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Background Trauma is the leading cause of death and disability worldwide, especially in the young population. Cardiac injuries are an independent predictor for a poor overall outcome after trauma. The aim of the present study was to analyze systemic inflammation as well as local cardiac inflammation after experimental limb-, neuro- and combined trauma in mice. Methods Male C57BL/6 mice received either a closed tibia fracture (Fx), isolated traumatic brain injury (TBI) or a combination of both (Fx ​+ ​TBI). Control animals underwent sham procedure. After 6 and 24 ​h, systemic levels of inflammatory mediators were analyzed, respectively. Locally, cardiac inflammation and cardiac structural alterations were investigated in left ventricular tissue of mice 6 and 24 ​h after trauma. Results Mice showed enhanced systemic inflammation after combined trauma, which was manifested by increased levels of KC, MCP-1 and G-CSF. Locally, mice exhibited increased expression of inflammatory cytokines (IL-1β, TNF) in heart tissue, which was probably mediated via toll-like receptor (TLR) signaling. Furthermore, mice demonstrated a redistribution of connexin 43 in cardiac tissue, which appeared predominantly after combined trauma. Besides inflammation and structural cardiac alterations, expression of glucose transporter 4 (GLUT4) mRNA was increased in the heart early after TBI and after combination of TBI and limb fracture, indicating a modification of energy metabolism. Early after combination of TBI and tibia fracture, nitrosative stress was increased, manifested by elevation of nitrotyrosine in cardiac tissue. Finally, mice showed a trend of increased systemic levels of cardiac troponin I and heart-fatty acid binding protein (HFABP) after combined trauma, which was associated with a significant decrease of troponin I and HFABP mRNA expression in cardiac tissue after TBI and combination of TBI and limb fracture. Conclusion Mice exhibited early cardiac alterations as well as alterations in cardiac glucose transporter expression, indicating a modification of energy metabolism, which might be linked to increased systemic- and local cardiac inflammation after limb-, neuro- and combined trauma. These cardiac alterations might predispose individuals for secondary cardiac damage after trauma that might compromise cardiac function after TBI and long bone fracture. Translational potential statement Injuries to the head and extremities frequently occur after severe trauma. In our study, we analyzed the effects of closed tibia fracture, isolated TBI, and the combination of both injuries with regard to the development of post-traumatic secondary cardiac injuries.

Background and purpose: The aim of the study was to determine the effects of post-traumatically released High Mobility Group Box-1 protein (HMGB1) and extracellular histones on cardiomyocytes (CM). We also evaluated a therapeutic option to capture circulating histones after trauma, using a hemadsorption filter to treat CM dysfunction. Experimental Approach: We evaluated cell viability, calcium handling and mitochondrial respiration of human cardiomyocytes in the presence of HMGB-1 and extracellular histones. In a translational approach, a hemadsorption filter was applied to either directly eliminate extracellular histones or to remove them from blood samples obtained from multiple injured patients. Key results: Incubation of human CM with HMGB-1 or histones is associated with changes in calcium handling, a reduction of cell viability and a substantial reduction of the mitochondrial respiratory capacity. Filtrating plasma from injured patients with a hemadsorption filter reduces histone concentration ex vivo and in vitro, depending on dosage. Conclusion and implications: Danger associated molecular patterns such as HMGB-1 and extracellular histones impair human CM in vitro. A hemadsorption filter could be a therapeutic option to reduce high concentrations of histones., publishedVersion

Polymicrobial sepsis (after cecal ligation and puncture, CLP) causes robust complement activation with release of C5a. Many adverse events develop thereafter and will be discussed in this review article. Activation of complement system results in generation of C5a which interacts with its receptors (C5aR1, C5aR2). This leads to a series of harmful events, some of which are connected to the cardiomyopathy of sepsis, resulting in defective action potentials in cardiomyocytes (CMs), activation of the NLRP3 inflammasome in CMs and the appearance of extracellular histones, likely arising from activated neutrophils which form neutrophil extracellular traps (NETs). These events are associated with activation of mitogen-activated protein kinases (MAPKs) in CMs. The ensuing release of histones results in defective action potentials in CMs and reduced levels of [Ca(2+)]i-regulatory enzymes including sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and Na(+)/Ca(2+) exchanger (NCX) as well as Na(+)/K(+)-ATPase in CMs. There is also evidence that CLP causes release of IL-1β via activation of the NLRP3 inflammasome in CMs of septic hearts or in CMs incubated in vitro with C5a. Many of these events occur after in vivo or in vitro contact of CMs with histones. Together, these data emphasize the role of complement (C5a) and C5a receptors (C5aR1, C5aR2), as well as extracellular histones in events that lead to cardiac dysfunction of sepsis (septic cardiomyopathy).

Platelets modulate the immune system following injury by interacting with CD4+ T regulatory cells (CD4+ Tregs). The underlying mechanisms remain unsolved. We hypothesize paracrine interactions via Tumor necrosis factor-alpha (TNFα)-, Toll like receptor-4 (TLR4)-, and Interleukin-10 (IL-10). In the murine burn injury model, CD4+ Treg activation pathways were selectively addressed using TNFR2-, TLR4- and IL-10-deficient mice. The CD4+ Treg signalling molecule PKC-θ was analyzed using phospho-flow cytometry to detect rapid cell activation. Thromboelastometry (ROTEM®) was used to assess platelet activation. Injury induced significant early activation of CD4+ Tregs, disruption of TNFR2 and TLR4 activation pathways resulted in lower activity. The disruption of IL-10 crosstalk had no significant impact. Selective disruption of paracrine interactions is associated with changes in posttraumatic hemostasis parameters. TNFR2- and TLR4-dependent pathways modulate the activation of CD4+ Tregs following trauma. In contrast, we did not observe a role of IL-10 in the posttraumatic activation of CD4+ Tregs. One sentence summary TLR4- and TNFR2-dependent mechanisms, but not IL-10-dependent pathways, modulate the anti-inflammatory response of CD4+ Tregs following trauma.

Polymicrobial sepsis in mice causes myocardial dysfunction after generation of the complement anaphylatoxin, complement component 5a (C5a). C5a interacts with its receptors on cardiomyocytes (CMs), resulting in redox imbalance and cardiac dysfunction that can be functionally measured and quantitated using Doppler echocardiography. In this report we have evaluated activation of MAPKs and Akt in CMs exposed to C5a in vitro and after cecal ligation and puncture (CLP) in vivo. In both cases, C5a in vitro caused activation (phosphorylation) of MAPKs and Akt in CMs, which required availability of both C5a receptors. Using immunofluorescence technology, activation of MAPKs and Akt occurred in left ventricular (LV) CMs, requiring both C5a receptors, C5aR1 and -2. Use of a water-soluble p38 inhibitor curtailed activation in vivo of MAPKs and Akt in LV CMs as well as the appearance of cytokines and histones in plasma from CLP mice. When mouse macrophages were exposed in vitro to LPS, activation of MAPKs and Akt also occurred. The copresence of the p38 inhibitor blocked these activation responses. Finally, the presence of the p38 inhibitor in CLP mice reduced the development of cardiac dysfunction. These data suggest that polymicrobial sepsis causes cardiac dysfunction that appears to be linked to activation of MAPKs and Akt in heart.—Fattahi, F., Kalbitz, M., Malan, E. A., Abe, E., Jajou, L., Huber-Lang, M. S., Bosmann, M., Russell, M. W., Zetoune, F. S., Ward, P. A. Complement-induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction.

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.

After severe trauma, the resulting excessive inflammatory response is countered by compensatory anti‐inflammatory mechanisms. The systemic inflammatory response to trauma enhanced by inappropriately timed surgical second hits may be detrimental for the patient. On the other hand, overwhelming anti‐inflammatory mechanisms may put patients at increased risk from secondary local and systemic infections. The ensuing sepsis and organ dysfunction due to immune dysregulation remain the leading causes of death after injury. To date, there are no clinically applicable techniques to monitor the pro‐/anti‐inflammatory immune status of the patients and the remaining ability to react to microbial stimuli. Therefore, in the present study, we used a highly standardized and easy‐to‐use system to draw peripheral whole blood from polytraumatized patients (ISS ≥ 32, n = 7) and to challenge it with bacterial lipopolysaccharide. Secreted cytokines were compared with those in samples from healthy volunteers. We observed a significant decrease in the release of monocyte‐derived mediators. Surprisingly, we detected stable or even increased concentrations of cytokines related to T cell maturation and function. For clinical practicability, we reduced the incubation time before supernatants were collected. Even after an abbreviated stimulation period, a stable release of almost all analysed parameters in patient blood could be detected. In conclusion, the data are indicative of a clinically well‐applicable approach to monitor the immune status in severely injured patients in a short time. This may be used to optimize the timing of necessary surgical interventions to avoid a boost of proinflammation and reduce risk of secondary infections., publishedVersion

Background Approximately 30,000 patients with blunt cardiac trauma are recorded each year in the United States. Blunt cardiac injuries after trauma are associated with a longer hospital stay and a poor overall outcome. Organ damage after trauma is linked to increased systemic release of pro-inflammatory cytokines and damage-associated molecular patterns. However, the interplay between polytrauma and local cardiac injury is unclear. Additionally, the impact of surgical intervention on this process is currently unknown. This study aimed to determine local cardiac immunological and structural alterations after multiple trauma. Furthermore, the impact of the chosen fracture stabilization strategy (reamed versus non-reamed femoral nailing) on cardiac alterations was studied. Experimental approach 15 male pigs were either exposed to multiple trauma (blunt chest trauma, laparotomy, liver laceration, femur fracture and haemorrhagic shock) or sham conditions. Blood samples as well as cardiac tissue were analysed 4 h and 6 h after trauma. Additionally, murine HL-1 cells were exposed to a defined polytrauma-cocktail, mimicking the pro-inflammatory conditions after multiple trauma in vitro. Results After multiple trauma, cardiac structural changes were observed in the left ventricle. More specifically, alterations in the alpha-actinin and desmin protein expression were found. Cardiac structural alterations were accompanied by enhanced local nitrosative stress, increased local inflammation and elevated systemic levels of the high-mobility group box 1 protein. Furthermore, cardiac alterations were observed predominantly in pigs that were treated by non-reamed intramedullary reaming. The polytrauma-cocktail impaired the viability of HL-1 cells in vitro, which was accompanied by a release of troponin I and HFABP. Discussion Multiple trauma induced cardiac structural alterations in vivo, which might contribute to the development of early myocardial damage (EMD). This study also revealed that reamed femoral nailing (reamed) is associated with more prominent immunological cardiac alterations compared to nailing without reaming (non-reamed). This suggests that the choice of the initial fracture treatment strategy might be crucial for the overall outcome as well as for any post-traumatic cardiac consequences., PLoS ONE, 15 (6), ISSN:1932-6203

Shock : injury, inflammation, and sepsis, laboratory and clinical approaches 54(6), 761-773 (2020). doi:10.1097/SHK.0000000000001536, Published by Biomedical Press, Augusta, Ga.

Background: The complement system is part of the innate immunity, is activated immediately after trauma and is associated with adult respiratory distress syndrome, acute lung injury, multiple organ failure, and with death of multiply injured patients. The aim of the study was to investigate the complement activation in multiply injured pigs as well as its effects on the heart in vivo and in vitro. Moreover, the impact of reamed vs. non-reamed intramedullary nailing was examined with regard to the complement activation after multiple trauma in pigs. Materials and Methods: Male pigs received multiple trauma, followed by femoral nailing with/without prior conventional reaming. Systemic complement hemolytic activity (CH-50 and AH-50) as well as the local cardiac expression of C3a receptor, C5a receptors1/2, and the deposition of the fragments C3b/iC3b/C3c was determined in vivo after trauma. Human cardiomyocytes were exposed to C3a or C5a and analyzed regarding calcium signaling and mitochondrial respiration. Results: Systemic complement activation increased within 6 h after trauma and was mediated via the classical and the alternative pathway. Furthermore, complement activation correlated with invasiveness of fracture treatment. The expression of receptors for complement activation were altered locally in vivo in left ventricles. C3a and C5a acted detrimentally on human cardiomyocytes by affecting their functionality and their mitochondrial respiration in vitro. Conclusion: After multiple trauma, an early activation of the complement system is triggered, affecting the heart in vivo as well as in vitro, leading to complement-induced cardiac dysfunction. The intensity of complement activation after multiple trauma might correlate with the invasiveness of fracture treatment. Reaming of the femoral canal might contribute to an enhanced “second hit” response after trauma. Consequently, the choice of fracture treatment might imply the clinical outcome of the critically injured patients and might be therefore crucial for their survival.

The aim of this study was to define the influence of trauma on cardiac glucose and fatty acid transport. The effects were investigated in vivo in a porcine mono- and polytrauma model and in vitro in human cardiomyocytes, which were treated simultaneously with different inflammatory substances, mimicking posttraumatic inflammatory conditions.In the porcine fracture- and polytrauma model, blood glucose concentrations were measured by blood gas analysis during an observation period of 72 h. The expression of cardiac glucose and fatty acid transporters in the left ventricle was determined by RT-qPCR and immunofluorescence. Cardiac and hepatic glycogen storage was examined. Furthermore, human cardiomyocytes were exposed to a defined trauma-cocktail and the expression levels of glucose- and fatty acid transporters were determined. Early after polytrauma, hyperglycemia was observed. After 48 and 72 h, pigs with fracture- and polytrauma developed hypoglycemia. The propofol demand significantly increased posttrauma. The hepatic glycogen concentration was reduced 72 h after trauma. Cardiac glucose and fatty acid transporters changed in both trauma models in vivo as well as in vitro in human cardiomyocytes in presence of proinflammatory mediators.Monotrauma as well as polytrauma changed the cardiac energy transport by altering the expression of glucose and fatty acid transporters. In vitro data suggest that human cardiomyocytes shift to a state alike myocardial hibernation preferring glucose as primary energy source to maintain cardiac function.

There is accumulating evidence during sepsis that cardiomyocyte (CM) homeostasis is compromised, resulting in cardiac dysfunction. An important role for complement in these outcomes is now demonstrated. Addition of C5a to electrically paced CMs caused prolonged elevations of intracellular Ca2+ concentrations during diastole, together with the appearance of spontaneous Ca2+ transients. In polymicrobial sepsis in mice, we found that three key homeostasis-regulating proteins in CMs were reduced: Na+/K+-ATPase, which is vital for effective action potentials in CMs, and two intracellular Ca2+ concentration regulatory proteins, that is, sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and the Na+/Ca2+ exchanger. Sepsis caused reduced mRNA levels and reductions in protein concentrations in CMs for all three proteins. The absence of either C5a receptor mitigated sepsis-induced reductions in the three regulatory proteins. Absence of either C5a receptor (C5aR1 or C5aR2) diminished development of defective systolic and diastolic echocardiographic/Doppler parameters developing in the heart (cardiac output, left ventricular stroke volume, isovolumic relaxation, E′ septal annulus, E/E′ septal annulus, left ventricular diastolic volume). We also found in CMs from septic mice the presence of defective current densities for Ik1, l-type calcium channel, and Na+/Ca2+ exchanger. These defects were accentuated in the copresence of C5a. These data suggest complement-related mechanisms responsible for development of cardiac dysfunction during sepsis.

The incidence of cardiac injury in immediate fatalities after blunt trauma remains underestimated, and reliable diagnostic strategies are still missing. Furthermore, clinical data concerning heart-specific troponin serum levels, injury severity score (ISS), catecholamine treatment and survival of patients on admission to the hospital have rarely been interrelated so far. Therefore, the object of the present study was to identify predictive parameters for mortality in the context of blunt cardiac injury. This retrospective observational study included 173 severely injured patients with an ISS ≥25 admitted to the University Hospital of Ulm, a level 1 trauma center, during 2009–2013 . Furthermore, 83 blunt trauma victims who died before hospital admission were subjected to postmortem examination at the Institute of Legal Medicine, University of Ulm, during 2009–2014. ISS, cardiac injury and associated thoracic injuries were determined in both groups. Furthermore, in the hospitalized patients, serum troponin and IL-6 levels were measured. Macroscopic heart injury was observed in 18 % of the patients who died at the scene and only in 1 % of the patients admitted to the hospital, indicating that macroscopic heart injury is associated with an immediate life-threatening condition. Troponin levels were elevated in 43 % of the patients after admission to the hospital. Moreover, troponin serum concentrations were significantly higher in patients treated with norepinephrine (26.4 ± 4 ng/l) and in non-survivors (84.9 ± 22.8 ng/l) compared to patients without catecholamines and survivors, respectively. Macroscopic heart injury was 20 times more frequent in non-survivors than in survivors. Serum troponin levels correlated with mortality after multiple injury and therefore may represent a valuable prognostic marker in trauma patients.

Objective Severe trauma often led to a systemic inflammatory response, accompanied by release of danger associated molecular patterns such as the high mobility group box-1 protein (HMGB-1) and extracellular histones. The aim of this study was to clarify the influence of HMGB-1 and extracellular histones on human cardiomyocytes, in context of posttraumatic cardiac dysfunction. Material and Methods Cell viability, mitochondrial respiration and calcium handling of human cardiomyocytes in presence of extracellular histones or HMGB-1 were evaluated. A hemadsorption filter was tested to eliminate extracellular histones in vitro and from blood samples of multiple trauma patients (mean injury severity score >16). Results Incubation of human cardiomyocytes with HMGB-1 or histones were associated with impaired calcium handling, mitochondrial respiration and reduction of cell viability. Furthermore, human cardiomyocytes release further cardio-depressive cytokines in the presence of extracellular histones and HMGB-1 such as IL-1b and TNF. Hemadsorption of human plasma after multiple trauma showed a significant reduction of histone levels. Furthermore, in vitro analysis of hemofiltration capacity detected a dose dependant reduction of extracellular histones. Conclusion Despite their positive intention as part of the immune reaction, HMGB-1 and extracellular histones are associated with detrimental effects on cardiomyocyte viability, calcium handling and mitochondrial function. Moreover, presence of DAMPs resulted in additional release of cardiodepressive cytokines. Therefore, in the context of tissue trauma cardiomyocytes are target and actor at the same time.

Background The complement system is part of the innate immunity, is activated immediately after trauma and is associated with ARDS, MOF and with death of multiply injured patients. The aim of the study was to investigate the complement activation in multiply injured pigs as well as its effects on the heart in vivo and in vitro. Moreover, the impact of reamed vs. non-reamed intramedullary nailing was examined with regard to the complement activation. Methods Male pigs received multiple trauma (blunt chest trauma, liver laceration, femur fracture, hemorrhagic shock), followed by femoral nailing with/without prior conventional reaming. Systemic complement hemolytic activity as well as the local cardiac expression of complement receptors was determined in vivo after trauma. Human cardiomyocytes were exposed to C3a or C5a and analyzed regarding calcium signaling and mitochondrial respiration. Results Systemic complement activation increased within 6h after trauma and was mediated via the classical and the alternative pathway. The expression of receptors for complement activation were altered locally in vivoin left ventricles. C3a and C5a acted detrimentally on human cardiomyocytes in vitro. Conclusion After multiple trauma, an early activation of the complement system is triggered, affecting the heart in vivo as well as in vitro, leading to complement-induced cardiac dysfunction. The intensity of complement activation after multiple trauma correlated with the invasiveness of fracture treatment. Consequently, the choice of fracture treatment might imply the clinical outcome of the critically injured patients and might be therefore crucial for their survival.

Cardiovascular disorders (CVD) and posttraumatic stress disorder (PTSD) are highly comorbid, but the underlying mechanisms are not fully understood. Chronic psychosocial stress was induced in male mice by chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for PTSD. Cardiac structure and function were assessed on day 20 of the CSC paradigm. Following CSC, mice were kept in different sensory contact modalities to the last aggressor for 30 days, and development of cardiac function and behavioral aspects were determined. Here we show that psychosocial trauma affects heart structure by disturbing cell-to-cell integrity of cardiomyocytes, causes tachycardia, disturbance of diurnal heart rate rhythmicity and behavioral deficits in a mouse model for PTSD. Structural and functional alterations were also found in cardiomyocytes upon in vitro treatment with pro-inflammatory cytokines typically increased after psychosocial trauma. Interestingly, sensory contact to the aggressor subsequent to psychosocial trauma prohibits functional and structural heart recovery, while isolation was beneficial for cardiac but detrimental for mental health. These findings contribute to our understanding of potential mechanisms underlying the high comorbidity of CVD and PTSD.

Polytrauma (PT) is a life-threatening disease and a major global burden of injury. Mesenchymal stromal cells (MSC) might be a therapeutic option for PT patients due to their anti-inflammatory and regenerative potential. We hypothesised that the inflammatory response of MSC is similar after exposure to selected trauma-relevant factors to sera from PT patients (PTS). Therefore, we investigated the effects of a mixture of defined factors, supposed to play a role on MSC in the early phase of PT. Additionally, in a translational approach we investigated the effect of serum from PT patients on MSC in vitro. MSC were incubated with a PT cocktail in physiological (PTCL) and supra-physiological (PTCH) concentrations or PTS. The effect on gene expression and protein secretion of MSC was analysed by RNA sequencing, ELISA and Multiplex assays of cell culture supernatant. Stimulation of MSC with PTCH, PTCL or IL1B led to significant up- or downregulation of 470, 183 and 469 genes compared to unstimulated MSC at 6 h. The intersection of differentially expressed genes in these groups was very high (92% overlap with regard to the PTCL group; treated for 6 h). Cytokine secretion profile of MSC revealed that IL1B mimics the effect of a more complex PT cocktail as well. However, there was only a minor proportion of overlapping differentially expressed genes between the MSC group stimulated with early times of PTS and the MSC group stimulated with PTCH, PTCL and IL1B. In conclusion, the effect of sera from PT patients on MSC activation cannot be simulated by the chosen trauma-relevant factors. Furthermore, we conclude that while IL1B might be useful to prime MSC prior to therapeutic application, it might not be as useful for the in vitro study of functional properties of MSC in the context of PT.

ObjectiveAsphyxia of newborns is a severe and frequent challenge of the peri- and postnatal period. The purpose of this study was to study early morphological, immunological and structural alterations in lung tissue after asphyxia and hemorrhage (AH).Methods44 neonatal piglets (age 32 hrs) underwent asphyxia and hemorrhage (AH) and were treated according to the international liaison committee of resuscitation (ILCOR) guidelines. For this study, 15 piglets (blood transfusion (RBC) n = 9; NaCl n = 6, mean age 31 hrs) were randomly picked. 4 hours after ROSC (return of spontaneous circulation), lung tissue and blood samples were collected.ResultsAn elevation of myeloperoxidase (MPO) activity was observed 4 hrs after AH accompanied by an increase of surfactant D after RBC treatment. After AH tight junction proteins Claudin 18 and junctional adhesion molecule 1 (JAM1) were down-regulated, whereas Occludin was increased. Furthermore, after AH and RBC treatment dephosphorylated active form of Connexin 43 was increased.ConclusionsAH in neonatal pigs is associated with early lung injury, inflammation and alterations of tight junctions (Claudin, Occludin, JAM-1) and gap junctions (Connexin 43) in lung tissue, which contributes to the development of lung edema and impaired function.

Background: Evidence for recommendations on the use of volume expansion during cardiopulmonary resuscitation in newborn infants is limited. Objectives: To develop a newborn piglet model with asphyxia, hemorrhage, and cardiac arrest to test different volume resuscitation on return of spontaneous circulation (ROSC). We hypothesized that immediate red cell transfusion reduces time to ROSC as compared to the use of an isotonic crystalloid fluid. Methods: Forty-four anaesthetized and intubated newborn piglets [age 32 h (12–44 h), weight 1,220 g (1,060–1,495g), Median (IQR)] were exposed to hypoxia and blood loss until asystole occurred. At this point they were randomized into two groups: (1) Crystalloid group: receiving isotonic sodium chloride (n = 22). (2) Early transfusion group: receiving blood transfusion (n = 22). In all other ways the piglets were resuscitated according to ILCOR 2015 guidelines [including respiratory support, chest compressions (CC) and epinephrine use]. One hour after ROSC piglets from the crystalloid group were randomized in two sub-groups: late blood transfusion and infusion of isotonic sodium chloride to investigate the effects of a late transfusion on hemodynamic parameters. Results: All animals achieved ROSC. Comparing the crystalloid to early blood transfusion group blood loss was 30.7 ml/kg (22.3–39.6 ml/kg) vs. 34.6 ml/kg (25.2–44.7 ml/kg), Median (IQR). Eleven subjects did not receive volume expansion as ROSC occurred rapidly. Thirty-three animals received volume expansion (16 vs. 17 in the crystalloid vs. early transfusion group). 14.1% vs. 10.5% of previously extracted blood volume in the crystalloid vs. early transfusion group was infused before ROSC. There was no significant difference in time to ROSC between groups [crystalloid group: 164 s (129–198 s), early transfusion group: 163 s (162–199 s), Median (IQR)] with no difference in epinephrine use. Conclusions: Early blood transfusion compared to crystalloid did not reduce time to ROSC, although our model included only a moderate degree of hemorrhage and ROSC occurred early in 11 subjects before any volume resuscitation occurred.

Postmenopausal females display a chronic inflammatory phenotype with higher levels of circulating pro-inflammatory cytokines. Furthermore, the inflammatory response to injury may be altered under estrogen-deficiency, because it was shown previously that estrogen-deficient mice displayed increased levels of the inflammatory cytokines Midkine (Mdk) and Interleukin-6 (IL-6) in the early fracture hematoma. Because a balanced immune response to fracture is required for successful bone regeneration, this might contribute to the delayed fracture healing frequently observed in osteoporotic, postmenopausal fracture patients. In this study, we aimed to investigate whether further cytokines in addition to Mdk and IL-6 might be affected by estrogen-deficiency after fracture in mice and whether these cytokines are also relevant during human fracture healing. Additionally, we aimed to investigate whether serum from male vs. female fracture patients affects osteogenic differentiation of human mesenchymal stem cells (MSCs). To address these questions, female mice were either sham-operated or ovariectomized (OVX) and subjected to standardized femur osteotomy. A broad panel of pro- and anti-inflammatory cytokines was determined systemically and locally in the fracture hematoma. In a translational approach, serum was collected from healthy controls and patients with an isolated fracture. Mdk and IL-6 serum levels were determined at day 0, day 14 and day 42 after fracture. Subgroup analysis was performed to investigate differences between male and female fracture patients after menopause. In an in vitro approach, human MSCs were cultured with the collected patient serum and osteogenic differentiation was assessed by qPCR and alkaline-phosphatase staining. Our results suggest an important role for the pro-inflammatory cytokines Mdk and IL-6 in the response to fracture in estrogen-deficient mice among all of the measured inflammatory mediators. Notably, both cytokines were also significantly increased in the serum of patients after fracture. However, only Mdk serum levels differed significantly between male and female fracture patients after menopause. MSCs cultivated with serum from female fracture patients displayed significantly reduced osteogenic differentiation, which was attenuated by Mdk-antibody treatment. In conclusion, our study demonstrated increased Mdk levels after fracture in OVX mice and female fracture patients after menopause. Because Mdk is a negative regulator of bone formation, this might contribute to impaired osteoporotic fracture healing., publishedVersion

ORTHOUNION is a multicentre, open, comparative, three-arm, randomized clinical trial (EudraCT number 2015-000431-32) to compare the efficacy, at one and two years, of autologous human bone marrow-derived expanded mesenchymal stromal cell (hBM-MSC) treatments versus iliac crest autograft (ICA) to enhance bone healing in patients with diaphyseal and/or metaphysodiaphyseal fracture (femur, tibia, and humerus) status of atrophic or oligotrophic nonunion (more than 9 months after the acute fracture, including recalcitrant cases after failed treatments). The primary objective is to determine if the treatment with hBM-MSCs combined with biomaterial is superior to ICA in obtaining bone healing. If confirmed, a secondary objective is set to determine if the dose of 100 × 106 hBM-MSCs is noninferior to that of 200 × 106 hBM-MSCs. The participants (n=108) will be randomly assigned to either the experimental low dose (n=36), the experimental high dose (n=36), or the comparator arm (n=36) using a central randomization service. The trial will be conducted in 20 clinical centres in Spain, France, Germany, and Italy under the same clinical protocol. The confirmation of superiority for the proposed ATMP in nonunions may foster the future of bone regenerative medicine in this indication. On the contrary, absence of superiority may underline its limitations in clinical use.

Osteosarcoma is the leading primary bone cancer in young adults and exhibits high chemoresistance rates. Therefore, characterization of both alternative treatment options and the underlying mechanisms is essential. Simvastatin, a cholesterol-lowering drug, has among its pleiotropic effects anticancer potential. Characterizing this potential and the underlying mechanisms in osteosarcoma is the subject of the present study. Human osteosarcoma cells (SaOS-2 and U2OS) were treated with simvastatin (4-66 µM) for 48 or 72 h. The effects of downstream substrate mevalonate (MA) or substrates for isoprenylation farnesyl pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) were evaluated using add-back experiments. Tumour growth using MTT assay, apoptosis, cell cycle and signalling cascades involved in simvastatin-induced manipulation were analysed. The results revealed that simvastatin dose-dependently inhibited cell growth. Simvastatin significantly induced apoptosis, increased the Bax/Bcl-2 ratio, and cleavage of caspase-3 and PARP protein. Simvastatin impaired cell cycle progression as shown by significantly increased percentages of cells in the G0/G1 phase and lower percentages of cells in the S phase. Gene expression levels of cell cycle-regulating genes (TP53, CDKN1A and CDK1) were markedly altered. These effects were not completely abolished by FPP, but were reversed by MA and GGPP. JNK and c-Jun phosphorylation was enhanced after simvastatin treatment, while those were abolished when either MA or GGPP were added. In conclusion, simvastatin acts primarily by reducing prenylation to induce apoptosis and reduce osteosarcoma cell growth. Particularly enhanced activation of c-Jun seems to play a pivotal role in osteosarcoma cell death.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

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.

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger-associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1β and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1β, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Ab-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K+, increased intracellular Ca2+ concentration, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1β levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models, suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug.

Sepsis, both in humans and in rodents, is associated with persistent immunosuppression accompanied by defects in innate immunity during the acute phase of sepsis. Mice were rendered septic by cecal ligation and puncture (CLP) followed by the induction of acute lung injury, employing distal airway deposition of IgG immune complexes, in order to quantitatively evaluate innate immune responses following the induction of sepsis. Suppression of innate immune responses in the lung occurred as early as 12 h after CLP and up to 21 days thereafter. The mechanism of innate immune defects included a reduced leak of albumin into the lungs together with reduced levels of tumor necrosis factor in bronchoalveolar lavage fluids and increased levels of interleukin-10 that were persistent. Bone marrow-derived neutrophils (polymorphonuclear neutrophils; PMNs) from CLP mice also had reduced levels of the activation marker CD11b and a depressed respiratory burst following stimulation in vitro. These results were not observed in mice with endotoxemia, where the innate inflammatory response was preserved. However, sustained lymphopenia was present in both models, suggesting differential regulation of innate and adaptive immunity in the two sepsis models. These data indicate that CLP induced a prolonged suppression of inflammatory responses both in the lung and systemically, as defined by bone marrow-derived PMN dysfunction.

Objective Severe blunt chest trauma in humans is associated with high mortality rates. Whereas lung tissue damage and lung inflammation after blunt chest trauma have extensively been investigated, the traumatic and posttraumatic effects on the heart remain poorly understood. Therefore, the purpose of this study was to define cardiac injury patterns in an experimental blunt chest trauma model in rats. Methods Experimental blunt chest trauma was induced by a blast wave in rats, with subsequent analysis of its effects on the heart. The animals were subjected either to a sham or trauma procedure. Systemic markers for cardiac injury were determined after 24 h and 5 days. Postmortem analysis of heart tissue addressed structural injury and inflammation 24 h and 5 days after trauma. Results Plasma levels of extracellular histones were elevated 24 h and 5 days after blunt chest trauma compared to sham-treated animals. In the heart, up-regulation of interleukin-1β 24 h after trauma and increased myeloperoxidase activity 24 h and 5 days after trauma were accompanied by reduced complement C5a receptor-1 expression 24 h after trauma. Histological analysis revealed extravasation of erythrocytes and immunohistochemical analysis alteration of the pattern of the gap-junction protein connexin 43. Furthermore, a slight reduction of α-actinin and desmin expression in cardiac tissue was found after trauma together with a minor increase in sarcoplasmatic/endoplasmatic reticlulum calcium-ATPase (SERCA) expression. Conclusions The clinically highly relevant rat model of blast wave-induced blunt chest trauma is associated with cardiac inflammation and structural alterations in cardiac tissue.

Background Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury. Methods 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak's multiple comparison test (significance, p≤ 0.05). Results In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt. Conclusions In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent.

Background Reversible, depressed cardiac function is frequently encountered during septic shock and commonly called septic cardiomyopathy. Previous studies demonstrated reduced ejection fraction and left ventricular dilatation in both humans and animal models. However, the majority of the studies in humans excluded pre-existing cardiac disease and animal studies were performed on healthy specimen and/or without vasopressor support during sepsis. In order to more closely mimic the actual patients’ conditions on intensive care units and to assess the influence of both cardiac comorbidity and vasopressor support on septic cardiomyopathy, we evaluated the left ventricular function in a porcine model of resuscitated septic shock with pre-existing atherosclerosis. Methods Hypercholesterolaemic, atherosclerotic pigs due to homozygous low-density lipoprotein receptor mutation and high-fat diet were anaesthetised and surgically instrumented. Faecal peritonitis was induced by inoculation of autologous faeces into the peritoneal cavity in n = 8 animals; n = 5 pigs underwent sham procedure. Sepsis resuscitation included administration of fluids and noradrenaline. Left ventricular function was analysed via pressure-conductance catheters before, 12 and 24 h after the induction of sepsis. Results The main findings were impaired ventricular dilatation (no significant change in the left ventricular end-diastolic volume) and unchanged ejection fraction in septic pigs with pre-existing atherosclerosis. The relaxation time constant τ decreased while dp/dtmax increased. Cardiac nitrotyrosine formation increased while expression of the endogenous hydrogen sulphide (H2S)-producing enzyme cystathionine γ-lyase (CSE) decreased. Conclusions The data of the present study are in conflict with previously published data from healthy animal models, most likely as a result of ongoing resuscitation including noradrenaline treatment or intrinsic pathophysiologic processes of the pre-existing atherosclerosis. Moreover, increased nitrotyrosine formation and decreased expression of CSE suggest the implication of augmented oxidative/nitrosative stress and/or reduced bioavailability of nitric oxide as well as diminished endogenous H2S release in the pathophysiology of septic cardiomyopathy. Electronic supplementary material The online version of this article (doi:10.1186/s40635-016-0089-y) contains supplementary material, which is available to authorized users.

Christian Ehrnthaller,1 Umme Amara,1 Sebastian Weckbach,1 Miriam Kalbitz,1 Markus Huber-Lang,1 Soheyl Bahrami21Department of Traumatology, Hand, Plastic, and Reconstructive Surgery, Center of Surgery, University of Ulm, Germany; 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, AustriaAbstract: Complement activation is involved in various diseases in which innate immunity plays a crucial role. However, its pathophysiological relevance is not clearly understood. Experimental models have been widely used to characterize the role of complement activation under different pathological conditions, such as hypoxemia, ischemia and reperfusion, tissue damage, and polymicrobial invasion. Screening of the complement status and function is, however, strongly dependent on the laboratory-specific techniques being used to sample and measure complement, making it difficult to compare the results found in different laboratories. Therefore, we evaluated complement function by measuring complement hemolytic activity (CH50) in various animal models of isolated ischemia reperfusion (I/R: kidney, liver, gut), hemorrhagic traumatic shock (HTS), endotoxic shock (LPS), and sepsis (CLP). Complement activation was less pronounced in isolated models of ischemia and reperfusion, whereas a strong complement response was observed early after HTS, CLP, and LPS. In summary, CH50 is a well-established, quick, and cost-effective screening method of complement function. However, because we obtained different results in clinically relevant animal models, further differentiation using specific complement factor analysis is necessary.Keywords: CH50, complement, hemorrhagic shock, inflammation, ischemia/reperfusion, sepsis

In response to severe tissue trauma several danger sensing and signalling cascades are activated, including the complement and the apoptosis systems. In polytrauma patients, both the early activation of the complement cascade with an excessive generation of the potent anaphylatoxin C5a and the induction of apoptosis have been shown to modulate the post-traumatic immune response. However, little is known about a direct interaction between the complement and apoptosis systems after severe tissue trauma. Therefore the focus of the present study was to elucidate the interplay between the central complement component C5 and the pro-apoptotic aspartic protease cathepsin D. In vivo, the cathepsin D plasma concentration of multiple injured patients was markedly increased when compared to healthy volunteers. In vitro incubation of C5 with cathepsin D resulted in a concentration- and time-dependent generation of C5a, which was inhibited by the aspartate protease inhibitor pepstatin A. Immunoblotting and sequencing analysis indicated that the C5 cleavage product represents the native form of human C5a, also exhibiting chemotactic activity for human neutrophils. In conclusion, these data show for the first time that cathepsin D is increased in plasma early after severe tissue injury. Furthermore, the results provide in vitro evidence of cleavage of C5 by an aspartic protease with subsequent generation of functional C5a, which represents a new path of complement activation.

Hintergrund Die Objective-structured-clinical-exam- (OSCE-)Methode ist eine etablierte Prufungsform, die theoretische und praktische Fahigkeiten uberpruft. Im Fach Chirurgie und Orthopadie wurde die OSCE bisher nur unzureichend evaluiert, insbesondere nicht durch die studentischen Testteilnehmer. Daher wurde in der vorliegenden Studie eine studentische OSCE-Evaluation durchgefuhrt.

As a crucial element of innate immunity, the complement cascade becomes activated after severe trauma. Regulation of the complement cascade and protection against complement-mediated tissue destruction is provided by a selection of soluble and membrane-bound complement regulatory proteins (CRegs). To date, the leukocyte expression profile of CRegs in multiple injured patients is unknown. In the present study, expression of CRegs and the C5a receptor (CD88) was analyzed on neutrophils, monocytes, and lymphocytes by flow cytometry. Whole blood samples were obtained from healthy volunteers (n = 16) or multiple injured patients (n = 12) on admission in the emergency department and 4, 12, 24, 120, and 240 h after trauma. The content of CRegs and CD88 on leukocytes was significantly altered posttrauma: CD55 (decay accelerating factor) displayed a time-dependent, elevated expression pattern on neutrophils and monocytes, but not on lymphocytes. CD59 (membrane attack complex inhibitor) expression was significantly increased on neutrophils and monocytes at the time of admission and after 5 to 10 days in lymphocytes. CD46 (membrane cofactor protein) was significantly down-regulated in all three cell types posttrauma. CD35 (complement receptor 1) expression on neutrophils was initially decreased, whereas monocytes presented a significant increase in CD35 expression. CD35 on lymphocyte remained unchanged throughout the observation period. CD88 expression was considerably reduced on leukocytes between 0 and 240 h after injury. CD59, CD46, and CD88 expression values on neutrophils reversely correlated with severity of injury. In summary, expression profiles of CRegs and CD88 on leukocytes are specifically altered after polytrauma in humans, indicating a trauma-induced "complementopathy."

INTRODUCTION Heart injury represents an independent predictor for a poor outcome after trauma. However, molecular mechanisms of depressed cardiac function after trauma remains elusive. METHODS Multiple trauma (PTHS) included a blast-wave induced blunt chest trauma, traumatic brain injury and closed femoral fracture as well as pressure controlled hemorrhagic shock. In vitro studies were performed using human cardiomyocytes (Cellular Dynamics). Data were analyzed by one-way ANOVA analysis of variance followed by Dunnett’s multiple comparison test. RESULTS 4h after PTHS in mice plasma concentration of pro-inflammatory cytokines such as MCP-1 and chemokine KC were significantly increased in comparison to sham treated animals. Plasma concentration of cardiac troponin levels were significantly increased early after experimental PTHS. HMGB1 staining provided significant increase in left ventricular tissue after PTHS. Distribution of the gap junction protein connexin 43 (Cx43) was was disintegrated in histological sections. In presence of TNF reactive oxygen species were significantly increased in human cardiomyocytes, indicating a redox imbalance indicative of an oxidative state. Furthermore, presence of TNF in cultured human cardiomyocytes was associated with increased expression of the NLRP3 inflammasome and increased concentration of troponin in supernatant, which is specific marker for cardiac cell damage. DISCUSSION The cellular and subcellular events demonstrated in this report may explain molecular mechanisms associated with cardiac dysfunction after trauma. Better understanding of cardiac damage and molecular mechanisms of cardiac dysfunction after trauma will improve patient care in future.

Scientific reports 6, 39659 (2016). doi:10.1038/srep39659, Published by Nature Publishing Group, London

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca2+]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains–containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca2+]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.—Kalbitz, M., Grailer, J. J., Fattahi, F., Jajou, L., Herron, T. J., Campbell, K. F., Zetoune, F. S., Bosmann, M., Sarma, J. V., Huber-Lang, M., Gebhard, F., Loaiza, R., Valdivia, H. H., Jalife, J., Russell, M. W., Ward, P. A. Role of extracellular histones in the cardiomyopathy of sepsis.

The paper “Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice” by Anja C. Bloom et al. in Bone 2016 (1) describes for the first time an increased femur length and cortical bone volume (with reduced mineral density) in male mice deficient in CD59a. It appears that the role of CD59a, which inhibits the assembly of the membrane attack complex (MAC) and thereby the terminal complement activation pathway (2), was rather underestimated in the past regarding bone tissue development and function.

In response to severe tissue trauma, several "molecular danger" sensing and signaling pathways are activated, especially the complement and the apoptosis cascade. Although possible crossroads between both systems have been proposed, little is known about the underlying molecular interactions. In this study a new interaction interface is presented for C3a and C5a generation by the pro-apoptotic factor granzyme B. In vitro incubation of the central human complement components C3 and C5 with the serine protease granzyme B resulted in a concentration-dependent production of the anaphylatoxins C3a and C5a. The so generated anaphylatoxin C5a was chemotactic active for isolated human neutrophils. In a translational approach, intracellular granzyme B concentration in leukocytes was determined early after severe tissue trauma. In comparison to healthy volunteers, multiple injured patients (less than one hour after trauma, Injury Severity Score 18, n = 5) presented a significant increase in granzmye B levels in neutrophils and lymphocytes. Thus, tissue trauma is associated with early activation of both, the complement and apoptosis system. The present data suggest a new form of interaction between the complement and the apoptosis system on the level of granzyme B that is capable to generate C3a and C5a independently of the established complement proteases.

In response to severe tissue trauma, several “molecular danger” sensing and signaling pathways are activated, especially the complement and the apoptosis cascade. Although possible crossroads between both systems have been proposed, little is known about the underlying molecular interactions. In this study a new interaction interface is presented for C3a and C5a generation by the pro-apoptotic factor granzyme B. In vitro incubation of the central human complement components C3 and C5 with the serine protease granzyme B resulted in a concentration-dependent production of the anaphylatoxins C3a and C5a. The so generated anaphylatoxin C5a was chemotactic active for isolated human neutrophils. In a translational approach, intracellular granzyme B concentration in leukocytes was determined early after severe tissue trauma. In comparison to healthy volunteers, multiple injured patients (less than one hour after trauma, Injury Severity Score > 18, n = 5) presented a significant increase in granzmye B levels in neutrophils and lymphocytes. Thus, tissue trauma is associated with early activation of both, the complement and apoptosis system. The present data suggest a new form of interaction between the complement and the apoptosis system on the level of granzyme B that is capable to generate C3a and C5a independently of the established complement proteases.

Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa–antithrombin (AT) and FXIa–AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa–C1INH and KK–C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa–AT, FXIa–AT, and kallikrein (KK)–AT but almost no C1INH complexes. In severe trauma patients, FXIIa–AT and FXIa–AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa–C1INH complexes were detected when the FXIIa–AT levels were low. No correlations were found between FXIIa–C1INH and FXIIa–AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa–AT and FXIIa–C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa–AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.

Reversible, depressed cardiac function is frequently encountered during septic shock and commonly called septic cardiomyopathy. Previous studies demonstrated reduced ejection fraction and left ventricular dilatation in both humans and animal models. However, the majority of the studies in humans excluded pre-existing cardiac disease and animal studies were performed on healthy specimen and/or without vasopressor support during sepsis. In order to more closely mimic the actual patients' conditions on intensive care units and to assess the influence of both cardiac comorbidity and vasopressor support on septic cardiomyopathy, we evaluated the left ventricular function in a porcine model of resuscitated septic shock with pre-existing atherosclerosis.Hypercholesterolaemic, atherosclerotic pigs due to homozygous low-density lipoprotein receptor mutation and high-fat diet were anaesthetised and surgically instrumented. Faecal peritonitis was induced by inoculation of autologous faeces into the peritoneal cavity in n = 8 animals; n = 5 pigs underwent sham procedure. Sepsis resuscitation included administration of fluids and noradrenaline. Left ventricular function was analysed via pressure-conductance catheters before, 12 and 24 h after the induction of sepsis.The main findings were impaired ventricular dilatation (no significant change in the left ventricular end-diastolic volume) and unchanged ejection fraction in septic pigs with pre-existing atherosclerosis. The relaxation time constant τ decreased while dp/dtmax increased. Cardiac nitrotyrosine formation increased while expression of the endogenous hydrogen sulphide (H2S)-producing enzyme cystathionine γ-lyase (CSE) decreased.The data of the present study are in conflict with previously published data from healthy animal models, most likely as a result of ongoing resuscitation including noradrenaline treatment or intrinsic pathophysiologic processes of the pre-existing atherosclerosis. Moreover, increased nitrotyrosine formation and decreased expression of CSE suggest the implication of augmented oxidative/nitrosative stress and/or reduced bioavailability of nitric oxide as well as diminished endogenous H2S release in the pathophysiology of septic cardiomyopathy.