Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

CD14highCD16+ monocytes are the main producers of Interleukin-10 following clinical heart transplantation.

Introduction: Following heart transplantation, a cascade of immunological responses is initiated influencing the clinical outcome and long-term survival of the transplanted patients. The anti-inflammatory cytokine interleukin-10 (IL-10) was shown to be elevated in the blood of heart transplant recipients directly after transplantation but the releasing cell populations and the composition of lymphocyte subsets following transplantation have not been thoroughly studied. Methods: We identified immune cells by immunophenotyping and analyzed intracellular IL-10 production in peripheral blood mononuclear cells (PBMC) of heart transplanted patients (n= 17) before, directly after and 24h post heart transplantation. The cells were stimulated with lipopolysaccharide or PMA/Ionomycin to enhance cytokine production within leukocytes in vitro. Results and discussion: We demonstrate that intermediate monocytes (CD14highCD16+), but not CD8+ T cells, CD4+ T cells, CD56+ NK cells or CD20+ B cells appeared to be the major IL-10 producers within patients PBMC following heart transplantation. Consequently, the absolute monocyte count and the ratio of intermediate monocytes to classical monocytes (CD14+CD16-) were specifically increased in comparison to pre transplant levels. Hence, this population of monocytes, which has not been in the focus of heart transplantation so far, may be an important modulator of clinical outcome and long-term survival of heart transplant recipients. Alteration of blood-circulating monocytes towards a CD14highCD16+ phenotype could therefore shift the pro-inflammatory immune response towards induction of graft tolerance, and may pave the way for the optimization of immunosuppression.

Pushing the boundaries of innovation: the potential of ex vivo organ perfusion from an interdisciplinary point of view.

Ex vivo machine perfusion (EVMP) is an emerging technique for preserving explanted solid organs with primary application in allogeneic organ transplantation. EVMP has been established as an alternative to the standard of care static-cold preservation, allowing for prolonged preservation and real-time monitoring of organ quality while reducing/preventing ischemia-reperfusion injury. Moreover, it has paved the way to involve expanded criteria donors, e.g., after circulatory death, thus expanding the donor organ pool. Ongoing improvements in EVMP protocols, especially expanding the duration of preservation, paved the way for its broader application, in particular for reconditioning and modification of diseased organs and tumor and infection therapies and regenerative approaches. Moreover, implementing EVMP for in vivo-like preclinical studies improving disease modeling raises significant interest, while providing an ideal interface for bioengineering and genetic manipulation. These approaches can be applied not only in an allogeneic and xenogeneic transplant setting but also in an autologous setting, where patients can be on temporary organ support while the diseased organs are treated ex vivo, followed by reimplantation of the cured organ. This review provides a comprehensive overview of the differences and similarities in abdominal (kidney and liver) and thoracic (lung and heart) EVMP, focusing on the organ-specific components and preservation techniques, specifically on the composition of perfusion solutions and their supplements and perfusion temperatures and flow conditions. Novel treatment opportunities beyond organ transplantation and limitations of abdominal and thoracic EVMP are delineated to identify complementary interdisciplinary approaches for the application and development of this technique.

Novel Size-Variable Dedicated Rodent Oxygenator for ECLS Animal Models-Introduction of the "RatOx" Oxygenator and Preliminary In Vitro Results.

The overall survival rate of extracorporeal life support (ECLS) remains at 60%. Research and development has been slow, in part due to the lack of sophisticated experimental models. This publication introduces a dedicated rodent oxygenator ("RatOx") and presents preliminary in vitro classification tests. The RatOx has an adaptable fiber module size for various rodent models. Gas transfer performances over the fiber module for different blood flows and fiber module sizes were tested according to DIN EN ISO 7199. At the maximum possible amount of effective fiber surface area and a blood flow of 100 mL/min, the oxygenator performance was tested to a maximum of 6.27 mL O2/min and 8.2 mL CO2/min, respectively. The priming volume for the largest fiber module is 5.4 mL, while the smallest possible configuration with a single fiber mat layer has a priming volume of 1.1 mL. The novel RatOx ECLS system has been evaluated in vitro and has demonstrated a high degree of compliance with all pre-defined functional criteria for rodent-sized animal models. We intend for the RatOx to become a standard testing platform for scientific studies on ECLS therapy and technology.

Immediate major dynamic changes in the T- and NK-cell subset composition after cardiac transplantation.

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3 wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4+ or CD8+ T cells and CD56dim CD16+ NK cells immediately after HTx linked to a decrease in naïve CD8+ and CM CD4+ T as well as CD56bright CD16- NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69+ CD25- and diminished CD69- CD25- CD4+ or CD8+ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.

Biohybrid lung Development: Towards Complete Endothelialization of an Assembled Extracorporeal Membrane Oxygenator.

Towards the establishment of a long-term lung-assist device to be used both as a bridge and as an alternative to lung transplantation according to final destination therapy, we develop the biohybrid lung (BHL) on the technical basis of contemporary extracorporeal membrane oxygenation (ECMO). Here, to overcome the significant drawbacks of ECMO, in particular the missing hemocompatibility of the artificial surfaces, all blood-contacting areas need to be endothelialized sufficiently. In continuation of our recent accomplishments, demonstrating the feasibility of establishing a physiological acting endothelial cell (EC) monolayer on the hollow fiber membranes (HFMs) of the ECMO in vitro, the next step towards BHL translation is the endothelialization of the complete oxygenator, consisting of HFMs and the surrounding housing. Therefore, we assessed EC seeding inside our model oxygenator (MOx), which simulated the conditions in the assembled HFM oxygenators in order to identify the most important factors influencing efficient endothelialization, such as cell seeding density, cell distribution, incubation time and culture medium consumption. Overall, upon adjusting the concentration of infused ECs to 15.2 × 104/cm2 and ensuring optimal dispersion of cells in the MOx, viable and confluent EC monolayers formed on all relevant surfaces within 24 h, even though they comprised different polymers, i.e., the fibronectin-coated HFMs and the polysulfone MOx housing. Periodic medium change ensured monolayer survival and negligible apoptosis rates comparable to the reference within the assembled system. By means of these results, revealing essential implications for BHL development, their clinical translation is coming one step closer to reality.

Determinants of survival in patients on extracorporeal membrane oxygenation therapy due to severe covid-19.

BACKGROUND: Severe acute respiratory distress syndrome (ARDS) due to Coronavirus Disease-19 (COVID-19) is associated with high mortality. Although survival on mechanical circulatory support has improved, determinants for better prognosis are still unclear. Here, we report on the outcome of our patient population with the need for mechanical circulatory support due to severe COVID-19 (sCOVID-19) induced ARDS. METHODS: All patients treated with extracorporeal membrane oxygenation (ECMO) for severe ARDS due to sCOVID-19 were analysed. Patients > 18 years of age at the time of initiation of ECMO were included. Pre-existing comorbidities, complications during ECMO implantation, and ECMO runtime were reviewed. The latency to intubation, proning, tracheotomy, and ECMO implantation was analysed. Furthermore, the survival and non-survival population were compared to determine factors in favour of a better outcome. RESULTS: In total, 85 patients were treated with veno-venous membrane oxygenation (vv-ECMO) for severe ARDS in our medical centre. The patient population was predominantly male (83.5%) with a mean patient age of 54.9 years. A history of cardiovascular disease (p = .01), smoking (p < .05), need for vasopressor- (p < .05), and renal replacement therapy (p < .001) was associated with a worse prognosis. Overall survival was 50%. The survival population was significantly younger (p = .004), had a significantly higher body weight (p = .02) and body mass index (BMI) (p = .01). Furthermore, survival was significantly better when vv-ECMO was initiated within 48 h after admission (p < .001). CONCLUSIONS: Pre-existing cardiovascular disease, higher age, history of nicotine abuse, and development of renal failure are associated with poor outcome. Early start of vv-ECMO therapy may lead to better survival in sCOVID-19 patients, although complications during ECMO therapy are associated with a worse prognosis.

The role of ex-situ perfusion for thoracic organs.

PURPOSE OF REVIEW: Ex-situ machine perfusion for both heart (HTx) and lung transplantation (LuTx) reduces ischemia-reperfusion injury (IRI), allows for greater flexibility in geographical donor management, continuous monitoring, organ assessment for extended evaluation, and potential reconditioning of marginal organs. In this review, we will delineate the impact of machine perfusion, characterize novel opportunities, and outline potential challenges lying ahead to improve further implementation. RECENT FINDINGS: Due to the success of several randomized controlled trials (RCT), comparing cold storage to machine perfusion in HTx and LuTx, implementation and innovation continues. Indeed, it represents a promising interface for organ-specific therapies targeting IRI, allo-immune responses, and graft reconditioning. These mostly experimental efforts range from genetic approaches and nanotechnology to cellular therapies, involving mesenchymal stem cell application. Despite tremendous potential, prior to clinical transition, more data is needed. SUMMARY: Collectively, machine perfusion constitutes the vanguard in thoracic organ transplantation research with extensive potential for expanding the donor pool, enhancing transplant outcomes as well as developing novel therapy approaches.

Towards Biohybrid Lung Development: Establishment of a Porcine In Vitro Model.

Lung transplantation (LTx) is the only curative therapy option for patients with end-stage lung diseases, though only available for chosen patients. To provide an alternative treatment option to LTx, we aim for the development of an implantable biohybrid lung (BHL) based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Crucial for long-lasting BHL durability is complete hemocompatibility of all blood contacting surfaces, which can be achieved by their endothelialization. In continuation to successful in vitro investigations using human endothelial cells (ECs), indicating general feasibility, the appropriate porcine in vivo model needs to be prepared and established to fill the translational data gap prior to patient's application. Therefore, isolation of porcine ECs from carotid arteries (pCECs) was established. Following, pCECs were used for HFM endothelialization and examined under static and dynamic conditions using cell medium or heparinized blood, to assess their proliferation capacity, flow resistance and activation state, especially under clinically relevant conditions. Additionally, comparative hemocompatibility tests between native and endothelialized HFMs were performed. Overall, pure pCECs formed a viable and confluent monolayer, which resisted applied flow conditions, in particular due to physiological extracellular matrix synthesis. Additionally, pCECs remained the non-inflammatory and anti-thrombogenic status, significantly improving the hemocompatibility of endothelialized HFMs. Finally, as relevant for reliable porcine to human translation, pCECs behaved in the same way as human ECs. Concluding, generated in vitro data justify further steps towards pre-clinical BHL examination, in particular BHL application to porcine lung injury models, reflecting the clinical scenario with end-stage lung-diseased patients.

Trends, Advantages and Disadvantages in Combined Extracorporeal Lung and Kidney Support From a Technical Point of View.

Extracorporeal membrane oxygenation (ECMO) provides pulmonary and/or cardiac support for critically ill patients. Due to their diseases, they are at high risk of developing acute kidney injury. In that case, continuous renal replacement therapy (CRRT) is applied to provide renal support and fluid management. The ECMO and CRRT circuits can be combined by an integrated or parallel approach. So far, all methods used for combined extracorporeal lung and kidney support present serious drawbacks. This includes not only high risks of circuit related complications such as bleeding, thrombus formation, and hemolysis, but also increase in technical workload and health care costs. In this sense, the development of a novel optimized artificial lung device with integrated renal support could offer important treatment benefits. Therefore, we conducted a review to provide technical background on existing techniques for extracorporeal lung and kidney support and give insight on important aspects to be addressed in the development of this novel highly integrated artificial lung device.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Identification of distinct secretory patterns and their regulatory networks of ischemia versus reperfusion phases in clinical heart transplantation.

BACKGROUND: Ischemia/reperfusion injury (IRI) is associated with inflammatory responses contributing to the development of primary graft dysfunction (PGD) and rejection. Here, we investigated the pathophysiology of IRI and the early phase after heart transplantation (HTx) regarding its cytokine/chemokine and endothelial networks. METHODS: Using multiplex technology, we assessed protein concentrations in plasma samples of HTx recipients (n = 11) pre-, postoperatively, 24 h and 3 weeks after HTx. The same proteins were quantified in organ storage solutions at the end of heart storage (n = 10). Unsupervised cluster, principal component analysis (PCA), K-nearest neighbor (KNN) network classifier analysis, ANOVA and Spearman correlation analyses were performed to identify specific patterns for IRI and individual kinetics of important soluble factors in HTx. RESULTS: Unique patterns of soluble factors were identified in plasma of HTx patients. KNN analysis defined IL-10, IL-6, sIL-6Rα, IL-1RA, IL-16, sVEGFR-1, IGFBP-1, HGF and sHer-2 as strongest signals directly post-Tx declining 24 hrs after HTx. By contrast, MIF, osteopontin (OPN), sVCAM-1 and sICAM-1, IGFBP-1, SCGF-ß, HGF were highly enriched in organ storage solutions, reflecting distinct ischemic (storage solution) vs. reperfusion (plasma) signatures. CONCLUSIONS: We identified specific inflammatory signatures for ischemic vs. reperfusion phases of HTx, associated with pro- as well as anti-inflammatory and endothelial biomarker candidates for IRI. These signatures might help to identify potential danger factors and their networks at both the ex situ (ischemic) as well as the reperfusion phase in the recipient after implantation.

Donor NK and T Cells in the Periphery of Lung Transplant Recipients Contain High Frequencies of Killer Cell Immunoglobulin-Like Receptor-Positive Subsets.

Introduction: For end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells. Methods: Peripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry. Results: Within the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells. Conclusion: Higher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.

Towards Biohybrid Lung: Induced Pluripotent Stem Cell Derived Endothelial Cells as Clinically Relevant Cell Source for Biologization.

In order to provide an alternative treatment option to lung transplantation for patients with end-stage lung disease, we aim for the development of an implantable biohybrid lung (BHL), based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. Complete hemocompatibility of all blood contacting surfaces is crucial for long-lasting BHL durability and can be achieved by their endothelialization. Autologous endothelial cells (ECs) would be the ideal cell source, but their limited proliferation potential excludes them for this purpose. As induced pluripotent stem cell-derived ECs enable the generation of a large number of ECs, we assessed and compared their capacity to form a viable and confluent monolayer on HFM, while indicating physiologic EC-specific anti-thrombogenic and anti-inflammatory properties. ECs were generated from three different human iPSC lines, and seeded onto fibronectin-coated poly-4-methyl-1-pentene (PMP) HFM. Following phenotypical characterization, ECs were analyzed for their thrombogenic and inflammatory behavior with or without TNFα induction, using FACS and qRT-PCR. Complementary, leukocyte- and platelet adhesion assays were carried out. The capacity of the iPSC-ECs to reendothelialize cell-free monolayer areas was assessed in a scratch assay. ECs sourced from umbilical cord blood (hCBECs) were used as control. iPSC-derived ECs formed confluent monolayers on the HFM and showed the typical EC-phenotype by expression of VE-cadherin and collagen-IV. A low protein and gene expression level of E-selectin and tissue factor was detected for all iPSC-ECs and the hCBECs, while a strong upregulation of these markers was noted upon stimulation with TNFα. This was in line with the physiological and strong induction of leukocyte adhesion detected after treatment with TNFα, iPSC-EC and hCBEC monolayers were capable of reducing thrombocyte adhesion and repopulating scratched areas. iPSCs offer the possibility to provide patient-specific ECs in abundant numbers needed to cover all blood contacting surfaces of the BHL with a viable, non-thrombogenic and non-inflammatory monolayer. iPSC-EC clones can differ in terms of their reendothelialization rate, and pro-inflammatory response. However, a less profound inflammatory response may even be advantageous for BHL application. With the proven ability of the seeded iPSC-ECs to reduce thrombocyte adhesion, we expect that thrombotic events that could lead to BHL occlusion can be avoided, and thus, justifies further studies on enabling BHL long-term application.

Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells.

Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.

Hemodynamic Assessment of Hollow-Fiber Membrane Oxygenators Using Computational Fluid Dynamics in Heterogeneous Membrane Models.

Extracorporeal membrane oxygenation (ECMO) has been used clinically for more than 40 years as a bridge to transplantation, with hollow-fiber membrane (HFM) oxygenators gaining in popularity due to their high gas transfer and low flow resistance. In spite of the technological advances in ECMO devices, the inevitable contact of the perfused blood with the polymer hollow-fiber gas-exchange membrane, and the subsequent thrombus formation, limits their clinical usage to only 2-4 weeks. In addition, the inhomogeneous flow in the device can further enhance thrombus formation and limit gas-transport efficiency. Endothelialization of the blood contacting surfaces of ECMO devices offers a potential solution to their inherent thrombogenicity. However, abnormal shear stresses and inhomogeneous blood flow might affect the function and activation status of the seeded endothelial cells (ECs). In this study, the blood flow through two HFM oxygenators, including the commercially available iLA® MiniLung Petite Novalung (Xenios AG, Germany) and an experimental one for the rat animal model, was modeled using computational fluid dynamics (CFD), with a view to assessing the magnitude and distribution of the wall shear stress (WSS) on the hollow fibers and flow fields in the oxygenators. This work demonstrated significant inhomogeneity in the flow dynamics of both oxygenators, with regions of high hollow-fiber WSS and regions of stagnant flow, implying a variable flow-induced stimulation on seeded ECs and possible EC activation and damage in a biohybrid oxygenator setting.

Towards Biohybrid Lung Development-Fibronectin-Coating Bestows Hemocompatibility of Gas Exchange Hollow Fiber Membranes by Improving Flow-Resistant Endothelialization.

To provide an alternative treatment option for patients with end-stage lung disease, we aim for biohybrid lung development (BHL) based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. For long-term BHL application, complete hemocompatibility of all blood-contacting surfaces is indispensable and can be achieved by their endothelialization. Indeed, albumin/heparin (AH) coated HFM enables initial endothelialization, but as inexplicable cell loss under flow conditions was seen, we assessed an alternative HFM coating using fibronectin (FN). Therefore, endothelial cell (EC) adherence and viability on both coated HFM were analyzed by fluorescence-based staining. Functional leukocyte and thrombocyte adhesion assays were performed to evaluate hemocompatibility, also in comparison to blood plasma coated HFM as a clinically relevant control. To assess monolayer resistance and EC behavior under clinically relevant flow conditions, a mock circulation setup was established, which also facilitates imitation of lung-disease specific blood gas settings. Besides quantification of flow-associated cell loss, endothelial responses towards external stimuli, like flow exposure or TNFα stimulation, were analyzed by qRT-PCR, focusing on inflammation, thrombus formation and extracellular matrix production. Under static conditions, both coated HFM enabled the generation of a viable, confluent, non-inflammatory and anti-thrombogenic monolayer. However, by means of homogenous FN coating, cell retention and physiologic gene regulation towards an improved hemocompatible-and extracellular matrix producing phenotype, was significantly superior compared to the inhomogeneous AH coating. In summary, our adaptable in-house FN coating secures the endothelial requirements for long-term BHL application and may promote monolayer establishment on all other blood contacting surfaces of the BHL (e.g., cannulae).

Hypothermic preservation of endothelialized gas-exchange membranes.

Endothelialization of the blood contacting surfaces of blood-contacting medical devices, such as cardiovascular prostheses or biohybrid oxygenators, represents a plausible strategy for increasing their hemocompatibility. Nevertheless, isolation and expansion of autologous endothelial cells (ECs) usually requires multiple processing steps and time to obtain sufficient cell numbers. This excludes endothelialization from application in acute situations. Off-the-shelf availability of cell-seeded biohybrid devices could be potentially facilitated by hypothermic storage. In this study, the survival of cord-blood-derived endothelial colony forming cells (ECFCs) that were seeded onto polymethylpentene (PMP) gas-exchange membranes and stored for up to 2 weeks in different commercially available and commonly used preservation media was measured. While storage at 4°C in normal growth medium (EGM-2) for 3 days resulted in massive disruption of the ECFC monolayer and a significant decline in viability, ECFC monolayers preserved in Chillprotec could recover after up to 14 days with negligible effects on their integrity and viability. ECFC monolayers preserved in Celsior, HTS-FRS, or Rokepie medium showed a significant decrease in viability after 7 days or longer periods. These results demonstrated the feasibility of hypothermic preservation of ECFC monolayers on gas-exchange membranes for up to 2 weeks, with potential application on the preservation of pre-endothelialized oxygenators and further biohybrid cardiovascular devices.

Organ Care System Lung resulted in lower apoptosis and iNOS expression in donor lungs.

Ischemia-reperfusion (IR) injury after lung transplantation is still today an important complication in up to 25% of patients. The Organ Care System (OCS) Lung, an advanced normothermic ex vivo lung perfusion system, was found to be effective in reducing primary graft dysfunction compared to standard organ care (SOC) but studies on tissue/molecular pathways that could explain these more effective clinical results are lacking. This observational longitudinal study aimed to investigate IR injury in 68 tissue specimens collected before and after reperfusion from 17 OCS and 17 SOC preserved donor lungs. Several tissue analyses including apoptosis evaluation and inducible nitric oxide synthase (iNOS) expression (by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction) were performed. Lower iNOS expression and apoptotic index were distinctive of OCS preserved tissues at pre- and post-reperfusion times, independently from potential confounding factors. Moreover, OCS recipients had lower acute cellular rejection at the first 6-month follow-up. In conclusion, IR injury, in terms of apoptosis and iNOS expression, was less frequent in OCS- than in SOC-preserved lungs, which could eventually explain a better clinical outcome. Further studies are needed to validate our data and determine the role of iNOS expression as a predictive biomarker of the complex IR injury mechanism.

Ex vivo limb perfusion for traumatic amputation in military medicine.

BACKGROUND: Limb loss has a drastic impact on a patient's life. Severe trauma to the extremities is common in current military conflicts. Among other aspects, "life before limb" damage control surgery hinders immediate replantation within the short post-traumatic timeframe, which is limited in part by the ischemic time for successful replantation. Ex vivo limb perfusion is currently being researched in animal models and shows promising results for its application in human limb replantation and allotransplantation. PRESENTATION OF THE HYPOTHESIS: The current lack of replantation possibilities in military operations with high rates of amputation can be addressed with the development of a portable ex vivo limb perfusion device, as there are several opportunities present with the introduction of this technique on the horizon. We hypothesize that ex vivo limb perfusion will enable overcoming the critical ischemic time, provide surgical opportunities such as preparation of the stump and limb, allow for spare-part surgery, enable rigorous antibiotic treatment of the limb, reduce ischemia-reperfusion injuries, enable a tissue function assessment before replantation, and enable the development of large limb transplant programs. TESTING THE HYPOTHESIS: Data from in vivo studies in porcine models are limited by the relatively short perfusion time of 24 h. In the military setting, notably longer perfusion times need to be realized. Therefore, future animal studies must focus especially on long-term perfusion, since this represents the military setting, considering the time for stabilization of the patient until evacuation to a tertiary treatment center. IMPLICATIONS OF THE HYPOTHESIS: The development and clinical introduction of ex vivo limb perfusion in the military setting could lead to a drastic reduction in the number of limb amputations among service members. Ex vivo limb perfusion enables replantation surgery in Role 4 facilities and changes the clinical setting from a highly urgent, life-threatening situation to a highly methodical, well-prepared starting point for optimal treatment of the wounded service member. With its introduction, the principle of "life before limb" will change to "life before limb before elective replantation/allotransplantation after ex vivo limb perfusion".