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Introduction and objectivesMesenchymal stem cells (MSC) have well-established anti-inflammatory properties and could potentially be used therapeutically in Acute Respiratory Distress Syndrome (ARDS).1 MSC conditioned medium (MSC-CM) has been found to reproduce the beneficial effects of MSCs.2 However, the impact of expansion on MSC secretome remains elusive. In the present study we assessed the expression of four potent MSC paracrine factors after prolonged in vitro culture, while investigating the effects of passaging on the in vitro properties of MSCs.MethodsHuman Bone marrow-derived MSCs were expanded in vitro in αMEM with 16% FBS. Conditioned medium was collected from each passage and stored at −40°C. Scratch assays were undertaken using A549 cells treated with MSC-CM and control media. Proliferation of A549 cells was assessed via BrdU assay. Gene expression of expanded MSCs was assessed by RT-qPCR. VEGF and Angiopoietin in the MSC-CM were quantified by ELISA.ResultsRT-qPCR revealed that MSCs express the anti-inflammatory genes PTGES2, FGF7 and ANGPT1. The expression of these genes doubled after 8 days in culture and subsequently decreased. (P < 0.0005, one way Anova, n = 2). The secretion of Angiopoietin and VEGF decreased with prolonged expansion (Figure 1). MSC-CM obtained after 8 days of culture induced more efficient wound healing compared to MSC-CM obtained following prolonged expansion (P < 0.005, one way Anova, n = 2). However, MSC-CM failed to affect the proliferation of A549 cells.ConclusionWe conclude that MSCs of early passages are more potent angiogenic inducers, while promoting wound healing in vitro. Thus, we provide important insights into the changes occurring after prolonged in vitro expansion, underlining the importance of using low passage MSCs in clinical trials for ARDS. In agreement with published data, we also found that MSCs do not induce cellular proliferation in the absence of stimulation.Abstract S11 Figure 1Levels of secreted VEGF decrease after prolonged in vitro culture (*P<0.05, ***P<0.0005, n = 2)ReferencesAntunes MA, et al. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem 2014;115:1023–1032.Kordelas L, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. LeukaemiaLeukemia 2014;28:970–973.

manner; no peptide, 2468.56mg/ml, 5 mg, 2181.7626.2 ng/ml (p¼0.006), 10 mg, 15766164.7 ng/ml (p¼0.001), and 20 and 30 mg completely prevented polymer formation in inclusions (p#0.001). Unrelated peptides had no effect. Elastase activity of AT in the supernatant from Z-AT cells was significantly reduced compared to M-AT cells; p#0.001, in keeping with the secretory defect due to retention of Z-AT in inclusion bodies. The elastase activity (and AT concentration) in the supernatant from Z-AT cells was restored by 20 mg 4M, O.D. 405 nm, Z-AT vs Z-AT + 20 mg 4M, 0.12960.009 vs 0.78860.054 respectively, (p#0.001), where a higher O.D. represents higher elastase activity. Functional activity of secreted AT following treatment with 4M was confirmed by its ability to form an SDS-stable complex with elastase as shown by immunoblot. RT-PCR showed that the ER accumulation of Z-AT induced cell stress; NF-kB activation, expression of protein kinase RNA (PKR)-like ER kinase (PERK), and IL-6 (100.4616 pg/ml) and IL-8 (2592.56575 pg/ ml), all of which could be abrogated effectively by 20 mg 4M (IL-6, 45.8628 pg/ml, p#0.001 and IL-8, 184.3629 pg/ml, p¼0.014). These findings are the first evidence that inhibitors of Z-AT polymerisation targeting s4A can prevent its cellular accumulation and deleterious effects. Importantly, this strategy was also able to improve plasma concentration of Z-AT. Acute exacerbations of COPD are the commonest cause of acute medical admissions in the UK and w50% are associated with bacterial infection. Alveolar macrophages (AM) normally clear inhaled bacteria but defective phagocytosis may lead to chronic colonisation and increased exacerbations. Monocyte-derived macrophages (MDM), used to model AM, were obtained from COPD, smoking and healthy subjects. MDM phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae (HI) or Streptococcus pneumoniae (SP) was measured by fluorimetry. MDM derived from all subjects showed equivalent ability to phagocytose beads, however, COPD and smoker MDM showed significantly reduced phagocytosis of bacteria. Phagocytosis of HI was reduced by 28% and 48% in COPD and smoker MDM respectively, compared to healthy, while SP phagocytosis was reduced by 32% and 52% in COPD and smoker MDM respectively, compared to healthy (Abstract S52 table 1). Having identified defective bacterial phagocytosis in smoker and COPD MDM, the next step was to elucidate the underlying mechanism. Cytoskeletal rearrangement was investigated, with COPD MDM showing significantly reduced phagocytosis of bacteria in comparison to healthy after pre-incubation with nocodazole (microtubule disruptor). Microtubules are involved in membrane trafficking of the phagolysosome and microtubule stability is necessary for effective phagocytosis. Tubulin is acetylated to form stable microtubules and is deacetylated by HDAC6 and Sirt2. COPD MDM showed reduced levels of acetylated tubulin compared to healthy MDM. Pre-incubation with epothilone B (10 nm) a microtubule stabiliser, improved HI phagocytos...

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