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.

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.

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.

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.

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.

Stable overexpression of p130/E2F4 affects the multipotential abilities of bone-marrow-derived mesenchymal stem cells.

Bone-marrow-derived mesenchymal stem cells (MSCs) have great potential in transplantation medicine due to their multiple advantages. However, the controlled differentiation of MSCs is one of the key aspects of effective clinical transplantation. Growing evidence suggests that the cell cycle plays an important role in regulating differentiation, while p130 and E2F4 are key to cell cycle checkpoints. The aim of the study is to evaluate the effects and mechanism of p130/E2F4 on the multidifferentiation of MSCs. Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3%-84.4%. p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC normal control (NC) cells. Similar results were also observed for p130 and E2F4 protein expression. After osteogenic or adipogenic differentiation, the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared with that in the MSC-NC group. However, the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation. Moreover, overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mouse MSCs (mMSCs). Moreover, overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCs. Our data suggest that cell cycle regulation may be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCs.

Effects of neurally adjusted ventilatory assist on air distribution and dead space in patients with acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Neurally adjusted ventilatory assist (NAVA) could improve patient-ventilator interaction; its effects on ventilation distribution and dead space are still unknown. The aim of this study was to evaluate the effects of varying levels of assist during NAVA and pressure support ventilation (PSV) on ventilation distribution and dead space in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). METHODS: Fifteen mechanically ventilated patients with AECOPD were included in the study. The initial PSV levels were set to 10 cmH2O for 10 min. Thereafter, the ventilator mode was changed to NAVA for another 10 min with the same electrical activity of the diaphragm as during PSV. Furthermore, the ventilation mode was switched between PSV and NAVA every 10 min in the following order: PSV 5 cmH2O; NAVA 50%; PSV 15 cmH2O; and NAVA 150% (relative to the initial NAVA support level). Ventilation distribution in the lung was evaluated in percentages in regions of interest (ROI) of four anteroposterior segments of equal height (ROI1 to ROI4 represents ventral, mid-ventral, mid-dorsal, and dorsal, respectively). Blood gases, ventilation distribution (electrical impedance tomography), diaphragm activity (B-mode ultrasonography), and dead space fraction (PeCO2 and PaCO2) were measured. RESULTS: The trigger and cycle delays were lower during NAVA than during PSV. The work of trigger was significantly lower during NAVA compared to PSV. The diaphragm activities based on ultrasonography were higher during NAVA compared to the same support level during PSV. The ventilation distribution in ROI4 increased significantly (P < 0.05) during NAVA compared to PSV (except for a support level of 50%). Similar results were found in ROI3 + 4. NAVA reduced dead space fraction compared to the corresponding support level of PSV. CONCLUSIONS: NAVA was superior to PSV in AECOPD for increasing ventilation distribution in ROI4 and reducing dead space. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02289573 . Registered on 12 November 2014.

The Vascular Endothelial Growth Factors-Expressing Character of Mesenchymal Stem Cells Plays a Positive Role in Treatment of Acute Lung Injury In Vivo.

Recently, mesenchymal stem cells (MSC) have been proved to be beneficial in acute respiratory distress syndrome (ARDS). Vascular endothelial growth factor (VEGF) is an important angiogenesis factor that MSC release. However, the precise role of VEGF-expressing character of MSC in the MSC treatment for ARDS remains obscure. Here, we firstly knocked down the gene VEGF in MSC (MSC-ShVEGF) with lentiviral transduction. Then we injected the MSC-ShVEGF to rats with lipopolysaccharide-induced acute lung injury (ALI) via the tail vein. Data showed that MSC transplantation significantly increased VEGF levels in the lung, reduced lung permeability, protected lung endothelium from apoptosis, facilitated VE-cadherin recovery, controlled inflammation, and attenuated lung injury. However, VEGF gene knockdown in MSC led to relatively insufficient VEGF expression in the injured lung and significantly diminished the therapeutic effects of MSC on ALI, suggesting an important role of VEGF-expressing behavior of MSC in the maintenance of VEGF in the lung and the MSC treatment for ALI. Hence, we conclude that MSC restores the lung permeability and attenuates lung injury in rats with ALI in part by maintaining a "sufficient" VEGF level in the lung and the VEGF-expressing character of MSC plays a positive role in the therapeutic effects of MSC on ARDS.

Why do Asian Americans academically outperform Whites? - The cultural explanation revisited.

We advocate an interactive approach to examining the role of culture and SES in explaining Asian Americans' achievement. We use Education Longitudinal Study (ELS) 2002 baseline data to test our proposition that the cultural orientation of Asian American families is different from that of white American families in ways that mediate the effects of family SES on children's academic achievement. The results support our hypothesis, indicating that: (1) SES's positive effects on achievement are stronger among white students than among Asian-Americans; (2) the association between a family's SES and behaviors and attitudes is weaker among Asian-Americans than among Whites; (3) a fraction of the Asian-White achievement gap can be accounted for by ethnic differences in behaviors and attitudes, particularly ethnic differences in family SES's effects on behaviors and attitudes. We find that Asian Americans' behaviors and attitudes are less influenced by family SES than those of Whites are and that this difference helps generate Asians' premium in achievement. This is especially evident at lower levels of family SES.

Novel avian-origin influenza A (H7N9) in critically ill patients in China*.

OBJECTIVES: In March 2013, human infection with a novel avian-origin reassortment influenza A (H7N9) virus was identified in China. A total of 26 cases were confirmed and treated in Jiangsu. All the patients had findings consistent with pneumonia and were admitted to an ICU, which pose a threat to human health. We aimed to provide the clinical features, treatment, and prognosis of the critically ill patients with H7N9 viral infection. DESIGN: A retrospective cohort study. SETTING: Eight closed ICUs in general hospitals distributed throughout the Jiangsu Provincial, China. PATIENTS: Patients infected with influenza A (H7N9) virus from March 20, 2013, through May 1, 2013, in Jiangsu Province were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Twenty-seven patients infected with H7N9 virus were identified in Jiangsu. Of these, 26 were hospitalized. The median age was 54.5 years, and 18 patients (69.2%) were men. The most common symptoms at the onset of illness were high fever and cough. White cell counts were normal or decreased. All the patients had findings consistent with pneumonia. Twenty-four patients (92.3%) developed acute respiratory distress syndrome, and 10 (38.5%) developed septic shock quickly after the onset of illness. Treatment with antiviral drugs was initiated in all the patients at a median of 8 days after the onset of illness. Mortality was 19.2% at 28 days and 30.8% at 90 days. Based on multiple logistic regression analysis, septic shock associated with severe hypoxemia was the only independent risk factor for mortality. CONCLUSIONS: Infection with novel avian-origin reassortment influenza A (H7N9) virus is characterized by high fever, cough, and severe respiratory failure and is associated with a high mortality. These data provide some general understandings for the early identification, ICU treatment, and short-term prognosis of hospitalized critical patients with H7N9.

A high mean arterial pressure target is associated with improved microcirculation in septic shock patients with previous hypertension: a prospective open label study.

INTRODUCTION: The effect of mean arterial pressure titration to a higher level on microcirculation in septic shock patients with previous hypertension remains unknown. Our goal is to assess the effect of mean arterial pressure titration to a higher level on microcirculation in hypertensive septic shock patients. METHODS: This is a single-center, open-label study. Hypertensive patients with septic shock for less than 24 hours after adequate fluid resuscitation and requiring norepinephrine to maintain a mean arterial pressure of 65 mmHg were enrolled. Mean arterial pressure was then titrated by norepinephrine from 65 mmHg to the normal level of the patient. In addition to hemodynamic variables, sublingual microcirculation was evaluated by sidestream dark field imaging. RESULTS: Nineteen patients were enrolled in the study. Increasing mean arterial pressure from 65 mmHg to normal levels was associated with increased central venous pressure (from 11 ± 4 to 13 ± 4 mmHg, P = 0.002), cardiac output (from 5.4 ± 1.4 to 6.4 ± 2.1 l/minute, P = 0.001), and central venous oxygen saturation (from 81 ± 7 to 83 ± 7%, P = 0.001). There were significant increases in small perfused vessel density (from 10.96 ± 2.98 to 11.99 ± 2.55 vessels/mm(2), P = 0.009), proportion of small perfused vessels (from 85 ± 18 to 92 ± 14%, P = 0.002), and small microvascular flow index (from 2.45 ± 0.61 to 2.80 ± 0.68, P = 0.009) when compared with a mean arterial pressure of 65 mmHg. CONCLUSIONS: Increasing mean arterial pressure from 65 mmHg to normal levels is associated with improved microcirculation in hypertensive septic shock patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01443494; registered 28 September 2011.

Stable genetic alterations of ß-catenin and ROR2 regulate the Wnt pathway, affect the fate of MSCs.

The Wnt pathways have been shown to be critical for the fate of mesenchymal stem cells (MSCs) in vitro, but their roles in MSCs in vivo remain poorly characterized due to the lack of stable alterations in their signaling. In the present study, we constructed long-term and stable mMSCs lines with activated and inactivated ß-catenin (the key molecule of the canonical Wnt signaling pathway) or ROR2 (the key molecule of the noncanonical Wnt5a/ROR2 signaling pathway) modifications with lentiviral vectors. We found that the transduction efficiencies mediated by the lentiviral vectors were 92.61-97.04% and were maintained over 20 passages of mMSCs. Transfection by lentiviral vectors not only regulated the mRNA and protein expression of ß-catenin or ROR2 but also regulated nuclear ß-catenin accumulation or the Wnt5a/JNK and Wnt5a/PKC pathways belonging to the canonical Wnt and noncanonical Wnt5a/ROR2 pathways, respectively. ß-Catenin or ROR2 gene overexpression promoted mMSC proliferation, migration and differentiation into osteoblasts, while inhibiting the adipogenic differentiation of mMSCs. In contrast, inactivation of the ß-catenin or ROR2 genes resulted in the opposite effects. Therefore, these results confirm that lentiviral vector transduction can facilitate sustained and efficient gene modification of the Wnt pathway in mMSCs. This study provides a method to investigate the effects of the Wnt pathway on the fate of mMSCs in vivo and for the further improvement of MSC-based therapies.

The effect of glutamine therapy on outcomes in critically ill patients: a meta-analysis of randomized controlled trials.

INTRODUCTION: Glutamine supplementation is supposed to reduce mortality and nosocomial infections in critically ill patients. However, the recently published reducing deaths due to oxidative stress (REDOX) trials did not provide evidence supporting this. This study investigated the impact of glutamine-supplemented nutrition on the outcomes of critically ill patients using a meta-analysis. METHODS: We searched for and gathered data from the Cochrane Central Register of Controlled Trials, MEDLINE, Elsevier, Web of Science and ClinicalTrials.gov databases reporting the effects of glutamine supplementation on outcomes in critically ill patients. We produced subgroup analyses of the trials according to specific patient populations, modes of nutrition and glutamine dosages. RESULTS: Among 823 related articles, eighteen Randomized Controlled Trials (RCTs) met all inclusion criteria. Mortality events among 3,383 patients were reported in 17 RCTs. Mortality showed no significant difference between glutamine group and control group. In the high dosage subgroup (above 0.5 g/kg/d), the mortality rate in the glutamine group was significantly higher than that of the control group (relative risk (RR) 1.18; 95% confidence interval (CI), 1.02 to 1.38; P = 0.03). In 15 trials, which included a total of 2,862 patients, glutamine supplementation reportedly affected the incidence of nosocomial infections in the critically ill patients observed. The incidence of nosocomial infections in the glutamine group was significantly lower than that of the control group (RR 0.85; 95% CI, 0.74 to 0.97; P = 0.02). In the surgical ICU subgroup, glutamine supplementation statistically reduced the rate of nosocomial infections (RR 0.70; 95% CI, 0.52 to 0.94; P = 0.04). In the parental nutrition subgroup, glutamine supplementation statistically reduced the rate of nosocomial infections (RR 0.83; 95% CI, 0.70 to 0.98; P = 0.03). The length of hospital stay was reported in 14 trials, in which a total of 2,777 patients were enrolled; however, the patient length of stay was not affected by glutamine supplementation. CONCLUSIONS: Glutamine supplementation conferred no overall mortality and length of hospital stay benefit in critically ill patients. However, this therapy reduced nosocomial infections among critically ill patients, which differed according to patient populations, modes of nutrition and glutamine dosages.

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 canonical wnt pathway promotes differentiation of mouse bone marrow-derived MSCs into type II alveolar epithelial cells, confers resistance to oxidative stress, and promotes their migration to injured lung tissue in vitro.

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow-derived MSCs (mMSCs) into AT II cells. Using a modified co-culture system with murine lung epithelial-12 (MLE-12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3ß and ß-catenin in the canonical wnt pathway were up-regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co-culture system to activate wnt/ß-catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/ß-catenin signaling promoted mMSCs migration towards ALI mouse-derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl-2/Bax induced by H(2) O(2), which simultaneously caused reduced GSK 3ß and ß-catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI.

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.

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.

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.

[The evaluation value of severity and prognosis of septic shock patients based on the arterial-to-venous carbon dioxide difference].

OBJECTIVE: To assess the value of central venous-to-arterial carbon dioxide difference [P((cv-a))CO2] in evaluation of disease severity and prognosis in patients with septic shock. METHODS: There were 45 consecutive resuscitated septic shock patients from April 2009 to October 2010 included immediately after their admission into our ICU. The patients were divided into low P((cv-a))CO2 group and high P((cv-a))CO2 group according to a threshold of 6 mm Hg (1 mm Hg = 0.133 kPa). All patients were treated by early goal directed therapy (EGDT). The parameters of hemodynamics, lactate clearance rate, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, the sequential organ failure assessment (SOFA) score, 6 h rate of EGDT achievement, the ICU mortality and 28 days in-hospital mortality were recorded for all patients. RESULTS: There were 30 patients in the low P((cv-a))CO2 group, and 15 in the high P((cv-a))CO2 group. There were no significant differences between low P((cv-a))CO2 and high P((cv-a))CO2 patients in age, APACHE II score and SOFA score (all P > 0.05). Compared with the high P((cv-a))CO2 group, the low P((cv-a))CO2 group had higher cardiac index (CI) and 24 h CI, higher delivery O2 (DO2) and 24 h DO2, higher central venous oxygen saturation (ScvO2) [(74 ± 9)% vs (67 ± 8)%], lower lactate [(3.4 ± 2.1) mmol/L vs (5.7 ± 4.5) mmol/L] and higher ΔSOFA score [(0.7 ± 1.8) vs (-0.4 ± 1.1)], lower 24 h SOFA score [(7.8 ± 2.0) vs (9.8 ± 2.0)], higher 6 h rate of EGDT achievement (83.3% vs 53.3%) (P < 0.05), however, there were no differences in 28 days mortality and ICU mortality between the two groups (P > 0.05). CONCLUSION: P((cv-a))CO2 might be an indicator for predicting the severity of patients with septic shock and evaluating tissue perfusion.

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.