Inhibitors for metallo-ß-lactamases from the B1 and B3 subgroups provide an avenue to combat a major mechanism of antibiotic resistance.

Metallo-ß-lactamases (MBLs) are a group of Zn(II)-dependent enzymes that pose a major threat to global health. They are linked to an increasing number of multi-drug resistant bacterial pathogens, but no clinically useful inhibitor is yet available. Since ß-lactam antibiotics, which are inactivated by MBLs, constitute ∼65% of all antibiotics used to treat infections, the search for clinically relevant MBL inhibitors is urgent. Here, derivatives of a 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1a) were synthesised and their inhibitory effects assessed against prominent representatives of the MBL family. Several compounds are potent inhibitors of each MBL tested, making them promising candidates for the development of broad-spectrum drug leads. In particular, compound 5f is highly potent across the MBL family, with Ki values in the low µM range. Furthermore, this compound also appears to display synergy in combination with antibiotics such as penicillin G, cefuroxime or meropenem. This molecule thus represents a promising starting point to develop new drugs to inhibit a major mechanism of antibiotic resistance.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Discovery of a Pyrimidinedione Derivative with Potent Inhibitory Activity against Mycobacterium tuberculosis Ketol-Acid Reductoisomerase.

New drugs aimed at novel targets are urgently needed to combat the increasing rate of drug-resistant tuberculosis (TB). Herein, the National Cancer Institute Developmental Therapeutic Program (NCI-DTP) chemical library was screened against a promising new target, ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. From this library, 6-hydroxy-2-methylthiazolo[4,5-d]pyrimidine-5,7(4H,6H)-dione (NSC116565) was identified as a potent time-dependent inhibitor of Mycobacterium tuberculosis (Mt) KARI with a Ki of 95.4 nm. Isothermal titration calorimetry studies showed that this inhibitor bound to MtKARI in the presence and absence of the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), which was confirmed by crystal structures of the compound in complex with closely related Staphylococcus aureus KARI. It is also shown that NSC116565 inhibits the growth of H37Ra and H37Rv strains of Mt with MIC50 values of 2.93 and 6.06 µm, respectively. These results further validate KARI as a TB drug target and show that NSC116565 is a promising lead for anti-TB drug development.

Discovery, Synthesis and Evaluation of a Ketol-Acid Reductoisomerase Inhibitor.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 µm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Šresolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.

Design, synthesis, biological evaluation and in silico studies of certain aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles as potent metallo-ß-lactamase inhibitors.

Based on a structure-guided approach, aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles were designed to target metallo-ß-lactamases (MBLs), using Klebsiella pneumoniaeNDM-1 as a model. The in vitro MBLs inhibition showed remarkable inhibition constant for most of the designed compounds at a low micromolar range (1.5-16.4 µM) against NDM-1, IMP-1 and AIM-1 MBLs. Furthermore, all compounds showed promising antibacterial activity against (K+, K1-K9) resistant clinical isolates of K. pneumoniae and were able to re-sensitize resistant K. pneumoniae (K5) strain towards meropenem and cefalexin. Besides, in vivo toxicity testing exhibited that the most active compound was non-toxic and well tolerated by the experimental animals orally up to 350 mg/kg and up to 125 mg/kg parenterally. The docking experiments on NDM-1 and IMP-1 rationalized the observed in vitro MBLs inhibition activity. Generally, this work presents a fruitful matrix to extend the chemical space for MBLs inhibition. This aids in tackling drug-resistance issues in antibacterial treatment.

Application of Piezo-Based Measuring System for Evaluation of Nucleic Acid-Based Drugs Influencing the Coagulation.

During open-heart surgery, the status of hemostasis has to be constantly monitored to quickly and reliably detect bleeding or coagulation disorders. In this study, a novel optimized piezo-based measuring system (PIEZ) for rheological monitoring of hemostasis was established. The applicability of the PIEZ for the evaluation of nucleic acid-based drugs influencing coagulation was analyzed. Thrombin aptamers such as NU172 might be used during extracorporeal circulation (ECC) in combination with a reduced heparin concentration or for patients with heparin-induced thrombocytopenia (HIT). Therefore, the effect of the coagulation inhibiting thrombin aptamer NU172 and the abrogation by its complementary antidote sequence (AD) were investigated by this rheological PIEZ system. After the addition of different NU172 concentrations, the coagulation of fresh human blood was analyzed under static conditions and using an in vitro rotation model under dynamic conditions (simulating ECC). The clotting times (CTs) detected by PIEZ were compared to those obtained with a medical reference device, a ball coagulometer. Additionally, after the circulation of blood samples for 30 min at 37 °C, blood cell numbers, thrombin markers (thrombin-antithrombin III (TAT) and fibrinopeptide A (FPA)) and a platelet activation marker (ß-thromboglobulin (ß-TG)) were analyzed by enzyme-linked immunosorbent assays (ELISAs). The increase of NU172 concentration resulted in prolonged CTs, which were comparable between the reference ball coagulometer and the PIEZ, demonstrating the reliability of the new measuring system. Moreover, by looking at the slope of the linear regression of the viscous and elastic components, PIEZ also could provide information on the kinetics of the coagulation reaction. The shear viscosity at the end of the measurements (after 300 s) was indicative of clot firmness. Furthermore, the PIEZ was able to detect the abrogation of coagulation inhibition after the equimolar addition of NU172 aptamer´s AD. The obtained results showed that the established PIEZ is capable to dynamically measure the hemostasis status in whole blood and can be applied to analyze nucleic acid-based drugs influencing the coagulation.

Anatomically Integrated In-Place Visualization of Patient Data for Cooperative Tasks with a Case Study on a Neurosurgical Ward.

The workflow in modern hospitals entails that the medical treatment of a patient is distributed between several physicians and nurses. This leads to intensive cooperation, which takes place under particular time pressure and requires efficient conveyance of relevant patient-related medical data to colleagues. This requirement is difficult to achieve with traditional data representation approaches. In this paper, we introduce a novel concept of anatomically integrated in-place visualization designed to engage with cooperative tasks on a neurosurgical ward by using a virtual patient's body as spatial representation of visually encoded abstract medical data. Based on the findings of our field studies, we provide a set of formal requirements and procedures for this kind of visual encoding. Moreover, we implemented a prototype on a mobile device that supports the diagnosis of spinal disc herniation and has been evaluated by 10 neurosurgeons. The physicians have assessed the proposed concept as beneficial, especially emphasizing the advantages of the anatomical integration such as intuitiveness and a better data availability due to providing all information at a glance. Particularly, four of nine respondents have stressed solely benefits of the concept, other four have mentioned benefits with some limitations and only one person has seen no benefits.

Guideline for allergological diagnosis of drug hypersensitivity reactions: S2k Guideline of the German Society for Allergology and Clinical Immunology (DGAKI) in cooperation with the German Dermatological Society (DDG), the Association of German Allergologists (ÄDA), the German Society for Pediatric Allergology (GPA), the German Contact Dermatitis Research Group (DKG), the German Society for Pneumology (DGP), the German Society of Otorhinolaryngology, Head and Neck Surgery, the Austrian Society of Allergology and Immunology (ÖGAI), the Austrian Society of Dermatology and Venereology (ÖGDV), the German Academy of Allergology and Environmental Medicine (DAAU), and the German Documentation Center for Severe Skin Reactions (dZh).

Not available.

Phosphorylation of vasodilator-stimulated phosphoprotein prevents platelet-neutrophil complex formation and dampens myocardial ischemia-reperfusion injury.

BACKGROUND: Recent work has suggested that the formation of platelet-neutrophil complexes (PNCs) aggravates the severity of inflammatory tissue injury. Given the importance of vasodilator-stimulated phosphoprotein (VASP) for platelet function, we pursued the role of VASP on the formation of PNCs and its impact on the extent of myocardial ischemia-reperfusion (IR) injury. METHODS AND RESULTS: In initial in vitro studies we found that neutrophils facilitated the movement of platelets across endothelial monolayers. Phosphorylation of VASP reduced the formation of PNCs and transendothelial movement of PNCs. During myocardial IR injury, VASP(-/-) animals demonstrated reduced intravascular formation of PNCs and reduced presence of PNCs within the ischemic myocardial tissue. This was associated with reduced IR injury. Studies using platelet transfer and bone marrow chimeric animals showed that hematopoietic VASP expression was crucial for the intravascular formation of PNCs the presence of PNCs within ischemic myocardial tissue and the extent of myocardial IR injury. Furthermore, phosphorylation of VASP on Ser153 or Ser235 reduced intravascular PNC formation and presence of PNCs within ischemic myocardial tissue. This finding was associated with reduced myocardial IR injury. CONCLUSION: Previously unappreciated, the phosphorylation of VASP performs a key function for the formation of PNCs that is crucially important for the extent of myocardial IR injury.

Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.

OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

Flow cytometry analysis of porcine platelets: optimized methods for best results.

Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.

Hemostatic wound dressings: Predicting their effects by in vitro tests.

BACKGROUND: Application of controlled in vitro techniques can be used as a screening tool for the development of new hemostatic agents allowing quantitative assessment of overall hemostatic potential. MATERIALS AND METHODS: Several tests were selected to evaluate the efficacy of cotton gauze, collagen, and oxidized regenerated cellulose for enhancing blood clotting, coagulation, and platelet activation. RESULTS: Visual inspection of dressings after blood contact proved the formation of blood clots. Scanning electron microscopy demonstrated the adsorption of blood cells and plasma proteins. Significantly enhanced blood clot formation was observed for collagen together with ß-thromboglobulin increase and platelet count reduction. Oxidized regenerated cellulose demonstrated slower clotting rates not yielding any thrombin generation; yet, led to significantly increased thrombin-anti-thrombin-III complex levels compared to the other dressings. As hemostyptica ought to function without triggering any adverse events, induction of hemolysis, instigation of inflammatory reactions, and initiation of the innate complement system were also tested. Here, cotton gauze provoked high PMN elastase and elevated SC5b-9 concentrations. CONCLUSIONS: A range of tests for desired and undesired effects of materials need to be combined to gain some degree of predictability of the in vivo situation. Collagen-based dressings demonstrated the highest hemostyptic properties with lowest adverse reactions whereas gauze did not induce high coagulation activation but rather activated leukocytes and complement.

Keratinocyte Growth Factor Modified Messenger RNA Accelerating Cell Proliferation and Migration of Keratinocytes.

Keratinocyte growth factor (KGF) plays a central role in wound healing as it induces cell proliferation and motility. The use of growth factors such as KGF is therefore viewed as a promising approach in wound therapy, although effective application remains a major problem because of inactivation and the resulting short half-life of applied growth factors in wound beds. Therefore, the rational of this study was to develop and investigate an innovative strategy to improve wound healing using an in vitro-transcribed modified KGF messenger RNA (mRNA). After transfection of cells, we evaluated the effects of the produced KGF protein on cell migration and reepithelialization of keratinocytes using a scratch assay. The results demonstrate that KGF-mRNA-transfected cells exhibited a high KGF protein release that is sufficient to significantly improve reepithelialization in the performed scratch assays. Transfection with growth factor mRNA therefore seems to be a promising therapeutic strategy, especially for difficult wounds, as it leads to a temporary increase of growth factor expression in the treated wound area without interfering with the DNA of the nucleus, as seen in gene therapeutic applications.

Low-thrombogenic fibrin-heparin coating promotes in vitro endothelialization.

Long-term performance of implanted cardiovascular grafts can be ensured if living endothelium overgrows their surface. Surface modifications to implants are therefore being sought that can encourage endothelialization while preventing thrombus formation until the natural endothelium is formed. In the present study, heparin was covalently attached to a fibrin mesh grown from a polyvinyl chloride (PVC) substrate surface by the catalytic action of surface immobilized thrombin on a fibrinogen solution. The coating prevented platelet activation, thrombin generation and clot formation, and reduced inflammatory reactions when exposed to fresh human whole blood circulating in a Chandler loop model. In addition, in vitro seeded human umbilical vein and human saphenous vein endothelial cells showed considerably enhanced attachment and proliferation on the coating. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2995-3005, 2017.

Generation of Large-Scale DNA Hydrogels with Excellent Blood and Cell Compatibility.

Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme-linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993-5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.

Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications.

Synthetically modified mRNA is a unique bioactive agent, ideal for use in therapeutic applications, such as cancer vaccination or treatment of single-gene disorders. In order to facilitate mRNA transfections for future therapeutic applications, there is a need for the delivery system to achieve optimal transfection efficacy, perform with durable stability, and provide drug safety. The objective of our study was to comprehensively analyze the use of 3ß-[N-(N',N'-dimethylaminoethane) carbamoyl](DC-Cholesterol)/dioleoylphosphatidylethanolamine (DOPE) liposomes as a potential transfection agent for modified mRNAs. Our cationic liposomes facilitated a high degree of mRNA encapsulation and successful cell transfection efficiencies. More importantly, no negative effects on cell viability or immune reactions were detected posttransfection. Notably, the liposomes had a long-acting transfection effect on cells, resulting in a prolonged protein production of alpha-1-antitrypsin (AAT). In addition, the stability of these mRNA-loaded liposomes allowed storage for 80 days, without the loss of transfection efficacy. Finally, comprehensive analysis showed that these liposomes are fully hemocompatible with fresh human whole blood. In summary, we present an extensive analysis on the use of DC-cholesterol/DOPE liposomes as mRNA delivery vehicles. This approach provides the basis of a safe and efficient therapeutic strategy in the development of successful mRNA-based drugs.

Comparison of pharmacokinetics and vasodilatory effect of nebulized and infused iloprost in experimental pulmonary hypertension: rapid tolerance development.

Aerosolized iloprost has been suggested for selective pulmonary vasodilatation in severe pulmonary hypertension, but its pharmacokinetic profile is largely unknown. In perfused rabbit lungs, continuous infusion of the thromboxane mimetic U46619 was employed for establishing stable pulmonary hypertension. Delivery of a total amount of 75, 300, and 900 ng of iloprost to the bronchoalveolar space by a 10 min-aerosolization maneuver caused a dose-dependent pulmonary vasodilatation. Similarly, dose-dependent appearance of iloprost in the recirculating perfusate was noted, with maximum intravascular concentrations of iloprost ranging at 140, 510, and 1163 pg/mL at the same time period. Comparing pharmacokinetics and pharmacodynamics in a more detailed fashion, the following aspects were of interest. (i) The bioavailability (i.e., the percentage of aerosolized iloprost appearing intravascularly) decreased from 76% at the lowest to 33% at the highest iloprost dosage. (ii) The pulmonary vasodilatory response commenced already during the nebulization maneuver and preceded the perfusate entry of iloprost. (iii) After 3-3.5 h, the pulmonary vasodilatory response to aerosolized iloprost had virtually completely leveled off, whereas approximately two-thirds of the maximum iloprost perfusate levels were still detectable. A corresponding loss of vasodilatory response was also noted in experiments with continuous iloprost perfusion for clamping of the intravascular concentration of this prostanoid. We conclude that aerosolized iloprost causes dose-dependent vasodilatation and iloprost entry into the vascular space in a pulmonary hypertension model. Limited bioavailability in the higher dose range may suggest active prostanoid transport processes, and the early pulmonary vasodilatory response appears to be independent of prostanoid entry into the vessel lumen. Surprisingly, rapid tolerance development to the vasodilatory effect of iloprost is noted, occurring even with fully maintained perfusate levels of this agent.

Hemostyptic property of chitosan: Opportunities and pitfalls.

BACKGROUND: Chitosan is used in a wide field of applications and therapies and has been reported to be an effective hemostyptic. The objective of this study was to provide further information about the use of chitosan as a hemostyptic agent also taking into focus its hemocompatible effects. METHODS: Human whole blood (n=5) was anticoagulated with heparin, treated with different chitosan concentrations (0, 2.5, 5, 7.5, 10, 12.5, 25 mg/mL) and incubated at 37°C for 30 minutes. Before and after incubation different parameters for coagulation and hemocompatibility were evaluated. RESULTS: Blood treated with high chitosan concentrations showed enhanced coagulation, which we evaluated with activated clotting time, activated partial thromboplastin time and concentration of thrombin-antithrombin complexes. Furthermore, we observed an activation of blood platelets, complement cascade and granulocytes in the groups treated with chitosan. CONCLUSION: Our data indicate that chitosan activates human blood coagulation and hence has good properties as a hemostyptic agent. However, inflammatory parameters were upregulated after direct contact with human blood indicating that systemic administration of chitosans should not be performed whereas the topical use of chitosan as a hemostypticum should not present any hazard with regard to adverse inflammatory reactions at the site of application.

In Vitro Evaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency.

In single-gene disorders, like alpha-1-antitrypsin deficiency (AATD), a gene mutation causes missing or dysfunctional protein synthesis. This, in turn, can lead to serious complications for the patient affected. Furthermore, single-gene disorders are associated with severe early-onset conditions and necessitate expensive lifelong care. Until nowadays, therapeutic treatment options are still limited, cost-intensive, or lack effectiveness. For these reasons, we aim to develop a novel mRNA-based therapeutic strategy for the treatment of single-gene disorders, such as AATD, which is based on the induction of de novo synthesis of the functional proteins. Therefore, an alpha-1-antitrypsin (AAT) encoding mRNA was generated by in vitro transcription. After in vitro delivery of the mRNA to different cells, protein expression and functionality, as well as adverse effects and mRNA serum stability, were analyzed. Our results show that the AAT mRNA-transfected cells express the AAT protein in high amounts within the first 24 h. Moreover, the expressed AAT protein is highly functional, since the activity of elastase is significantly inhibited. Our data also show that mRNA concentrations up to 1 µg per 150,000 cells have no adverse effects on cell viability and immune activation. Furthermore, the encapsulated AAT encoding mRNA is stable and functional in human serum for up to 30 min. Overall, the proposed project provides an innovative, highly promising, and safe therapeutic approach and, thus, promises a novel progress in the treatment of single-gene disorders, whereby affected patients could greatly benefit.

First In Vivo Results of a Novel Pediatric Oxygenator with an Integrated Pulsatile Pump.

Extracorporeal membrane oxygenation (ECMO) is a pivotal bridge to recovery for cardiopulmonary failure in children. Besides its life-saving quality, it is often associated with severe system-related complications, such as hemolysis, inflammation, and thromboembolism. Novel oxygenator and pump systems may reduce such ECMO-related complications. The ExMeTrA oxygenator is a newly designed pediatric oxygenator with an integrated pulsatile pump minimizing the priming volume and reducing the surface area of blood contact. The aim of our study was to investigate the feasibility and safety of this new ExMeTrA (expansion mediated transport and accumulation) oxygenator in an animal model. During 6 h of extracorporeal circulation (ECC) in pigs, parameters of the hemostatic system including coagulation, platelets and complement activation, and flow rates were investigated. A nonsignificant trend in C3 consumption, thrombin-antithrombin-III (TAT) complex formation and a slight trend in hemolysis were detected. During the ECC, the blood flow was constantly at 500 ml/min using only flexible silicone tubes inside the oxygenator as pulsatile pump. Our data clearly indicate that the hemostatic markers were only slightly influenced by the ExMeTrA oxygenator. Additionally, the oxygenator showed a constant quality of blood flow. Therefore, this novel pediatric oxygenator shows the potential to be used in pediatric and neonatal support with ECMO.