A J Simpson

Evaluation of the effect of diagnostic methodology on the reported incidence of ventilator-associated pneumonia

Background: The optimal method for diagnosing ventilator-associated pneumonia (VAP) is controversial and its effect on reported incidence uncertain. This study aimed to model the impact of using either endotracheal aspirate or bronchoalveolar lavage on the reported incidence of pneumonia and then to test effects suggested from theoretical modelling in clinical practice. Methods: A three-part single-centre study was undertaken. First, diagnostic performance of aspirate and lavage were compared using paired samples from 53 patients with suspected VAP. Secondly, infection surveillance data were used to model the potential effect on pneumonia incidence and antibiotic use of using exclusively aspirate or lavage to investigate suspected pneumonia (643 patients; 110 clinically suspected pneumonia episodes). Thirdly, a practice change initiative was undertaken to increase lavage use; pneumonia incidence and antibiotic use were compared for the 12 months before and after the change. Results: Aspirate overdiagnosed VAP compared with lavage (89% vs 21% of clinically suspected cases, p<0.0001). Modelling suggested that changing from exclusive aspirate to lavage diagnosis would decrease reported pneumonia incidence by 76% (95% CI 67% to 87%) and antibiotic use by 30% (95% CI 20% to 42%). After the practice change initiative, lavage use increased from 37% to 58%. Although clinically suspected pneumonia incidence was unchanged, microbiologically confirmed VAP decreased from 18 to 9 cases per 1000 ventilator days (pu200a=u200a0.001; relative risk reduction 0.61 (95% CI 0.46 to 0.82)), and mean antibiotic use fell from 9.1 to 7.2 antibiotic days (21% decrease, pu200a=u200a0.08). Conclusions: Diagnostic technique impacts significantly on reported VAP incidence and potentially on antibiotic use.

Combined dysfunctions of immune cells predict nosocomial infection in critically ill patients

BACKGROUNDnNosocomial infection occurs commonly in intensive care units (ICUs). Although critical illness is associated with immune activation, the prevalence of nosocomial infections suggests concomitant immune suppression. This study examined the temporal occurrence of immune dysfunction across three immune cell types, and their relationship with the development of nosocomial infection.nnnMETHODSnA prospective observational cohort study was undertaken in a teaching hospital general ICU. Critically ill patients were recruited and underwent serial examination of immune status, namely percentage regulatory T-cells (Tregs), monocyte deactivation (by expression) and neutrophil dysfunction (by CD88 expression). The occurrence of nosocomial infection was determined using pre-defined, objective criteria.nnnRESULTSnNinety-six patients were recruited, of whom 95 had data available for analysis. Relative to healthy controls, percentage Tregs were elevated 6-10 days after admission, while monocyte HLA-DR and neutrophil CD88 showed broader depression across time points measured. Thirty-three patients (35%) developed nosocomial infection, and patients developing nosocomial infection showed significantly greater immune dysfunction by the measures used. Tregs and neutrophil dysfunction remained significantly predictive of infection in a Cox hazards model correcting for time effects and clinical confounders {hazard ratio (HR) 2.4 [95% confidence interval (CI) 1.1-5.4] and 6.9 (95% CI 1.6-30), respectively, P=0.001}. Cumulative immune dysfunction resulted in a progressive risk of infection, rising from no cases in patients with no dysfunction to 75% of patients with dysfunction of all three cell types (P=0.0004).nnnCONCLUSIONSnDysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

Pulmonary and systemic effects of mononuclear leukapheresis

Background and Objectivesu2002 There is increasing evidence that monocytes play a key role in the pathogenesis of acute lung inflammation. Mononuclear cell (MNC) leukapheresis can be used to remove large numbers of monocytes from circulating blood; however, the detailed characteristics of monocyte subpopulations removed by MNC leukapheresis, and the biological effects on the lung, remain incompletely defined.

S79 Potential diagnostic significance of neutrophil proteases in ventilator-associated pneumonia

Introduction and Objectives The clinical diagnosis of ventilator-associated pneumonia (VAP) remains notoriously difficult, as several non-infective conditions mimic VAP. Microbiological confirmation of the diagnosis using conventional cultures typically takes 48–72u2005h. Identification of molecules measurable within a short time frame and closely associated with microbiologically confirmed VAP is therefore highly desirable. VAP is associated with significant influx of activated neutrophils into the alveolar space. We postulated that extracellular neutrophil proteases in bronchoalveolar lavage fluid (BALF) may reliably identify VAP in suspected cases. Methods Fifty-four intubated and mechanically ventilated patients in the intensive care unit developed clinically suspected VAP and were recruited. Bronchoalveolar lavage (BAL) was performed using a standardised protocol. An aliquot of BALF was sent to the diagnostic microbiology laboratory for quantitative culture, with confirmation of VAP defined as growth of a pathogen(s) at >104 colony forming units/ml. Remaining BALF was centrifuged. The following neutrophil-specific proteases were assayed in cell-free BALF supernatant—matrix metalloproteinase (MMP)-8 and MMP-9 by Luminex assay, and human neutrophil elastase (HNE) by enzyme-linked immunosorbent assay. Urea was simultaneously measured in serum and BALF, and used to correct for the dilution of epithelial lining induced by BAL. Receiver operating characteristic (ROC) curves were constructed and optimal specificity and sensitivity for each marker calculated. Results Eleven patients (20%) had confirmed VAP. For HNE (cut off 670ng/ml) the ROC area under curve (AUC) was 0.87 (p<0.0001), sensitivity 93%, specificity 79%. For MMP-8 (13u2005ng/ml), ROC AUC was 0.81 (p<0.005), sensitivity 91%, specificity 63%. For MMP-9 (22u2005ng/ml), ROC AUC was 0.79 (p<0.005), sensitivity 82%, specificity 63%. Conclusions Neutrophil proteases are strongly associated with confirmed infection in cases of suspected VAP. The values for HNE, in particular, compare extremely favourably with any previously published equivalent values. These data suggest that neutrophil protease concentrations in BALF deserve further attention as potentially diagnostic markers for VAP. They further suggest that neutrophil proteases, inappropriately released into the extracellular space, may contribute to the pathophysiology of VAP.

S104 Monocyte influx accompanies the early neutrophilic inflammation seen in bronchoalveolar lavage fluid following lipopolysaccharide inhalation

Introduction Acute lung injury (ALI) has a mortality rate of over 30%, with no proven pharmacological treatment. Inhalation of lipopolysaccharide (LPS) in healthy volunteers induces transient inflammation resembling that found in patients with ALI. Inhaled LPS causes neutrophilia that is detectable in bronchoalveolar lavage fluid (BALF) and blood, but its effect on BALF and blood monocyte populations is not well established. Methods 12 healthy volunteers were recruited and randomly allocated to receive either 60u2005μg of inhaled LPS or saline (n=6 each arm). Clinical parameters, including temperature, and any reported symptoms were recorded. Full blood counts were taken at baseline and 2, 4, 6, 8 and 24u2005h post-inhalation. BAL was performed at 8u2005h. BALF cell populations were analysed morphologically using cytospins and cytometrically by flow cytometry after staining for cell surface markers (alveolar macrophages: CD163, CD206, CCR5; neutrophils/monocytes: HLA-DR, CD14, CD16). Results 4 LPS volunteers developed pyrexia, two reported cough and one myalgia. The mean maximal increment in temperature was significantly greater in the LPS arm (p=0.047). Compared to saline inhalation, LPS caused a peripheral blood neutrophilia (p=0.006) that was evident from 4u2005h and greatest at 8u2005h. There was no significant difference in peripheral blood monocyte counts between treatment arms at any point measured (p=0.87). Although mean total alveolar macrophage numbers were similar between the two groups, their relative proportion in the LPS volunteers was significantly reduced due to the expansion in neutrophil and monocyte populations. Flow cytometry revealed a 24-fold expansion of the neutrophil population following LPS (in parallel with morphological data). These neutrophils were distinguishable by HLA-DR-/CD14-/CD16+ staining. There was a concomitant similar rise in the population of HLA-DR+/CD14+/CD16- ‘classical’ monocytes. Further analysis of these monocytes revealed that macrophage cell surface marker expression was absent. Conclusion Morphological analysis of BAL fluid in previous LPS inhalation studies has consistently suggested that there is no change in the monocyte population. Using flow cytometry enables a more detailed analysis. This study is the first to clearly demonstrate that an early expansion in the monocyte population accompanies the neutrophil influx seen in BALF 8u2005h following inhalation of LPS.

S81 Primary type II alveolar epithelial cells respond differentially to bacterial virulence factors

Introduction and objectives The pathogens most commonly implicated in ventilator-associated pneumonia (VAP) are Pseudomonas aeruginosa and Staphylococcus aureus. Although a florid inflammatory response characteristically occurs in the alveolar space in VAP, the underlying mechanisms remain unclear, partly owing to a lack of adequate models of alveolar injury. We therefore sought to characterise the response of primary human type II alveolar epithelial (ATII) cells to virulence factors from these pathogens. Methods Primary ATII cells were derived from seven patients undergoing surgical resection for lung cancer. Lung tissue was refrigerated overnight; flushed with saline; incubated with trypsin; diced; incubated with DNAse I; and strained/filtered. Macrophages and fibroblasts were removed by adherence. The resulting cell population was centrifuged, washed, resuspended and plated onto tissue culture plates pre-coated with type I bovine collagen at 2×106u2005cells/ml. When cells achieved confluence medium was replenished and the following were added for 24u2005h: 100u2005ng/ml P aeruginosa lipopolysaccharide (LPS); 10u2005ug/ml S aureus lipoteichoic acid (LTA); 10u2005ug/ml S aureus peptidoglycan (PGN); 10u2005ng/ml human recombinant tumour necrosis factor alpha (TNFα); or control medium. Supernatant was aspirated at 24u2005h and cytokines were measured by cytometric bead array. Results Interleukin (IL)-1β, IL-6, IL-8 IL-10, IL-12p70 and TNFα were all detectable in control medium at 24u2005h. None of the measured cytokines were significantly altered by application of LPS or LTA. In contrast, PGN induced a significant rise in concentrations of IL-1β, IL-6, IL-8 IL-10 and TNFα. Addition of TNFa induced a significant increase in IL-6, IL-8 and IL-10. The only cytokine to be uniformly uninfluenced by stimulation was IL-12p70. Conclusions In our hands primary ATII cells appeared to be unresponsive to P aeruginosa LPS or to S aureus LTA. By contrast, S aureus PGN provoked a brisk and significant inflammatory response simultaneously affecting a range of cytokines. These data suggest that ATII cells have strikingly different responses to individual bacterial virulence factors. They further suggest that PGN (but not LTA) contributes, at least in part, to the florid inflammatory response seen in Staphylococcal pneumonia.

S125 Ly6Chi circulating monocytes direct alternatively activated, pro-fibrotic, lung macrophage regulation of pulmonary fibrosis

Introduction and objectives Idiopathic pulmonary fibrosis (IPF) remains one of the few respiratory conditions for which there are no effective therapies. The role of monocytes and macrophages in IPF has been disputed as anti-inflammatory therapies produce questionable benefit. Corticosteroids, however, actually induce an alternatively activated, pro-fibrotic, macrophage phenotype. We sought to determine whether monocytes and macrophages play a role in disease pathogenesis in an attempt to explain why current hypotheses and anti-inflammatory therapies have produced limited clinical benefit despite years of research. Methods Using multiple in vivo depletional strategies, backed up by an adoptive transfer technique, we extensively investigated the role of monocytes and macrophages during lung fibrogenesis. We performed studies on samples from patients with IPF in an attempt to determine the translational importance of our findings. Results Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (p=0.0079), fibrosis score (p=0.0051), and qPCR for surrogate markers of fibrosis Col1 (p=0.0083) and a-smooth muscle actin (p=0.0349). There was an associated reduction in expression of markers of alternative macrophage activation, Ym1 (p=0.0179), and Arginase1. This reduction was confirmed by immunohistochemistry (IHC) for Ym1 (p=0.0233). IHC on lung macrophages from patients with IPF demonstrated the novel finding of expression of the human alternative macrophage marker CD163. Depletion of Ly6Chi circulating monocytes reduced pulmonary fibrosis (p=0.0052). Adoptive transfer of Ly6Chi BMDMS during fibrogenesis exacerbated pulmonary fibrosis (p=0.0304). Furthermore, depletion of circulating Ly6Chi monocytes lead to a subsequent reduction in the number of Ym1-positive alternatively activated lung macrophages (p=0.0310) with a concomitant reduction in the expression of Ym1 and Arginase1. Conclusions We have demonstrated that monocytes and macrophages do modulate pulmonary fibrosis and suggest that Ly6Chi monocytes (possible fibrocyte precursors) are precursors of alternatively activated, pro-fibrotic, lung macrophages. These findings could link the ‘inflammatory’ and aberrant wound healing hypotheses and explain the lack of effectiveness of corticosteroids in treating IPF. By enhancing our understanding of the pathogenesis of this dreadful disease, our results may enable new therapeutic targets to be developed, facilitate targeted cell-based therapy, and bring hope to one of the longstanding enigmas of respiratory medicine.

S50 Evaluation of secretory leucoprotease inhibitor (SLPI) as an anti-inflammatory therapy for donor lung inflammation

Introduction Donor lung inflammation, reflected by high concentrations of interleukin-8 in bronchoalveolar lavage (BAL) and an imbalance between pro-inflammatory IL-6 and anti inflammatory IL-10 in tissue, correlates with poor graft function and reduced survival after human lung transplantation. Secretory leucoprotease inhibitor (SLPI) is an anti-protease abundant in the lung. SLPI inhibits neutrophil elastase and down regulates inflammatory cytokine transcription via the NFκB pathway. We investigated the effect of SLPI on inflammatory mediators, in a rat model of brain death induced lung injury. Methods Brain death (BD) was induced in anesthetised ventilated male Wistar rats (n=16) by rapid inflation of an intracranial balloon, the balloon was not inflated in non-BD sham animals. Rats received intra-tracheal human recombinant SLPI (400u2005ng/g) (n=8) or saline (control n=8, sham n=9) at 1u2005h. The experiment was terminated at 5u2005h. Serum samples were taken at 0, 1, 3 and 5u2005h, and BAL from one lung was taken for cytokine analysis. The second lung was used for wet/dry ratio and Q-PCR analysis. Results CXCL1, TNF-α, IFN-γ and IL-6 were significantly higher in BAL and serum of control than sham rats, demonstrating that BD induced lung inflammation in this model. There was no change in lung wet-dry ratio between SLPI treated and control groups. Surprisingly, CXCL1 levels were higher in the BAL of SLPI treated rats than controls (p=0.05), however no significant difference was detected for any other cytokine. There was a non-significant trend towards a higher number of CD45+ leucocytes in BAL in SLPI treated rats compared to controls. CXCL1 mRNA was also increased 1.5-fold in the SLPI treated group compared to controls (p<0.05). Conclusions In this study, SLPI does not appear to have an anti-inflammatory effect in the rat lung, and may exacerbate inflammation as seen by an increased concentration of the chemokine CXCL1. This surprising effect may be due to the short time course of this experiment where the initial effect of SLPI may be pro-inflammatory. Given the small window of opportunity available to treat donor lungs we believe that SLPI is an inappropriate intervention for use in lung transplantation.Abstract S50 Figure 1

S124 Macrophage deletion of vHL results in alternative activation and enhanced lung fibrosis independent of HIF-1

Background Hypoxia-inducible factor (HIF-1) is a master regulator of the cellular hypoxic response and has been implicated in the pathogenesis of inflammatory and fibrotic disease including IPF. Aims To study the role of hypoxia and HIF-1 activation in macrophages in the i.t. bleomycin-induced lung fibrosis model. Methods The i.t. bleomycin model was used to study the effect of HIF-1 manipulation in mice. The primary end-point was lung collagen content at day 24 post i.t. bleomycin instillation. The HIF-1α inducer dimethyloxallyl glycine (DMOG) was administered i.p. on days 14, 17 and 21. The role of myeloid-HIF-1 activity in lung fibrosis was determined using mice in which either HIF-1α or vHL (the dominant negative-regulator of HIF-1α) was selectively knocked out of lysosyme M expressing cells (LysM-Cre-Hif-1 and Cre-LysM-vHL). Lung tissue hypoxia was determined using Hypoxyprobe-1TM administered on day 24. Alternative activation status of HIF-1 null and vHL null macrophages was studied in bone-marrow derived cells from LysM-Cre-Hif-1 and Cre-LysM-vHL mice. Results Pharmacological induction of HIF-1 in the late period of the bleomycin model with i.p. dimethyloxallyl glycine (DMOG) resulted in significantly enhanced lung collagen (mean±s.e.mμg/lung) on day 24 compared to controls (193±15 vs 152±8, p<0.05, n>7 per gp). Hypoxyprobe-1 staining in the bleomycin-injured lung revealed hypoxic alveolar macrophages even in areas of lung distant to patches if severe fibrosis, implying a role for hypoxic/HIF-1 expressing alveolar macrophages in lung fibrosis. However, lung collagen content was identical in myeloid-cell Hif-1 null mice and wild-type litter-mate controls (276±23 vs 277±22, n=8 per gp). In contrast, myeloid-cell vHL-null mice exhibited significantly enhanced lung collagen deposition versus controls (373±36 vs 282±54, p<0.05, n>9 per gp). Isolated vHL-null macrophages exhibited enhanced expression of the alternative activation markers YM-1, mannose receptor, arginase-1 and FIZZ-1. Conclusions vHL deletion in macrophages enhances alternative activation and promotes lung fibrosis independent of HIF-1.

P254 Investigating HMGB1 as a potential inflammatory mediator in brain death induced lung damage

Background Donor lungs are particularly susceptible to the haemodynamic instability and systemic inflammation which occurs following induction of brain death (BD). Increased donor lung inflammation with IL-6 or IL-8 is associated with poor post-transplant outcomes. Donor LPS pre-conditioning significantly ameliorates the lung inflammation after BD in a rat model suggesting that TLR-4 ligation following BD contributes to donor lung inflammation, even in the absence of LPS. We hypothesised that HMGB1, an alarmin released from damaged tissue and immune cells and known TLR-4 ligand, may act as an inflammatory mediator in BD induced lung injury. Methods BD was induced in a rat model using rapid intra-cranial balloon inflation and bronchoalveolar lavage (BAL), serum and lung tissue were collected and compared with sham operated controls rats. The HMGB1 concentration in the rat serum and BAL was measured by ELISA. Real-time PCR was used to assess HMGB1 mRNA expression in lung tissue following BD. HMGB1 immunolocalisation studies were performed on BD and sham rat lung tissue. Finally HMGB1 staining was assessed in lung tissue from human BD donors and normal controls. Results The BAL HMGB1 concentration was significantly higher in the BD group (965±302u2009ng/ml) than in the sham group (655 ± 274u2009ng/ml) (p=0.0172). There was however no difference in HMGB1 gene expression between the two groups. HMGB1 positive staining was visualised in a nuclear and extra-nuclear location and was dispersed throughout the rat and human lung with a greater density around the bronchioles. There was a significantly higher area of positive staining in BD rat lung tissue than sham tissue (p=0.0345). No difference was seen in the human BD lungs compared to controls (Abstract P254 Figure 1).Abstract P254 Figure 1 Conclusions HMGB1 is likely to be released passively from lung cells suffering sustained damage during BD. As HMGB1 gene transcription is unchanged up to 5u2009h following BD, the lung tissue is most likely releasing presynthesised cellular HMGB1. This study provides evidence of the presence of an alarmin which is likely to potentiate inflammation in the donor lung via the TLR-4 pathway. If release occurs early following BD, HMGB1 could be an important initiating mediator in donor lung inflammation.