Kev Dhaliwal

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron Oxide Feasibility and Safety in Humans

Background— Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice–compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. Methods and Results— Peripheral blood mononuclear cells 1–5×109 were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 108 SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 109 SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. Conclusions— Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration— URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.

C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kδ. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

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

BACKGROUND Nosocomial 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. METHODS A 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. RESULTS Ninety-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). CONCLUSIONS Dysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

A novel subpopulation of monocyte-like cells in the human lung after lipopolysaccharide inhalation

The co-ordinated recruitment of monocyte subpopulations, neutrophils and regulatory T-cells (Tregs) during the early stages of human acute lung inflammation remains poorly understood. We therefore performed a detailed characterisation of these lineages in the blood and lungs in a model of human acute lung inflammation. Healthy volunteers inhaled lipopolysaccharide (LPS) or saline (n=6 for each group). Blood was collected at 0, 2, 4, 6 and 8 h and bronchoscopy with bronchoalveolar lavage (BAL) performed at 8 h. Multiparameter flow cytometry was used to characterise monocyte subpopulations, neutrophils and Tregs in the blood and lung. Inhalation of LPS was associated with significant blood and BAL fluid neutrophilia. Blood populations of monocyte subpopulations and Tregs were unaltered by LPS. In contrast, LPS induced an accumulation of a pulmonary monocyte-like cell (PMLC) population, which was further subdivided into “inducible” CD14++CD16- and “resident” CD14++CD16+ subsets. Inducible PMLCs were significantly increased following LPS inhalation (p=0.0046), whereas resident PMLCs were unchanged. In addition, we noted a significant decrease in Tregs in BAL fluid with LPS inhalation (p=0.027). The early stages of LPS-induced inflammation in humans is characterised by pulmonary accumulation of a novel inducible monocyte-like subpopulation, accompanied by significant changes in both neutrophil and Treg numbers.

The use of a portable digital thoracic suction Thopaz drainage system for the management of a persistent spontaneous secondary pneumothorax in a patient with underlying interstitial lung disease.

We present the case of a 68-year-old woman who presented in extremis with a secondary pneumothorax with a past history of severe idiopathic pulmonary fibrosis. Following insertion of a 32F intercostal drain, she developed a persistent broncho-pleural fistula and became dependent on negative-pressure wall-mounted suction to prevent respiratory compromise. She declined definitive surgical intervention and was therefore managed conservatively. After adhering to the wall-mounted suction method for 49 days, we obtained for use a portable digital thoracic drainage system previously used only in the cardiothoracic postoperative patient. This electronically delivered, negative-pressure drainage system induced radiographic improvement within 24 h, and allowed the patient to mobilise for the first time since admission. The patient was discharged home with the Thopaz drain in situ 8 weeks after placing it, and the drain was removed successfully with a resolved pneumothorax 20 weeks after her initial presentation.

T4 Optically detectable antimicrobial peptides enable the immediate detection of bacteria and fungi in the lung

Introduction The immediate detection of pathogens in the lungs of patients with unexplained pulmonary opacities in the intensive care unit would represent a significant advance in their management. An optical imaging strategy, including the endobronchial administration of bacterial specific Smartprobes, would confer a number of advantages over conventional techniques such as bronchoalveolar lavage, principally real-time detection to immediately inform antimicrobial therapy. The aims of this study were to fluorescently label and iteratively develop anti-microbial peptides to image bacteria in situ in the lung using fibered confocal fluorescence microscopy (FCFM). Methods Antimicrobial peptides (AMP) have been synthesised on a dendrimeric scaffold (AMP-1) and conjugated to an environmentally sensitive fluorophore called NBD, following the continuous development a linear counterpart. A further construct consists of an AMP with gram-selectivity conjugated to the NBD fluorophore (AMP-2). These are combined with FCFM to allow distal alveolar imaging at micron resolution in an ex vivo ovine model of bacterial infection. Results AMP-1 demonstrates bacterial binding affinity in a concentration dependent manner and labels a diverse panel of bacteria, including a panel consisting of >70% of ventilator-associated pneumonia causing organisms and the pathogenic fungi Aspergillus fumigatus. AMP-1 demonstrates significantly higher fluorescence over isomolar linear equivalents for E. coli, K. pneumoniae, P. aeruginosa, MSSA, A. baumannii and S. pneumoniae (all p < 0.01), is selective for bacteria over mammalian cells and has improved chemical stability over the linear equivalent when incubated with bronchoaoveolar lavage from patients with acute respiratory distress syndrome. Furthermore, AMP-1 can label E. coli, K. pneumoniae, P. aeruginosa and MSSA in situ in an ex vivo ovine model when instilled endobronchially and imaged with FCFM (pin vitro and remains selective for gram-negative bacteria over mammalian cells. In the ex-vivo model AMP-2 selectively labels the gram-negative bacterial segments (P. aeruginosa, K. pneumonia and E. coli) over the gram-positive (MSSA, MRSA and S. pneumoniae) or control pulmonary segments (all p < 0.05). Conclusions A Smartprobe/FCFM strategy to immediately detect bacteria with gram selectivity in size relevant pre-clinical models is described, and are undergoing first-in-man translation.

Pulmonary and systemic effects of mononuclear leukapheresis

Background and Objectives  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.

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron Oxide

Background— Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice–compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. Methods and Results— Peripheral blood mononuclear cells 1–5×109 were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 108 SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 109 SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. Conclusions— Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration— URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.

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 48–72 h. 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 (13 ng/ml), ROC AUC was 0.81 (p<0.005), sensitivity 91%, specificity 63%. For MMP-9 (22 ng/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 60 μg 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 24 h post-inhalation. BAL was performed at 8 h. 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 4 h and greatest at 8 h. 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 8 h following inhalation of LPS.