Publications by authors named "Masao Takata"

62 Publications

Secreted Extracellular Cyclophilin A is a Novel Mediator of Ventilator Induced Lung Injury.

Am J Respir Crit Care Med 2021 Apr 13. Epub 2021 Apr 13.

Imperial College London, Anaesthetics & Intensive Care, London, United Kingdom of Great Britain and Northern Ireland;

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator induced lung injury. Extracellular Cyclophilin A is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to COVID-19 infection.

Objectives: Here we explore the involvement of extracellular Cyclophilin A in the pathophysiology of ventilator-induced lung injury.

Methods: Mice were ventilated with low or high tidal volume for up to 3 hours, with or without blockade of extracellular Cyclophilin A signalling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretch to explore the cellular source of extracellular Cyclophilin A, and Cyclophilin A levels were measured in bronchoalveolar lavage fluid from acute respiratory distress syndrome patients, to evaluate clinical relevance.

Measurements And Main Results: High tidal volume ventilation in mice provoked a rapid increase in soluble Cyclophilin A levels in the alveolar space, but not plasma. In vivo ventilation and in vitro stretch experiments indicated alveolar epithelium as the likely major source. In vivo blockade of extracellular Cyclophilin A signalling substantially attenuated physiological dysfunction, macrophage activation and matrix metalloproteinases. Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated levels of extracellular Cyclophilin A within bronchoalveolar lavage.

Conclusions: Cyclophilin A is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. Extracellular Cyclophilin A represents an exciting novel target for pharmacological intervention.
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http://dx.doi.org/10.1164/rccm.202009-3545OCDOI Listing
April 2021

Intra-alveolar neutrophil-derived microvesicles are associated with disease severity in COPD.

Am J Physiol Lung Cell Mol Physiol 2021 01 4;320(1):L73-L83. Epub 2020 Nov 4.

Division of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom.

Despite advances in the pathophysiology of chronic obstructive pulmonary disease (COPD), there is a distinct lack of biochemical markers to aid clinical management. Microvesicles (MVs) have been implicated in the pathophysiology of inflammatory diseases including COPD, but their association to COPD disease severity remains unknown. We analyzed different MV populations in plasma and bronchoalveolar lavage fluid (BALF) taken from 62 patients with mild to very severe COPD (51% male; mean age: 65.9 yr). These patients underwent comprehensive clinical evaluation (symptom scores, lung function, and exercise testing), and the capacity of MVs to be clinical markers of disease severity was assessed. We successfully identified various MV subtype populations within BALF [leukocyte, polymorphonuclear leukocyte (PMN; i.e., neutrophil), monocyte, epithelial, and platelet MVs] and plasma (leukocyte, PMN, monocyte, and endothelial MVs) and compared each MV population to disease severity. BALF neutrophil MVs were the only population to significantly correlate with the clinical evaluation scores including forced expiratory volume in 1 s, modified Medical Research Council dyspnea score, 6-min walk test, hyperinflation, and gas transfer. BALF neutrophil MVs, but not neutrophil cell numbers, also strongly correlated with BODE index. We have undertaken, for the first time, a comprehensive evaluation of MV profiles within BALF/plasma of COPD patients. We demonstrate that BALF levels of neutrophil-derived MVs are unique in correlating with a number of key functional and clinically relevant disease severity indexes. Our results show the potential of BALF neutrophil MVs for a COPD biomarker that tightly links a key pathophysiological mechanism of COPD (intra-alveolar neutrophil activation) with clinical severity/outcome.
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http://dx.doi.org/10.1152/ajplung.00099.2020DOI Listing
January 2021

Monocytes mediate homing of circulating microvesicles to the pulmonary vasculature during low-grade systemic inflammation.

J Extracell Vesicles 2020 5;9(1):1706708. Epub 2020 Jan 5.

Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, Chelsea & Westminster Hospital, London, UK.

Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6C and Ly6C monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6C monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6C monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6C monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6C subset monocytes, become a dominant target cell population for MVs during systemic inflammation, with significant implications for the function and targeting of endogenous and therapeutically administered MVs, lending novel insights into the pathophysiology of pulmonary vascular inflammation.
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http://dx.doi.org/10.1080/20013078.2019.1706708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968433PMC
January 2020

Microvesicles as new therapeutic targets for the treatment of the acute respiratory distress syndrome (ARDS).

Expert Opin Ther Targets 2019 11 22;23(11):931-941. Epub 2019 Nov 22.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital, London, UK.

: Acute respiratory distress syndrome (ARDS) is a heterogeneous and multifactorial disease; it is a common and devastating condition that has a high mortality. Treatment is limited to supportive measures hence novel pharmacological approaches are necessary. We propose a new direction in ARDS research; this means moving away from thinking about individual inflammatory mediators and instead investigating how packaged information is transmitted between cells. Microvesicles (MVs) represent a novel vehicle for inter-cellular communication with an emerging role in ARDS pathophysiology.: This review examines current approaches to ARDS and emerging MV research. We describe advances in our understanding of microvesicles and focus on their pro-inflammatory roles in airway and endothelial signaling. We also offer reasons for why MVs are attractive therapeutic targets.: MVs have a key role in ARDS pathophysiology. Preclinical studies must move away from simple models toward more realistic scenarios while clinical studies must embrace patient heterogeneity. Microvesicles have the potential to aid identification of patients who may benefit from particular treatments and act as biomarkers of cellular status and disease progression. Understanding microvesicle cargoes and their cellular interactions will undoubtedly uncover new targets for ARDS.
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http://dx.doi.org/10.1080/14728222.2019.1692816DOI Listing
November 2019

CCR2 mediates the adverse effects of LPS in the pregnant mouse.

Biol Reprod 2020 02;102(2):445-455

Imperial College Parturition Research Group, Academic Department of Obstetrics & Gynaecology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK.

In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-μg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL.
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http://dx.doi.org/10.1093/biolre/ioz188DOI Listing
February 2020

Ventilation following established ARDS: a preclinical model framework to improve predictive power.

Thorax 2019 12 5;74(12):1120-1129. Epub 2019 Jul 5.

Department of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, London, UK

Background: Despite advances in understanding the pathophysiology of acute respiratory distress syndrome, effective pharmacological interventions have proven elusive. We believe this is a consequence of existing preclinical models being designed primarily to explore biological pathways, rather than predict treatment effects. Here, we describe a mouse model in which both therapeutic intervention and ventilation were superimposed onto existing injury and explored the impact of β-agonist treatment, which is effective in simple models but not clinically.

Methods: Mice had lung injury induced by intranasal lipopolysaccharide (LPS), which peaked at 48 hours post-LPS based on clinically relevant parameters including hypoxaemia and impaired mechanics. At this peak of injury, mice were treated intratracheally with either terbutaline or tumour necrosis factor (TNF) receptor 1-targeting domain antibody, and ventilated with moderate tidal volume (20 mL/kg) to induce secondary ventilator-induced lung injury (VILI).

Results: Ventilation of LPS-injured mice at 20 mL/kg exacerbated injury compared with low tidal volume (8 mL/kg). While terbutaline attenuated VILI within non-LPS-treated animals, it was ineffective to reduce VILI in pre-injured mice, mimicking its lack of clinical efficacy. In contrast, anti-TNF receptor 1 antibody attenuated secondary VILI within pre-injured lungs, indicating that the model was treatable.

Conclusions: We propose adoption of a practical framework like that described here to reduce the number of ultimately ineffective drugs reaching clinical trials. Novel targets should be evaluated alongside interventions which have been previously tested clinically, using models that recapitulate the (lack of) clinical efficacy. Within such a framework, outperforming a failed pharmacologic should be a prerequisite for drugs entering trials.
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http://dx.doi.org/10.1136/thoraxjnl-2019-213460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858882PMC
December 2019

Osteopontin mediates necroptosis in lung injury after transplantation of ischaemic renal allografts in rats.

Br J Anaesth 2019 Oct 28;123(4):519-530. Epub 2019 Jun 28.

Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK. Electronic address:

Background: Respiratory complications after surgery are associated with morbidity and mortality. Acute lung injury can result from the systemic inflammatory response after acute kidney injury. The mechanisms behind this remote injury are not fully understood. In this study, a renal transplantation model was used to investigate remote lung injury and the underlying molecular mechanisms, especially the role of osteopontin (OPN).

Methods: In vitro, human lung epithelial cell line (A549) and monocyte/macrophage cell line (U937) were challenged with tumour necrosis factor-alpha (TNF-α) in combination with OPN. In vivo, the Fischer rat renal grafts were extracted and stored in 4°C University of Wisconsin preserving solution for up to 16 h, and transplanted into Lewis rat recipients. Lungs were harvested on Day 1 after grafting for further analysis.

Results: Renal engraftment was associated with pathological changes and an increase in TNF-α and interleukin-1 beta in the lung of the recipient. OPN, endoplasmic reticulum (ER) stress, and necroptosis were increased in both the recipient lung and A549 cells challenged with TNF-α. Exogenous OPN exacerbated lung injury and necroptosis. Suppression of OPN through siRNA reduced remote lung injury by mitigation of ER stress, necroptosis, and the inflammatory response.

Conclusions: Renal allograft transplant triggers recipient remote lung injury, which is, in part, mediated by OPN signalling. This study may provide a molecular basis for strategies to be developed to treat such perioperative complications.
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http://dx.doi.org/10.1016/j.bja.2019.05.041DOI Listing
October 2019

LPS-Induced Hypotension in Pregnancy: The Effect of Progesterone Supplementation.

Shock 2020 02;53(2):199-207

Imperial College Parturition Research Group, Chelsea and Westminster Hospital, London, UK.

Our previous work has shown that pregnancy exacerbates the hypotensive response to both infection and lipopolysaccharide (LPS). The high levels of progesterone (P4) associated with pregnancy have been suggested to be responsible for the pregnancy-induced changes in the cardiovascular response to infection. Here, we test the hypothesis that P4 supplementation exacerbates the hypotensive response of the maternal cardiovascular to LPS.Female CD1 mice had radiotelemetry probes implanted to measure hemodynamic function noninvasively and were time-mated. From day 14 of pregnancy, mice received either 10 mg of P4 or vehicle alone per day and on day 16, intraperitoneal LPS (10 μg of serotype 0111:B4) was injected. In two identically treated cohorts of mice, tissue and serum (for RNA, protein studies) were collected at 6 and 12 h.Administration of LPS resulted in a fall in blood pressure in vehicle treated, but not P4 supplemented mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors and in both circulating and tissue inflammatory cell numbers, but with reduced left ventricular expression of cytokines in P4-supplemented mice. However, left ventricular expression of markers of cardiac dysfunction and apoptosis were similar.This study demonstrates that P4 supplementation prevented LPS-induced hypotension in pregnant mice in association with reduced myocardial inflammatory cytokine gene expression. These observations suggest that rather than being detrimental, P4 supplementation has a protective effect on the maternal cardiovascular response to sepsis.
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http://dx.doi.org/10.1097/SHK.0000000000001343DOI Listing
February 2020

ATP redirects cytokine trafficking and promotes novel membrane TNF signaling microvesicles.

FASEB J 2019 05 18;33(5):6442-6455. Epub 2019 Feb 18.

Section of Anaesthetics, Pain Medicine, and Intensive Care, Faculty of Medicine, Chelsea and Westminster Hospital, Imperial College London, London, United Kingdom.

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF , producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling microvesicles.
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http://dx.doi.org/10.1096/fj.201802386RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463909PMC
May 2019

Mechanical Ventilation in Mice: Does Longer Equal Better?

Am J Respir Cell Mol Biol 2019 02;60(2):137-138

1 Anaesthetics, Pain Medicine and Intensive Care Imperial College London London, United Kingdom and.

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http://dx.doi.org/10.1165/rcmb.2018-0308EDDOI Listing
February 2019

Profiling inflammatory markers in patients with pneumonia on intensive care.

Sci Rep 2018 10 3;8(1):14736. Epub 2018 Oct 3.

Section of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, London, UK.

Clinical investigations lack predictive value when diagnosing pneumonia, especially when patients are ventilated and develop ventilator associated pneumonia (VAP). New tools to aid diagnosis are important to improve outcomes. This pilot study examines the potential for a panel of inflammatory mediators to aid in the diagnosis. Forty-four ventilated patients, 17 with pneumonia and 27 with brain injuries, eight of whom developed VAP, were recruited. 51 inflammatory mediators, including cytokines and oxylipins, were measured in patients' serum using flow cytometry and mass spectrometry. The mediators could separate patients admitted to ICU with pneumonia compared to brain injury with an area under the receiver operating characteristic curve (AUROC) 0.75 (0.61-0.90). Changes in inflammatory mediators were similar in both groups over the course of ICU stay with 5,6-dihydroxyeicosatrienoic and 8,9-dihydroxyeicosatrienoic acids increasing over time and interleukin-6 decreasing. However, brain injured patients who developed VAP maintained inflammatory profiles similar to those at admission. A multivariate model containing 5,6-dihydroxyeicosatrienoic acid, 8,9-dihydroxyeicosatrienoic acid, intercellular adhesion molecule-1, interleukin-6, and interleukin-8, could differentiate patients with VAP from brain injured patients without infection (AUROC 0.94 (0.80-1.00)). The use of a selected group of markers showed promise to aid the diagnosis of VAP especially when combined with clinical data.
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http://dx.doi.org/10.1038/s41598-018-32938-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170441PMC
October 2018

Progesterone, the maternal immune system and the onset of parturition in the mouse.

Biol Reprod 2018 03;98(3):376-395

Imperial College Parturition Research Group, Academic Department of Obstetrics & Gynaecology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK.

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.
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http://dx.doi.org/10.1093/biolre/iox146DOI Listing
March 2018

The response of the innate immune and cardiovascular systems to LPS in pregnant and nonpregnant mice.

Biol Reprod 2017 Aug;97(2):258-272

Imperial College Parturition Research Group, Chelsea and Westminster Hospital, 369 Fulham Road, London, UK.

Sepsis is the leading cause of direct maternal mortality, but there are no data directly comparing the response to sepsis in pregnant and nonpregnant (NP) individuals. This study uses a mouse model of sepsis to test the hypothesis that the cardiovascular response to sepsis is more marked during pregnancy. Female CD1 mice had radiotelemetry probes implanted and were time mated. NP and day 16 pregnant CD-1 mice received intraperitoneal lipopolysaccharide (LPS; 10 μg, serotype 0111: B4). In a separate study, tissue and serum (for RNA, protein and flow cytometry studies), aorta and uterine vessels (for wire myography) were collected after LPS or vehicle control administration. Administration of LPS resulted in a greater fall in blood pressure in pregnant mice compared to NP mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors, and circulating leukocytes, but with a greater monocyte and lesser neutrophil margination in the lungs of pregnant mice. Baseline markers of cardiac dysfunction and apoptosis as well as cytokine expression were higher in pregnant mice, but the response to LPS was similar in both groups as was the ex vivo assessment of vascular function. In pregnant mice, nonfatal sepsis is associated with a more marked hypotensive response but not a greater immune response. We conclude that endotoxemia induces a more marked hypotensive response in pregnant compared to NP mice. These changes were not associated with a more marked systemic inflammatory response in pregnant mice, although monocyte lung margination was greater. The more marked hypotensive response to LPS may explain the greater vulnerability to some infections exhibited by pregnant women.
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http://dx.doi.org/10.1093/biolre/iox076DOI Listing
August 2017

High-Fat Feeding Protects Mice From Ventilator-Induced Lung Injury, Via Neutrophil-Independent Mechanisms.

Crit Care Med 2017 Aug;45(8):e831-e839

All authors: Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom.

Objective: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury.

Design: In vivo study.

Setting: University research laboratory.

Subjects: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls.

Interventions: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes.

Measurements And Main Results: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells.

Conclusions: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.
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http://dx.doi.org/10.1097/CCM.0000000000002403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511730PMC
August 2017

Intravascular donor monocytes play a central role in lung transplant ischaemia-reperfusion injury.

Thorax 2018 04 7;73(4):350-360. Epub 2017 Apr 7.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK.

Rationale: Primary graft dysfunction in lung transplant recipients derives from the initial, largely leukocyte-dependent, ischaemia-reperfusion injury. Intravascular lung-marginated monocytes have been shown to play key roles in experimental acute lung injury, but their contribution to lung ischaemia-reperfusion injury post transplantation is unknown.

Objective: To define the role of donor intravascular monocytes in lung transplant-related acute lung injury and primary graft dysfunction.

Methods: Isolated perfused C57BL/6 murine lungs were subjected to warm ischaemia (2 hours) and reperfusion (2 hours) under normoxic conditions. Monocyte retention, activation phenotype and the effects of their depletion by intravenous clodronate-liposome treatment on lung inflammation and injury were determined. In human donor lung transplant samples, the presence and activation phenotype of monocytic cells (low side scatter, 27E10+, CD14+, HLA-DR+, CCR2+) were evaluated by flow cytometry and compared with post-implantation lung function.

Results: In mouse lungs following ischaemia-reperfusion, substantial numbers of lung-marginated monocytes remained within the pulmonary microvasculature, with reduced L-selectin and increased CD86 expression indicating their activation. Monocyte depletion resulted in reductions in lung wet:dry ratios, bronchoalveolar lavage fluid protein, and perfusate levels of RAGE, MIP-2 and KC, while monocyte repletion resulted in a partial restoration of the injury. In human lungs, correlations were observed between pre-implantation donor monocyte numbers/their CD86 and TREM-1 expression and post-implantation lung dysfunction at 48 and 72 hours.

Conclusions: These results indicate that lung-marginated intravascular monocytes are retained as a 'passenger' leukocyte population during lung transplantation, and play a key role in the development of transplant-associated ischaemia-reperfusion injury.
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http://dx.doi.org/10.1136/thoraxjnl-2016-208977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870457PMC
April 2018

If We Ask a Mouse about Biotrauma, Will It Give Us a Sensible Answer?

Anesthesiology 2017 05;126(5):766-767

From the Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, London, United Kingdom.

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http://dx.doi.org/10.1097/ALN.0000000000001606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398376PMC
May 2017

Inhibition of TNF Receptor p55 By a Domain Antibody Attenuates the Initial Phase of Acid-Induced Lung Injury in Mice.

Front Immunol 2017 13;8:128. Epub 2017 Feb 13.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital , London , UK.

Background: Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment.

Methods: We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting "dummy" dAb, 1 or 4 h before acid instillation.

Results: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase.

Conclusion: These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.
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http://dx.doi.org/10.3389/fimmu.2017.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304467PMC
February 2017

Shape of a slowly rotating star measured by asteroseismology.

Sci Adv 2016 Nov 16;2(11):e1601777. Epub 2016 Nov 16.

Center for Space Science, NYUAD Institute, New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, UAE.; New York University, New York, NY 10012, USA.

Stars are not perfectly spherically symmetric. They are deformed by rotation and magnetic fields. Until now, the study of stellar shapes has only been possible with optical interferometry for a few of the fastest-rotating nearby stars. We report an asteroseismic measurement, with much better precision than interferometry, of the asphericity of an A-type star with a rotation period of 100 days. Using the fact that different modes of oscillation probe different stellar latitudes, we infer a tiny but significant flattening of the star's shape of Δ/ = (1.8 ± 0.6) × 10. For a stellar radius that is 2.24 times the solar radius, the difference in radius between the equator and the poles is Δ = 3 ± 1 km. Because the observed Δ/ is only one-third of the expected rotational oblateness, we conjecture the presence of a weak magnetic field on a star that does not have an extended convective envelope. This calls to question the origin of the magnetic field.
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http://dx.doi.org/10.1126/sciadv.1601777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5262460PMC
November 2016

Circulating Microvesicles Are Elevated Acutely following Major Burns Injury and Associated with Clinical Severity.

PLoS One 2016 9;11(12):e0167801. Epub 2016 Dec 9.

Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery & Cancer, Imperial College London, London, United Kingdom.

Microvesicles are cell-derived signaling particles emerging as important mediators and biomarkers of systemic inflammation, but their production in severe burn injury patients has not been described. In this pilot investigation, we measured circulating microvesicle levels following severe burns, with severe sepsis patients as a comparator group. We hypothesized that levels of circulating vascular cell-derived microvesicles are elevated acutely following burns injury, mirroring clinical severity due to the early onset and prevalence of systemic inflammatory response syndrome (SIRS) in these patients. Blood samples were obtained from patients with moderate to severe thermal injury burns, with severe sepsis, and from healthy volunteers. Circulating microvesicles derived from total leukocytes, granulocytes, monocytes, and endothelial cells were quantified in plasma by flow cytometry. All circulating microvesicle subpopulations were elevated in burns patients on day of admission (day 0) compared to healthy volunteers (leukocyte-microvesicles: 3.5-fold, p = 0.005; granulocyte-microvesicles: 12.8-fold, p<0.0001; monocyte-microvesicles: 20.4-fold, p<0.0001; endothelial- microvesicles: 9.6-fold, p = 0.01), but decreased significantly by day 2. Microvesicle levels were increased with severe sepsis, but less consistently between patients. Leukocyte- and granulocyte-derived microvesicles on day 0 correlated with clinical assessment scores and were higher in burns ICU non-survivors compared to survivors (leukocyte MVs 4.6 fold, p = 0.002; granulocyte MVs 4.8 fold, p = 0.003). Mortality prediction analysis of area under receiver operating characteristic curve was 0.92 (p = 0.01) for total leukocyte microvesicles and 0.85 (p = 0.04) for granulocyte microvesicles. These findings demonstrate, for the first time, acute increases in circulating microvesicles following burns injury in patients and point to their potential role in propagation of sterile SIRS-related pathophysiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148002PMC
July 2017

P311 induces the transdifferentiation of epidermal stem cells to myofibroblast-like cells by stimulating transforming growth factor β1 expression.

Stem Cell Res Ther 2016 12 1;7(1):175. Epub 2016 Dec 1.

Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China.

Background: Epithelial to mesenchymal transition, especially to myofibroblasts, plays an important role in wound healing, fibrosis, and carcinogenesis. Epidermal stem cells (EpSCs) are responsible for epidermal renewal and wound re-epithelialization. However, it remains unclear whether and how EpSCs transdifferentiate into myofibroblasts or myofibroblast-like cells (MFLCs). Here, we provide the first evidence showing that P311 induces EpSC to MFLC transdifferentiation (EpMyT) via TGFβ1/Smad signaling.

Methods: Wound healing and mesenchymal features were observed in the P311 KO and P311 WT mouse model of superficial second-degree burns. After the primary human or mouse EpSCs were forced to highly express P311 using an adenoviral vector, EpMyT was observed by immunofluorescence, real-time PCR, and western blot. The activity of TGFβ1 and Smad2/3 in EpSCs with different P311 levels was observed by western blot. The TβRI/II inhibitor LY2109761 and Smad3 siRNA were applied to block the EpMyT in P311-overexpressing EpSCs and exogenous TGFβ1 was to restore the EpMyT in P311 KO EpSCs. Furthermore, the mechanism of P311 regulating TGFβ1 was investigated by bisulfite sequencing PCR, luciferase activity assay, and real-time PCR.

Results: P311 KO mouse wounds showed delayed re-epithelialization and reduced mesenchymal features. The human or mouse EpSCs with overexpressed P311 exhibited fusiform morphological changes, upregulated expression of myofibroblast markers (α-SMA and vimentin), and downregulated expression of EpSC markers (β1-integrin and E-cadherin). P311-expressing EpSCs showed decreased TGFβ1 mRNA and increased TGFβ1 protein, TβRI/II mRNA, and activated Smad2/3. Moreover, LY2109761 and Smad3 siRNA reversed P311-induced EpMyT. Under the stimulation of exogenous TGFβ1, the phosphorylation of Smad2 and Smad3 in P311 KO EpSCs was significantly lower than that in P311 WT EpSCs and the EpMyT in P311 KO EpSCs was restored. Furthermore, P311 enhanced the methylation of TGFβ1 promoter and increased activities of TGFβ1 5'/3' untranslated regions (UTRs) to stimulate TGFβ1 expression. P311α-SMA cells and P311vimentin cells were observed in the epidermis of human burn wounds. Also, P311 was upregulated by IL-1β, IL-6, TNFα, and hypoxia.

Conclusions: P311 is a novel TGFβ1/Smad signaling-mediated regulator of transdifferentiation in EpSCs during cutaneous wound healing. Furthermore, P311 might stimulate TGFβ1 expression by promoting TGFβ1 promoter methylation and by activating the TGFβ1 5'/3' UTR.
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http://dx.doi.org/10.1186/s13287-016-0421-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131552PMC
December 2016

Loss of epithelial Gq and G11 signaling inhibits TGFβ production but promotes IL-33-mediated macrophage polarization and emphysema.

Sci Signal 2016 10 25;9(451):ra104. Epub 2016 Oct 25.

Division of Respiratory Medicine, University of Nottingham, Nottingham, U.K.

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling links hundreds of G protein-coupled receptors with four G protein signaling pathways. Two of these, one mediated by G and G (G) and the other by G and G (G), are implicated in the force-dependent activation of transforming growth factor-β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore, precise control of alveolar TGFβ activation is essential for alveolar homeostasis. We investigated the involvement of the G and G pathways in epithelial cells in generating active TGFβ and regulating alveolar inflammation. Mice deficient in both Gα and Gα developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired G signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar G signaling maintains alveolar homeostasis and likely independently increases TGFβ activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of G signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.
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http://dx.doi.org/10.1126/scisignal.aad5568DOI Listing
October 2016

The Local and Systemic Immune Response to Intrauterine LPS in the Prepartum Mouse.

Biol Reprod 2016 12 19;95(6):125. Epub 2016 Oct 19.

Imperial College Parturition Research Group, Academic Department of Obstetrics & Gynaecology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, United Kingdom

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta.
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http://dx.doi.org/10.1095/biolreprod.116.143289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333944PMC
December 2016

Alveolar macrophage-derived microvesicles mediate acute lung injury.

Thorax 2016 11 10;71(11):1020-1029. Epub 2016 Jun 10.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK.

Background: Microvesicles (MVs) are important mediators of intercellular communication, packaging a variety of molecular cargo. They have been implicated in the pathophysiology of various inflammatory diseases; yet, their role in acute lung injury (ALI) remains unknown.

Objectives: We aimed to identify the biological activity and functional role of intra-alveolar MVs in ALI.

Methods: Lipopolysaccharide (LPS) was instilled intratracheally into C57BL/6 mice, and MV populations in bronchoalveolar lavage fluid (BALF) were evaluated. BALF MVs were isolated 1 hour post LPS, assessed for cytokine content and incubated with murine lung epithelial (MLE-12) cells. In separate experiments, primary alveolar macrophage-derived MVs were incubated with MLE-12 cells or instilled intratracheally into mice.

Results: Alveolar macrophages and epithelial cells rapidly released MVs into the alveoli following LPS. At 1 hour, the dominant population was alveolar macrophage-derived, and these MVs carried substantive amounts of tumour necrosis factor (TNF) but minimal amounts of IL-1β/IL-6. Incubation of these mixed MVs with MLE-12 cells induced epithelial intercellular adhesion molecule-1 (ICAM-1) expression and keratinocyte-derived cytokine release compared with MVs from untreated mice (p<0.001). MVs released in vitro from LPS-primed alveolar macrophages caused similar increases in MLE-12 ICAM-1 expression, which was mediated by TNF. When instilled intratracheally into mice, these MVs induced increases in BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05).

Conclusions: We demonstrate, for the first time, the sequential production of MVs from different intra-alveolar precursor cells during the early phase of ALI. Our findings suggest that alveolar macrophage-derived MVs, which carry biologically active TNF, may play an important role in initiating ALI.
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http://dx.doi.org/10.1136/thoraxjnl-2015-208032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099194PMC
November 2016

Heme Oxygenase-1 Mediates Neuroprotection Conferred by Argon in Combination with Hypothermia in Neonatal Hypoxia-Ischemia Brain Injury.

Anesthesiology 2016 07;125(1):180-92

From the Faculty of Medicine, Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom (H.Z., S.M., S.K., Y.T.C., M.T., D.M.); Department of Anesthesiology, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China (Q.L.); Division of Imaging Sciences and Biomedical Engineering, Centre for the Developing Brain, King's College London, St. Thomas' Hospital, London, United Kingdom (H.H.); Perinatal Center, Institute of Physiology and Neurosciences and Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden (H.H.); and Reproductive Biology, Department of Surgery and Cancer, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom (M.R.J.).

Background: Hypoxic-ischemic encephalopathy is a major cause of mortality and disability in the newborn. The authors investigated the protective effects of argon combined with hypothermia on neonatal rat hypoxic-ischemic brain injury.

Methods: In in vitro studies, rat cortical neuronal cell cultures were challenged by oxygen and glucose deprivation for 90 min and exposed to 70% Ar or N2 with 5% CO2 balanced with O2, at 33°C for 2 h. Neuronal phospho-Akt, heme oxygenase-1 and phospho-glycogen synthase kinase-3β expression, and cell death were assessed. In in vivo studies, neonatal rats were subjected to unilateral common carotid artery ligation followed by hypoxia (8% O2 balanced with N2 and CO2) for 90 min. They were exposed to 70% Ar or N2 balanced with oxygen at 33°, 35°, and 37°C for 2 h. Brain injury was assessed at 24 h or 4 weeks after treatment.

Results: In in vitro studies, argon-hypothermia treatment increased phospho-Akt and heme oxygenase-1 expression and significantly reduced the phospho-glycogen synthase kinase-3β Tyr-216 expression, cytochrome C release, and cell death in oxygen-glucose deprivation-exposed cortical neurons. In in vivo studies, argon-hypothermia treatment decreased hypoxia/ischemia-induced brain infarct size (n = 10) and both caspase-3 and nuclear factor-κB activation in the cortex and hippocampus. It also reduced hippocampal astrocyte activation and proliferation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway through LY294002 attenuated cerebral protection conferred by argon-hypothermia treatment (n = 8).

Conclusion: Argon combined with hypothermia provides neuroprotection against cerebral hypoxia-ischemia damage in neonatal rats, which could serve as a new therapeutic strategy against hypoxic-ischemic encephalopathy.
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http://dx.doi.org/10.1097/ALN.0000000000001128DOI Listing
July 2016

In vivo compartmental analysis of leukocytes in mouse lungs.

Am J Physiol Lung Cell Mol Physiol 2015 Oct 7;309(7):L639-52. Epub 2015 Aug 7.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined "interstitial" leukocyte populations during models of inflammatory lung diseases.
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http://dx.doi.org/10.1152/ajplung.00140.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593833PMC
October 2015

Revised Baux Score and updated Charlson comorbidity index are independently associated with mortality in burns intensive care patients.

Burns 2015 Nov 14;41(7):1420-7. Epub 2015 Jul 14.

Magill Department of Anaesthesia, Intensive Care and Pain Management, Chelsea and Westminster Hospital, London, United Kingdom; Imperial College London Faculty of Medicine, London, United Kingdom. Electronic address:

Purpose: The purpose of the current study was to utilise established scoring systems to analyse the association of (i) burn injury severity, (ii) comorbid status and (iii) associated systemic physiological disturbance with inpatient mortality in patients with severe burn injuries admitted to intensive care.

Methods: Case notes of all patients with acute thermal injuries affecting ≥15% total body surface area (TBSA) admitted to the Burns Intensive Care Unit (BICU) at Chelsea and Westminster Hospital during a 10-year period were retrospectively reviewed. Revised Baux Score, Belgian Outcome in Burn Injury (BOBI) Score, Abbreviated Burn Severity Index (ABSI), APACHE II Score, Sequential Organ Failure Assessment (SOFA) Score and Updated Charlson Comorbidity Index (CCI) were computed for each patient and analysed for association with inpatient mortality.

Results: Ninety mechanically ventilated patients (median age 45.7 years, median % TBSA burned 36.5%) were included. 72 patients had full thickness burns and 35 patients had inhalational injuries. Forty-four patients died in hospital while 46 survived to discharge. In a multivariate logistic regression model, only the Revised Baux Score (p<0.001) and updated CCI (p=0.014) were independently associated with mortality. This gave a ROC curve with area under the curve of 0.920. On multivariate cox regression survival analysis, only the Revised Baux Score (p<0.001) and the updated CCI (p=0.004) were independently associated with shorter time to death.

Conclusion: Our data suggest that the Revised Baux Score and the updated CCI are independently associated with inpatient mortality in patients admitted to intensive care with burn injuries affecting ≥15% TBSA. This emphasises the importance of comorbidities in the prognosis of patients with severe burn injuries.
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http://dx.doi.org/10.1016/j.burns.2015.06.009DOI Listing
November 2015

Influence of glutathione-S-transferase (GST) inhibition on lung epithelial cell injury: role of oxidative stress and metabolism.

Am J Physiol Lung Cell Mol Physiol 2015 Jun 10;308(12):L1274-85. Epub 2015 Apr 10.

Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, London, United Kingdom; Department of Anaesthetics, Royal Brompton and Harefield NHS Foundation Trust, Harefield Hospital, Harefield, Middlesex, United Kingdom; Department of Anaesthesia and Intensive Therapy, Semmelweis University, Budapest, Hungary;

Oxidant-mediated tissue injury is key to the pathogenesis of acute lung injury. Glutathione-S-transferases (GSTs) are important detoxifying enzymes that catalyze the conjugation of glutathione with toxic oxidant compounds and are associated with acute and chronic inflammatory lung diseases. We hypothesized that attenuation of cellular GST enzymes would augment intracellular oxidative and metabolic stress and induce lung cell injury. Treatment of murine lung epithelial cells with GST inhibitors, ethacrynic acid (EA), and caffeic acid compromised lung epithelial cell viability in a concentration-dependent manner. These inhibitors also potentiated cell injury induced by hydrogen peroxide (H2O2), tert-butyl-hydroperoxide, and hypoxia and reoxygenation (HR). SiRNA-mediated attenuation of GST-π but not GST-μ expression reduced cell viability and significantly enhanced stress (H2O2/HR)-induced injury. GST inhibitors also induced intracellular oxidative stress (measured by dihydrorhodamine 123 and dichlorofluorescein fluorescence), caused alterations in overall intracellular redox status (as evidenced by NAD(+)/NADH ratios), and increased protein carbonyl formation. Furthermore, the antioxidant N-acetylcysteine completely prevented EA-induced oxidative stress and cytotoxicity. Whereas EA had no effect on mitochondrial energetics, it significantly altered cellular metabolic profile. To explore the physiological impact of these cellular events, we used an ex vivo mouse-isolated perfused lung model. Supplementation of perfusate with EA markedly affected lung mechanics and significantly increased lung permeability. The results of our combined genetic, pharmacological, and metabolic studies on multiple platforms suggest the importance of GST enzymes, specifically GST-π, in the cellular and whole lung response to acute oxidative and metabolic stress. These may have important clinical implications.
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http://dx.doi.org/10.1152/ajplung.00220.2014DOI Listing
June 2015

Monocyte Tumor Necrosis Factor-α-Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation.

Crit Care Med 2015 Jul;43(7):1375-85

1Section of Anesthetics, Pain Medicine and Intensive Care, Imperial College London, London, United Kingdom. 2Department of Critical Care, Imperial College Healthcare NHS Trust, London, United Kingdom.

Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α-converting enzyme baseline and inducible activity profiles.

Design: Observational clinical study.

Setting: Mixed surgical/medical teaching hospital ICU.

Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome.

Interventions: None.

Measurements And Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α-converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients' monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α-converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α-converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α-converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α-converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α-converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide.

Conclusions: Monocyte tumor necrosis factor-α-converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α-converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis.
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http://dx.doi.org/10.1097/CCM.0000000000000992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467590PMC
July 2015

Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation.

Am J Physiol Lung Cell Mol Physiol 2015 May 13;308(9):L912-21. Epub 2015 Mar 13.

Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom

Mechanical ventilation, through overdistension of the lung, induces substantial inflammation that is thought to increase mortality among critically ill patients. The mechanotransduction processes involved in converting lung distension into inflammation during this ventilator-induced lung injury (VILI) remain unclear, although many cell types have been shown to be involved in its pathogenesis. This study aimed to identify the profile of in vivo lung cellular activation that occurs during the initiation of VILI. This was achieved using a flow cytometry-based method to quantify the phosphorylation of several markers (p38, ERK1/2, MAPK-activated protein kinase 2, and NF-κB) of inflammatory pathway activation within individual cell types. Anesthetized C57BL/6 mice were ventilated with low (7 ml/kg), intermediate (30 ml/kg), or high (40 ml/kg) tidal volumes for 1, 5, or 15 min followed by immediate fixing and processing of the lungs. Surprisingly, the pulmonary endothelium was the cell type most responsive to in vivo high-tidal-volume ventilation, demonstrating activation within just 1 min, followed by the alveolar epithelium. Alveolar macrophages were the slowest to respond, although they still demonstrated activation within 5 min. This order of activation was specific to VILI, since intratracheal lipopolysaccharide induced a very different pattern. These results suggest that alveolar macrophages may become activated via a secondary mechanism that occurs subsequent to activation of the parenchyma and that the lung cellular activation mechanism may be different between VILI and lipopolysaccharide. Our data also demonstrate that even very short periods of high stretch can promote inflammatory activation, and, importantly, this injury may be immediately manifested within the pulmonary vasculature.
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http://dx.doi.org/10.1152/ajplung.00048.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4421782PMC
May 2015

The role of ex vivo lung perfusion in lung transplantation.

J Intensive Care Soc 2015 Feb 9;16(1):58-63. Epub 2014 Dec 9.

Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Chelsea and Westminster Hospital NHS Foundation Trust, Imperial College, London, UK.

Whilst lung transplantation is a viable solution for end-stage lung disease, donor shortages, donor lung inflammation and perioperative lung injury remain major limitations. Ex vivo lung perfusion has emerged as the next frontier in lung transplantation to address and overcome these limitations, with multicentre clinical trials ongoing in the UK, rest of Europe and North America. Our research seeks to identify the poorly understood cellular and molecular mechanisms of primary graft dysfunction through the development of an isolated perfused lung model of transplantation and investigation of the role of pulmonary inflammation in this paradigm.
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http://dx.doi.org/10.1177/1751143714554062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593288PMC
February 2015