Rationale: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer.Objectives: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects.Methods: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice.Measurements and Main Results: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSCderived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects.Conclusions: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSCderived EVs ameliorate lung injury in vivo.

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte‐derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)‐like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210–2223

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Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications.

BackgroundStandardized Nucleic Acid Quantification for SEQuencing (SNAQ-SEQ) is a novel method that utilizes synthetic DNA internal standards spiked into each sample prior to next generation sequencing (NGS) library preparation. This method was applied to analysis of normal appearing airway epithelial cells (AEC) obtained by bronchoscopy in an effort to define a somatic mutation field effect associated with lung cancer risk. There is a need for biomarkers that reliably detect those at highest lung cancer risk, thereby enabling more effective screening by annual low dose CT. The purpose of this study was to test the hypothesis that lung cancer risk is characterized by increased prevalence of low variant allele frequency (VAF) somatic mutations in lung cancer driver genes in AEC.MethodsSynthetic DNA internal standards (IS) were prepared for 11 lung cancer driver genes and mixed with each AEC genomic (g) DNA specimen prior to competitive multiplex PCR amplicon NGS library preparation. A custom Perl script was developed to separate IS reads and respective specimen gDNA reads from each target into separate files for parallel variant frequency analysis. This approach identified nucleotide-specific sequencing error and enabled reliable detection of specimen mutations with VAF as low as 5 × 10− 4 (0.05%). This method was applied in a retrospective case-control study of AEC specimens collected by bronchoscopic brush biopsy from the normal airways of 19 subjects, including eleven lung cancer cases and eight non-cancer controls, and the association of lung cancer risk with AEC driver gene mutations was tested.ResultsTP53 mutations with 0.05–1.0% VAF were more prevalent (p < 0.05) and also enriched for tobacco smoke and age-associated mutation signatures in normal AEC from lung cancer cases compared to non-cancer controls matched for smoking and age. Further, PIK3CA and BRAF mutations in this VAF range were identified in AEC from cases but not controls.ConclusionsApplication of SNAQ-SEQ to measure mutations in the 0.05–1.0% VAF range enabled identification of an AEC somatic mutation field of injury associated with lung cancer risk. A biomarker comprising TP53, PIK3CA, and BRAF somatic mutations may better stratify individuals for optimal lung cancer screening and prevention outcomes.

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The purpose of this retrospective study was to evaluate the long-term outcome in dogs with left atrial rupture secondary to myxomatous mitral valve disease. Eleven client-owned dogs met the inclusion criteria for the study. Median age was 11.6 yr (range, 8.3-17.8 yr), and median weight was 5.8 kg (range, 3.8-15.2 kg). Of the 11 dogs, 10 survived the initial 24 hr after diagnosis and 5 of the dogs were still alive at the conclusion of data collection. The median survival of all dogs was 203 days. Dogs with no previous history of congestive heart failure (CHF) at the time of diagnosis had a significantly longer median survival time (345 days) compared with dogs with a previous history of CHF (160 days, P = 0.0038). Outcome of dogs with myxomatous mitral valve disease presenting with left atria rupture was more favorable than previously reported, and dogs without previous CHF survived the longest.

An empirical equation containing three constants is suggested to express the solubilities of gases at various temperatures. Lithium chloride, and probably also barium chloride, lanthanum chloride, and hydrochloric acid, cause a salting-out effect which is uniformly less than that expected when the ionic size is compared with that of sodium chloride. Potassium chloride behaves normally. Potassium iodide causes specific decreases in salting-out as compared with sodium chloride and this effect increases with increasing molecular size. The salting-out of gases generally diminishes with rise in temperature. The partial molar heat capacity of the gas is affected, the greatest diminution being for lithium chloride and the least for potassium iodide. It is therefore suggested that the effect of ions on the structure of the solvent plays an important part in the salting-out effect.