Minimum protamine dose required to neutralize heparin in cardiac surgery: a single-centre, prospective, observational cohort study.

PURPOSE: Excess protamine contributes to coagulopathy following cardiopulmonary bypass (CPB) and may increase blood loss and transfusion requirements. The primary aim of this study was to find the least amount of protamine necessary to neutralize residual heparin following CPB using the gold standard assays of anti-IIa and anti-Xa activity. Secondary objectives were to evaluate whether the post-CPB activated clotting time could be used as a surrogate marker for quantifying heparin neutralization. METHODS: Twenty-eight consecutive patients undergoing elective cardiac surgery were enrolled. Protamine administration was standardized through an infusion pump at 25 mg·min-1. Blood samples were withdrawn prior to and following administration of 150, 200, 250, and 300 mg protamine and analyzed for activated clotting time and anti-IIa and -Xa activity. RESULTS: Following a mean (standard deviation) cumulative heparin dose of 67,700 (19,400) units and a CPB duration of 113 (71) min, protamine requirements varied widely. Eight out of 25 (32%) patients showed complete neutralization of anti-IIa and -Xa activity at the first sampling point (150 mg protamine; protamine:heparin ratio, 0.3 [0.1]). A protamine:heparin ratio of 0.5 (0.2) was sufficient for heparin neutralization in > 90% of patients. After CPB, a low to mid-range activated clotting time correlated well with anti-IIa and -Xa activity. CONCLUSIONS: The protamine:heparin ratio required to neutralize residual unfractionated heparin (UFH) following CPB is variable. A protamine:heparin ratio of 0.3 was sufficient to neutralize UFH in some patients, while a ratio of 0.5 is sufficient to neutralize both residual anti-IIa and -Xa activity in most patients. Larger studies are necessary to confirm these findings and evaluate their clinical implications. STUDY REGISTRATION: ClinicalTrials.gov (NCT03787641); registered 26 December 2018.

RéSUMé: OBJECTIF: L'excès de protamine contribue à la coagulopathie après la circulation extracorporelle (CEC) et peut augmenter les pertes de sang et les besoins transfusionnels. L'objectif principal de cette étude était de déterminer la quantité minimale de protamine nécessaire pour neutraliser l'héparine résiduelle post-CEC en utilisant les tests de référence de l'activité anti-IIa et anti-Xa. Les objectifs secondaires consistaient à évaluer si le temps de coagulation activé post-CEC pouvait être utilisé comme marqueur de substitution pour quantifier la neutralisation de l'héparine. MéTHODE: Vingt-huit patients consécutifs bénéficiant d'une chirurgie cardiaque non urgente ont été recrutés. L'administration de protamine par une pompe à perfusion à 25 mg·min-1 a été normalisée. Des échantillons de sang ont été prélevés avant et après l'administration de 150, 200, 250 et 300 mg de protamine et analysés pour déterminer le temps de coagulation activé et l'activité anti-IIa et -Xa. RéSULTATS: Après une dose cumulative moyenne (écart type) d'héparine de 67 700 (19 400) unités et une durée de CEC moyenne de 113 (71) min, les besoins en protamine variaient considérablement. Huit patients sur 25 (32 %) ont affiché une neutralisation complète de l'activité anti-IIa et -Xa au premier point de prélèvement (150 mg de protamine; rapport protamine : héparine, 0,3 [0,1]). Un rapport protamine/héparine de 0,5 (0,2) était suffisant pour la neutralisation de l'héparine chez > 90 % des patients. Après la CEC, un temps de coagulation activé bas à moyen était bien corrélé avec l'activité anti-IIa et -Xa. CONCLUSION: Le rapport protamine : héparine nécessaire pour neutraliser l'héparine non fractionnée (HNF) résiduelle suivant une CEC est variable. Un rapport protamine : héparine de 0,3 était suffisant pour neutraliser l'HNF chez certains patients, tandis qu'un rapport de 0,5 est suffisant pour neutraliser à la fois l'activité résiduelle des anti-IIa et celle des anti-Xa chez la plupart des patients. Des études plus vastes sont nécessaires pour confirmer ces résultats et évaluer leurs implications cliniques. ENREGISTREMENT DE L'éTUDE: ClinicalTrials.gov (NCT03787641); enregistrée le 26 décembre 2018.

Exploring and reconciling discordance between documented and preferred resuscitation preferences for hospitalized patients: a quality improvement study.

PURPOSE: A discordance, predominantly towards overtreatment, exists between patients' expressed preferences for life-sustaining interventions and those documented at hospital admission. This quality improvement study sought to assess this discordance at our institution. Secondary objectives were to explore if internal medicine (IM) teams could identify patients who might benefit from further conversations and if the discordance can be reconciled in real-time. METHODS: Two registered nurses were incorporated into IM teams at a tertiary hospital to conduct resuscitation preference conversations with inpatients either specifically referred to them (group I, n = 165) or randomly selected (group II, n = 164) from 1 August 2016 to 31 August 2018. Resuscitation preferences were documented and communicated to teams prompting revised resuscitation orders where appropriate. Multivariable logistic regression was used to determine potential risk factors for discordance. RESULTS: Three hundred and twenty-nine patients were evaluated with a mean (standard deviation) age of 80 (12) and Charlson Comorbidity Index Score of 6.8 (2.6). Discordance was identified in 63/165 (38%) and 27/164 (16%) patients in groups I and II respectively. 42/194 patients (21%) did not want cardiopulmonary resuscitation (CPR) and 15/36 (41%) did not prefer intensive care unit (ICU) admission, despite these having been indicated in their initial preferences. 93% (84/90) of patients with discordance preferred de-escalation of care. Discordance was reconciled in 77% (69/90) of patients. CONCLUSION: Hospitalized patients may have preferences documented for CPR and ICU interventions contrary to their preferences. Trained nurses can identify inpatients who would benefit from further in-depth resuscitation preference conversations. Once identified, discordance can be reconciled during the index admission.

RéSUMé: OBJECTIF: Il existe une discordance, qui tend surtout vers un sur-traitement, entre les préférences exprimées par les patients pour les interventions de maintien de la vie et celles documentées lors de l'admission à l'hôpital. Cette étude d'amélioration de la qualité avait pour objectif d'évaluer cette discordance au sein de notre institution. Les objectifs secondaires de notre étude étaient d'explorer la possibilité que les équipes de médecine interne (MI) identifient les patients qui pourraient bénéficier de conversations approfondies et de voir si la discordance pouvait être corrigée en temps réel. MéTHODE: Deux infirmières ont intégré des équipes de MI dans un hôpital tertiaire pour discuter avec les patients hospitalisés de leurs préférences en matière de réanimation entre le 1er août 2016 et le 31 août 2018; les patients leur étaient soit spécifiquement référés (groupe I, n = 165), ou sélectionnés au hasard (groupe II, n = 164). Les préférences en matière de réanimation ont été documentées et communiquées aux équipes, entraînant une révision des ordonnances de réanimation, le cas échéant. La régression logistique multivariée a été utilisée afin de déterminer les facteurs de risque potentiels de discordance. RéSULTATS: Trois cent vingt-neuf patients ont été évalués, d'un âge moyen (écart type) de 80 ans (12) et avec un score de 6,8 (2,6) à l'Indice de comorbidité de Charlson. Une discordance a été identifiée chez 63/165 (38 %) et 27/164 (16 %) patients dans les groupes I et II, respectivement. Au total, 42/194 patients (21 %) ne souhaitaient pas de réanimation cardiorespiratoire (RCR) et 15/36 (41 %) préféraient ne pas être admis à l'unité de soins intensifs (USI), malgré une mention dans leurs préférences initiales. Parmi les patients chez lesquels une discordance a été notée, 93 % (84/90) ont préféré une désescalade des soins. La discordance a pu être corrigée pour 77 % (69/90) des patients. CONCLUSION: La documentation des patients hospitalisés pourrait indiquer des préférences pour des interventions de RCR et d'admission à l'USI contraires aux véritables préférences. Des infirmières formées à cet effet peuvent identifier les patients hospitalisés qui bénéficieraient d'une conversation approfondie sur leurs préférences en matière de réanimation. Une fois identifiée, une discordance peut être corrigée lors de l'admission initiale.

Protamine requirements in cardiac surgery: effect of changes in the heparin reference standard.

OBJECTIVE: UFH (unfractionated heparin) and protamine are integral to cardiac surgery, and inappropriate dosing can predispose to coagulopathy and hemorrhage. The FDA (Food and Drug Administration) recently has instituted changes to UFH formulation and it is not known if this has influenced its susceptibility to neutralization by protamine. Hence, the authors sought to compare 2 commercial preparations of UFH (old and new) with regard to their neutralization by protamine in patients undergoing cardiopulmonary bypass (CPB). DESIGN: Prospective, observational, cohort study. SETTING: Tertiary care university hospital and associated research laboratory PARTICIPANTS: Twenty adult patients undergoing elective cardiac surgery with CPB. INTERVENTIONS: Blood samples were drawn preinduction, prior to, and 5 and 30 minutes following protamine, and 0 and 2 hours after ICU admission. Protamine titration assays were conducted in vitro on samples drawn prior to and following protamine administration. Anti-IIa and anti-Xa activity were assayed in all samples. RESULTS: Anti-IIa and anti-Xa activity were detected ubiquitously at all time points following CPB, and there were no differences in susceptibility to protamine neutralization between the 2 groups. In vitro protamine titration studies revealed that anti-IIa was more resistant to protamine neutralization compared to anti-Xa activity. CONCLUSIONS: The 'old' and 'new' formulations of UFH evaluated in this study were similar in their susceptibility to protamine neutralization. Circulating UFH is detected as early as 5 minutes after protamine administration and anti-IIa is more resistant to protamine neutralization as compared to anti-Xa activity. Further studies are required to quantify the precise dose of protamine following CPB.

Effect of cardiopulmonary bypass on thrombin generation and protein C pathway.

OBJECTIVE: The purpose of this study was to evaluate the mechanisms of cardiopulmonary bypass (CPB)-induced dysregulation between thrombin and its regulatory anticoagulant activated protein C (APC). DESIGN: A prospective observational cohort study. SETTING: A tertiary care university hospital and associated research laboratory. PATIENTS: Twenty patients undergoing elective coronary artery bypass surgery with (n = 10) or without CPB (n = 10). INTERVENTIONS: Blood samples were collected at 7 time points: preinduction; after heparin; 1 hour after the institution of CPB (or the completion of distal anastomoses in off-CPB group); after protamine; and at 0, 4, and 18 hours in the Intensive care unit (ICU). Samples were analyzed for prothrombin fragments (F1+2), thrombin-antithrombin complexes, protein C (PC), APC, soluble thrombomodulin (sTM), and soluble endothelial protein C receptor (sEPCR). MEASUREMENTS AND MAIN RESULTS: F1+2 levels increased significantly 1 hour after the initiation of CPB in comparison with baseline (2.7 ± 0.5 v 0.5 ± 0.2 nmol/L, p < 0.001) (mean ± standard deviation) and remained elevated until 4 hours after ICU admission (p < 0.001). In contrast, APC levels did not show any significant changes over time in either group. sEPCR, sTM, and PC levels did not change during CPB although sEPCR decreased significantly after the termination of CPB compared with baseline in the CPB group. CONCLUSIONS: Exposure to CPB is associated with a distinct thrombin surge that continues postoperatively for 4 hours. The impaired ability to generate APC reflects a complex process that is not associated with increased levels of sEPCR and thrombomodulin during CPB. Further studies are required to evaluate the regulation of the host APC response in cardiac surgery.

Evaluation of tricuspid and pulmonary valves using epicardial and transesophageal echocardiography--a comparative study.

OBJECTIVE: To compare measurements obtained by transesophageal echocardiography (TEE) and epicardial echocardiography (EE) for evaluation of the tricuspid valve (TV) and pulmonary valve (PV). DESIGN: Prospective observational. SETTING: University hospital. PARTICIPANTS: Patients undergoing elective coronary artery bypass grafting with or without aortic valve replacement. INTERVENTIONS: After routine intraoperative TEE, EE was performed to compare measurements obtained by the 2 methods. MEASUREMENTS AND MAIN RESULTS: After institutional review board approval, 25 patients >18 years old were recruited. Biases with EE versus TEE for E and A waves were 11.9 cm/second (95% confidence interval [CI], 48.2 to -24.4) and 6.8 cm/second (95% CI, 28 to -15), respectively, and for E/A ratio was 0.08 (95% CI, 1.2 to -1). Pulmonary velocity bias was 57.94 cm/second (95% CI, 192.9 to -76.98), with higher values using EE. Bias for pulmonary trunk diameter was -0.31 cm (95% CI, 1.5 to -2.1). For quality of images, means were 2.4 (standard deviation [SD], 1.0) for EE and 2.3 (SD, 0.57) with TEE for TV and 2.4 (SD, 1.0) with EE and 2.5 (SD, 1.0) with TEE for PV. For the number of leaflets visualized, means were 2.2 (SD, 1.0) with EE and 2.5 (SD, 0.5) with TEE for TV and 2.5 (SD, 0.5) for EE and 1.3 (SD, 1.1) with TEE for PV. CONCLUSIONS: There was good agreement for Doppler measurements across TVs; however, measurements across PVs were significantly higher with EE versus TEE. TV Doppler measurements were difficult to acquire even for surgeons experienced in epiaortic scanning.

Evolution of practice patterns in the management of acute respiratory distress syndrome: A secondary analysis of two successive randomized controlled trials.

PURPOSE: We sought to examine changes in acute respiratory distress syndrome (ARDS) management over a 12-year period of two successive randomized trials. METHODS: Analyses included baseline data, from eligible patients, prior to influence of trial protocols, and daily study data, from randomized patients, of variables not determined by trial protocols. Mixed linear regressions examined changes in practice year-on-year. RESULTS: A total of 2376 patients met the inclusion criteria. Over the 12-year period, baseline tidal volume index decreased (9.0 to 7.0 ml/kg, p < 0.001), plateau pressures decreased (30.8 to 29.0 cmH2O, p < 0.05), and baseline positive end-expiratory pressures increased (10.8 to 13.2 cmH2O, p < 0.001). Volume-controlled ventilation declined from 29.4 to 14.0% (p < 0.01). Use of corticosteroids increased (baseline: 7.7 to 30.3%; on study: 32.6 to 61.2%; both p < 0.001), as did neuromuscular blockade (baseline: 12.3 to 24.5%; on study: 55.5 to 70.0%; both p < 0.01). Inhaled nitric oxide use increased (24.9 to 65.8%, p < 0.05). We observed no significant change in prone positioning (16.2 to 18.9%, p = 0.70). CONCLUSIONS: Clear trends were apparent in tidal volume, airway pressures, ventilator modes, adjuncts and rescue therapies. With the exception of prone positioning, and outside the context of rescue therapy, these trends appear consistent with the evolving literature on ARDS management.

Modulation of neutrophil apoptosis by murine pulmonary microvascular endothelial cell inducible nitric oxide synthase.

Neutrophils contribute significantly to ALI (acute lung injury) through adhesion to pulmonary microvascular endothelial cells (PMEC), trans-PMEC migration and alveolar infiltration. Trans-PMEC migration delays expression of neutrophil apoptosis, which promotes intra-alveolar neutrophil survival and neutrophil mediated ALI. We assessed the role of neutrophil vs PMEC inducible nitric oxide (NO) synthase (iNOS) in modulating neutrophil apoptosis. Apoptosis of wild-type vs iNOS-/- neutrophils was quantified by microscopy and FACS annexin-V binding. In a murine model of ALI, neutrophils isolated by BAL(broncho-alveolar lavage) from iNOS-/- mice had increased expression of apoptosis after 24h culture ex vivo than wild-type neutrophils (15.2±3.3 vs 3.0±0.4%, mean±sd, p<0.01). Apoptosis rates of isolated bone marrow iNOS+/+ vs iNOS-/- neutrophils were similar under basal and LPS/IFN-γ stimulation, and following LPS/IFN-γ-stimulated trans-PMEC migration. Apoptosis of both iNOS+/+ and iNOS-/- neutrophils was inhibited by trans-PMEC migration only across iNOS+/+ PMEC (1.6±0.3 and 1.5±0.3%, respectively; p<0.05 for each vs non-migrated neutrophils) but not across iNOS-/- PMEC (4.3±1 and 3.1±0.6%, respectively). PMEC iNOS-dependent inhibition of neutrophil apoptosis was independent of changes in neutrophil caspase-3 activity. We conclude that PMEC iNOS, but not neutrophil iNOS, has an important inhibitory effect on neutrophil apoptosis during trans-PMEC neutrophil migration, which is independent of caspase-3 activity. Further studies will define the mechanism of PMEC iNOS-dependent inhibition of neutrophil apoptosis and assess the potential relevance of this phenomenon in human neutrophils and ALI.

Diagnosis and Management of Acute Respiratory Distress Syndrome in a Time of COVID-19.

Acute respiratory distress syndrome (ARDS) remains a serious illness with significant morbidity and mortality, characterized by hypoxemic respiratory failure most commonly due to pneumonia, sepsis, and aspiration. Early and accurate diagnosis of ARDS depends upon clinical suspicion and chest imaging. Coronavirus disease 2019 (COVID-19) is an important novel cause of ARDS with a distinct time course, imaging and laboratory features from the time of SARS-CoV-2 infection to hypoxemic respiratory failure, which may allow diagnosis and management prior to or at earlier stages of ARDS. Treatment of ARDS remains largely supportive, and consists of incremental respiratory support (high flow nasal oxygen, non-invasive respiratory support, and invasive mechanical ventilation), and avoidance of iatrogenic complications, all of which improve clinical outcomes. COVID-19-associated ARDS is largely similar to other causes of ARDS with respect to pathology and respiratory physiology, and as such, COVID-19 patients with hypoxemic respiratory failure should typically be managed as other patients with ARDS. Non-invasive respiratory support may be beneficial in avoiding intubation in COVID-19 respiratory failure including mild ARDS, especially under conditions of resource constraints or to avoid overwhelming critical care resources. Compared to other causes of ARDS, medical therapies may improve outcomes in COVID-19-associated ARDS, such as dexamethasone and remdesivir. Future improved clinical outcomes in ARDS of all causes depends upon individual patient physiological and biological endotyping in order to improve accuracy and timeliness of diagnosis as well as optimal targeting of future therapies in the right patient at the right time in their disease.

Elevated activated partial thromboplastin time does not correlate with heparin rebound following cardiac surgery.

PURPOSE: Exposure to cardiopulmonary bypass (CPB) is associated with postoperative coagulopathy and hemorrhage. Recent literature indicates that heparin rebound occurs almost universally following cardiac surgery. We conducted this pilot study to evaluate if the presence of residual circulating heparin following cardiac surgery can be diagnosed by elevation of activated partial thromboplastin time (APTT). METHOD: After obtaining Research Ethics Board approval, blood samples from 30 patients receiving heparin for CPB were evaluated at the time of intensive care unit admission and 2, 4, and 6 hr thereafter. Activated clotting time, whole blood heparin concentration (Hepcon HMS Plus, Medtronic), anti-Xa levels, and APTT were measured at each time point. Samples with prolonged APTT were subjected to mechanistic studies with heparin adsorption and 1:1 mixing. RESULTS: Anti-Xa was elevated in 52 of the 120 blood samples (0.08 +/- 0.08 U . mL(-1), mean +/- SD). APTT was elevated in 49 (40.8%) samples with an average of 51.4 +/- 31.9 sec. At all time points, the APTT correlated poorly with anti-Xa levels with correlation coefficients ranging from -0.26 to -0.05. Mean APTT was modestly, but not significantly, associated with total dose of protamine with r = 0.34 (CI: -0.03, 0.62). After 1:1 mixing studies, APTT returned to normal in most (82%) samples tested. CONCLUSION: Circulating residual heparin is commonly presented following cardiac surgery and does not correlate with APTT. Considering that mixing studies normalize APTT in most samples, elevated APTT following CPB may reflect deficiency of coagulation factors or presence of a coagulation inhibitor such as protamine. Further studies are required to confirm this observation.

The role of epicardial echocardiography in the measurement of transvalvular flow velocities during aortic valve replacement.

OBJECTIVE: The purpose of this study was to compare transvalvular flow velocities obtained by transesophageal echocardiography and epicardial echocardiography (EE) during aortic valve replacement (AVR). DESIGN: Prospective observational study. SETTING: University hospital. PARTICIPANTS: Patients undergoing AVR for aortic stenosis. INTERVENTIONS: After institutional review board approval, 17 patients undergoing AVR consented. Deep transgastric (deep TG LAX) and transgastric long-axis (TG LAX) views and epicardial aortic valve long-axis views (S8 probe) were obtained on a SONOS 5500 (Phillips Medical Systems, Bothell, WA) before and after AVR. Transvalvular flow velocity and velocity time integral (VTI) were recorded via each technique. Measurements were made offline by 2 independent reviewers. Agreement between measurements made by different views was evaluated by using Bland-Altman analysis. MEASUREMENTS AND MAIN RESULTS: The epicardial probe was well tolerated. Quality images were obtained in all patients with TEE and 30 of 34 studies via epicardial scanning. The mean bias for peak velocities derived through EE and deep TG LAX was 96.3 cm/s (95% confidence interval [CI], 51.1-141.4) before AVR and 58 cm/s (95% CI, 32.4-83.7) after AVR. The mean bias for peak velocities between EE and TG LAX was 70 cm/s (95% CI, 31.1-108.9) before and 84.7 cm/s (95% CI, 55.6-113.7) after AVR. Similar results were obtained for VTI. CONCLUSIONS: Peak transaortic valve velocities and VTI measured with epicardial echocardiography are higher in comparison to measurements via TEE in patients undergoing AVR. The precise role of epicardial echocardiography in the comprehensive echocardiographic examination of patients undergoing aortic valve replacement needs further evaluation.

Perioperative coagulation management and blood conservation in cardiac surgery: a Canadian Survey.

OBJECTIVE: To determine which strategies are currently used for (anti)coagulation management and blood conservation during cardiac surgery in Canada. DESIGN: Institutional survey. SETTING: University hospital. PARTICIPANTS: All sites performing cardiac surgery in Canada. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The response rate was 85%. Anticoagulation with heparin is monitored routinely through the activated coagulation time (ACT). Less than 10% of centers use heparin concentrations (Hepcon HMS, Medtronic), thromboelastography, or other point-of-care tests perioperatively. Eighty percent of centers routinely use tranexamic acid as the primary antifibrinolytic agent; however aprotinin until recently, was used more commonly for patients at increased risk for bleeding. Retrograde autologous prime is commonly used (62%); however, cell savers are uncommon for routine patients undergoing cardiac surgery (29%). Although most hospitals use a hematocrit of 20% to 21% for transfusing red blood cells, more than 50% of intensive care units do not have written guidelines for the administration of protamine, fresh frozen plasma, platelets, or factor VIIa. At least one third of centers do not audit their transfusion practices regularly. CONCLUSIONS: The majority of Canadian institutions do not use point-of-care tests other than ACT. Most institutions do not have algorithms for management of bleeding following cardiac surgery and at least 30% do not monitor their transfusion practice perioperatively. Cardiac surgery patients in Canada may benefit from a standardized approach to blood conservation in the perioperative period.

Pulmonary microvascular albumin leak is associated with endothelial cell death in murine sepsis-induced lung injury in vivo.

Sepsis is a systemic inflammatory response that targets multiple components of the cardiovascular system including the microvasculature. Microvascular endothelial cells (MVEC) are central to normal microvascular function, including maintenance of the microvascular permeability barrier. Microvascular/MVEC dysfunction during sepsis is associated with barrier dysfunction, resulting in the leak of protein-rich edema fluid into organs, especially the lung. The specific role of MVEC apoptosis in septic microvascular/MVEC dysfunction in vivo remains to be determined. To examine pulmonary MVEC death in vivo under septic conditions, we used a murine cecal ligation/perforation (CLP) model of sepsis and identified non-viable pulmonary cells with propidium iodide (PI) by intravital videomicroscopy (IVVM), and confirmed this by histology. Septic pulmonary microvascular Evans blue (EB)-labeled albumin leak was associated with an increased number of PI-positive cells, which were confirmed to be predominantly MVEC based on specific labeling with three markers, anti-CD31 (PECAM), anti-CD34, and lectin binding. Furthermore, this septic death of pulmonary MVEC was markedly attenuated by cyclophosphamide-mediated depletion of neutrophils (PMN) or use of an anti-CD18 antibody developed for immunohistochemistry but shown to block CD18-dependent signaling. Additionally, septic pulmonary MVEC death was iNOS-dependent as mice lacking iNOS had markedly fewer PI-positive MVEC. Septic PI-positive pulmonary cell death was confirmed to be due to apoptosis by three independent markers: caspase activation by FLIVO, translocation of phosphatidylserine to the cell surface by Annexin V binding, and DNA fragmentation by TUNEL. Collectively, these findings indicate that septic pulmonary MVEC death, putatively apoptosis, is a result of leukocyte activation and iNOS-dependent signaling, and in turn, may contribute to pulmonary microvascular barrier dysfunction and albumin hyper-permeability during sepsis.

Human alveolar epithelial cells attenuate pulmonary microvascular endothelial cell permeability under septic conditions.

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), are characterised by high-protein pulmonary edema and severe hypoxaemic respiratory failure due to increased permeability of pulmonary microvascular endothelial cells (PMVEC). Alveolar epithelial cells (AEC) contribute importantly to normal alveolar function, and AEC dysfunction in ALI/ARDS is associated with worse outcomes. We hypothesized that AEC can modulate human PMVEC barrier function, and investigated the effects of AEC presence on human PMVEC barrier under septic conditions in vitro. PMVEC isolated from human lung were treated in vitro with septic stimulation (lipopolysaccharide [LPS], a mixture of clinically-relevant cytokines [cytomix], or plasma from patients with severe sepsis), and the trans-PMVEC leak of Evans Blue dye-labeled albumin assessed. PMVEC septic responses were compared in the presence/absence of co-cultured A549 epithelial cell line or primary human AEC. Septic stimulation with LPS, cytomix, or septic plasma induced marked PMVEC hyper-permeability (10.2±1.8, 8.9±2.2, and 3.7±0.2 fold-increase vs. control, respectively, p<0.01 for all). The presence of A549 cells or primary human AEC in a non-contact co-culture model attenuated septic PMVEC hyper-permeability by 39±4% to 100±3%, depending on the septic stimulation (p<0.05). Septic PMVEC hyper-permeability was also attenuated following treatment with culture medium conditioned by previous incubation with either naïve or cytomix-treated A549 cells (p<0.05), and this protective effect of A549 cell-conditioned medium was both heat-stable and transferable following lipid extraction. Cytomix-stimulated PMN-dependent PMVEC hyper-permeability and trans-PMVEC PMN migration were also inhibited in the presence of A549 cells or A549 cell-conditioned medium (p<0.05). Human AEC appear to protect human PMVEC barrier function under septic conditions in vitro, through release of a soluble mediator(s), which are at least partly lipid in nature. This study suggests a scientific and potential clinical therapeutic importance of epithelial-endothelial cross talk in maintaining alveolar integrity in ALI/ARDS.

Specific role of neutrophil inducible nitric oxide synthase in murine sepsis-induced lung injury in vivo.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes importantly to acute lung injury (ALI), but the specific contribution of neutrophil iNOS has not been defined. Thus, we defined the role of neutrophils and specifically neutrophil iNOS in a murine model of septic ALI. Four hours after cecal ligation/perforation, ALI was characterized by increases in pulmonary neutrophil infiltration (tissue myeloperoxidase activity, bronchoalveolar lavage neutrophils), microvascular leak of Evans blue (EB) dye-labeled albumin, and oxidant stress (8-isoprostane levels). Septic ALI was neutrophil dependent, as pretreatment with anti-CD18 before cecal ligation/perforation significantly (P < 0.05) attenuated septic increases in pulmonary myeloperoxidase (39 ± 11 vs. 85 ± 14 mU/mg protein), bronchoalveolar lavage neutrophils (0.5% ± 0.2% vs. 2.1% ± 0.6%), microvascular EB-albumin leak (1.3 ± 0.3 vs. 2.6 ± 0.7 µg EB/g per minute), and 8-isoprostane content (74 ± 15 vs. 115 ± 16 pg/mg protein). The role of neutrophil iNOS was assessed by creation of neutrophil-iNOS chimeric mice: iNOS(+/+) versus iNOS(-/-) mice were bone marrow depleted by irradiation and selectively reconstituted with iNOS(+/+) versus iNOS(-/-) neutrophils. Cecal ligation/perforation resulted in significant septic ALI in + to - neutrophil-iNOS chimeric mice (iNOS(+/+) neutrophils in iNOS(-/-) mice), but not in - to + neutrophil depleted-reconstituted mice (iNOS(-/-) neutrophils in iNOS(+/+) mice). There were no significant differences between iNOS(+/+) and iNOS(-/-) neutrophils in phagocytosis, respiratory burst, or CD11a/b/CD18 surface expression, although septic shedding of CD62L was blunted in iNOS(-/-) neutrophils. Neutrophil iNOS contributes importantly to murine septic ALI in vivo, but not simply through a change in neutrophil phenotype. We speculate that neutrophil iNOS may modulate neutrophil-endothelial interactions in complex fashion, including regulation of transendothelial neutrophil migration and pulmonary neutrophil infiltration.