the improvement of prognosis in critically ill adult patients. Tweet: Synbiotic prevents nosocomial infection in critically ill adults, while total parenteral nutrition has the adverse curative.

Nucleotide polymorphism in ARDS outcome: a whole exome sequencing association study.

BACKGROUND: Genetic locus were identified associated with acute respiratory distress syndrome (ARDS). Our goal was to explore the associations between genetic variants and ARDS outcome, as well as subphenotypes. METHODS: This was a single-center, prospective observational trial enrolling adult ARDS patients. After baseline data were collected, blood samples were drawn to perform whole exome sequencing, single nucleotide polymorphism (SNP)/insertion-deletion to explore the quantitative and functional associations between genetic variants and ICU outcome, clinical subphenotypes. Then the lung injury burden (LIB), which was defined as the ratio of nonsynonymous SNP number per megabase of DNA, was used to evaluate its value in predicting ARDS outcome. RESULTS: A total of 105 ARDS patients were enrolled in the study, including 70 survivors and 35 nonsurvivors. Based on the analysis of a total of 65,542 nonsynonymous SNP, LIB in survivors was significantly higher than nonsurvivors [1,892 (1,848-1,942)/MB versus 1,864 (1,829-1,910)/MB, P=0.018], while GO analysis showed that 60 functions were correlated with ARDS outcome, KEGG enrichment analysis showed that SNP/InDels were enriched in 13 pathways. Several new SNPs were found potentially associated with ARDS outcome. Analysis of LIB was used to determine its outcome predicting ability, the area under the ROC curve of which was only 0.6103, and increase to 0.712 when combined with APACHE II score. CONCLUSIONS: Genetic variants are associated with ARDS outcome and subphenotypes; however, their prognostic value still need to be verified by larger trials. TRIAL REGISTRATION: Clinicaltrials.gov NCT02644798. Registered 20 April 2015.

An optimized method for the induction and purification of mouse bone marrow dendritic cells.

Dendritic cells (DCs) play an essential role in the initiation of adaptive immune responses, but they are rare in all organs. The traditional methods used to increase the yield and purity of DCs are the early removal of granulocyte culture medium and the isolation of high-purity DCs by magnetic-activated cell sorting (MACS). This study provides a more rapid and economical optimization method to obtain more high-purity DCs. (i) We harvested 18% more bone marrow (BM) cells by using forceps to crack the epiphysis instead of cutting it with scissors during BM cell extraction. (ii) When the cells in the culture medium that is discarded on day 3 in the traditional method were centrifuged and then added back to the petri dish, the DC yield on day 5 increased by 61%. (iii) On the third day, the addition of fresh medium and the retention of the original medium rather than discarding it increased the number of DCs harvested on the fifth day by 137%. (i-iii) The improved method cost an average of 74% less than the conventional method and yielded the same number and function of cells. (iv) The initial number of BM cells was increased by 15% in 4-week-old mice compared with 8-week-old mice. (v) The Percoll density centrifugation (PDS) method was used to purify DCs on day 6 after induction, and the purity of the DCs was greater than 90%, which showed no significant difference from the MACS method. However, the yield of the PDS method increased by 21%. In addition, the PDS method has a lower cost, with an average purification cost of 4 CNY ($0.58) compared with 648 CNY ($93.25) for MACS, reducing the cost by 99%. Therefore, high-purity and high-yield DCs can be rapidly obtained through a five-step improvement in the process of BM cell extraction, induction and purification.

Catalyst-free one-pot, four-component approach for the synthesis of di- and tri-substituted N-sulfonyl formamidines.

A straightforward one-pot, multicomponent approach was developed to synthesize di- and tri-substituted N-sulfonyl formamidines from sulfonyl chlorides, NaN3, ethyl propiolate, and primary/secondary amines under mild conditions without catalysts or additives. Structural analysis of the di-substituted sulfonyl formamidines indicated formation of the E-syn/anti isomeric form. Tri-substituted analogues only formed E-isomers.

Role of immunodeficiency in Acinetobacter baumannii associated pneumonia in mice.

BACKGROUND: Acinetobacter baumannii (A. baumannii) has become one of the most important opportunistic pathogens inducing nosocomial pneumonia and increasing mortality in critically ill patients recently. The interaction between A. baumannii infection and immune response can influence the prognosis of A. baumannii related pneumonia. The target of the present study was to investigate the role of immunodeficiency in A. baumannii induced pneumonia. METHODS: Male BALB/c mice were randomly divided into the normal immunity control (NIC) group, normal immunity infection (NIA) group, immune compromised control (CIC) group, and immune compromised infection (CIA) group (n = 15 for each group). Intraperitoneal injection of cyclophosphamide and intranasal instillation of A. baumannii solution were used to induce compromised immunity and murine pneumonia, respectively. The mice were sacrificed at 6 and 24 h later and the specimens were collected for further tests. Seven-day mortality of mice was also assessed. RESULTS: After A. baumannii stimulation, the recruitment of neutrophils in mice with normal immunity increased sharply (P = 0.030 at 6 h), while there was no significant raise of neutrophil counts in mice with compromised immune condition (P = 0.092 at 6 h, P = 0.772 at 24 h). The Th cell polarization presented with pulmonary interleukin (IL)-4 and interferon (IFN)-γ level in response to the A. baumannii in CIA group were significantly depressed in comparison with in NIA group (IFN-γ: P = 0.003 at 6 h; P = 0.001 at 24 h; IL-4: P < 0.001 at 6 h; P < 0.001 at 24 h). The pulmonary conventional dendritic cell accumulation was even found to be inhibited after A. baumannii infection in immunocompromised mice (P = 0.033). Correspondingly, A. baumannii associated pneumonia in mice with compromised immunity caused more early stage death, more severe histopathological impairment in lung. CONCLUSION: A. baumannii could frustrate the immune response in immunocompromised conditions, and this reduced immune response is related to more severe lung injury and worse outcome in A. baumannii induced pneumonia.

Therapeutic Effects of Hyaluronic Acid in Peritonitis-Induced Sepsis in Mice.

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 µg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Therapeutic potential of mesenchymal stem/stromal cell-derived secretome and vesicles for lung injury and disease.

Introduction: The acute respiratory distress syndrome (ARDS) is a devastating clinical condition common in patients with respiratory failure. Based largely on numerous preclinical studies and recent Phase I/II clinical trials, administration of stem cells, specifically mesenchymal stem or stromal cells (MSC), as a therapeutic for acute lung injury (ALI) holds great promise. However, concern for the use of stem cells, specifically the risk of iatrogenic tumor formation, remains unresolved. Accumulating evidence now suggest that stem cell-derived conditioned medium (CM) and/or extracellular vesicles (EV) might constitute compelling alternatives.Areas covered: The current review focuses on the preclinical studies testing MSC CM and/or EV as treatment for ALI and other inflammatory lung diseases.Expert opinion: Clinical application of MSC or their secreted CM may be limited by the cost of growing enough cells, the logistic of MSC storage, and the lack of standardization of what constitutes MSC CM. However, the clinical application of MSC EV remains promising, primarily due to the ability of EV to maintain the functional phenotype of the parent cell as a therapeutic. However, utilization of MSC EV will also require large-scale production, the cost of which may be prohibitive unless the potency of the EV can be increased.

Therapeutic Effects of Hyaluronic Acid in Bacterial Pneumonia in Ex Vivo Perfused Human Lungs.

Rationale: Recent studies have demonstrated that extracellular vesicles (EVs) released during acute lung injury (ALI) were inflammatory.Objectives: The current study was undertaken to test the role of EVs induced and released from severe Escherichia coli pneumonia (E. coli EVs) in the pathogenesis of ALI and to determine whether high-molecular-weight (HMW) hyaluronic acid (HA) administration would suppress lung injury from E. coli EVs or bacterial pneumonia.Methods:E. coli EVs were collected from the perfusate of an ex vivo perfused human lung injured with intrabronchial E. coli bacteria for 6 hours by ultracentrifugation and then given intrabronchially or intravenously to naive human lungs. One hour later, HMW HA was instilled into the perfusate (n = 5-6). In separate experiments, HMW HA was given after E. coli bacterial pneumonia (n = 6-10). In vitro experiments were conducted to evaluate binding of EVs to HMW HA and uptake of EVs by human monocytes.Measurements and Main Results: Administration of HMW HA ameliorated the impairment of alveolar fluid clearance, protein permeability, and acute inflammation from E. coli EVs or pneumonia and reduced total bacteria counts after E. coli pneumonia. HMW HA bound to E. coli EVs, inhibiting the uptake of EVs by human monocytes, an effect associated with reduced TNFα (tumor necrosis factor α) secretion. Surprisingly, HMW HA increased E. coli bacteria phagocytosis by monocytes.Conclusions: EVs induced and released during severe bacterial pneumonia were inflammatory and induced ALI, and HMW HA administration was effective in inhibiting the uptake of EVs by target cells and decreasing lung injury from E. coli EVs or bacterial pneumonia.

Can non-cognitive skills compensate for background disadvantage? -- the moderation of non-cognitive skills on family socioeconomic status and achievement during early childhood and early adolescence.

Combining the theory of resources substitution and recent evidence on the importance of children's non-cognitive skills from social sciences, this study asks whether family socioeconomic status' effects on achievement are contingent on or moderated by children's non-cognitive skills. I address this question from a longitudinal perspective by focusing on two developmental stages: early childhood and early adolescence. To overcome the methodological challenges involved in answering these questions, I use Structural Nested Mean Models (SNMM), a recent development in statistical methods. Using data from Early Childhood Longitudinal Study (ECLS), I test the hypothesis that higher non-cognitive skills will reduce family SES's effects on achievement in a longitudinal setting. The results corroborate the hypothesis, indicating that non-cognitive skills will moderate family SES's effects, and higher non-cognitive skills will lessen family SES's effects on achievement. In addition, such moderation effects are significant during both focal developmental stages of early childhood and early adolescence.

Therapeutic effects of human mesenchymal stem cell microvesicles in an ex vivo perfused human lung injured with severe E. coli pneumonia.

BACKGROUND: We previously reported that microvesicles (MVs) released by human mesenchymal stem cells (MSC) were as effective as the cells themselves in both Escherichia coli lipopolysaccharide and live bacteria-induced acute lung injury (ALI) mice models. However, it remained unclear whether the biological effect of MSC MV can be applied to human ALI. METHODS: In the current study, we tested the therapeutic effects of MSC MVs in a well-established ex vivo perfused human model of bacterial pneumonia. Using human donor lungs not used for transplantation, we instilled E. coli bacteria intrabronchially and, 1 hour later, administered MSC MVs into the perfusate as therapy. RESULTS: After 6 hours, instillation of E. coli bacteria caused influx of inflammatory cells, which resulted in significant inflammation, lung protein permeability and pulmonary oedema formation. Administration of MSC MV significantly increased alveolar fluid clearance and reduced protein permeability and numerically lowered the bacterial load in the injured alveolus. The beneficial effect on bacterial killing was more pronounced with pretreatment of MSCs with a Toll-like receptor 3 agonist, polyinosinic:polycytidylic acid (Poly (I:C)), prior to the isolation of MVs. Isolated human alveolar macrophages had increased antimicrobial activity with MSC MV treatment in vitro as well. Although oxygenation and lung compliance levels were similar between injury and treatment groups, administration of MSC MVs numerically decreased median pulmonary artery pressure at 6 hours. CONCLUSIONS: In summary, MSC MVs increased alveolar fluid clearance and reduced lung protein permeability, and pretreatment with Poly (I:C) enhanced the antimicrobial activity of MVs in an ex vivo perfused human lung with severe bacteria pneumonia.