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Abstract Background A sepsis-like syndrome is known to occur after cardiac arrest, leading to cerebral infiltration by white blood cells (WBC). We hypothesized that pharmacological sequestration of WBC, and more specifically lymphocytes within lymphoid tissues, could reduce the cerebral infiltration by these inflammatory cells and subsequent acute brain injury in a porcine model of cardiac arrest. Lymphocyte sequestration was induced by the sphingosine-1 phosphate receptors agonist fingolimod. Methods In a first set of experiments, anesthetized pigs underwent a sham instrumentation with no cardiac arrest (n = 4). They received an administration of fingolimod (1 mg/kg, i.v.) in order to confirm its effect on WBC. In a second set of experiments, animals randomly received fingolimod or saline two hours prior to an episode of ventricular fibrillation (14 min) with subsequent resuscitation (n = 6 in each group). Neurological injury was assessed 24 h after resuscitation. Results In the first set of experiments, WBC and blood lymphocyte counts were significantly reduced by − 61 ± 10% and − 75 ± 6% two hours after fingolimod administration. In the second set of experiments, blood lymphocyte counts, but not WBC, were also significantly reduced after cardiac arrest in Fingolimod vs Control group. However, most cytokine blood levels were not different among groups, including Interleukin (IL)-1ra, IL-8 or IL-18 blood levels. A difference was only observed for IL-6, which decreased in Fingolimod vs Control (e.g., 5.6 ± 4.8 vs 59.4 ± 20.6 pg/ml at 2 h after cardiac arrest, respectively; p = 0.126). Neurofilament light chain (NFL) blood levels were not different among groups (57 ± 25 vs 84 ± 41 pg/ml in Fingolimod vs Control at 6 h after resuscitation, respectively). After awakening, 3 and 2 animals were prematurely euthanized for ethical reasons due to recurrent seizures in Fingolimod and Control groups, respectively. At Day 1, neurological dysfunction score was not different between groups (87 ± 7 vs 87 ± 5% in Fingolimod vs Control, respectively). Conversely, a decrease in the number of CD3 + cells was observed in the brain of surviving animals in Fingolimod vs Control group (3.10 ± 0.50 vs 7.53 ± 0.57 CD3 + cells/field, respectively; p = 0.0286). Conclusion Fingolimod-induced WBC sequestration, and more specifically lymphocytes sequestration, did not improve clinical neurological dysfunction following cardiac arrest although it reduced cerebral infiltration by lymphocytes.

Abstract Duchenne muscular dystrophy (DMD) patients exhibit a late left ventricular systolic dysfunction preceded by an occult phase, during which myocardial fibrosis progresses and some early functional impairments can be detected. These latter include electrocardiographic (ECG) and heart rate variability (HRV) abnormalities. This longitudinal study aimed at describing the sequence of ECG and HRV abnormalities, using Holter ECG in the GRMD (Golden retriever muscular dystrophy) dog model, known to develop a DMD-like disease, including cardiomyopathy. Most of the known ECG abnormalities described in DMD patients were also found in GRMD dogs, including increased heart rate, prolonged QT and shortened PR intervals, ventricular arrhythmias, and several of them could be detected months before the decrease of fractional shortening. The HRV was impaired like in DMD patients, one of the earliest evidenced abnormalities being a decrease in the very low frequency (VLF) component of the power spectrum. This decrease was correlated with the further reduction of fractional shortening. Such decreased VLF probably reflects impaired autonomic function and abnormal vasomotor tone. This study provides new insights into the knowledge of the GRMD dog model and DMD cardiomyopathy and emphasizes the interest to monitor the VLF power in DMD patients, still unexplored in this disease, whilst it is highly predictive of deleterious clinical events in many other pathological conditions.

Background Brain injury is one of the most serious complications after cardiac arrest (CA). To prevent this phenomenon, rapid cooling with total liquid ventilation (TLV) has been proposed in small animal models of CA (rabbits and piglets). Here, we aimed to determine whether hypothermic TLV can also offer neuroprotection and mitigate cerebral inflammatory response in large animals. Methods and Results Anesthetized pigs were subjected to 14 minutes of ventricular fibrillation followed by cardiopulmonary resuscitation. After return of spontaneous circulation, animals were randomly subjected to normothermia (control group, n=8) or ultrafast cooling with TLV (TLV group, n=8). In the latter group, TLV was initiated within a window of 15 minutes after return of spontaneous circulation and allowed to reduce tympanic, esophageal, and bladder temperature to the 32 to 34 °C range within 30 minutes. After 45 minutes of TLV, gas ventilation was resumed, and hypothermia was maintained externally until 3 hours after CA, before rewarming using heat pads (0.5 °C–1 °C/h). After an additional period of progressive rewarming for 3 hours, animals were euthanized for brain withdrawal and histological analysis. At the end of the follow‐up (ie, 6 hours after CA), histology showed reduced brain injury as witnessed by the reduced number of Fluroro‐Jade C‐positive cerebral degenerating neurons in TLV versus control. IL (interleukin)‐1ra and IL‐8 levels were also significantly reduced in the cerebrospinal fluid in TLV versus control along with cerebral infiltration by CD3+ cells. Conversely, circulating levels of cytokines were not different among groups, suggesting a discrepancy between local and systemic inflammatory levels. Conclusions Ultrafast cooling with TLV mitigates neuroinflammation and attenuates acute brain lesions in the early phase following resuscitation in large animals subjected to CA.

Summary: Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified.

Background: Post-cardiac arrest (CA) shock is associated with multiple organ failure, including acute kidney injury, and is the leading cause of early death among patient successfully resuscitated from CA. Arginine-vasopressin (AVP) may be an interesting therapeutic alternative or complement to noradrenaline (NAD) to both control shock and preserve regional, especially renal, organ perfusions. Methods: 18 swine (24–39 kg) were submitted to 14 min of ventricular fibrillation and cardio-pulmonary resuscitation. After return of spontaneous circulation (ROSC), animals randomly received either AVP, NAD or AVP-NAD combination for maintaining a targeted mean arterial pressure of 70 ± 5 mmHg for 6 h. Haemodynamic and biological parameters, including kidney function biomarkers and diuresis, were monitored throughout the follow-up. Results: Targeted mean arterial pressure was successfully obtained in the NAD (n = 6) and the AVP-NAD (n = 6) groups, but not in the AVP group (n = 6), where 4 animals died. As compared to NAD alone, renal blood flow (2.9 ± 1.15 vs 4.36 ± 0.64 mL//kg/min in NAD and AVP-NAD groups) and diuresis were higher in the AVP-NAD group. This was associated with a reduction of carotid blood flow and a more severe metabolic acidosis during the first 3 h of follow-up in the AVP-NAD group as compared to NAD group. Conclusion: Combination of AVP and NAD improved renal perfusion and diuresis but reduced carotid blood flow as compared to NAD alone in a porcine model of post-resuscitation syndrome. AVP alone failed to manage shock and led to mortality.

Purpose Bladder cancer is a complex disease with a wide range of outcomes. Clinicopathological factors only partially explain the variability between patients in prognosis and treatment response. There is a need for large cohorts collecting extensive data and biological samples to: (1) investigate gene-environment interactions, pathological/molecular classification and biomarker discovery; and (2) describe treatment patterns, outcomes, resource use and quality of life in a real-world setting.Participants COBLAnCE (COhort to study BLAdder CancEr) is a French national prospective cohort of patients with bladder cancer recruited between 2012 and 2018 and followed for 6 years. Data on patient and tumour characteristics, treatments, outcomes and biological samples are collected at enrolment and during the follow-up.Findings to date We describe the cohort at enrolment according to baseline surgery and tumour type. In total, 1800 patients were included: 1114 patients with non-muscle-invasive bladder cancer (NMIBC) and 76 patients with muscle-invasive bladder cancer (MIBC) had transurethral resection of a bladder tumour without cystectomy, and 610 patients with NMIBC or MIBC underwent cystectomy. Most patients had a solitary lesion (56.3%) without basement membrane invasion (71.7% of Ta and/or Tis). Half of the patients with cystectomy were stage ≤T2 and 60% had non-continent diversion. Surgery included local (n=298) or super-extended lymph node dissections (n=11) and prostate removal (n=492). Among women, 16.5% underwent cystectomy and 81.4% anterior pelvectomy.Future plans COBLAnCE will be used for long-term studies of bladder cancer with focus on clinicopathological factors and molecular markers. It will lead to a much-needed improvement in the understanding of the disease. The cohort provides valuable real-world data, enabling researchers to study various research questions, assess routine medical practices and guide medical decision-making.

Background & Aims: Mitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia–reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI. Methods: Derivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best in vitro was selected for in vivo efficacy evaluation in a mouse model of hepatic IRI. Results: The two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI in vivo by reducing hepatocyte necrosis and apoptosis. Conclusions: We identified a novel cyclophilin inhibitor with strong mitoprotective properties both in vitro and in vivo that represents a promising candidate for cellular protection in hepatic IRI. Impact and Implications: Hepatic ischaemia–reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both in vitro and in vivo, making it an attractive lead compound for subsequent clinical development.

Summary: Background: Coronary microvascular obstruction also known as no-reflow phenomenon is a major issue during myocardial infarction that bears important prognostic implications. Alterations of the microvascular network remains however challenging to assess as there is no imaging modality in the clinics that can image directly the coronary microvascular vessels. Ultrasound Localization Microscopy (ULM) imaging was recently introduced to map microvascular flows at high spatial resolution (∼10 μm). In this study, we developed an approach to image alterations of the microvascular coronary flow in ex vivo perfused swine hearts. Methods: A porcine model of myocardial ischemia-reperfusion was used to obtain microvascular coronary alterations and no-reflow. Four female hearts with myocardial infarction in addition to 6 controls were explanted and placed immediately in a dedicated preservation and perfusion box manufactured for ultrasound imaging. Microbubbles (MB) were injected into the vasculature to perform Ultrasound Localization Microscopy (ULM) imaging and a linear ultrasound probe mounted on a motorized device was used to scan the heart on multiple slices. The coronary microvascular anatomy and flow velocity was reconstructed using dedicated ULM algorithms and analyzed quantitatively. Findings: We were able to image the coronary microcirculation of ex vivo swine hearts at a resolution of tens of microns and measure flow velocities ranging from 10 mm/s in arterioles up to more than 200 mm/s in epicardial arteries. Under different aortic perfusion pressures, we measured in large arteries of a subset of control hearts an increase of flow velocity from 31 ± 11 mm/s at 87 mmHg to 47 ± 17 mm/s at 132 mmHg (N = 3 hearts, P

Background High‐mobility group box 1 (HMGB1) is a major promotor of ischemic injuries and aseptic inflammatory responses. We tested its inhibition on neurological outcome and systemic immune response after cardiac arrest (CA) in rabbits. Methods and Results After 10 minutes of ventricular fibrillation, rabbits were resuscitated and received saline (control) or the HMGB1 inhibitor glycyrrhizin. A sham group underwent a similar procedure without CA. After resuscitation, glycyrrhizin blunted the successive rises in HMGB1, interleukin‐6, and interleukin‐10 blood levels as compared with control. Blood counts of the different immune cell populations were not different in glycyrrhizin versus control. After animal awakening, neurological outcome was improved by glycyrrhizin versus control, regarding both clinical recovery and histopathological damages. This was associated with reduced cerebral CD4+ and CD8+ T‐cell infiltration beginning 2 hours after CA. Conversely, granulocytes' attraction or loss of microglial cells or cerebral monocytes were not modified by glycyrrhizin after CA. These modifications were not related to the blood–brain barrier preservation with glycyrrhizin versus control. Interestingly, the specific blockade of the HMGB1 receptor for advanced glycation end products by FPS‐ZM1 recapitulated the neuroprotective effects of glycyrrhizin. Conclusions Our findings support that the early inhibition of HMGB1‐signaling pathway prevents cerebral chemoattraction of T cells and neurological sequelae after CA. Glycyrrhizin could become a clinically relevant therapeutic target in this situation.

Abstract Background Extracorporeal cardiopulmonary resuscitation (E-CPR) is used for the treatment of refractory cardiac arrest. However, the optimal target to reach for mean arterial pressure (MAP) remains to be determined. We hypothesized that MAP levels critically modify cerebral hemodynamics during E-CPR and tested two distinct targets (65–75 vs 80–90 mmHg) in a porcine model. Methods Pigs were submitted to 15 min of untreated ventricular fibrillation followed by 30 min of E-CPR. Defibrillations were then delivered until return of spontaneous circulation (ROSC). Extracorporeal circulation was initially set to an average flow of 40 ml/kg/min. The dose of epinephrine was set to reach a standard or a high MAP target level (65–75 vs 80–90 mmHg, respectively). Animals were followed during 120-min after ROSC. Results Six animals were included in both groups. During E-CPR, high MAP improved carotid blood flow as compared to standard MAP. After ROSC, this was conversely decreased in high versus standard MAP, while intra-cranial pressure was superior. The pressure reactivity index (PRx), which is the correlation coefficient between arterial blood pressure and intracranial pressure, also demonstrated inverted patterns of alteration according to MAP levels during E-CPR and after ROSC. In standard-MAP, PRx was transiently positive during E-CPR before returning to negative values after ROSC, demonstrating a reversible alteration of cerebral autoregulation during E-CPR. In high-MAP, PRx was negative during E-CPR but became sustainably positive after ROSC, demonstrating a prolonged alteration in cerebral autoregulation after ROSC. It was associated with a significant decrease in cerebral oxygen consumption in high- versus standard-MAP after ROSC. Conclusions During early E-CPR, MAP target above 80 mmHg is associated with higher carotid blood flow and improved cerebral autoregulation. This pattern is inverted after ROSC with a better hemodynamic status with standard versus high-MAP.

Abstract Background The administration of epinephrine in the management of non-traumatic cardiac arrest remains recommended despite controversial effects on neurologic outcome. The use of resuscitative endovascular balloon occlusion of the aorta (REBOA) could be an interesting alternative. The aim of this study was to compare the effects of these 2 strategies on return of spontaneous circulation (ROSC) and cerebral hemodynamics during cardiopulmonary resuscitation (CPR) in a swine model of non-traumatic cardiac arrest. Results Anesthetized pigs were instrumented and submitted to ventricular fibrillation. After 4 min of no-flow and 18 min of basic life support (BLS) using a mechanical CPR device, animals were randomly submitted to either REBOA or epinephrine administration before defibrillation attempts. Six animals were included in each experimental group (Epinephrine or REBOA). Hemodynamic parameters were similar in both groups during BLS, i.e., before randomization. After epinephrine administration or REBOA, mean arterial pressure, coronary and cerebral perfusion pressures similarly increased in both groups. However, carotid blood flow (CBF) and cerebral regional oxygenation saturation were significantly higher with REBOA as compared to epinephrine administration (+ 125% and + 40%, respectively). ROSC was obtained in 5 animals in both groups. After resuscitation, CBF remained lower in the epinephrine group as compared to REBOA, but it did not achieve statistical significance. Conclusions During CPR, REBOA is as efficient as epinephrine to facilitate ROSC. Unlike epinephrine, REBOA transitorily increases cerebral blood flow and could avoid its cerebral detrimental effects during CPR. These experimental findings suggest that the use of REBOA could be beneficial in the treatment of non-traumatic cardiac arrest.

Immune cells contribute to adverse remodeling following myocardial infarction. Here the authors show in mice and pigs that CD8+ lymphocytes release Granzyme B in the infarcted heart leading to cardiomyocyte death, enhanced inflammation and deterioration of cardiac function.

Background: Understanding and effectively treating dystrophin-deficient cardiomyopathy is of high importance for Duchenne muscular dystrophy (DMD) patients due to their prolonged lifespan. We used two-dimensional speckle tracking echocardiography to analyze more deeply the non-uniformity of myocardial strain within the left ventricle during the progression of cardiomyopathy in golden retriever muscular dystrophy (GRMD) dogs. Methods: The circumferential strain (CS) and longitudinal strain (LS) of left ventricular (LV) endocardial, middle and epicardial layers were analyzed from three parasternal short-axis views and three apical views, respectively, in GRMD (n = 22) and healthy control dogs (n = 7) from 2 to 24 months of age. Results: In GRMD dogs, despite normal global systolic function (normal LV fractional shortening and ejection fraction), a reduction in systolic CS was detected in the three layers of the LV apex but not in the LV middle-chamber and base at 2 months of age. This spatial heterogeneity in CS progressed with age, whereas a decrease in systolic LS could be detected early at 2 months of age in the three layers of the LV wall from three apical views. Conclusions: Analyzing the evolution of myocardial CS and LS in GRMD dogs reveals spatial and temporal non-uniform alterations of LV myocardial strain, providing new insights into the progression of dystrophin-deficient cardiomyopathy in this relevant model of DMD.

Abstract Background Contrasting to the well documented tyrosine kinase inhibitor (TKI)-induced hypertension, little is known on their intrinsic vasomotor effects. We investigated the vasomotor effects of sorafenib, a widely used multikinase inhibitor in the treatment of hepatocellular and renal cell carcinoma and tested the hypothesis that sildenafil, a phosphodiesterase-5 (PDE-5) inhibitor, could represent a pharmacological strategy for the treatment of TKI-induced hypertension. Methods Concentration-response curves of sorafenib were constructed in endothelium-intact or denuded precontracted rat aorta, in the presence or absence of several inhibitors. Acute intravenous effects of sorafenib on arterial blood pressure were also investigated in anaesthetized rats. Finally, rats were chronically treated with sorafenib during 4 weeks in the presence and absence of sildenafil. Results In endothelium intact aortic ring, sorafenib induced a potent concentration-dependent relaxation of precontracted rat aorta. Removal of the endothelium shifted the concentration-response curve of sorafenib to the right and significantly reduced its maximal effects, demonstrating that sorafenib-induced vasorelaxation is endothelium-dependent and endothelium-independent. Inhibition of the different pathways implicated in the endothelium-dependent and independent vasorelaxation revealed that the endothelium-dependent effects of sorafenib result mainly from the activation of prostaglandin and the nitric oxide (NO) pathways. The endothelium-independent vasodilatory effects of sorafenib may result mainly from the activation of Na/K-ATPase and soluble guanylate cyclase. These vasodilatory effects observed in vitro were confirmed by the decrease in arterial blood pressure observed during acute administrations of sorafenib in anesthetized rats. Finally, and most importantly, we report here for the first time that chronic administration of sorafenib in rats induced an increase in SBP that was abolished by sildenafil. Conclusion The multikinase inhibitor sorafenib induced in vitro vasorelaxation of large conductance artery, primary by activating soluble guanylate cyclase. Its chronic administration led to arterial blood hypertension that was counteracted by a PDE-5 inhibitor, sildenafil. Our results suggest that targeting the cGMP pathway including NO signalling might be an interesting pharmacological strategy for the treatment of TKI-induced hypertension.

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity.

Background Total liquid ventilation (TLV) has been shown to prevent neurological damage though ultrafast cooling in animal models of cardiac arrest. We investigated whether its neuroprotective effect could be explained by mitigation of early inflammatory events. Methods and Results Rabbits were submitted to 10 minutes of ventricular fibrillation. After resuscitation, they underwent normothermic follow‐up (control) or ultrafast cooling by TLV and hypothermia maintenance for 3 hours (TLV). Immune response, survival, and neurological dysfunction were assessed for 3 days. TLV improved neurological recovery and reduced cerebral lesions and leukocyte infiltration as compared with control (eg, neurological dysfunction score=34±6 versus 66±6% at day 1, respectively). TLV also significantly reduced interleukin‐6 blood levels during the hypothermic episode (298±303 versus 991±471 pg/mL in TLV versus control at 3 hours after resuscitation, respectively), but not after rewarming (752±563 versus 741±219 pg/mL in TLV versus control at 6 hours after resuscitation, respectively). In vitro assays confirmed the high temperature sensitivity of interleukin‐6 secretion. Conversely, TLV did not modify circulating high‐mobility group box 1 levels or immune cell recruitment into the peripheral circulation. The link between interleukin‐6 early transcripts (8 hours). Conclusions The neuroprotective effect of hypothermic TLV was associated with a mitigation of humoral interleukin‐6 response. A temperature‐dependent attenuation of immune cell reactivity during the early phase of the post–cardiac arrest syndrome could explain the potent effect of rapid hypothermia. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT00999583.

Background: Adjusting drug therapy under veno-venous extracorporeal membrane oxygenation (VV ECMO) is challenging. Although impaired pharmacokinetics (PK) under VV ECMO have been reported for sedative drugs and antibiotics, data about amiodarone are lacking. We evaluated the pharmacokinetics of amiodarone under VV ECMO both in vitro and in vivo. Methods: In vitro: Amiodarone concentration decays were compared between closed-loop ECMO and control stirring containers over a 24 h period. In vivo: Potassium-induced cardiac arrest in 10 pigs with ARDS, assigned to either control or VV ECMO groups, was treated with 300 mg amiodarone injection under continuous cardiopulmonary resuscitation. Pharmacokinetic parameters Cmax, Tmax AUC and F were determined from both direct amiodarone plasma concentrations observation and non-linear mixed effects modeling estimation. Results: An in vitro study revealed a rapid and significant decrease in amiodarone concentrations in the closed-loop ECMO circuitry whereas it remained stable in control experiment. In vivo study revealed a 32% decrease in the AUC and a significant 42% drop of Cmax in the VV ECMO group as compared to controls. No difference in Tmax was observed. VV ECMO significantly modified both central distribution volume and amiodarone clearance. Monte Carlo simulations predicted that a 600 mg bolus of amiodarone under VV ECMO would achieve the amiodarone bioavailability observed in the control group. Conclusions: This is the first study to report decreased amiodarone bioavailability under VV ECMO. Higher doses of amiodarone should be considered for effective amiodarone exposure under VV ECMO.

Background Absolute hyperemic coronary blood flow and microvascular resistances can be measured by continuous thermodilution with a dedicated infusion catheter. We aimed to determine the mechanisms of this hyperemic response in animal. Methods and Results Twenty open chest pigs were instrumented with flow probes on coronary arteries. The following possible mechanisms of saline‐induced hyperemia were explored compared with maximal hyperemia achieve with adenosine by testing: (1) various infusion rates; (2) various infusion content and temperature; (3) NO production inhibition with L‐arginine methyl ester and endothelial denudation; (4) effects of vibrations generated by rotational atherectomy and of infusion through one end‐hole versus side‐holes. Saline infusion rates of 5, 10 and 15 mL/min did not reach maximal hyperemia as compared with adenosine. Percentage of coronary blood flow expressed in percent of the coronary blood flow after adenosine were 48±17% at baseline, 57±18% at 5 mL/min, 65±17% at 10 mL/min, 82±26% at 15 mL/min and 107±18% at 20 mL/min. Maximal hyperemia was observed during infusion of both saline at body temperature and glucose 5%, after endothelial denudation, l‐arginine methyl ester administration, and after stent implantation. The activation of a Rota burr in the first millimeters of the epicardial artery also induced maximal hyperemia. Maximal hyperemia was achieved by infusion through lateral side‐holes but not through an end‐hole catheter. Conclusions Infusion of saline at 20 mL/min through a catheter with side holes in the first millimeters of the epicardial artery induces maximal hyperemia. The data indicate that this vasodilation is related neither to the composition/temperature of the indicator nor is it endothelial mediated. It is suggested that it could be elicited by epicardial wall vibrations.

Background: Total liquid ventilation (TLV) of the lungs could provide radically new benefits in critically ill patients requiring lung lavage or ultra-fast cooling after cardiac arrest. It consists in an initial filling of the lungs with perfluorocarbons and subsequent tidal ventilation using a dedicated liquid ventilator. Here, we propose a new paradigm for a lung-conservative TLV using pulmonary volumes of perfluorocarbons below functional residual capacity (FRC). Methods and findings: Using a dedicated technology, we showed that perfluorocarbon end-expiratory volumes could be maintained below expected FRC and lead to better respiratory recovery, preserved lung structure and accelerated evaporation of liquid residues as compared to complete lung filling in piglets. Such TLV below FRC prevented volutrauma through preservation of alveolar recruitment reserve. When used with temperature-controlled perfluorocarbons, this lung-conservative approach provided neuroprotective ultra-fast cooling in a model of hypoxic-ischemic encephalopathy. The scale-up and automating of the technology confirmed that incomplete initial lung filling during TLV was beneficial in human adult-sized pigs, despite larger size and maturity of the lungs. Our results were confirmed in aged non-human primates, confirming the safety of this lung-conservative approach. Interpretation: This study demonstrated that TLV with an accurate control of perfluorocarbon volume below FRC could provide the full potential of TLV in an innovative and safe manner. This constitutes a new paradigm through the tidal liquid ventilation of incompletely filled lungs, which strongly differs from the previously known TLV approach, opening promising perspectives for a safer clinical translation. Fund: ANR (COOLIVENT), FRM (DBS20140930781), SATT IdfInnov (project 273). Keywords: Liquid ventilation, Critical care, Therapeutic hypothermia, Biomedical engineering

Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) support leads to complex pharmacokinetic alterations, whereas adequate drug dosing is paramount for efficacy and absence of toxicity in critically ill patients. Amikacin is a major antibiotic used in nosocomial sepsis, especially for these patients. We aimed to describe amikacin pharmacokinetics on V-A ECMO support and to determine relevant variables to improve its dosing. All critically ill patients requiring empirical antimicrobial therapy, including amikacin for nosocomial sepsis supported or not by V-A ECMO, were included in a prospective population pharmacokinetic study. This population pharmacokinetic analysis was built with a dedicated software, and Monte Carlo simulations were performed to identify doses achieving therapeutic plasma concentrations. Thirty-nine patients were included (control n = 15, V-A ECMO n = 24); 215 plasma assays were performed and used for the modeling process. Patients received 29 (24–33) and 32 (30–35) mg/kg of amikacin in control and ECMO groups, respectively. Data were best described by a two-compartment model with first-order elimination. Inter-individual variabilities were observed on clearance, central compartment volume (V1), and peripherical compartment volume (V2). Three significant covariates explained these variabilities: Kidney Disease Improving Global Outcomes (KDIGO) stage on amikacin clearance, total body weight on V1, and ECMO support on V2. Our simulations showed that the adequate dosage of amikacin was 40 mg/kg in KDIGO stage 0 patients, while 25 mg/kg in KDIGO stage 3 patients was relevant. V-A ECMO support had only a secondary impact on amikacin pharmacokinetics, as compared to acute kidney injury.

Abstract Background Experimental studies suggest that morphine may protect the myocardium against ischemia-reperfusion injury by activating salvage kinase pathways. The objective of this two-center, randomized, double-blind, controlled trial was to assess potential cardioprotective effects of intra-coronary morphine in patients with ST-segment elevation myocardial infarction (STEMI) referred for primary percutaneous intervention. Methods Ninety-one patients with STEMI were randomly assigned to intracoronary morphine (1 mg) or placebo at reperfusion of the culprit coronary artery. The primary endpoint was infarct size/left ventricular mass ratio assessed by magnetic resonance imaging on day 3–5. Secondary endpoints included the areas under the curve (AUC) for troponin T and creatine kinase over three days, left ventricular ejection fraction assessed by echocardiography on days 1 and 6, and clinical outcomes. Results Infarct size/left ventricular mass ratio was not significantly reduced by intracoronary morphine compared to placebo (27.2% ± 15.0% vs. 30.5% ± 10.6%, respectively, p = 0.28). Troponin T and creatine kinase AUCs were similar in the two groups. Morphine did not improve left ventricular ejection fraction on day 1 (49.7 ± 10.3% vs. 49.3 ± 9.3% with placebo, p = 0.84) or day 6 (48.5 ± 10.2% vs. 49.0 ± 8.5% with placebo, p = 0.86). The number of major adverse cardiac events, including stent thrombosis, during the one-year follow-up was similar in the two groups. Conclusions Intracoronary morphine at reperfusion did not significantly reduce infarct size or improve left ventricular systolic function in patients with STEMI. Presence of comorbidities in some patients may contribute to explain these results. Trial registration ClinicalTrials.gov, NCT01186445 (date of registration: August 23, 2010).

Abstract Background Ultrafast cooling by total liquid ventilation (TLV) provides potent cardio- and neuroprotection after experimental cardiac arrest. However, this was evaluated in animals with no initial lung injury, whereas out-of-hospital cardiac arrest is frequently associated with early-onset pneumonia, which may lead to acute respiratory distress syndrome (ARDS). Here, our objective was to determine whether hypothermic TLV could be safe or even beneficial in an aspiration-associated ARDS animal model. Methods ARDS was induced in anesthetized rabbits through a two-hits model including the intra-tracheal administration of a pH = 1 solution mimicking gastric content and subsequent gaseous non-protective ventilation during 90 min (tidal volume [Vt] = 10 ml/kg with positive end-expiration pressure [PEEP] = 0 cmH2O). After this initial period, animals either received lung protective gas ventilation (LPV; Vt = 8 ml/kg and PEEP = 5 cmH2O) under normothermic conditions, or hypothermic TLV (TLV; Vt = 8 ml/kg and end-expiratory volume = 15 ml/kg). Both strategies were applied for 120 min with a continuous monitoring of respiratory and cardiovascular parameters. Animals were then euthanized for pulmonary histological analyses. Results Eight rabbits were included in each group. Before randomization, all animals elicited ARDS with arterial oxygen partial pressure over inhaled oxygen fraction ratios (PaO2/FiO2) below 100 mmHg, as well as decreased lung compliance. After randomization, body temperature rapidly decreased in TLV versus LPV group (32.6 ± 0.6 vs. 38.2 ± 0.4 °C after 15 min). Static lung compliance and gas exchanges were not significantly different in the TLV versus LPV group (PaO2/FiO2 = 62 ± 4 vs. 52 ± 8 mmHg at the end of the procedure, respectively). Mean arterial pressure and arterial bicarbonates levels were significantly higher in TLV versus LPV. Histological analysis also showed significantly lower inflammation in TLV versus LPV group (median histological score = 3 vs. 4.5/5, respectively; p = 0.03). Conclusion Hypothermic TLV can be safely induced in rabbits during aspiration-associated ARDS. It modified neither gas exchanges nor respiratory mechanics but reduced lung inflammation and hemodynamic failure in comparison with LPV. Since hypothermic TLV was previously shown to provide neuro- and cardio protective effects after cardiac arrest, these findings suggest a possible use of TLV in the settings of cardiac arrest-associated ARDS.

Abstract Opening of the mitochondrial permeability transition pore (mPTP) plays a major role in cell death during cardiac ischaemia-reperfusion. Adult isolated rodent cardiomyocytes are valuable cells to study the effect of drugs targeting mPTP. This study investigated whether the use of Ca2+ ionophores (A23187, ionomycin and ETH129) represent a reliable model to study inhibition of mPTP opening in cardiomyocytes. We monitored mPTP opening using the calcein/cobalt fluorescence technique in adult rat and wild type or cyclophilin D (CypD) knock-out mice cardiomyocytes. Cells were either treated with Ca2+ ionophores or subjected to hypoxia followed by reoxygenation. The ionophores induced mPTP-dependent swelling in isolated mitochondria. A23187, but not ionomycin, induced a decrease in calcein fluorescence. This loss could not be inhibited by CypD deletion and was explained by a direct interaction between A23187 and cobalt. ETH129 caused calcein loss, mitochondrial depolarization and cell death but CypD deletion did not alleviate these effects. In the hypoxia-reoxygenation model, CypD deletion delayed both mPTP opening and cell death occurring at the time of reoxygenation. Thus, Ca2+ ionophores are not suitable to induce CypD-dependent mPTP opening in adult murine cardiomyocytes. Hypoxia-reoxygenation conditions appear therefore as the most reliable model to investigate mPTP opening in these cells.

[This corrects the article DOI: 10.1371/journal.pone.0137339.].

Argon inhalation attenuates multiorgan failure (MOF) after experimental ischemic injury. We hypothesized that this protection could involve decreased High Mobility Group Box 1 (HMGB1) systemic release. We investigated this issue in an animal model of MOF induced by aortic cross-clamping. Anesthetized rabbits were submitted to supra-coeliac aortic cross-clamping for 30 min, followed by 300 min of reperfusion. They were randomly divided into three groups (n = 7/group). The Control group inhaled nitrogen (70%) and oxygen (30%). The Argon group was exposed to a mixture of argon (70%) and oxygen (30%). The last group inhaled nitrogen/oxygen (70/30%) with an administration of the HMGB1 inhibitor glycyrrhizin (4 mg/kg i.v.) 5 min before aortic unclamping. At the end of follow-up, cardiac output was significantly higher in Argon and Glycyrrhizin vs. Control (60 ± 4 and 49 ± 4 vs. 33 ± 8 mL/kg/min, respectively). Metabolic acidosis was attenuated in Argon and Glycyrrhizin vs. Control, along with reduced amount of norepinephrine to reverse arterial hypotension. This was associated with reduced interleukin-6 and HMGB1 plasma concentration in Argon and Glycyrrhizin vs. Control. End-organ damages were also attenuated in the liver and kidney in Argon and Glycyrrhizin vs. Control, respectively. Argon inhalation reduced HMGB1 blood level after experimental aortic cross-clamping and provided similar benefits to direct HMGB1 inhibition.

BackgroundExtracorporeal cardiopulmonary resuscitation (ECPR) is widely proposed for the treatment of refractory cardiac arrest. It should be associated with coronary angiography if coronary artery disease is suspected. However, the prioritization of care remains unclear in this situation. Our goal was to determine whether coronary reperfusion should be instituted as soon as possible in such situations in a pig model. Methods and ResultsAnesthetized pigs were instrumented and submitted to coronary artery occlusion and ventricular fibrillation. After 5 minutes of untreated cardiac arrest, conventional cardiopulmonary resuscitation (CPR) was started. Fifteen minutes later, ECPR was initiated for a total duration of 240 minutes. Animals randomly underwent either early or late coronary reperfusion at 20 or 120 minutes of ECPR, respectively. This timing was adapted to the kinetic of infarct extension in pigs. Return of spontaneous circulation was determined as organized electrocardiogram rhythm with systolic arterial pressure above 80 mm Hg. During conventional CPR, hemodynamic parameters were not different between groups. Carotid blood flow then increased by 70% after the onset of ECPR in both groups. No animal (0 of 7) elicited return of spontaneous circulation after late reperfusion versus 4 of 7 after early reperfusion (P=0.025). The hemodynamic parameters, such as carotid blood flow, were also improved in early versus late reperfusion groups (113±20 vs 43±17 mL/min after 240 minutes of ECPR, respectively; P=0.030), along with infarct size decrease (71±4% vs 84±2% of the risk zone, respectively; P=0.013). ConclusionsEarly reperfusion improved hemodynamic status and facilitated return of spontaneous circulation in a porcine model of ischemic cardiac arrest treated by ECPR.

Serial invasive endomyocardial biopsies (EMB) remain the gold standard for acute cellular rejection (ACR) diagnosis. However histological grading has several limitations. We aimed to explore the value of myocardial Gene Expression Profiling (GEP) for diagnosing and identifying predictive biomarkers of ACR.A case-control study nested within a retrospective heart transplant patients cohort included 126 patients with median (IQR) age 50 (41-57) years and 111 (88%) males. Among 1157 EMB performed, 467 were eligible (i.e, corresponding to either ISHLT grade 0 or ≥3A), among which 36 were selected for GEP according to the grading: 0 (CISHLT, n = 13); rejection ≥3A (RISHLT, n = 13); 0 one month before ACR (BRISHLT, n = 10).We found 294 genes differentially expressed between CISHLT and RISHLT, mainly involved in immune activation, and inflammation. Hierarchical clustering showed a clear segregation of CISHLT and RISHLT groups and heterogeneity of GEP within RISHLT. All EMB presented immune activation, but some RISHLT EMB were strongly subject to inflammation, whereas others, closer to CISHLT, were characterized by structural modifications with lower inflammation level. We identified 15 probes significantly different between BRISHLT and CISHLT, including the gene of the muscular protein TTN. This result suggests that structural alterations precede inflammation in ACR. Linear Discriminant Analysis based on these 15 probes was able to identify the histological status of every 36 samples.Myocardial GEP is a helpful method to accurately diagnose ACR, and predicts rejection one month before its histological occurrence. These results should be considered in cardiac allograft recipients' care.

Chronic obstructive pulmonary disease (COPD) is often associated with age-related systemic abnormalities that adversely affect the prognosis. Whether these manifestations are linked to the lung alterations or are independent complications of smoking remains unclear.To look for aging-related systemic manifestations and telomere shortening in COPD patients and smokers with minor lung destruction responsible for a decline in the diffusing capacity for carbon monoxide (DLCO) corrected for alveolar volume (KCO).Cross-sectional study in 301 individuals (100 with COPD, 100 smokers without COPD, and 101 nonsmokers without COPD).Compared to control smokers, patients with COPD had higher aortic pulse-wave velocity (PWV), lower bone mineral density (BMD) and appendicular skeletal muscle mass index (ASMMI), and shorter telomere length (TL). Insulin resistance (HOMA-IR) and glomerular filtration rate (GFR) were similar between control smokers and COPD patients. Smokers did not differ from nonsmokers for any of these parameters. However, smokers with normal spirometry but low KCO had lower ASMMI values compared to those with normal KCO. Moreover, female smokers with low KCO, had lower BMD and shorter TL compared to those with normal KCO.Aging-related abnormalities in patients with COPD are also found in smokers with minor lung dysfunction manifesting as a KCO decrease. Decreased KCO might be useful, particularly among women, for identifying smokers at high risk for aging-related systemic manifestations and telomere shortening.

INTRODUCTION:In autism spectrum disorders (ASD), complex gene-environment interactions contribute to disease onset and progress. Given that gastro-intestinal dysfunctions are common in ASD, we postulated involvement of microbial dysbiosis in ASD and investigated, under a case-control design, the influence of DNA polymorphisms in the CLEC7A gene that encodes a pivotal fungal sensor, Dectin-1. MATERIAL AND METHODS:DNAs from 478 ASD patients and 351 healthy controls (HC) were analyzed for the CLEC7A rs16910631G/A and rs2078178 A/G single nucleotide polymorphisms (SNPs). Differences in the distribution of allele, genotype and haplotype by Chi-square testing and nonparametric analysis by Kruskal-Wallis/Mann-Whitney tests, where appropriate, were performed. The free statistical package R.2.13 software was used for the statistical analysis. RESULTS:We found that the CLEC7A rs2078178 G allele and GG genotype were more prevalent in HC as compared to ASD but failed to reach statistical significance for the latter (pc = 0.01, 0.06 respectively). However, after phenotype-based stratification, the CLEC7A rs2078178 G allele and GG genotype were found to be significantly more frequent in the Asperger group as compared to other ASD subsets (pc = 0.02, 0.01), a finding reinforced by haplotype analysis (rs2078178/rs16910631 G-G/G-G) (pc = 0.002). Further, intellectual quotient (IQ)-based stratification of ASD patients revealed that IQ values increase linearly along the CLEC7A rs2078178 AA, AG and GG genotypes (p = 0.05) and in a recessive manner (GG vs. AA+AG p = 0.02), further confirmed by haplotype distribution (CLEC7A rs2078178-16910631; A-G/A-G, A-G/G-G and G-G/G-G, p = 0.02, G-G/G-G vs. others, p = 0.01). CONCLUSION:Our data suggest that the genetic diversity of CLEC7A gene influences the ASD phenotype by behaving as a disease specifier and imply that the genetic control of innate immune response could determine the ASD phenotype.