Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death

Background

Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.

Methods

C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.

Results

Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.

Conclusions

These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

     

Cytotoxic lymphocytes in COPD airways: increased NK cells associated with disease,iNKT and NKT-like cells with current smoking

Background

Cytotoxic lymphocytes are increased in the airways of COPD patients. Whether this increase is driven primarily by the disease or by smoking is not clear, nor whether it correlates with the rate of decline in lung function.

Methods

Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study according to pre-determined criteria; 12 with COPD and a rapid decline in lung function (loss of FEV₁?≥?60?ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV₁?≤?30?ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry.

Results

In BAL fluid, the proportions of NK, iNKT and NKT-like cells all increased with pack-years. Within the COPD group, NK cells – but not iNKT or NKT-like cells – were significantly elevated also in subjects that had quit smoking. In contrast, current smoking was associated with a marked increase in iNKT and NKT-like cells but not in NK cells. Rate of lung function decline did not significantly affect any of the results.

Conclusions

In summary, increased proportions of NK cells in BAL fluid were associated with COPD; iNKT and NKT-like cells with current smoking but not with COPD. Interestingly, NK cell percentages did not normalize in COPD subjects that had quit smoking, indicating that these cells might play a role in the continued disease progression seen in COPD even after smoking cessation.

Trial registration

Clinicaltrials.gov identifier NCT02729220.

     

Down-regulation of microRNA-126-5p contributes to overexpression of VEGFA in lipopolysaccharide-induced acute lung injury

Objectives

To investigate the role of microRNA-126-5p (miR-126-5p) in acute lung injury induced by bronchial instillation of lipopolysaccharide (LPS), and to explore the potential target(s) of miR-126-5p in acute lung injury.

Results

In the mice with LPS-induced acute lung injury, the level of miR-126-5p in the pulmonary tissues was decreased by 41 % whilst pulmonary vascular endothelial growth factor-A (VEGFA) doubled in its mRNA content and increased threefold in its protein level. Similar results were observed in the alveolar type II (ATII) cells treated with LPS. By using luciferase reporter assay, we found that miR-126-5p inhibited VEGFA expression by targeting its 3′-untranslated region. In addition, overexpression of miR-126-5p attenuated LPS-induced reduction of epithelial sodium channel and aquaporin 1 in ATII cells

Conclusions

MiR-126-5p was down-regulated in LPS-induced acute lung injury in mice. Thus overexpression of miR-126-5p may alleviate acute lung injury by down-regulating VEGFA.

     

Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells

Background

This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs).

Methods

The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 μg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs.

Results

Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 μg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059), p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion.

Conclusions

Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.

     

Beneficial effects of inhaled NO on apoptotic pneumocytes in pulmonary thromboembolism model

Background

Lung ischemia–reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. Inhaled NO may be useful in treating acute and chronic pulmonary thromboembolism (PTE) due to the biological effect property of NO.

Methods

A PTE canine model was established through selectively embolizing blood clots to an intended right lower lobar pulmonary artery. PaO2/FiO2, the mPAP and PVR were investigated at the time points of 2, 4, 6 hours after inhaled NO. Masson’s trichrome stain, apoptotic pneumocytes and lung sample ultrastructure were also investigated among different groups.

Results

The PaO2/FiO2 in the Inhaled NO group increased significantly when compared with the Reperfusion group at time points of 4 and 6 hours after reperfusion, mPAP decreased significantly at point of 2 hours and the PVR decreased significantly at point of 6 hours after reperfusion. The amounts of apoptotic type II pneumocytes in the lower lobar lung have negative correlation trend with the arterial blood PaO2/FiO2 in Reperfusion group and Inhaled NO group. Inhaled nitric oxide given at 20 ppm for 6 hours can significantly alleviate the LIRI in the model.

Conclusions

Dramatic physiological improvements are seen during the therapeutic use of inhaled NO in pulmonary thromboembolism canine model. Inhaled NO may be useful in treating LIRI in acute or chronic PTE by alleviating apoptotic type II pneumocytes. This potential application warrants further investigation.

     

Efficient expression of functional human coagulation factor IX in stably-transfected <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells; comparison with the mammalian CHO system

Objective

To compare stably-transfected Drosophila melanogaster S2 and mammalian Chinese hamster ovary (CHO) cells for the expression and secretion efficiency of biologically-active human coagulation factor IX (hFIX).

Result

Selection of an hFIX-expressing cell line derived from stably-transfected S2 cells was performed over 2 weeks, while the same procedure required 2 months for stably-transfected CHO cells. Furthermore, the selected S2 cell line was superior in producing of total hFIX protein (70 % increase), biologically-active hFIX (35 % increase), and specific hFIX activity (20 % increase) relative to the selected CHO cell line. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated that up to 90 % of the hFIX expressed by S2 cells was γ-carboxylated versus 79 % of CHO-expressed hFIX. Inhibition of N-glycosylation by tunicamycin indicated that N-glycosylation of S2-expressed hFIX had occurred to a similar extent as in the CHO-produced hFIX.

Conclusion

The Drosophila S2 cell system is an attractive candidate for the efficient production of recombinant hFIX as it has the potential of significantly reducing the cell development time, while producing functional hFIX.

     

Proteomic identification of moesin upon exposure to acrolein

Background

Acrolein (allyl Aldehyde) as one of smoke irritant exacerbates chronic airway diseases and increased in sputum of patients with asthma and chronic obstructive lung disease. But underlying mechanism remains unresolved. The aim of study was to identify protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to acrolein.

Methods

A proteomic approach was used to determine the different expression of proteins at 8 h and 24 h after treatment of acrolein 30 nM and 300 nM to HMVEC-L. Treatment of HMVEC-L with acrolein 30 nM and 300 nM altered 21 protein spots on the two-dimensional gel, and these were then analyzed by MALDI-TOF MS.

Results

These proteins included antioxidant, signal transduction, cytoskeleton, protein transduction, catalytic reduction. The proteins were classified into four groups according to the time course of their expression patterns such as continually increasing, transient increasing, transient decreasing, and continually decreasing. For validation immunohistochemical staining and Western blotting was performed on lung tissues from acrolein exposed mice. Moesin was expressed in endothelium, epithelium, and inflammatory cells and increased in lung tissues of acrolein exposed mice compared with sham treated mice.

Conclusions

These results indicate that some of proteins may be an important role for airway disease exacerbation caused by acrolein exposure.

     

Serum metabolomic markers for traumatic brain injury: a mouse model

Introduction

Traumatic brain injury (TBI) is physical injury to brain tissue that temporarily or permanently impairs brain function.

Objectives

Evaluate the use of metabolomics for the development of biomarkers of TBI for the diagnosis and timing of injury onset.

Methods

A validated model of closed injury TBI was employed using 10 TBI mice and 8 sham operated controls. Quantitative LC–MS/MS metabolomic analysis was performed on the serum.

Results

Thirty-six (24.0 %) of 150 metabolites were altered with TBI. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) analyses revealed clear segregation between TBI versus control sera. The combination of methionine sulfoxide and the lipid PC aa C34:4 accurately diagnosed TBI, AUC (95 % CI) 0.85 (0.644–1.0). A combination of metabolite markers were highly accurate in distinguishing early (4 h post TBI) from late (24 h) TBI: AUC (95 % CI) 1.0 (1.0–1.0). Spermidine, which is known to have an antioxidant effect and which is known to be metabolically disrupted in TBI, was the most discriminating biomarker based on the variable importance ranking in projection (VIP) plot. Several important metabolic pathways were found to be disrupted including: pathways for arginine, proline, glutathione, cysteine, and sphingolipid metabolism.

Conclusion

Using serum metabolomic analysis we were able to identify novel putative serum biomarkers of TBI. They were accurate for detecting and determining the timing of TBI. In addition, pathway analysis provided important insights into the biochemical mechanisms of brain injury. Potential clinical implications for diagnosis, timing, and monitoring brain injury are discussed.

     

Enhanced succinic acid production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> with increasing the available NADH supply and glucose consumption rate by decreasing H<Superscript>+</Superscript>-ATPase activity

Objectives

To enhance succinic acid production in Corynebacterium glutamicum by increasing the supply of NADH and the rate of glucose consumption by decreasing H⁺-ATPase activity.

Results

A mutant of C. glutamicum NC-3-1 with decreased H⁺-ATPase activity was constructed. This increased the rate of glycolysis and the supply of NADH. Fermentation of C. glutamicum NC-3-1 gave 39 % higher succinic acid production (113 and 81 g/l), a 29 % higher succinic acid yield (0.94 and 0.73 g succinic acid/g glucose) and decreased by-products formation compared to that of C. glutamicum NC-3 in 5 l bioreactor.

Conclusion

The point mutation in C. glutamicum NC-3-1 increased the rate of glycolysis and resulted in higher succinic acid production, higher succinic acid yield and significantly decreased formation of by-products.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

New insights into the mechanistic action of methyldehydrodieugenol B towards <Emphasis Type="Italic">Leishmania</Emphasis> (L.) <Emphasis Type="Italic">infantum</Emphasis> via a multiplatform based untargeted metabolomics approach

Introduction

Leishmaniasis is a parasitic neglected disease affecting millions of people worldwide. Clinical practice resorts to long and costly treatments with a therapeutic arsenal limited to highly toxic drugs, often associated to adverse side effects. Additionally, resistant strains are reported to be increasing.

Aim

In this work, the mechanistic action of a drug candidate (methydehydrodieugenol B), isolated from twigs of Nectandra leucantha, towards Leishmania infantum was studied by a global metabolomics approach using GC-MS and RPLC-MS platforms.

Method

L. infantum promastigotes were grown in culture medium for 72 h and treated with methydehydrodieugenol B at 58.18 μg.mL-1 concentration; after 48 h treatment, enzyme activity was quenched, cells washed and frozen until analysis. For GC-MS analysis (Fiehn’s method), 1:1 methanol:water extracts were prepared and derivatized with O-methoxyamine in pyridine at room temperature for 90 min, followed by silylation with BSTFA/1% TMCS at 40 °C for 30 min. Pure methanolic extracts were also prepared and analyzed directly by RPLC-MS with a acetonitrile/water mobile phase acidulated with formic acid and gradient elution.

Result

Several amino acids, fatty acids, carbohydrates, and glycerolipids were found as discriminant metabolites, mostly decreased in treated samples. Due to the complexity of the parasite metabolism and the great diversity of altered metabolites, a multi-target mechanism was assigned to the drug candidate, where changes in the cell energy sources and in the lipid composition of the parasite plasma membrane were prominent.

Conclusion

These results contributed to elucidate the broad action of methyldehydrodieugenol B against Leishmania, paving the way in the search of novel alternative therapies.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> endoglycosidase and sugar oxazoline

Objectives

To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.

Results

Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CC^N180H) were employed. pH 7.5 was ideal for both SG-oxazoline’s stability and Endo-CC’s transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 μg GlcNAc-RNase B, 200 μg SG-oxazoline and 3 μg Endo-CC^N180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min.

Conclusions

This transglycosylation method using SG-oxazoline and Endo-CC^N180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

     

EGF regulates tyrosine phosphorylation and membrane-translocation of the scaffold protein Tks5

Background

Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.

Results

Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.

Conclusions

Our results identify Tks5 as a novel component of the EGF signaling pathway.

     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

Harvesting <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> via rapid sedimentation induced by combined coagulants and tapered shear

Objectives

In this study, a rapid sedimentation induced by combined coagulants and gradual shear was developed to harvest Chlorella vulgaris.

Results

The microalgal harvesting efficiency was observably promoted by the synergistic effect between FeCl₃ and PAM, especially in the first 10 min. A higher harvesting efficiency, 95.61%, could be achieved within approximately 3 min due to the large and dense flocs generated by the combined coagulants. In contrast, the efficiencies were only 54.25 and 60.20% with FeCl₃ and PAM, independently. When coagulation was performed under gradually reduced shear (from 50 to 30 rpm), smaller clusters or cells filled the pores of the aggregates via interception, which caused the flocs to become larger and more compact.

Conclusions

The sedimentation time was shortened to 30 s for microalgal coagulation induced by the simultaneous use of combined coagulants and tapered shear, providing an effective approach to harvesting microalgae.

     

Accumulation of Fascin<Superscript>+</Superscript> cells during experimental autoimmune neuritis

Background

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles.

Methods

Here we have studied by immunohistochemistry the spatiotemporal accumulation of Fascin?+?cells in sciatic nerves of EAN rats.

Results

A robust accumulation of Fascin?+?cell was observed in the peripheral nervous system of EAN which was correlated with the severity of neurological signs in EAN.

Conclusion

Our results suggest a pathological role of Fascin in EAN.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticphatology.diagnomx.eu/vs/6734593451114811

     

Improvement of uridine production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by metabolic engineering

Objectives

To construct a Bacillus subtilis strain for improved uridine production.

Results

The AAG_2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation.

Conclusion

This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

     

Characterization of a <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain for reduction of citrulline accumulation during soy sauce fermentation

Objectives

To reduce the amount of citrulline produced by arginine-consuming bacteria in the moromi mash during soy sauce production.

Results

Bacillus amyloliquefaciens JY06, a salt-tolerant strain with high arginine consumption ability and low citrulline accumulation capacity, was isolated from moromi mash. The concentration of citrulline was decreased from 26.8 to 5.1 mM and ethyl carbamate in soy sauce, after sterilization, decreased from 97 to 17 μg kg^?1 when B. amyloliquefaciens JY06 was added during fermentation. The aroma of the sauce was improved by increasing the ester content.

Conclusions

B. amyloliquefaciens JY06 is a beneficial bacterium that can be used in soy sauce fermentation to eliminate ethyl carbonate and enhance the flavor of the sauce.