Complement C5a Functions as a Master Switch for the pH Balance in Neutrophils Exerting Fundamental Immunometabolic Effects

During sepsis, excessive activation of the complement system with generation of the anaphylatoxin C5a results in profound disturbances in crucial neutrophil functions. Moreover, because neutrophil activity is highly dependent on intracellular pH (pHi), we propose a direct mechanistic link between complement activation and neutrophil pHi In this article, we demonstrate that in vitro exposure of human neutrophils to C5a significantly increased pHi by selective activation of the sodium/hydrogen exchanger. Upstream signaling of C5a-mediated intracellular alkalinization was dependent on C5aR1, intracellular calcium, protein kinase C, and calmodulin, and downstream signaling regulated the release of antibacterial myeloperoxidase and lactoferrin. Notably, the pH shift caused by C5a increased the glucose uptake and activated glycolytic flux in neutrophils, resulting in a significant release of lactate. Furthermore, C5a induced acidification of the extracellular micromilieu. In experimental murine sepsis, pHi of blood neutrophils was analogously alkalinized, which could be normalized by C5aR1 inhibition. In the clinical setting of sepsis, neutrophils from patients with septic shock likewise exhibited a significantly increased pHi These data suggest a novel role for the anaphylatoxin C5a as a master switch of the delicate pHi balance in neutrophils resulting in profound inflammatory and metabolic changes that contribute to hyperlactatemia during sepsis

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Astrocytomes intramédullaires : analyse rétrospective française multicentrique

The authors report the results of a multicenter retrospective series with a long-term follow-up and the analysis of clinical, radiological, surgical data as well as the complementary treatments in patients with intramedullary astrocytomas (IA). We performed a retrospective analysis of all the patients with IA operated on between 1984 and 2011 at 7 French centers (Kremlin-Bicêtre, Lille, Lyon, Marseille, Montpellier, Nice, and Nîmes). The minimum follow-up was 12 months. The clinical evaluation was based on the McCormick scale (MCS) results from the pre- and postoperative period. Data from 95 patients with a pathologically confirmed diagnosis of IA were considered: 54 patients were treated at the Neurosurgical Department of Kremlin-Bicêtre Hospital, 8 were treated at Lille and 33 were treated in the south region of France. The epidemiological analysis was performed on the whole cohort of patients while follow-up considerations were made solely on the 54 patients managed at Kremlin-Bicêtre Hospital to obtain homogeneous data. The average age at diagnosis was 35.6 years without significant gender difference (47 % men for 53 % women). The age at first clinical manifestation was 33.7 years. The average duration of the symptoms before the diagnosis was 22.9 months. Neuropathic pain was the principal revealing symptom (76 % of cases). The localization of IA was thoracic in 40 %, purely cervical in 28.4 %. Complete removal was achieved in 29.5 % of cases when considering the whole cohort and in 38 % of cases treated at Bicêtre Hospital. The histological distribution recorded was: grade 1 in 35 %; grade 2 in 35 %; grade 3 in 22 % and grade 4 in 8 %. During the early postoperative period (3 months) a worsening of functional capacity was observed with an increase in the frequencies of ranks 3 and 4 of MCS in 18.4 %. At 5 years follow-up, the frequencies of ranks 1 and 2 were increased. The application of a Cox model for the determination of the relative risk of death for IA grade 1 and 2 (66 patients) showed a probability of survival at 5 years of 78.6 % (CI 95 %: 68.6 %-87.6 %). Survival at 10 years is to 76.8 % (CI 95 %: 62.3 %-84.2 %). Surgery is indicated if the patient is symptomatic or the tumor increases in size. A radical excision remains the mainstay of treatment, while searching to preserve the motor function. A total resection was however only possible in 38 % of cases. A regular postoperative follow-up is compulsory and the adjuvant treatment is based on chemotherapy and radiotherapy according to the histological type

Complement C5a Alters the Membrane Potential of Neutrophils during Hemorrhagic Shock

Background. Polymorphonuclear granulocytes (PMN) play a crucial role in host defense. Physiologically, exposure of PMN to the complement activation product C5a results in a protective response against pathogens, whereas in the case of systemic inflammation, excessive C5a substantially impairs neutrophil functions. To further elucidate the inability of PMN to properly respond to C5a, this study investigates the role of the cellular membrane potential of PMN in response to C5a. Methods. Electrophysiological changes in cellular and mitochondrial membrane potential and intracellular pH of PMN from human healthy volunteers were determined by flow cytometry after exposure to C5a. Furthermore, PMN from male Bretoncelles-Meishan-Willebrand cross-bred pigs before and three hours after severe hemorrhagic shock were analyzed for their electrophysiological response. Results. PMN showed a significant dose- and time-dependent depolarization in response to C5a with a strong response after one minute. The chemotactic peptide fMLP also evoked a significant shift in the membrane potential of PMN. Acidification of the cellular microenvironment significantly enhanced depolarization of PMN. In a clinically relevant model of porcine hemorrhagic shock, the C5a-induced changes in membrane potential of PMN were markedly diminished compared to healthy littermates. Overall, these membrane potential changes may contribute to PMN dysfunction in an inflammatory environment

Early structural changes of the heart after experimental polytrauma and hemorrhagic shock

<div><p>Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction.</p></div

Histological and humoral markers of cardiac damage.

<p>(A) Plasma levels of cardiac troponin I (cTnI) as a marker of cardiac damage from animals 4 h after the insult of polytrauma and hemorrhagic shock (PTHS; n = 8) and native controls (CTRL, n = 4). Results are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. (B) Representative images of immunohistochemical (IHC) assessment of tissue cleaved caspase 3. Magnification: 100x (bar: 50 μm). (C) Representative hematoxylin and eosin stained sections of cardiac tissue, showing no signs of marked histomorphological changes. Magnifications: 100x (upper images, bar: 50 μm) and 200x (lower images, bar: 100 μm) for each group. (D) Representative images and densitometric analysis of IHC preparations of tissue high-mobility group box 1 (HMGB1) showing increased signal in samples from PTHS animals. Magnification: 100x (bar: 50 μm). For histological evaluation: n = 5 (PTHS); n = 5 (CTRL). DSR: density sum red. Experimental means were compared for statistical significance using the unpaired t-test (D) and Mann-Whitney rank sum test (A). *: p<0.05.</p

Plasma levels of inflammatory mediators and hemoglobin.

<p>Levels of (A) haemoglobin (Hb) in blood and (B) macrophage chemotactic protein 1 (MCP-1), (C) keratinocyte chemoattractant (KC) and (D) complement component C3a in plasma of animals 4 h after infliction of polytrauma and hemorrhagic shock (PTHS; n = 7 for Hb, n = 8 for MCP-1, KC and C3a) and native control animals (CTRL; n = 8 for Hb, n = 4 for MCP-1, n = 5 for C3a and KC). Results (B, C, D) are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. For statistical comparison of experimental means, unpaired t-tests (A, C, D) and Mann-Whitney rank sum test (B) were performed. *: p<0.05.</p