Vitamin D deficiency is associated with increased risk of ARDS post-oesophagectomy. We recruited patients to a double-blind, randomised controlled trial of high dose Vitamin D supplementation 3–14 days pre-oesophagectomy.79 patients were randomised to receive placebo or 300,000 IU oral Vitamin D liquid 3–14 days prior to oesophagectomy. Blood samples were collected pre-dose, post-dose (pre-op) and post-op and analysed for 25-OH and 1,25-dOH Vitamin D, inflammatory cells and cytokines. Broncho-alveolar lavage fluid was collected at the end of the operation. PICCO biomarkers of alveolar capillary damage (EVLWI and PVPI) were measured pre- and post-op.Pre-operative supplementation with Vitamin D was well tolerated with no SUSARs and significantly increased circulating 25-OH and 1,25-OH Vitamin D (p < 0.0001). This was associated with reduced systemic inflammation (IL-6 (p = 0.02) and IL-8 (p = 0.002)) and an increase in circulatory Treg (p = 0.027).Changes in PICCO biomarkers were lower in supplemented patients suggesting lower perioperative alveolar oedema (EVLWI p = 0.05, PVPI p = 0.04). This did not result in a significant difference in oxygenation at 24 h.Post-op, systemic and alveolar alarmin (IL-1B, IL-6, IL-8) response was similar in treated and untreated patients but the systemic release of IL-1ra (p = 0.046), sTNFR-1 (p = 0.05) and s-TNFR-2 (p = 0.02) were elevated in treated patients. There was also evidence of decreased alveolar macrophage efferocytosis in patients with Vitamin D deficiency (p = 0.003).Clinical diagnoses of ARDS were significantly lower in this cohort than in previous cohorts, but the study was not powered to detect that outcome. Mortality post-operative was not significantly different at 30 or 90 days but there is a significant difference after 300 days of follow-up (placebo 33% mortality, Vitamin D 8% mortality p = 0.033).In conclusion, vitamin D supplementation was a safe, well tolerated pre-operative intervention that reduced systemic inflammation and biomarkers of alveolar oedema. With evidence of enhanced anti-inflammatory mechanism that may have influenced longer term post-operative survival, Vitamin D deficiency should be identified and treated in patients at risk of ARDS.

Introduction and objectivesElectronic cigarette usage or “vaping” has risen exponentially in recent years in smokers and ex-smokers. Published data suggests that vaping e-cigarette liquid (ECL) may not be as benign as propounded by e-cigarette companies which are increasingly owned by “big tobacco”. Much of the current literature has focused on the effect of non-vaporised ECL – such studies do not fully reflect the exposure of the user, as the process of vaping causes chemical changes in ECL. To investigate the effect of unvaped ECL and vaped e-cig condensate (ECVC) using our novel system, with and without nicotine, on alveolar macrophage (AM) viability and immune responses.MethodsWe developed a novel method to produce ECVC to allow direct comparison with unvaped ECL. Nicotine concentration as assessed by GFID was 31 mg/ml in ECL and 26 mg/ml in ECVC. AMs were obtained from lung resection tissue and treated with ECVC/ECL ± nicotine. Cell viability was assessed by cell titre aqueous assay, apoptosis, necrosis and markers of macrophage phenotype (CD68, CD80, CD163, CD206) were assessed by flow cytometry. IL-8 release by AMs was assessed by ELISA.ResultsAM culture with ECL or ECVC resulted in dose dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL (0.8% ECVC vs 5 %ECL, n = 6). 24 hour culture with 1% ECVC resulted in a 5fold increase in AM apoptosis and 2 fold increase in necrosis compared with 1%ECL (p = 0.079, n = 5). Nicotine containing ECVC caused more apoptosis vs nicotine free ECVC (27.2% vs 13.4%, (p = 0.0079,n = 4). Culture with 0.6%ECVC significantly increased supernatant levels of IL-8 compared with 1% ECL (p = 0.015, n = 4). ECVC was also found to affect macrophage phenotype, showing both nicotine dependent/independent regulations of markers of macrophage m1/m2 polarisation (CD80 p = 0.0357, CD163 p = 0.0179, CD206 p = 0.0357, n = 6).ConclusionsOur novel system creates ECVC which is sterile, minimises loss of nicotine and prevents dilution of the vapour. Vaped E-cigarette condensate is significantly more toxic to AMs than non-vaped e cigarette liquid. Furthermore, ECVC with nicotine is significantly more toxic than ECVC without Nicotine. Effects shown on inflammatory cytokine production and markers of macrophage polarisation indicate both nicotine dependent and independent effects of ECVC on alveolar macrophages.

The acute respiratory distress syndrome (ARDS) is characterised by exaggerated alveolar inflammation. Vitamin D deficiency in an LPS induced murine model of ARDS results in exaggerated alveolar inflammation. However the role of vitamin D deficiency in pulmonary inflammation in humans is unclear. We hypothesised that in healthy volunteers with vitamin D deficiency, pulmonary inflammation would be increased following LPS inhalation.MethodsHealthy volunteers inhaled 50 micrograms of LPS and six hours later underwent bronchoalveolar lavage for measurement of cytokines. Plasma was collected at baseline and one day post LPS inhalation for measurement of vitamin D.Results28 participants were included. The mean age of volunteers was 26.2 +/- 5.5 years. All 28 patients were vitamin D deficient (plasma levels <50 nmol/l), with 89% (25/28) patients having severe vitamin D deficiency (<25 nmol/l). Vitamin D levels were significantly higher after LPS inhalation (p < 0.002). Levels of IL-1β in BALF were significantly higher in those with severe deficiency than those with mild/moderate deficiency (Figure 1; p = 0.04). Levels of IL-6, IL-8 or TNF-α did not differ between groups.Abstract S67 Figure 1Bronchoalveolar lavage fluid (BALF) levels of IL-1 beta were significantly elevated in volunteers with severe plasma vitamin D deficiency (<25 nmol/l) compared to those with mild or moderate deficiency (25–50 nmol/l)ConclusionsVitamin D deficiency was highly prevalent in this population of healthy volunteers. The rise in vitamin D levels post LPS exposure may represent mobilisation of vitamin D from fat stores during inflammation though vitamin D metabolism and kinetics are complex and may differ in healthy volunteers and the critically ill. Severe deficiency correlated with increased alveolar inflammation.

Ageing is associated with increased episodes of sepsis and poorer outcomes. Statins are associated with improved outcomes during infection. We aimed to characterise the impact of age and acute severe infection on key neutrophil functions, assess whether physiologically relevant doses of simvastatin altered neutrophil functions and if benefits were seen, when during a septic episode statins could be utilised. Methods Neutrophils from healthy volunteers and patients with lower respiratory tract infections (LRTI), pneumonia and sepsis were assessed for migratory accuracy, phagocytosis and neutrophil extracellular trap production before and after in-vitro treatment with simvastatin. Healthy elderly volunteers received 80mg simvastatin or placebo in a cross over double-blind randomised controlled trial and neutrophil functions were assessed. Data presented is for migration. Results Neutrophils from healthy subjects (n = 70, aged 21-94) demonstrated preserved neutrophil movement) (R 2 = -0.48, p < 0.0001) towards chemoattractants (data shown for IL-8). Neutrophil chemotaxis decreased after 60yrs (comparing <35 to >65yrs: mean difference (MD)1.25µm/min, p = 0.02).There was a progressive decrease in neutrophil chemotaxis in old patients with a LRTI, pneumonia and severe sepsis (MD compared to healthy control; LRTI (n = 10), 0.7µm/min, p = 0.04; pneumonia (n = 5), MD1.1µm/min, p = 0.02; sepsis (n = 22) MD1.6µm/min, p = 0.01) with "septic neutrophils" unable to mount targeted chemotaxis. Improvements to baseline were seen following recovery.In-vitro treatment of neutrophils from healthy older people with simvastatin (1mM) restored "old" neutrophil chemotaxis to that of "young" cells. Simvastatin also restored neutrophil migration from old patients with LRTI and pneumonia to baseline but not in patients with sepsis.Two weeks of oral simvastatin 80mg once daily therapy in healthy old volunteers (Age>65,n = 20) increased the accuracy of neutrophil migration (MD1.68µm/min, p = 0.02) replicating benchwork. Conclusions "Elderly" neutrophil function is compromised in health, and deteriorates during infective episodes, in accordance with the severity of the insult. Migratory accuracy can be improved with simvastatin therapy however neutrophil function in sepsis patients cannot be modulated during short term in-vitro therapy. Our data suggest statin therapy might be a preventative or an early adjuvant intervention rather than a treatment in established sepsis. We are testing whether simvastatin 80mg for seven days modifies neutrophil responses in elderly patients with pneumonia and sepsis (SNOOPI Trial).

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