[show abstract][hide abstract] ABSTRACT: Complement factor C5a is a potent pro-inflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (L-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex. In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multi-organ failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture, NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.Molecular Therapy (2013); doi:10.1038/mt.2013.178.
[show abstract][hide abstract] ABSTRACT: Oral examinations have been a crucial format in ancient and modern assessment to evaluate and guarantee quality of medical education and thereby to secure patient safety. To achieve a high level of quality in the oral part of the final examination of medical students, a training program for oral examiners at the Medical Faculty of Ulm (Germany) has been established since 2007.However, little is known about the attitude of the examiners in regard to the impact of this training program and of oral examinations as instruments to ensure patient safety.
All 367 academic clinicians from operative and non-operative disciplines, attending the one-day examiner training program at the University of Ulm between 2007 and 2012 have been asked to answer an online survey (EvaSys 5.0). Focus of the survey was to find out in which respect the examiners profited from the trainings, if the training effects were discipline-dependent, and to which degree the oral examinations could contribute to patient safety. Statistical analysis was performed using the t-test for independent samples. Results were considered statistically significant when p < 0.05.
A total of 63 participants answered the survey, but in 4 cases the questionnaire was not fully completed (with single items missing). More than half of the study participants (n = 34/59; 58%) have experienced (at least sometimes or rarely) candidates that they deemed incompetent and perhaps even dangerous to the patients' health who nevertheless passed the oral exam successfully. The majority of participants were convinced that oral examinations using concrete clinical cases could significantly contribute to patient safety, if grading is based on clear criteria and if examinations as well as grading are performed more critically. The impact of the training program was rated significantly stronger by surgeons than by non-surgeons in several categories. These categories included "strengths and weaknesses of oral examinations", "reliability", "validity", "competence in grading", "critical grading", and "departmental improvements" concerning oral examinations.
In respect to patient safety, it seems crucial to prevent incompetent candidates from passing the oral examination. The present study indicates the importance to continue and to develop our examiner trainings, with main emphasis on concrete clinical problems and a criteria-based critical grading system for oral examinations. Since the impact of the training was particularly high for colleagues from the operative disciplines, the training program should be offered especially in surgical departments.
[show abstract][hide abstract] ABSTRACT: The complement system, as part of innate immunity, is activated immediately after trauma in response to various pathogen- and danger-associated molecular patterns (PAMPs and DAMPs), and helps to eliminate microorganisms and damaged cells. However, recent data indicate an extended role of complement far beyond pure "killing", which includes regulation of the cytokine/chemokine network, influencing physiological barriers, interaction with the coagulation cascade, and even involvement with bone metabolism and repair. Complement-induced hyper-activation and dysfunction reveal the dark side of this system, leading to complications such as sepsis, multiple-organ dysfunction, delayed fracture healing, and unfavorable outcome. Thus, the present review focuses on less known regulatory roles of the complement system after trauma and during fracture healing, rather than on its bacterial and cellular "killing functions". In particular, various complement crosstalks after trauma, including the coagulation cascade and apoptosis system, appear to be crucially involved early after trauma. Long-term effects of complement on tissue regeneration after fracture and bone turnover are also considered, providing new insights into innate immunity in local and systemic complement-driven effects after trauma.
Seminars in Immunology 06/2013; 25(1):73-78. · 5.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 (preHP2) has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes (IgGIC). A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide (LPS) model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for tight junction proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 kDa and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist, AT-1002, intensified ALI. Zonulin both in vitro and in vivo induced generation of C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.
[show abstract][hide abstract] ABSTRACT: During experimental sepsis, excessive generation of the anaphylatoxin C5a results in reduction of the C5a receptor (C5aR) on neutrophils. These events have been shown to result in impaired innate immunity. However, the regulation and fate of C5aR on neutrophils during sepsis are largely unknown. In contrast to 30 healthy volunteers, 60 patients in septic shock presented evidence of complement activation with significantly increased serum levels of C3a, C5a, and C5b-9. In the septic shock group, the corresponding decrease in complement hemolytic activity distinguished survivors from nonsurvivors. Neutrophils from patients in septic shock exhibited decreased C5aR expression, which inversely correlated with serum concentrations of C-reactive protein (CRP) and clinical outcome. In vitro exposure of normal neutrophils to native pentameric CRP led to a dose- and time-dependent loss of C5aR expression on neutrophils, whereas the monomeric form of CRP, as well as various other inflammatory mediators, failed to significantly alter C5aR levels on neutrophils. A circulating form of C5aR (cC5aR) was detected in serum by immunoblotting and a flow-based capture assay, suggestive of an intact C5aR molecule. Levels of cC5aR were significantly enhanced during septic shock, with serum levels directly correlating with lethality. The data suggest that septic shock in humans is associated with extensive complement activation, CRP-dependent loss of C5aR on neutrophils, and appearance of cC5aR in serum, which correlated with a poor outcome. Therefore, cC5aR may represent a new sepsis marker to be considered in tailoring individualized immune-modulating therapy.
The Journal of Immunology 03/2013; · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Direct acute lung injury (ALI) is still associated with a high mortality, whereas the underlying pathomechanisms are not yet fully understood. In this regard, epithelial cell death in the lungs has been attributed an important role in the pathogenesis of this clinical entity. Based on this background here, we hypothesized that signaling through Fas and tumor necrosis factor receptor 1 (TNFR-1) is involved in mediating apoptosis and inflammation in chest trauma induced septic ALI.
Male C57BL/6 mice (wild-type [WT]), male mutant mice expressing nonfunctional Fas receptor (B6.MRL-Faslpr/J [lpr]) (lpr) and male TNFR-1-deficient mice (TNFR-1) were subjected to a model of direct ALI consisting of blunt chest trauma followed by cecal ligation and puncture.Cytokine/chemokine concentrations of plasma, bronchoalveolar lavage (BAL) fluids, and lung tissue were investigated as well as BAL protein and lung myeloperoxidase. Lung histology was assessed; lung caspase 3, TUNEL-positive cells, and apoptotic polymorphonuclear neutrophil were measured, followed by a survival study.
Cytokine/chemokine levels in plasma, BAL, and lung tissue were markedly increased in WT animals following ALI, whereas lpr and TNFR-1 showed significantly decreased levels. BAL protein levels were substantially elevated following ALI, but lpr animals presented markedly diminished protein levels compared with WT and TNFR-1animals. Lung myeloperoxidase level was only increased 12 hours after ALI in WT animals, whereas lung myeloperoxidase levels in lpr and TNFR-1 animals were not increased compared with sham. Lung histology revealed beneficial effects in lpr and TNFR-1. Lung active caspase 3 after ALI was substantially decreased in lpr and TNFR-1 mice compared with WT. Interestingly, an early but not persisting survival benefit was observed in lpr and TNFR-1 animals.
Pathomechanistically, Fas and TNFR-1 signaling contributed to the apoptotic and inflammatory response in a clinically relevant double-hit model of trauma-induced septic ALI. Moreover, this was associated with a temporary survival benefit.
The journal of trauma and acute care surgery. 03/2013; 74(3):792-800.
[show abstract][hide abstract] ABSTRACT: We recently demonstrated that a blunt chest trauma, a strong inducer of the posttraumatic systemic inflammatory response and one of the most critical injuries in polytrauma patients, significantly delayed fracture healing in rats, possibly by the interaction of the systemic inflammation with early regeneration processes locally at the fracture site. The underlying cellular mechanisms, however, have as yet remained unknown. Therefore, the aim of this study was to analyze the cellular and morphologic composition of the early fracture callus after a blunt chest trauma.
Rats received an osteotomy of the right femur stabilized by an external fixator in combination with a blunt chest trauma or not. The animals were killed after 3, 7, and 35 days, and the fracture calli were analyzed histologically for new tissue formation, polymorphonuclear leucocytes, macrophages, osteoclasts, and the presence of the proinflammatory cytokine interleukin 6.
The blunt chest trauma considerably increased the number of polymorphonuclear leucocytes in the callus by Day 3 compared with animals with isolated fractures. The number of macrophages was significantly reduced by the thoracic trauma at Days 3 and 7. The number of osteoclasts was not changed at any postoperative time point. After 3 days, the blunt chest trauma led to a significantly stronger interleukin 6 staining within the periosteal callus in zones of intramembranous ossification. During the time of cortical bridging at Day 35, the amount of newly formed bone was significantly decreased after blunt chest trauma.
Our results suggest that the systemic posttraumatic inflammation induced by a thoracic trauma disturbed the inflammatory balance during the early healing stage by altering the recruitment of inflammatory cells and cytokine expression locally at the fracture site and thus impaired fracture healing. These findings provide new insights in the pathomechanisms of impaired fracture healing in patients experiencing severe trauma.
The journal of trauma and acute care surgery. 02/2013; 74(2):531-7.
[show abstract][hide abstract] ABSTRACT: The exact alterations of the immune system after polytrauma leading to sepsis and multiple-organ failure are poorly understood. Thus, the early local and systemic inflammatory and apoptotic response was characterized in a new polytrauma model and compared with the alterations seen after single or combined injuries.
Anesthetized C57BL/6 mice were subjected to either blunt bilateral chest trauma (Tx), closed head injury, right femur fracture including contralateral soft tissue injury, or a combination of injuries (PTx). After 2 hours or 6 hours, animals were sacrificed, and the systemic as well as the local pulmonary immune response (bronchoalveolar lavage [BAL]/plasma cytokines, lung myeloperoxidase [MPO] activity, and alveolocapillary barrier dysfunction) were evaluated along with lung/brain apoptosis (lung caspase 3 Western blotting, immunohistochemistry, and polymorphonuclear leukocytes [PMN] Annexin V).
Hemoglobin, PO2 saturation, and pH did not differ between the experimental groups. Local BAL cytokines/chemokines were significantly increased in almost all groups, which included Tx. There was no further enhancement of this local inflammatory response in the lungs in case of PTx. At 2 hours, all groups except sham and closed head injury alone revealed an increased activity of lung MPO. However, 6 hours after injury, lung MPO remained increased only in the PTx group. Increased BAL protein levels were found, reflecting enhanced lung leakage in all groups with Tx 6 hours after trauma. Only after PTx was neutrophil apoptosis significantly decreased, whereas lung caspase 3 and plasma interleukin 6/keratinocyte chemoattractant (KC) were substantially increased.
The combination of different injuries leads to an earlier systemic inflammatory response when compared with the single insults. Interestingly, only after PTx but not after single or double hits was lung apoptosis increased, and PMN apoptosis was decreased along with a prolonged presence of neutrophils in the lungs, which may therefore represent a possible pathomechanism for lung injury after polytrauma.
The journal of trauma and acute care surgery. 02/2013; 74(2):489-98.
[show abstract][hide abstract] ABSTRACT: Activation of Fas signaling is a potentially important pathophysiological mechanism in the development of septic acute lung injury (ALI). However, so far the optimal targets within this signaling cascade remain elusive. Thus, we tested the hypothesis that in vivo gene silencing of Fas, Fas-associated via death domain (FADD), or caspase 3 by intratracheal administration of small interfering RNA would ameliorate ALI in a clinically relevant double-hit mouse model of trauma induced septic lung injury. Male C57Bl/6 mice received small interfering (Fas, FADD, caspase 3) or control RNA 24 h before and 12 h after blunt chest trauma or sham procedures. Polymicrobial sepsis was induced by cecal ligation and puncture 24 h after chest trauma. Twelve or 24 h later, lung tissue, plasma, and bronchoalveolar lavage fluid were harvested. During ALI, lung apoptosis (active caspase 3 Western blotting, TUNEL staining) was substantially increased when compared with sham. Silencing of caspase 3 or FADD both markedly reduced pulmonary apoptosis. Fas- and FADD-small interfering RNA administration substantially decreased lung cytokine concentration, whereas caspase 3 silencing did not reduce lung inflammation. In addition, Fas silencing markedly decreased lung neutrophil infiltration. Interestingly, only in response to caspase 3 silencing, ALI-induced lung epithelial barrier dysfunction was substantially improved, and histological appearance was beneficially affected. Taken together, downstream inhibition of lung apoptosis via caspase 3 silencing proved to be superior in mitigating ALI when compared with upstream inhibition of apoptosis via Fas or FADD silencing, even in the presence of additional anti-inflammatory effects. This indicates a major pathophysiological role of lung apoptosis and suggests the importance of other than Fas-driven apoptotic pathways in trauma-induced septic ALI.
[show abstract][hide abstract] ABSTRACT: The C3bot1 protein (∼23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.
Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.
In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.
PLoS ONE 01/2013; 8(1):e54517. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way.
Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the µM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro.
These results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.
PLoS ONE 01/2013; 8(6):e66099. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Study Goals: It is known that the manifold limitations of oral and practical examinations can be improved by specific training. With the help of an online survey, our present study analyzes the effects that can be achieved by the training conducted at the University of Ulm for examiners in the final medical examination, the long-lasting impact of the training, and differences among participant subgroups. Method: All 367 participants in the training at Ulm (2007- 2012) were contacted via email. Sixty-three persons responded to the survey that included 28 items concerning demographic data, effectiveness, and sustainability. Results: Six main effects of the training were identified (meaning effects rated with a grade of 1 or 2 on a 6-point scale by two thirds of the participants, with 1="applicable" and 6="not applicable"; cumulated percentage of answers of 1 or 2 in parentheses): Conscious handling of strengths and weaknesses of oral examinations (71%),Knowledge of factors contributing to the reliability of oral/practical examinations (76%),Knowledge of factors contributing to the validity of oral/practical examinations (75%),Improvement of competence in task construction (68%),Improvement of competence in respect to examination formalities (75%),Implementation of the concept of "structured oral examinations" (a priori planning of examination subjects, tasks, levels of expectation and grading criteria) (86%). The responses of participants trained more than two years ago were not significantly different from the answers given by recently trained persons. This is an argument for the sustainability of the training effects. Furthermore, participants without relevant prior experience in oral/practical examinations profited significantly more from the trainings, especially in the areas of stress reduction, confidence in grading, and competence in critical discrimination of grading. Conclusion: The positive and sustained effects of the examiner training argue for continuing the training program, especially for inexperienced examiners. Expansion of the successful training program to include the first medical exam should be considered.
GMS Zeitschrift für medizinische Ausbildung. 01/2013; 30(3):Doc36.
[show abstract][hide abstract] ABSTRACT: There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.
PLoS ONE 01/2013; 8(11):e81341. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.
PLoS ONE 01/2013; 8(5):e64454. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Attenuating the sepsis-induced systemic inflammatory response, with subsequent homeostatic imbalance, has for years been one of the main tasks in sepsis related research. Complement and the TLR family constitute two important upstream sensor and effector-systems of innate immunity. Although they act as partly independent branches of pattern recognition, recent evidence indicate a considerable cross-talk implying that they can either compensate, synergize or antagonize each other. Combined inhibition of these pathways is therefore a particularly interesting approach with a profound anti-inflammatory potential. In previous preclinical studies, we demonstrated that targeting the key molecules C3 or C5 of complement and CD14 of the TLR family had a vast anti-inflammatory effect on Gram-negative bacteria-induced inflammation and sepsis. In this review, we elucidate the significance of these key molecules as important targets for intervention in sepsis and systemic inflammatory response syndrome. Finally, we argue that a combined inhibition of complement and CD14 represent a potential general treatment regimen, beyond the limit of sepsis, including non-infectious systemic inflammation and ischemia reperfusion injury.