Overcoming roadblocks in clinical innovation via high fidelity simulation: use of a phantom simulator to achieve FDA and IRB approval of a clinical trial of fetal embolization of vein of Galen malformations.

BACKGROUND: Vein of Galen malformation (VOGM) is a rare, life-threatening vascular malformation in neonates and is treated with embolization. However, even at the most experienced centers, patients face high mortality and morbidity. In utero treatment options have been limited by lack of animal models or simulations. OBJECTIVE: To create a novel ultrasound phantom simulator for a preclinical feasibility study of in utero fetal intervention for VOGM. METHODS: Novel phantoms were designed and built in two configurations of spherical and windsock shape from cryogel material to mimic the salient vasculature of the fetal VOGM, based on real-patient fetal MR imaging dimensions. Critical anatomy was realistically mimicked within this model and transtorcular ultrasound-guided coil deployment was simulated. Each phantom model was assessed before and after treatment to evaluate coil mass deposition within the target. RESULTS: The two phantoms underwent pretreatment T2-weighted MR imaging assessment, ultrasound-guided embolization, post-treatment MR and fluoroscopic imaging, and visual inspection of the sliced phantoms for target embolization verification. Postoperative MR scans confirmed realistic compact deposition of the coil masses within the central cavity. Phantom embolization results were submitted as part of the institutional review board and US Food and Drug Administration investigative device exemption approval for a first-in-humans clinical trial of fetal intervention for VOGM. CONCLUSIONS: A phantom simulator for fetal intervention of VOGM produces lifelike results during trial interventions, removing obstacles to feasibility and safety evaluations, typically precluded by non-availability of appropriate animal models. The study provides a proof of concept for potentially wider applications of medical simulation to enable novel procedural advancements in neurointerventions.

Macrophage protease-activated receptor 2 regulates fetal liver erythropoiesis in mice.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

Gut Microbiota Restricts NETosis in Acute Mesenteric Ischemia-Reperfusion Injury.

OBJECTIVE: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-ß) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. CONCLUSIONS: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes.

BACKGROUND: Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. METHODS: In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. RESULTS: In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS. CONCLUSIONS: miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.

Lumbar Spine Response of Computational Finite Element Models in Multidirectional Spaceflight Landing Conditions.

The goals of this study are to compare the lumbar spine response variance between the hybrid III, test device for human occupant restraint (THOR), and global human body models consortium simplified 50th percentile (GHBMC M50-OS) finite element models and evaluate the sensitivity of lumbar spine injury metrics to multidirectional acceleration pulses for spaceflight landing conditions. The hybrid III, THOR, and GHBMC models were positioned in a baseline posture within a generic seat with side guards and a five-point restraint system. Thirteen boundary conditions, which were categorized as loading condition variables and environmental variables, were included in the parametric study using a Latin hypercube design of experiments. Each of the three models underwent 455 simulations for a total of 1365 simulations. The hybrid III and THOR models exhibited similar lumbar compression forces. The average lumbar compression force was 45% higher for hybrid III (2.2 ± 1.5 kN) and 51% higher for THOR (2.0 ± 1.6 kN) compared to GHBMC (1.3 ± 0.9 kN). Compared to hybrid III, THOR sustained an average 64% higher lumbar flexion moment and an average 436% higher lumbar extension moment. The GHBMC model sustained much lower bending moments compared to hybrid III and THOR. Regressions revealed that lumbar spine responses were more sensitive to loading condition variables than environmental variables across all models. This study quantified the intermodel lumbar spine response variations and sensitivity between hybrid III, THOR, and GHBMC. Results improve the understanding of lumbar spine response in spaceflight landings.

Cardiothoracic Morphology Measures in Heart Failure Patients to Inform Device Designs.

PURPOSE: Approximately 5.7 million people in the US are affected by congestive heart failure. This study aimed to quantitatively evaluate cardiothoracic morphology and variability within a cohort of heart failure patients for the purpose of optimally engineering cardiac devices for a variety of heart failure patients. METHODS: Co-registered cardiac-gated and non-gated chest computed tomography (CT) scans were analyzed from 20 heart failure patients (12 males; 8 females) who were primarily older adults (79.5 ± 8.8 years). Twelve cardiothoracic measurements were collected and compared to study sex and left ventricular (LV) ejection fraction (EF) type differences in cardiothoracic morphology. RESULTS: Four measures were significantly greater in males compared to females: LV long-axis length, LV end diastolic diameter (LVEDD) at 50% length of the LV long-axis, the minimal distance between the sternum and heart, and the angle between the LV long-axis and coronal plane. Four measures were significantly greater in patients with reduced EF compared to preserved LV: LV long-axis length, LVEDD at 50% length of the LV long-axis, left ventricular volume normalized by body surface area, and the angle between the mitral valve plane and LV long-axis. CONCLUSIONS: These cardiothoracic morphology measurements are important to consider in the design of cardiac devices for heart failure management (e.g. cardiac pacemakers, ventricular assist devices, and implantable defibrillators), since morphology differs by sex and ejection fraction.

Pelvic and Lower Gastrointestinal Tract Anatomical Characterization of the Average Male.

OBJECTIVE: Colorectal surgeons report difficulty in positioning surgical devices in males, particularly those with a narrower pelvis. The objectives of this study were to (1) characterize the anatomy of the pelvis and surrounding soft tissue from magnetic resonance and computed tomography scans from 10 average males (175 cm, 78 kg) and (2) develop a model representing the mean configuration to assess variability. METHODS: The anatomy was characterized from existing scans using segmentation and registration techniques. Size and shape variation in the pelvis and soft tissue morphology was characterized using the Generalized Procrustes Analysis to compute the mean configuration. RESULTS: There was considerable variability in volume of the psoas, connective tissue, and pelvis and in surface area of the mesorectum, pelvis, and connective tissue. Subject height was positively correlated with mesorectum surface area (P = .028, R2 = 0.47) and pelvis volume ( P = .041, R2 = 0.43). The anterior-posterior distance between the inferior pelvic floor muscle and pubic symphysis was positively correlated with subject height ( P = .043, r = 0.65). The angle between the superior mesorectum and sacral promontory was negatively correlated with subject height ( P = .042, r = -0.65). The pelvic inlet was positively correlated with subject weight ( P = .001, r = 0.89). CONCLUSIONS: There was considerable variability in organ volume and surface area among average males with some correlations to subject height and weight. A physical trainer model created from these data helped surgeons trial and assess device prototypes in a controllable environment.

Multidirection Validation of a Finite Element 50th Percentile Male Hybrid III Anthropomorphic Test Device for Spaceflight Applications.

The use of anthropomorphic test devices (ATDs) for calculating injury risk of occupants in spaceflight scenarios is crucial for ensuring the safety of crewmembers. Finite element (FE) modeling of ATDs reduces cost and time in the design process. The objective of this study was to validate a Hybrid III ATD FE model using a multidirection test matrix for future spaceflight configurations. Twenty-five Hybrid III physical tests were simulated using a 50th percentile male Hybrid III FE model. The sled acceleration pulses were approximately half-sine shaped, and can be described as a combination of peak acceleration and time to reach peak (rise time). The range of peak accelerations was 10-20 G, and the rise times were 30-110 ms. Test directions were frontal (-GX), rear (GX), vertical (GZ), and lateral (GY). Simulation responses were compared to physical tests using the correlation and analysis (CORA) method. Correlations were very good to excellent and the order of best average response by direction was -GX (0.916±0.054), GZ (0.841±0.117), GX (0.792±0.145), and finally GY (0.775±0.078). Qualitative and quantitative results demonstrated the model replicated the physical ATD well and can be used for future spaceflight configuration modeling and simulation.

The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation.

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of "neutrophil extracellular traps" (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.

Neutrophil Extracellular Traps as a Drug Target to Counteract Chronic and Acute Inflammation.

Neutrophil extracellular traps (NET), extruded decondensated chromatin entangled with neutrophil proteases, have been first identified in neutrophils stimulated with bacteria or phorbol myristate acetate (PMA) via activation of NADPH oxidase and the generation of reactive oxygen species. Although the first findings demonstrated the beneficial role of NET formation by trapping the bacteria and limiting their dissemination, numerous studies in the recent decade revealed the multifunctional aspects of NET formation which manifests itself not only in the context of anti-microbial effect but also as a pathological trigger. Uncontrolled and exaggerated NET formation or inability to digest and remove NET have been reported in thrombosis, auto-immune diseases, cancer or even in infertility. Studies are ongoing to disclose the role of NET in different pathological situations and most importantly, NET regulation via compounds that either interfere with NET formation or target NET components such as DNA or neutrophil proteases. Although the final product of NET formation seems to be quite common i.e. DNA entangled with proteases, stimuli that induce NET have a wide range of varieties and the involved pathways are diverse too. Therefore, in every pathological condition, it is necessary to consider carefully the type of stimulus and the signaling pathways in order to target the disease more specifically. Here we briefly summarize some (out of many) NET triggers/pathways and discuss the potential interventions in the pathological situations.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.

In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation.

In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.

Multicenter analysis of CIREN occupant lumbar bone mineral density and correlation with age and fracture incidence.

OBJECTIVE: This study aimed to quantify lumbar volumetric bone mineral density (vBMD) for 873 seriously injured Crash Injury Research and Engineering Network (CIREN) motor vehicle crash occupants (372 male, 501 female) from 8 centers using phantomless computed tomography scans and to associate vBMD with age, fracture incidence, and osteopenia/osteoporosis diagnoses. The novelty of this work is that it associates vBMD with region of injury by applying an established method for vBMD measurement using phantomless computed tomography (CT). METHODS: A validated phantomless CT calibration method that uses patient-specific fat and muscle measurements to calibrate vBMD measured from the L1-L5 trabeculae was applied on 873 occupants from various CIREN centers. CT-measured lumbar vBMD < 145 mg/cc is indicative of osteopenia using a published threshold. CIREN occupant lumbar vBMD in milligrams per cubic centimeter was regressed against age, osteopenia/osteoporosis comorbidities, height, weight, body mass index (BMI), and the incidence of fracture in vertebral (cervical, thoracic, lumbar) and rib/sternum regions. RESULTS: Among the 873 occupants analyzed, 11% (92 occupants) were diagnosed as osteopenic in CIREN. Of these 92 occupants, 42% (39 occupants) had normal vBMD measures (≥145 mg/cc), suggesting possible misclassification in CIREN. Of the 134 occupants classified as osteopenic in vBMD analysis, 60% were not classified as osteopenic in CIREN, suggesting undiagnosed osteopenia, and 40% were correctly classified in CIREN. Age was negatively correlated with vBMD (P <.0001) and occupants with <145 mg/cc vBMD sustained a median number of 2 rib/sternum fractures compared to a median value of 0 rib/sternum fractures for the ≥145 mg/cc vBMD group (P <.0001). Vertebral fracture analysis revealed that the thoracolumbar region was the most common region of injury in the spine. Though the incidence of fracture was not significantly different in the thoracic (10% versus 6%, P =.122) and lumbar (16% versus 13%, P =.227) regions between the 2 bone quality groups, the proportion of thoracolumbar fractures was significantly higher in occupants with <145 mg/cc vBMD versus occupants with ≥145 mg/cc vBMD (24% versus 17%, P =.043). CONCLUSIONS: Low lumbar vertebral bone quality is associated with an increased number of rib/sternum fractures and a greater incidence of thoracolumbar vertebral body fractures within the CIREN population analyzed.

Suppression of Neutrophil-Mediated Tissue Damage-A Novel Skill of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016;34:2393-2406.

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.

Association of neutrophil extracellular traps with endometriosis-related chronic inflammation.

OBJECTIVE: To study if neutrophil extracellular traps (NETs) are present in the peritoneal fluid of endometriosis patients. NETs play a crucial role in fighting against microorganisms. However, exaggerated NET production may lead to tissue damage in their vicinity in pathological conditions. Our study evaluates the presence of NETs in endometriosis peritoneal fluid. STUDY DESIGN: Peritoneal fluid (PF) was collected in a case-control study from 52 women, who underwent either diagnostic or operative laparoscopy. The control group consisted of 17 women with infertility, chronic pelvic pain, simple or functional cysts or irregular bleeding. The endometriosis group, altogether 35 patients, comprised 19 patients with stage I and II and 16 patients with stage III and IV endometriosis. First we tested whether the PF is able to stimulate NET production. Neutrophils from healthy volunteers were treated with the PF of endometriosis patients and controls and NETs were detected with Sytox orange extracellular DNA dye and immunofluorescence microscopy. Then we evaluated if NETs were already present in the collected PF using the specific myeloperoxidase (MPO)-DNA capture ELISA method, based on the MPO associated with the NET scaffold. RESULTS: The PF of endometriosis patients did not stimulate NET release from healthy granulocytes. However, pre-existent NETs could be detected in 17 endometriosis patients out of 35 (49%). In contrary, in the control group NETs were present in only 3 patients out of 17 (18%), (p=0.03, OR: 4.4). Moreover, the quantification of NETs showed a significantly higher amount of NETs in endometriosis compared to the controls (0.097 vs. 0.02, p=0.04). CONCLUSION: This is the first study, which evaluated and described the presence of NETs in the PF of endometriosis patients. Our study shows, that NETs may be involved in the complex pathophysiology of endometriosis.

Characterization of rapid neutrophil extracellular trap formation and its cooperation with phagocytosis in human neutrophils.

Neutrophils, as the first cellular line of innate host defense, employ phagocytosis and formation of neutrophil extracellular traps (NETs) to combat infections. Classical NET formation induced by phorbol myristate acetate requires several hours to complete. However, recent studies demonstrated rapid NET formation in neutrophils upon stimulation by platelets, Staphylococcus aureus or fungal products. Here we describe that antibody- or complement-induced phagocytosis triggers rapid NET formation. In contrast to classical NETosis, chemical inhibition of NADPH oxidase as well as using NADPH oxidase-deficient patient neutrophils did not affect rapid NET formation. Although phagocytosis and rapid NET formation may not be the prerequisite of each other, cooperation of phagocytosis and rapid NET formation may be essential to improve the efficiency of defense mechanisms in combating disseminating bacteria. Dissecting the differential mechanisms of NET formation is crucial to develop novel therapeutic strategies for infectious and auto-immune diseases where NETs play an essential role.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1ß. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1ß or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

Internalization of Bacillus intermedius ribonuclease (BINASE) induces human alveolar adenocarcinoma cell death.

Ribonuclease (RNase) treatment represents a novel mechanism based approach to anticancer therapy as an alternative to the DNA damaging drugs commonly used in clinical practice. Apart from their ribonucleolytic activity, cytotoxic effects have attracted a considerable attention to RNases because of their potential as selective agents for treatment of certain malignancies. Among these enzymes, Binase, an RNase from Bacillus intermedius, has shown promising results. Here, we have found that binase selectively attacked human A549 alveolar adenocarcinoma cells to trigger an apoptotic response, whereas normal lung epithelial cells LEK were not affected by the ribonuclease. The tumor transformation led to the modification of certain cellular characteristics causing cell sensitivity to binase. Although a general mode for RNases cytotoxicity includes their penetration into the cell, translocation to the cytosol and degradation of ribonucleic acid, many aspects of this process have not been fully elucidated. Our data revealed the following time-dependent changes induced by binase in A549 cells: (a) fast permanent internalization of the enzyme during the first hours of treatment; (b) temporary increase in cellular permeability for macromolecules during the 4-6 h of treatment; (c) apoptotic alterations in population after 24 h and (d) DNA fragmentation and cell death after 72 h of treatment with binase. Elucidation of these molecular strategies used by this promising toxin provides us essential information for the development of new anticancer drugs.

Fighting against the dark side of neutrophil extracellular traps in disease: manoeuvres for host protection.

PURPOSE OF REVIEW: This review presents a concise summary of the role of neutrophil extracellular traps (NET) in several pathological situations associated with adverse effects of NET. Different strategies are indicated to dampen the unfavourable consequences of NET formation and function. RECENT FINDINGS: A major extracellular antimicrobial mechanism of neutrophils is induced by formation of NET, in which the microorganisms are trapped within the released chromatin from neutrophils and killed by highly concentrated, NET-entangled antimicrobial proteins. In recent years, exaggerated NET formation and the subsequent complications for the host organism have been reported in several pathological conditions. It has been shown that interfering with NET structure and/or components either via DNase to disrupt the DNA backbone of NET or via antihistone approaches to target the major proteins in NET can diminish the pathological symptoms in such diseases. SUMMARY: On the basis of the type of the disease and its severity as well as the outcome of NET generation, different therapeutic strategies may be considered to dampen the inauspicious consequences of exaggerated NET formation and function. Applying combinations of compounds that aim to decrease the adverse activity of DNA and NET-associated proteins/enzymes may increase the efficiency of treatment.