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Jayakody Arachchillage DR, Laffan M, Khanna S, et al., 2020, Frequency of thrombocytopenia and heparin induced thrombocytopenia in patients receiving extracorporeal membrane oxygenation compared to cardiopulmonary bypass and the limited sensitivity of pre-test probability score, Critical Care Medicine, Vol: 48, Pages: e371-e379, ISSN: 0090-3493
Objectives:To ascertain:i)the frequency of thrombocytopeniaand heparininducedthrombocytopenia (HIT), ii)positive predictive value (PPV) of the pre-test probability score (PTPS) in identifying HIT iii)clinical outcome of HITin adult patients receiving veno-venous (VV)-extracorporeal membraneoxygenation(ECMO)or veno-arterial (VA)-ECMO, comparedto cardiopulmonary bypass (CPB). Design: A single-centre, retrospective, observational cohort study from January 2016 to April 2018Setting: Tertiary referral centre for cardiac and respiratory failure Patients:Patients who received ECMOfor>48hrs or had CPB during specified period Interventions: None.Measurements and Main Results:Clinical and laboratory data were collected retrospectively. PTPS and HIT testing results were collected prospectively. Mean age (standard deviation) of the EMCO and CPB cohorts were 45.4 (±15.6) and 64.9 (±13), p< 0.00001.Median duration of CPB was 4.6 [2-16.5]hrs compared to 170.4[70-1008] hrson ECMO.Moderateand severethrombocytopenia were more common in ECMO compared to CPB throughout (p<0.0001).Thrombocytopenia increased in CPB patients on day2 but was 4normal in 83% compared to 42.3 % ofECMOpatients at day 10. Patients on ECMO also followed a similar pattern of platelet recoveryfollowing cessation of ECMO. The incidences of HIT in ECMO and CPB were 6.4% (19/298) and 0.6% (18/2998) respectivelyp<0.0001). There was no difference in prevalence of HIT in patients on VV-ECMO (9/156, 5.7%) vs VA-ECMO (11/142, 7.7%), p=0.81. The PPV of the PTPS in identifying HIT in patients post-CPB and on ECMO were 56.25% (18/32) and 25% (15/60) respectively. Mortalitywas not different with (6/19, 31.6%) or without (89/279, 32.2%) HIT in patients on ECMO,p=0.79. Conclusions Thrombocytopenia is already common at ECMO initiation. HIT is more frequent in both VV-and VA-
Tavazzi G, Dammassa V, Corradi F, et al., 2020, Correlation between echocardiographic and hemodynamic variables in cardiothoracic intensive care unit, Journal of Cardiothoracic and Vascular Anesthesia, Vol: 34, Pages: 1263-1269, ISSN: 1053-0770
Objectivesthe echocardiographic indices have not been validated in critically ill population. We investigated the correlation between some echocardiographic and hemodynamics parameters.DesignProspective Spontaneous non-interventional observational study.SettingAdult cardiothoracic intensive care unit, single center (Royal Brompton Hospital, London UK).ParticipantsConsecutive adult patients admitted to cardiothoracic intensive care unit for severe respiratory failure, primary cardio-circulatory failure and post-aortic surgery.InterventionsClinical, hemodynamic parameters (stroke volume – SV, cardiac output – CO, mean arterial pressure – MAP, and cardiac power index – CPI) and echocardiographic indices of ventricular function (left ventricular total isovolumic time – t-IVT, mitral annular plane systolic excursion – MAPSE, and left ventricular fraction – LVEF) were evaluated offline.Measurements and main results117 patients were studied (age 57.2 ± 19; 60.6% male). t-IVT showed an inverse correlation with SV, CO, MAP and CPI (respectively r: -67%; -38%; -45%; -51%). MAPSE exhibited a positive correlation with SV, CO, MAP and CPI (respectively r: 43%; 44%; 34%; 31%). LVEF did not show any correlation. In the multivariate analysis the association of t-IVT and hemodynamics was confirmed for SV, CO, MAP and CPI with the highest partial correlation between t-IVT and MAP (R = -58%).ConclusionsMAPSE and t-IVT are two reproducible and reliable echocardiographic indices of systolic function and ventricular efficacy associated with hemodynamic variables in cardiothoracic critically ill patients, while LVEF did not show any correlation.
O'Dea K, Tan YY, Shah S, et al., 2020, Monocytes mediate homing of circulating microvesicles to the pulmonaryvasculature during low-grade systemic inflammation, Journal of Extracellular Vesicles, Vol: 9, Pages: 1-16, ISSN: 2001-3078
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 Ly6Chigh and Ly6Clow 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 Ly6Chigh 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 Ly6Chigh 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 Ly6Chigh monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6Chigh subset monocytes, become a dominant
Handslip R, Mumby S, Calfee CS, et al., 2020, Necrosome complex release and necroinflammation in hyper-inflammatory ARDS, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, Pages: 1-2, ISSN: 1073-449X
Tripathi B, Khan S, Arora S, et al., 2019, Burden and trends of arrhythmias in hypertrophic cardiomyopathy and its impact of mortality and resource utilization, JOURNAL OF ARRHYTHMIA, Vol: 35, Pages: 612-625, ISSN: 1880-4276
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Tripathi B, Kumar V, Pitiliya A, et al., 2019, Trends in Incidence and Outcomes of Pregnancy-Related Acute Myocardial Infarction (From a Nationwide Inpatient Sample Database), AMERICAN JOURNAL OF CARDIOLOGY, Vol: 123, Pages: 1220-1227, ISSN: 0002-9149
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Derry J, Patel BV, Adcock IM, et al., 2018, Comparison of mechanisms of repair and regeneration in experimental models of acute respiratory distress syndrome, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A39-A39, ISSN: 0040-6376
Background The acute respiratory distress syndrome (ARDS) is a clinical condition with multiple aetiologies. Previous work has presented two relevant, distinct animal models of ARDS. A murine model of aspiration using acid, in which the lung injury begins to resolve after 48 hours, and another of infection using LPS, that continues to deteriorate. It has been suggested that alveolar epithelial regeneration is implicated in disease progression to different extents between the models.We hypothesised that the wnt and retinoic acid pathways are involved in regeneration and resolution of these murine models of ARDS, and that there is greater activation of these pathways in the aspiration compared to the infection model. Currently, management of ARDS is irrespective of lung injury aetiology, and these mechanisms may prove relevant for pathway modulation for future therapy.Methods Mice were administered either 25 µl intratracheal 0.1 M hydrochloric acid (aspiration model, n=5), 25 µg LPS (infection model, n=6) or sham treated (controls, n=6) and sacrificed at 48 hours. Lung tissue was homogenised. mRNA and protein levels of regenerative pathway genes were quantified, through reverse-transcription polymerase chain reaction and Western blotting.Results Significant upregulation of all 9 regenerative genes studied was seen in the acid model compared to controls. Rac-2 mRNA (wnt pathways) was the only regenerative gene in the LPS-injured mice to show a significant upregulation compared to controls (67.39±25.73 vs 0.32±0.13 ΔCTx1010p<0.05). Significant differences between the acid and LPS models were seen in the mRNA levels of Daam-2 (wnt pathways, figure 1) and Retinol Binding Protein-1 (RBP-1) from the retinoic acid pathway (26.130±2.812 vs 4.893±0.564 ΔCTx105, p<0.05). There were inter-model differences in protein levels, most apparent in β-catenin (β-catenin dependen
Arachchillage DJ, Khanna S, Vandenbriele C, et al., 2018, Incidence of Thrombocytopenia and Heparin induced thrombocytopenia in patients receiving Extracorporeal Membrane Oxygenation (ECMO) compared to cardiopulmonary bypass and the limited sensitivity of pre-test probability score, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: American Society of Hematology, ISSN: 0006-4971
Patel BV, Barrett NA, Vuylsteke A, 2018, ECMO for severe acute respiratory distress syndrome, New England Journal of Medicine, Vol: 379, Pages: 1090-1091, ISSN: 0028-4793
Patel BV, Barrett NA, Vuylsteke A, 2018, Correspondance: ECMO for severe acute respiratory distress syndrome, New England Journal of Medicine, Vol: 379, Pages: 1090-1093, ISSN: 0028-4793
Arachchillage DRJ, Passariello M, Laffan M, et al., 2018, Intracranial haemorrhage and early mortality in patients receiving extracorporeal membrane oxygenation for severe respiratory failure, Seminars in Thrombosis and Hemostasis, Vol: 44, Pages: 276-286, ISSN: 0094-6176
Intracranial hemorrhage (ICH) is a serious complication in patients receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO) and is associated with high mortality. It is unknown whether ICH may be a consequence of the ECMO or of an underlying disease. The authors first aimed to assess the incidence of ICH at initiation and during the course of VV-ECMO and its associated mortality. The second aim was to identify clinical and laboratory measures that could predict the development of ICH in severe respiratory failure. Data were collected from a total number of 165 patients receiving VV-ECMO from January, 2012 to December, 2016 in a single tertiary center and treated according to a single protocol. Only patients who had a brain computed tomography within 24 hours of initiation of ECMO (n = 149) were included for analysis. The prevalence and incidence of ICH at initiation and during the course of VV-ECMO (at median 9 days) were 10.7% (16/149) and 5.2% (7/133), respectively. Thrombocytopenia and reduced creatinine clearance (CrCL) were independently associated with increased risk of ICH on admission; odds ratio (95% confidence interval): 22.6 (2.6–99.5), and 10.8 (5.6–16.2). Only 30-day (not 180-day) mortality was significantly higher in patients with ICH on admission versus those without (37.5% [6/16] vs 16.4% [22/133]; p = 0.03 and 43.7% [7/16] vs 26.3% [35/133]; p = 0.15, respectively). Reduced CrCL and thrombocytopenia were associated with ICH at initiation of VV-ECMO. The higher incidence of ICH at initiation suggests it is more closely related to the severity of the underlying lung injury than to the VV-ECMO itself. ICH at VV-ECMO initiation was associated with early mortality.
Tatham KC, O'Dea KP, Romano R, et al., 2018, Intravascular donor monocytes play a central role in lung transplant ischaemia-reperfusion injury, Thorax, Vol: 73, Pages: 350-360, ISSN: 1468-3296
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.
Nakamura H, Wilson MR, Yao K, et al., 2018, Modulation Of Mechanical Ventilation-Induced Alveolar Epithelial Cell Death Signaling By Underlying Lung Inflammation, American Thoracic Society 2018, Publisher: American Thoracic Society
Pavasini R, Cirillo C, Campo G, et al., 2017, Extracorporeal Circulatory Support in Acute Coronary Syndromes: A Systematic Review and Meta-Analysis, Critical Care Medicine, Vol: 45, Pages: E1173-E1183, ISSN: 0090-3493
Objective: Extracorporeal circulatory support (ECS) is a life-saving technique and its use isincreasing in acute coronary syndromes (ACS). A meta-analysis on pooled event rate of short-termmortality and complications of ACS patients treated with ECS was performed.Data sources: Articles were searched in MEDLINE, Cochrane Library, Google Scholar andBiomed Central.Study selection: Inclusion criteria: observational studies on ACS patients treated with ECS.Primary outcome: short-term mortality. Secondary outcomes: ECS-related complications, causes ofdeath, long-term mortality and bridge therapy.Data extraction: Sixteen articles were selected. Data about clinical characteristics, ACS diagnosisand treatment, ECS setting, outcome definitions and event rate were retrieved from the articles.Random-effect meta-analytic pooling was performed reporting results as a summary point estimateand 95% confidence interval.Data synthesis: A total of 739 patients were included (mean age 59.8 ±2.9). The event rate ofshort-term mortality was 58% (95% CI, 51-64%), 6-month mortality was affecting 24% (95%CI,5%-63%) of 1-month survivors and 1-year mortality 17% (95%CI, 6%-40%) of 6-month survivors.The event rates of ECS-related complications were: acute renal failure 41%, bleeding 25%,neurologic damage in survivors 21%, sepsis/infections 21% and leg ischemia 12%. Between causesof death, multiorgan failure and brain death affected respectively 40% and 27% of patients. Bridgeto ventricular assistance device was offered to 14% of patients and 7% received a transplant.Conclusions: There is still a high rate of short-term mortality and complications in ACS patientstreated with ECS. New studies are needed to optimize and standardize ECS.
Lee N, Lawrence D, Patel B, et al., 2017, HIV-related Pneumocystis jirovecii pneumonia managed with caspofungin and veno-venous extracorporeal membrane oxygenation rescue therapy., BMJ Case Rep, Vol: 2017
Patients with pneumocystis pneumonia have a risk of progressing to acute respiratory failure necessitating admission to intensive care. The case described is of a patient with a newly diagnosed HIV infection presenting with pneumocystis pneumonia. Despite initiating the appropriate pharmacological treatment the patient's clinical condition deteriorated, and required both rescue pharmacological therapy with echinocandins as well as respiratory support with extracorporeal membrane oxygenation therapy. The patient recovered well on ventilator and circulatory support despite a long weaning process complicated by sequelae common to pneumocystis pneumonia. Following initialisation of antiretroviral therapy and step-down from an intensive care setting, the patient required further prolonged hospital stay for rehabilitation and mental health support before being discharged. This case reviews the novel pharmacological therapies and respiratory support strategies used in cases of pneumocystis pneumonia, including the clinical and psychological sequelae that may follow.
Wilson MR, Petrie JE, Shaw MW, et al., 2017, High fat feeding protects mice from ventilator-induced lung injury, via neutrophil-independent mechanisms, Critical Care Medicine, Vol: 45, Pages: e831-e839, ISSN: 1530-0293
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.
Patel BV, Yao K, Nakamura H, et al., 2017, Distinct Differences In Alveolar Epithelial Cell Death Signalling Between Models Of Ards, International Conference of the American-Thoracic-Society (ATS), Publisher: American Thoracic Society, ISSN: 1073-449X
Xu H, Halacli B, Banya W, et al., 2017, Can we predict Veno-Venous extra-corporeal membrane oxygenation duration?, European Journal of Heart Failure, Vol: 19, Pages: 48-48, ISSN: 1388-9842
Halacli B, Xu H, Banya W, et al., 2017, Are Risk Scores Applicable in Tertiary Veno-Venous Extra-Corporeal Membrane Oxygenation Centers?, EuroELSO 2017, Publisher: Wiley, Pages: 36-49, ISSN: 1567-4215
Tan YY, O'Dea KP, Soni S, et al., 2017, Enhanced Recognition and Internalisation of Microvesicles by Lung-Marginated, Ly-6C(high) Monocytes During Endotoxaemia, Annual Meeting of the American-Society-for-Pharmacology-and-Experimental-Therapeutics (ASPET) at Experimental Biology Meeting, Publisher: FEDERATION AMER SOC EXP BIOL, ISSN: 0892-6638
Wilson MR, Wakabayashi K, Bertok S, et al., 2017, Inhibition of TNF receptor p55 by a domain antibody attenuates the initial phase of acid-induced lung injury in mice, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224
Background: Tumor necrosis factor-α (TNF) is strongly implicated in the development ofacute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has beenhampered by its complex biology. TNF signals through two receptors, p55 and p75, whichplay differential roles in pulmonary edema formation during ARDS. We have recentlyshown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator36induced lung injury. In the current study we explored the efficacy of this antibody in mousemodels 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 aresolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally orintranasally with p55-targeting dAb or non-targeting ‘dummy’ dAb, 1 or 4 hours before acidinstillation.Results: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratorysystem elastance, pulmonary inflammation and edema in both the ventilated and resolvingmodels. Pretreatment with p55-targeting dAb significantly attenuated physiological markersof ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in theventilated model, with signs that altered cytokine production and leukocyte recruitmentpersisted beyond the very acute phase.Conclusions: These results demonstrate that the p55-targeting dAb attenuates lung injury andedema formation in models of ARDS induced by acid aspiration, with protection from asingle dose lasting up to 24 hours. Together with our previous data, the current study lends support towards the clinical targeting of p55 for patients with, or at risk of ARDS.
Berry M, Patel BV, Brett SJ, 2017, New consensus definitions for sepsis and septic shock: implications for treatment strategies and drug development?, Drugs, Vol: 77, Pages: 353-361, ISSN: 1179-1950
Sepsis continues to escape a precise diagnostic definition. The most recent consensus definition, termed Sepsis-3, highlights the importance of the maladaptive and potentially life threatening host response to infection. After briefly reviewing the history and epidemiology of sepsis, we go onto describe some of the challenges encountered classifying such a heterogenous disease state. In the context of these new definitions for sepsis and septic shock, we explore current as well as potentially novel therapies and conclude by mentioning some of the controversies of this most recent framework.Key points: The third international consensus definitions for Sepsis and Septic Shock take into account the latest pathophysiological understanding of sepsis, highlighting the importance of the life-threatening organ dysfunction caused by a dysregulated host response to infection.These advances in understanding the complexities of sepsis are not mirrored pharmacologically, where fluids, antibiotics and vasoactive medications continue to form the mainstay of therapy.Sepsis-3 aims to improve both the accuracy of the nomenclature of sepsis as well as improve clinical care. Important questions remain in regards to the clinical applicability of this latest framework.
Patel K, Patel B, Mishra M, et al., 2017, Privacy Issues in Big Data, 2nd International Conference for Convergence in Technology (I2CT), Publisher: IEEE, Pages: 259-264
Tatham KC, O'Dea KP, Romana R, et al., 2016, Retention And Activation Of Donor Vascular Monocytes In Transplanted Lungs Suggests A Central Role In Primary Graft Dysfunction, American Thoracic Society Conference 2016
Tatham KC, O'Dea KP, Romano R, et al., 2016, Retention And Activation Of Donor Vascular Monocytes In Transplanted Lungs Suggests A Central Role In Primary Graft Dysfunction, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Wilson M, Petrie J, Shaw M, et al., 2016, High Fat Feeding Protects Mice From Ventilator-Induced Lung Injury Via A Neutrophil-Independent Mechanism, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Patel BV, Bellingan G, 2016, Repair and recovery mechanisms following critical illness, Oxford Textbook of Critical Care, Editors: Webb, Angus, Finfer, Gattinoni, Singer
Mistry JN, Vakharia K, Tailor D, et al., 2016, Software and Hardware Implementation of Five level Neutral Point Clamped Inverter, International Conference on Electrical, Electronics, and Optimization Techniques (ICEEOT), Publisher: IEEE, Pages: 3913-3917
Patel BV, Tatham KC, Wilson MR, et al., 2015, In vivo compartmental analysis of leukocytes in mouse lungs, American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol: 309, Pages: L639-L652, ISSN: 1522-1504
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 labelling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labelling with fluorophore-conjugated anti-CD45 antibodies, and lung single cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labelling 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 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 labelling 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.
Patel BV, Tatham KC, Wilson MR, et al., 2015, In Vivo Compartmental Labeling of the Mouse Lung, American Thoracic Society, Publisher: ATS Journals, Pages: A3926-A3926, ISSN: 1073-449X
ShareModerators: G.N. Maksym, PhD, R.S. Harris, MD, J.C. Sieren, PhDSession Info: Mini Symposium, [C18] STATE OF PLAY IN RESPIRATORY STRUCTURE AND FUNCTION: SEEING IS BELIEVINGDay/Date: Tuesday, May 19, 2015Session Time: 9:30 AM - 11:30 AMRoom: Mile High Ballroom 2C/3C (Lower Level)Location: Colorado Convention CenterIn Vivo Compartmental Labeling of the Mouse Lung, [Publication Page: A3926]B.V. Patel, MBBS MRCP FRCA PhD, K.C. Tatham, MBBS, M.R. Wilson, PhD, K.P. O'Dea, PhD, M. Takata, MDLondon/UKRationaleCurrent methods for compartmental analyses of lungs using bronchoalveolar lavage (BAL) and/or lung perfusion have significant limitations for accurate determination of location and phenotype of lung leukocytes. BAL retrieves only <25% of intra-alveolar cells (1) and lung perfusion fails to remove vascular marginated cell populations (2). We present a novel method of in vivo antibody labelling to facilitate the accurate compartmental investigation of mouse lung leukocytes by flow cytometry.MethodsAnesthetized C57BL6 mice underwent tracheostomy and venous cannulation. An anti-CD45 antibody (PE-conjugated) was injected intravenously and allowed to circulate for 5 minutes. Mice were then exsanguinated and lungs immediately flooded intratracheally with another anti-CD45 antibody (PE-Cy5-conjugated). Lungs were harvested, and single cell suspensions prepared for flow cytometric analysis using a panel of myeloid markers. This in vivo labelling protocol was also applied to mice exposed to acid-aspiration induced lung injury (3). To confirm the separation between the vascular and interstitial cell populations, a group of mice underwent intravenous labelling alone followed by lung perfusion for 15 minutes using an isolated-perfused lung apparatus (2).ResultsThe combined in vivo intravenous and intratracheal CD45 labelling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung by flow cytometry, with <0.3% of leukocytes staining
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