Alastair Proudfoot

Human models of acute lung injury

Acute lung injury (ALI) is a syndrome that is characterised by acute inflammation and tissue injury that affects normal gas exchange in the lungs. Hallmarks of ALI include dysfunction of the alveolar-capillary membrane resulting in increased vascular permeability, an influx of inflammatory cells into the lung and a local pro-coagulant state. Patients with ALI present with severe hypoxaemia and radiological evidence of bilateral pulmonary oedema. The syndrome has a mortality rate of approximately 35% and usually requires invasive mechanical ventilation. ALI can follow direct pulmonary insults, such as pneumonia, or occur indirectly as a result of blood-borne insults, commonly severe bacterial sepsis. Although animal models of ALI have been developed, none of them fully recapitulate the human disease. The differences between the human syndrome and the phenotype observed in animal models might, in part, explain why interventions that are successful in models have failed to translate into novel therapies. Improved animal models and the development of human in vivo and ex vivo models are therefore required. In this article, we consider the clinical features of ALI, discuss the limitations of current animal models and highlight how emerging human models of ALI might help to answer outstanding questions about this syndrome.

Nuclear Factor κ-B Is Activated in the Pulmonary Vessels of Patients with End-Stage Idiopathic Pulmonary Arterial Hypertension

Objectives To assess activation of the inflammatory transcription factor NF-kappa B (NF-κB) in human idiopathic pulmonary arterial hypertension (PAH). Background Idiopathic PAH is a severe progressive disease characterized by pulmonary vascular remodeling and excessive proliferation of vascular cells. Increasing evidence indicates that inflammation is important in disease pathophysiology. Methods NF-κB-p65 and CD68, CD20 and CD45 were measured by immunohistochemistry and confocal microscopy on lung specimens from patients with idiopathic PAH (n = 12) and controls undergoing lung surgery (n = 14). Clinical data were recorded for all patients including invasive pulmonary hemodynamics for the PAH patients. Immunohistochemical images were analyzed by blinded observers to include standard pulmonary vascular morphometry; absolute macrophage counts/mm2 and p65-positivity (p65+) using composite images and image-analysis software; and cytoplasmic:nuclear p65+ of individual pulmonary arterial endothelial and smooth muscle cells (PASMC) in 10–20 pulmonary arteries or arterioles per subject. The expression of ET-1 and CCL5 (RANTES) in whole lung was determined by RT-qPCR. Results Macrophage numbers were increased in idiopathic PAH versus controls (49.0±4.5 vs. 7.95±1.9 macrophages/100 mm2, p<0.0001): these macrophages demonstrated more nuclear p65+ than in macrophages from controls (16.9±2.49 vs. 3.5±1.25%, p<0.001). An increase in p65+ was also seen in perivascular lymphocytes in patients with PAH. Furthermore, NF-κB activation was increased in pulmonary arterial endothelial cells (62.3±2.9 vs. 14.4±3.8, p<0.0001) and PASMC (22.6±2.3 vs. 11.2±2.0, p<0.001) in patients with PAH versus controls, with similar findings in arterioles. Gene expression of both ET-1 mRNA ((0.213±0.069 vs. 1.06±0.23, p<0.01) and CCL5 (RANTES) (0.16±0.045 vs. 0.26±0.039, p<0.05) was increased in whole lung homogenates from patients with PAH. Conclusions NF-κB is activated in pulmonary macrophages, lymphocytes, endothelial and PASMC in patients with end-stage idiopathic PAH. Future research should determine whether NF-κB activation is a driver or bystander of pulmonary vascular inflammation and if the former, its potential role as a therapeutic target.

The pulmonary endothelium in acute respiratory distress syndrome: insights and therapeutic opportunities

The pulmonary endothelium is a dynamic, metabolically active layer of squamous endothelial cells ideally placed to mediate key processes involved in lung homoeostasis. Many of these are disrupted in acute respiratory distress syndrome (ARDS), a syndrome with appreciable mortality and no effective pharmacotherapy. In this review, we consider the role of the pulmonary endothelium as a key modulator and orchestrator of ARDS, highlighting advances in our understanding of endothelial pathobiology and their implications for the development of endothelial-targeted therapeutics including cell-based therapies. We also discuss mechanisms to facilitate the translation of preclinical data into effective therapies including the application of biomarkers to phenotype patients with ARDS with a predominance of endothelial injury and emerging biotechnologies that could enhance delivery, discovery and testing of lung endothelial-specific therapeutics.

Translational research: what does it mean, what has it delivered and what might it deliver?

Purpose of reviewIn this article, we review recent developments in translational research in the fields of acute lung injury, acute kidney injury and sepsis with a focus on emerging biomarkers and outline future advances in the field. Recent findingsThere is currently a significant and unmet need for high quality translational research in critical care. The emergence of ‘-omics’ technologies and sophisticated imaging techniques have resulted in a rapid growth of emerging biomarkers. Biomarkers would ideally provide early and reliable endpoints for proof of concept in clinical trials and inform clinical decision making through earlier and more precise diagnosis and risk stratification. SummaryDespite significant investment in basic science and time-consuming clinical trials, the majority of pharmacological interventions developed for critical illness have yet to translate into measurable clinical benefit. Future validation and qualification of emerging biomarkers allied to advances in pharmacogenomic profiling have the potential to provide valuable clinical information while accurately phenotyping patients enrolled in future clinical trials.

Prevalence, natural history, and time-dependent outcomes of a multi-center North American cohort of out-of-hospital cardiac arrest extracorporeal CPR candidates

AIM Estimate prevalence of ECPR-eligible subjects in a large, North American, multi-center cohort, describe natural history with conventional resuscitation, and predict optimal timing of transition to ECPR. METHODS Secondary analysis of clinical trial enrolling adults with non-traumatic OHCA. Primary outcome was survival to discharge with favorable outcome (mRS 0-3). Subjects were additionally classified as survival with unfavorable outcome (mRS 4-5), ROSC without survival (mRS 6), or without ROSC. We plotted subject accrual as a function of resuscitation duration (CPR onset to return of spontaneous circulation (ROSC) or termination of resuscitation), and estimated time-dependent probabilities of ROSC and mRS 0-3 at discharge. Adjusted logistic regression models tested the association between resuscitation duration and survival with mRS 0-3. RESULTS Of 11,368 subjects, 1237 (10.9%; 95%CI 10.3-11.5%) were eligible for ECPR, Of these, 778 (63%) achieved ROSC, 466 (38%) survived to discharge, and 377 (30%) had mRS 0-3 at discharge. Half with eventual mRS 0-3 achieved ROSC within 8.8min (95%CI 8.3-9.2min) of resuscitation, and 90% within 21.0min (95%CI 19.1-23.7min). Time-dependent probabilities of ROSC and mRS 0-3 declined over elapsed resuscitation, and the likelihood of additional cases with mRS 0-3 beyond 20min was 8.4% (95%CI 5.9-11.0%). Resuscitation duration was independently associated with survival to discharge with mRS 0-3 (OR 0.95; 95%CI 0.92-0.97). CONCLUSION Approximately 11% of subjects were eligible for ECPR. Only one-third survived to discharge with favorable outcome. Performing 9-21min of conventional resuscitation captured most ECPR-eligible subjects with eventual mRS 0-3 at hospital discharge.

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury

Background Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects. Methods We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin. Results Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro (P<0.01 and P<0.001, respectively), and also significantly attenuated inflammation and signs of lung injury in non-human primates (P<0.01 in all cases). In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release (P<0.01 in all cases) and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples. Conclusion These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome. Trial registration number ClinicalTrials.gov NCT01587807.

von Willebrand factor disruption and continuous-flow circulatory devices

Bleeding events remain a significant and frequent complication of continuous-flow left ventricular assist devices (VADs). von Willebrand factor (VWF) is critical to hemostasis by acting as a bridging molecule at sites of vascular injury for normal platelet adhesion as well as promoting platelet aggregation under conditions of high shear. Clinical and experimental data support a role for acquired von Willebrand disease in VAD bleeding episodes caused by shear-induced qualitative defects in VWF. Pathologic shear induces VWF unfolding and proteolysis of large multimers into smaller less hemostatic multimers via ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). This review outlines the pathobiology of VWF disruption in the context of VADs as well as current diagnostic and management strategies of the associated acquired von Willebrand disease.

Killing without collateral damage: new hope for sepsis therapy.

Sepsis is a common condition, and more than 19 million cases of severe sepsis (i.e. cases where organ dysfunction has occurred as a result of sepsis) occur each year1. Despite many decades of research, the mortality rate remains approximately 30%, and survivors are often left with a significant morbidity burden, including neurocognitive impairment, neuromuscular weakness and persistent organ dysfunction2. End-organ dysfunction and injury ensues in part as a result of innate immune cells (including neutrophils) failing to control pathogen invasion, which leads to microbial proliferation, release of inflammatory mediators, endothelial dysfunction, and tissue injury. On page xxx of this issue, Ruchaud-Sparagano and colleagues present novel data investigating the hypothesis that monophosphoryl lipid A (MPLA), a derivative of lipopolysaccharide (LPS), can ameliorate the release of cytotoxic mediators from neutrophils without impairing bacterial clearance3. Maintenance of efficient neutrophil phagocytic and bactericidal function in the absence of significant release of cytotoxic mediators would be an attractive therapeutic strategy for septic patients. Using clinically relevant in vitro models, the authors show that exposing neutrophils to MPLA, in contrast to LPS, does not result in the release of reactive oxygen species and myeloperoxidase. In contrast to LPS, pre-exposure of MPLA treated neutrophils to a blocking antibody for TLR4 augmented myeloperoxidase release, suggesting that LPS and MPLA differentially modulates TLR4. Despite ameliorated production of superoxide and myeloperoxidase, MPLA exposed neutrophils retained P.aeruginosa killing abilities, implying that bacterial killing may not be related to the amount of enzymes released by neutrophils, and intriguingly that superoxide independent killing mechanisms may exist, at least in MPLA exposed neutrophils. Ruchaud-Sparagano and colleagues also observed no difference in the release of TNFα, IL-1β and IL-10 from MPLA and LPS treated neutrophils. However, there was a significant reduction in the release of the potent neutrophil chemoattractant IL-8 from MPLA treated cells, mediated via the PI3kinase pathway. LPS exposure was also found to increase neutrophil cell surface TLR4 expression, a phenomenon that did not occur with MPLA. The contrasting effects of MPLA and LPS are summarized in Table One Table one Effects of LPS and MPLA on human neutrophils The observations that administration of MPLA after LPS exposure reduced superoxide and myeloperoxidase release, and that MPLA abrogated increased TLR4 cell surface expression in LPS treated neutrophils whilst preserving efficient bacterial killing, support the notion that MPLA may hold promise as adjunctive therapy for Gram negative sepsis. Persistent activation of TLR pathways, as occurs in the early stages of sepsis, has been associated with excessive release of inflammatory cytokines and the development of tissue injury in sepsis4,5. In contrast to the current paper, a study of 20 patients treated with a synthetic lipid A analogue prior to intravenous endotoxin, demonstrated decreased serum concentrations of the pro-inflammatory cytokines TNFα, IL-1β, IL-8 and IL-6, in the presence of increased circulating neutrophil numbers6. It would seem reasonable to speculate that reduced IL-8 release, coupled with preserved chemotaxis in MPLA treated cells, to other chemoattractants such as LTB4, may serve to optimize neutrophil recruitment to sites of pathogen invasion without the sequellae of exuberant, mediator-induced tissue injury. The current work also seeks to determine the mechanistic basis of the contrasting responses of neutrophils to LPS and MPLA, demonstrating the differential signaling responses of LPS and MPLA downstream of TLR4. TLR4 activation can lead to activation of the MyD88 and TRIF pathways. IL-8 production requires recruitment of MyD88 to TLR4; based on the ameliorated concentrations of IL-8 produced by MPLA exposed neutrophils, it is plausible that the differential TLR4 effects of LPS and MPLA may be mediated via differential activation of pathways downstream of TLR4. LPS, but not MPLA, triggered IκB kinase (IKK) phosphorylation, which results from MyD88 activation, whereas both LPS and MPLA induced interferon regulatory factor 3 (IRF3) activation, which is downstream of TRIF. Both LPS and MPLA were able to trigger p38 MAPK phosphorylation (an event downstream from both MyD88 and TRIF), and LPS, but not MPLA induced JNK phosphorylation, which occurs downstream from MyD88, and to a less extent TRIF. Thus this differential activation of MyD88 and TRIF likely explains the different effects of LPS and MPLA on TLR4 up-regulation. Sepsis is a heterogeneous and poorly defined clinical syndrome. The continued absence of sepsis therapies suggests that our knowledge of the underlying biology of sepsis remains limited, and a paucity of mechanistic and preclinical data to support the interventions trialled, continues to hamper the rational development of therapeutic interventions for sepsis (e.g.7,8,9). Attempts to target (and antagonise) single molecules implicated in sepsis have proved futile in clinical trials, reinforcing the notion that therapies that modulate the body’s innate immune response, such as MPLA, may be more likely to demonstrate clinical benefit as adjuncts to standard therapy (antibiotics). We should therefore welcome preclinical human data such as these that inform the field of neutrophil biology and may lead to the development of new therapies for this widespread and often devastating condition.

Tricuspid valvular dynamics and 3-dimensional geometry in awake and anesthetized sheep

Objectives Clinical and experimental tricuspid valve physiology data are derived predominantly from anesthetized subjects, but normal tricuspid valve geometry and dynamics may be altered by general anesthesia and mechanical ventilation. We set out to investigate 3‐dimensional geometry and dynamics of the tricuspid valve complex in awake and anesthetized sheep. Methods While on cardiopulmonary bypass and with the heart beating, 6 adult sheep (50 ± 8 kg) underwent implantation of 6 sonomicrometry crystals around the tricuspid annulus. One crystal was implanted on the anterior, posterior, and septal papillary muscle tips, 4 on the right ventricular free wall and 1 on its apex. Pressure transducers were placed in both ventricles. Sonomicrometry and pressure transducer cables were externalized to subcutaneous buttons. After 7 days of recovery, hemodynamic and sonomicrometry data were recorded with animals awake and anesthetized. Results Hemodynamic parameters did not differ between groups. Tricuspid annular area contraction decreased with anesthesia (16.4% ± 4.2% vs 11.2% ± 3.2%, P = .047) as did tricuspid annular perimeter contraction (8.1% ± 2.2% vs 5.4% ± 1.7%, P = .050), predominantly due to reduced contraction of the septal annulus (10.5% ± 2.9% vs 7.5% ± 3.5%, P = .019). Tricuspid annular height did not differ between groups. Minimal distance from anterior, posterior, and septal papillary muscle tips to the annular plane did not change with anesthesia. Regional right ventricle free wall contraction was depressed under anesthesia in anterior (16.3% ± 3.1% vs 12.3% ± 2.2%, P = .027) and lateral (14.9% ± 1.3% vs 11.5% ± 2.8%, P = .016) segments, whereas the posterior remained unchanged. Conclusions General anesthesia did not alter tricuspid annular or subvalvular 3‐dimensional geometry but reduced right ventricular contraction and tricuspid annular dynamics. Graphical abstract Figure. No Caption available.

ARDS, up close and personal

The acute respiratory distress syndrome (ARDS) was first reported in a case series from Denver in 1967.1 Forty-five years later, the syndrome was reborn: the ‘Berlin’ definition captures patients with a mortality of 24% rising to 48% in the group of patients with the most severe respiratory failure.2 Survivors commonly suffer from muscle weakness and neuropsychiatric problems, such that fewer than 50% have returned to work 12 months after leaving intensive care.3 ARDS is important both clinically and financially. It is a not uncommon contributor to the deaths of critically ill patients of all ages4 and survivors commonly suffer from muscle weakness and neuro-psychiatric problems, such that fewer than 50% have returned to work 12 months after leaving intensive care.3 ,5 The onset of ARDS can be ascribed to two processes resulting from acute inflammation or injury at the lungs gas exchange surface: the alveolar–capillary membrane. These processes increase the permeability of the membrane associated with the recruitment of neutrophils into the airspace. The resulting acute inflammatory exudate inactivates surfactant, leading to collapse and consolidation with progressive loss of functioning lung. This would be well tolerated physiologically if the inflammatory process did not paralyse the lungs means of preventing ventilation–perfusion mismatch: hypoxic pulmonary vasoconstriction. The combination of these factors causes profound hypoxaemia accompanied by pulmonary oedema. The latter mechanism accounts for the fact that as a rule patients with ARDS are considerably more hypoxaemic than those with heart failure with similar radiographic appearances. Since its recognition, much has been learnt about …