Einstein BIH Visiting Fellow project, funded by Stiftung Charité
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The Stiftung Charité will fund our project “Vascular Composite Tissue Allotransplantation (VCA): An integrated, multidisciplinary basic and clinical research program for hand and uterus transplantation” (Einstein BIH Visiting Fellowship) within the framework of the Private Excellence Initiative Johanna Quandt for two more years!

The Charité has a long tradition as an international leader in transplantation. Prior to starting our Einstein BIH funded project in February 2017, Vascular Composite Tissue Allotransplantation (VCA) was neither object of scientific investigations, nor offered to patients. As an Einstein BIH Visiting Fellow Prof. Stefan G. Tullius, Harvard Medical School, ignited both: a basic research group in this field and a clinical research transplant program. During the first three years of our multidisciplinary basic and clinical research program, we have been able to implement complex small animal models (mouse hindlimb, heart, skin transplant models); a rat uterus transplant model is currently established. Those models offer unique opportunities to address basic research questions of translational relevance including: organ-specific alloimmune responses, immunogenicity, and the maternal-fetal interface in uterus transplantation.
An enthusiast clinical, multi-disciplinary has been established, led and mentored by Prof. Tullius that has brought preparatory surgical exercises and clinical algorithms for VCA at the Charité on the way.

Stiftung Charité is an independent charitable foundation. It was endowed in 2005 by entrepreneur Johanna Quandt, who entrusted it with the mission of supporting the innovative potential and excellence of Berlin’s university medicine, which can look back on a rich tradition in medical research and patient care. Thereby, the foundation is active in two focal areas: promoting technology transfer between the laboratory and the clinic as well as improving the framework conditions for innovation and entrepreneurship in medicine. Since 2014, Stiftung Charité is also funding the life sciences in Berlin by its Private Excellence Initiative Johanna Quandt.
Development of a rat liver machine perfusion system for normothermic and subnormothermic conditions
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Tissue Engineering Part A accepted our paper on the "Development of a rat liver machine perfusion system for normothermic and subnormothermic conditions" (Tissue Eng Part A. 2019 Jul 31. doi: 10.1089/ten.TEA.2019.0152. [Epub ahead of print[) !
Ex vivo liver machine perfusion is a promising alternative for preservation of liver grafts from extended criteria donors. Small animal models can be used to evaluate different perfusion conditions. We here describe the development of a miniaturized ex vivo machine perfusion system for rat liver grafts, evaluating cell-free and erythrocyte-based perfusion solutions, subnormothermic and normothermic temperatures and dialysis. A perfusion chamber was designed after a suitable liver position was identified. Normothermic ex vivo liver perfusion (NEVLP) required supplementation of erythrocytes to reduce cell damage. Perfusion with erythrocytes led to rising potassium levels after 12 hours (NEVLP, 16.2mmol/l, interquartile range (IQR) 5.7 and subnormothermic ex vivo liver perfusion (SNEVLP), 12.8 mmol/l, IQR 3.5), which were normalized by dialysis using a laboratory dialysis membrane (NEVLP, 6.2 mmol/l, IQR 0.5 and SNEVLP, 5.3 mmol/l, IQR 0.1; p=0.004). Livers treated with NEVLP conditions showed higher bile production (18.52mg/h/g, IQR 8.2) compared to livers perfused under SNEVLP conditions (0.4 mg/h/g, IQR 1.2, p=0.01). Reducing the perfusion volume from 100ml to 50ml allowed for higher erythrocytes concentrations, leading to significantly lower transaminases (15.75 U/l/ml, IQR 2.29 vs. 5.97 U/l/ml, IQR 18.07, p=0.002). In conclusion, a well-designed perfusion system, appropriate oxygen carriers, dialysis, and miniaturization of the perfusion volume are critical features for successful miniaturized ex vivo liver machine perfusion.

Authors are M. Nösser, J.M.G.V. Gassner, S. Moosburner, D. Wyrwal, F. Claussen, K.H. Hillebrandt, R. Horner, P. Tang, A. Reutzel-Selke, D. Polenz, R. Arsenic, J. Pratschke, I.M. Sauer, and N. Raschzok.
TEBURU – our latest bioreactor system
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Three‐dimensional tissue cultures are important models for the study of cell‐cell and cell‐matrix interactions, as well as, to investigate tissue repair and reconstruction pathways. Therefore, we designed a reproducible and easy to handle printable bioreactor system (Teburu), that is applicable for different approaches of pathway investigation and targeted tissue repair using human tissue slices as a three‐dimensional cell culture model. Here, we definitively describe Teburu as a controlled environment to reseed a 500‐µm thick decellularized human liver slice using human mesenchymal stroma cells. During a cultivation period of eight days, Teburu, as a semi‐open and low consumption system, was capable to maintain steady pH and oxygenation levels. Its combination with additional modules delivers an applicability for a wide range of tissue engineering approaches under optimal culture conditions.

"Teburu—Open source 3D printable bioreactor for tissue slices as dynamic three‐dimensional cell culture models" was published in Artif Organs. 2019 Jun 18. doi: 10.1111/aor.13518. [Epub ahead of print]. Authors are A. Daneshgar, P. Tang, C. Remde, M. Lommel, S. Moosburner, U. Kertzscher, O. Klein, M. Weinhart, J. Pratschke, I.M. Sauer, and K.H. Hillebrandt.
EUROSTARS project
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Within the Eurostars project „Comprehensive qualitative/quantitative multi-pathogen IVD workflow for immunocompromised patients (Im-compr-IVD)“ an IVD workflow covering sample preparation up to clinical relevant diagnosis of infection in immunocompromised patients will be developed. Deliverables are:
  • Design, development and preclinical validation of QIC-Finder assay with novel primers and probes for qualitative and quantitative screening of 23 pathogens (bacterial, viral, fungal and parasitic);
  • Detection instrumentation and interpretation software optimized for assay performance;
  • Instructions for high quality DNA/RNA extraction from plasma.
Partners are Pathofinder (Netherlands, Coordinator), Ella Biotech (Germany), IT-IS International Ltd. (United Kingdom) and Charité - Universitätsmedizin Berlin.

Eurostars projects are co-funded by EUREKA member countries and the European Union Horizon 2020 Framework program.
DFG Research Grant for PD Dr. Moritz Schmelzle
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Priv.-Doz. Dr. Moritz Schmelzle receives additional financial support for his project "CD39-dependent regulation of innate immune responses and modulation of exacerbated sterile inflammation in acute-on-chronic liver failure" (DFG Research Grant 299534341, SCHM2661/3-2).
Acute on chronic liver failure (ACLF) is defined as an acute hepatic insult in patients with chronic liver disease and is characterized by high death rates. Systemic inflammation is considered a hallmark of ACLF and can be linked to progression of liver failure and clinical deterioration. Criteria for ACLF and the systemic inflammatory response syndrome (SIRS) substantially overlap and support the assumption of mechanistic similarities between both syndromes. Thus, modulating inflammation and the linked immune responses in ACLF might help to restore homeostasis and improve of regenerative capacities of the injured liver.We hypothesize that the catalyzed hydrolysis of purinergic damage-associated molecular patterns (DAMPS), such as extracellular ATP, by the ectonucleotidase CD39 is crucially involved in the regulation of innate immune responses and the modulation of exacerbated sterile inflammation in ACLF. We here aim to describe characteristics and functions of monocyte subsets in ACLF and to investigate implications of purinergic signaling. We further plan to investigate the therapeutical relevance of non-classical monocytes and the immune-type ectonucleotidase CD39 in experimental ACLF. Finally, we will evaluate the clinical significance of cellular and non-cellular immune responses in ACLF patients enrolled in the GRAFT Trial.
Priv.-Doz. Dr. Felix Krenzien & Priv.-Doz. Dr. Christian Benzing
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Today Dr. Felix Krenzien and Dr. Christian Benzing received they post-doctoral lecturing qualification (Habilitation) at Charité – Universitätsmedizin Berlin.
The title of Felix Krenzien's Habilitationsschrift is "Der differenzierte Einfluss der Seneszenz auf die Organtransplantation und Leberteilresektion", Christian Benzing focussed on the "Untersuchung der gesundheitsbezogenen Lebensqualität und der psychischen Gesundheit nach Lebertransplantation".

Congratulations !
Two new BIH Charité Junior Clinician Scientists
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Dr. Karl Hillebrandt and Dr. Matthäus Felsenstein successfully applied for the BIH Charité Junior Clinician Scientist Program. Karl Hillebrandt will continue his work on human decellularized liver slices as 3D platform for in vitro models of cholangiocellular carcinoma. Matthäus Felsenstein focusses on derivation of normal pancreatic duct cells from human primary tissue and their stepwise genetic modification in vitro using CRISPR/Cas9 .

Congratulations!
Strategies based on organ decellularization and recellularization
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Transplantation is the only curative treatment option available for patients suffering from end-stage organ failure, improving their quality of life and long-term survival. However, because of organ scarcity, only a small number of these patients actually benefit from transplantation. Alternative treatment options are needed to address this problem. The technique of whole-organ decellularization and recellularization has attracted increasing attention in the last decade. Decellularization includes the removal of all cellular components from an organ, while simultaneously preserving the micro and macro anatomy of the extracellular matrix. These bioscaffolds are subsequently repopulated with patient-derived cells, thus constructing a personalized neo-organ and ideally eliminating the need for immunosuppression. However, crucial problems have not yet been satisfyingly addressed and remain to be resolved, such as organ and cell sources.

In this paper "Strategies based on organ decellularization and recellularization" (Transpl Int. 2019; 32(6):571-585), we focus on the actual state of organ de- and recellularization, as well as the problems and future challenges. Authors are K.H. Hillebrandt, H. Everwien, N. Haep, E. Keshi, J. Pratschke, and I.M. Sauer.
Impact of Percoll purification on isolation of primary human hepatocytes
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Scientific Reports published our paper "Impact of Percoll purification on isolation of primary human hepatocytes" (Sci Rep. 2019 Apr 25;9(1):6542). Authors are R. Horner, J.G.M.V. Gassner, M. Kluge, P. Tang, S. Lippert, K.H. Hillebrandt, S. Moosburner, A. Reutzel-Selke, J. Pratschke, I.M. Sauer, and N. Raschzok.

Research and therapeutic applications create a high demand for primary human hepatocytes. The limiting factor for their utilization is the availability of metabolically active hepatocytes in large quantities. Centrifugation through Percoll, which is commonly performed during hepatocyte isolation, has so far not been systematically evaluated in the scientific literature. 27 hepatocyte isolations were performed using a two-step perfusion technique on tissue obtained from partial liver resections. Cells were seeded with or without having undergone the centrifugation step through 25% Percoll. Cell yield, function, purity, viability and rate of bacterial contamination were assessed over a period of 6 days. Viable yield without Percoll purification was 42.4 × 106 (SEM ± 4.6 × 106) cells/g tissue. An average of 59% of cells were recovered after Percoll treatment. There were neither significant differences in the functional performance of cells, nor regarding presence of non-parenchymal liver cells. In five cases with initial viability of <80%, viability was significantly increased by Percoll purification (71.6 to 87.7%, p = 0.03). Considering our data and the massive cell loss due to Percoll purification, we suggest that this step can be omitted if the initial viability is high, whereas low viabilities can be improved by Percoll centrifugation.
Immunologic cellular characteristics of the tumour microenvironment of HCC
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"Immunologic cellular characteristics of the tumour microenvironment of hepatocellular carcinoma drive patient outcomes" is available via World J Surg Oncol. 2019; 17(1):97

Anti-tumour immune competence has an impact in hepatocarcinogenesis and success of anti-cancer therapies. Tumour-infiltrating lymphocytes (TILs) and monocytes/macrophages (TAMs) are proposed to have significance in cancer. However, there is only limited data concerning their impact on patient outcome and survival in hepatocellular carcinoma (HCC).
Frequencies of CD68+, CD163+ M2-polarized TAMs and TILs were measured in de novo HCC tumours in non-cirrhosis (n = 58) using immunohistology and correlated to patients' clinicopathological characteristics and survival rates.
Patients with tumours marked by appearance of TILs and CD68+ TAMs showed an improved 1-, 3- and 5-year recurrence-free survival (all p ≤ 0.05). CD68+ TAMs were associated with reduced incidence of recurrent and multifocal disease. Conversely, CD163+ TAMs were associated with multifocal HCC and lymphangiosis carcinomatosa (all p ≤ 0.05).
TILs and CD68+ TAMs are associated with multiple tumour characteristics and patient survival in HCC. However, there is only scarce data about the biology underlying their mechanistic involvement in human tumour progression. Thus, experimental data on functional links might help develop novel immunologic checkpoint inhibitor targets for liver cancer.

Authors are G. Atanasov, K. Dino, K. Schierle, C. Dietel, G. Aust, J. Pratschke, D. Seehofer, M. Schmelzle, H.M. Hau.
Dr. med. Martin Kluge
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Martin Kluge is wearing excellent suits and successfully defended his doctoral thesis magna cum laude! He examined the effects of the magnetic field of magnetic resonance imaging (MRI) systems on cells labeled with micrometer-sized iron oxide particles.

Congratulations!

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Cytokine production of human CD4+ memory T cells
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Nature Communication accepted the manuscript "Progressive expression of killer-like receptors and GPR56 defines the cytokine production of human CD4+ memory T cells" for publication. Authors are Kim-Long Truong, Stephan Schlickeiser, Katrin Vogt, David Boës, Katarina Stanko, Christine Appelt, Mathias Streitz, Gerald Grütz, Nadja Stobutzki, Christian Meisel, Christina Iwert, Stefan Tomiuk, Julia Polansky, Andreas Pascher, Nina Babel, Ulrik Stervbo, Igor Sauer, Undine Gerlach, and Birgit Sawitzki.

All memory T cells mount an accelerated response on antigen reencouter, but significant functional heterogeneity is present within the respective memory T cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, KLRB1, KLRG1, GPR56 and KLRF1, help define “low”, “high” or “exhausted” cytokine producers within human peripheral and intra-hepatic CD4+ memory T cells. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with reduced TNF/IFN-γ production and T cell exhaustion. Lastly, TCRbeta repertoire analysis and in vitro differentiation support a regulated, successive expression for these markers during CD4+ memory T cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.
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Critical Care for Potential Liver Transplant Candidates
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The book Critical Care for Potential Liver Transplant Candidates
(D. Bezinover and F. Saner [Eds.]) focuses on patients with end-stage-liver disease (ESLD) who could possibly qualify for liver transplant. This patient cohort raises many problems: who should be treated and also, when is it too late for transplant? The authors are all dedicated experts in the field of ESLD/liver transplantation, but from different disciplines with different views of the problem.
In the past 15 years many things have changed in the treatment for these patients: cardiac assessment, treatment of porto-pulmonary hypertension, hemodynamics, coagulation assessment and management, diagnosis of kidney failure, and the timing of dialysis. These issues are comprehensively discussed in this book, in order to provide physicians starting in the field of transplantation an overview of different areas of concern.
This book is aimed at specialists and trainees in critical care, hepatology, anesthesia, surgery, and nephrology.

N. Raschzok, K.H. Hillebrandt and I.M. Sauer contributed with the chapter "Liver Assist Systems for Bridging to Transplantation: Devices and Concepts".

More information via this link.
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DFG grant for Linda Feldbrügge
Dr. Linda Feldbrügge receives a research grant from the Deutsche Forschungsgemeinschaft (DFG) for her project "Purinergic regulation of inflammation in liver fibrosis by ectonucleoside triphosphate diphosphohydrolase-3 (ENTPD3)“.

ENTPD3, expressed by macrophages and various other cell types, modulates inflammation and tissue regeneration by scavenging extracellular ATP and ADP. As demonstrated by her preliminary work, ENTPD3 appears to play a deleterious role in liver fibrosis. Her new project aims to define the mechanisms of ENTPD3 mediated modulation of macrophage function and regulation of liver fibrosis, and test their relevance in human liver fibrosis.

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Dr. med. Antje Butter
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Today, Antje Butter successfully defended her doctoral thesis magna cum laude!
Congratulations!

Antje was involved in basic research with respect to liver decellularization and recellularization. A proprietary, customized bioreactor was established to repopulate decellularized rat livers with primary rat hepatocytes via the hepatic artery and to subsequently evaluate graft morphology and function during 7 days of ex vivo perfusion. More information via this link.
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Percoll purification after isolation of Primary Human Hepatocytes
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The manuscript "Isolation of Primary Human Hepatocytes: Is Percoll Purification Really Necessary?" was accepted for publication in Scientific Reports.
Authors are Rosa Horner, Jospeh G.M.V. Gassner, Martin Kluge M, Peter Tang, Steffen Lippert, Karl H. Hillebrandt, Simon Moosburner, Anja Reutzel-Selke, Johann Pratschke, Igor M. Sauer and Nathanael Raschzok.

Research and therapeutic applications create a high demand for primary human hepatocytes. The limiting factor for their utilization is the availability of metabolically active hepatocytes in large quantities. Centrifugation through Percoll, which is commonly performed during hepatocyte isolation, has so far not been systematically evaluated in the scientific literature. 27 hepatocyte isolations were performed using a two-step perfusion technique on tissue obtained from partial liver resections. Cells were seeded with or without having undergone the centrifugation step through 25% Percoll. Cell yield, function, purity, viability and rate of bacterial contamination were assessed over a period of 6 days. Viable yield without Percoll purification was 42.4 x 106 (SEM ± 4.6 x 106) cells/g tissue. An average of 59% of cells were recovered after Percoll treatment. There were neither significant differences in the functional performance of cells, nor regarding presence of non-parenchymal liver cells. In five cases with initial viability of <80%, viability was significantly increased by Percoll purification (71.6 to 87.7%, p=0.03). Considering our data and the massive cell loss due to Percoll purification, we suggest that this step can be omitted if the initial viability is high, whereas low viabilities can be improved by Percoll centrifugation.
Read More
Critical Care for Potential Liver Transplant Candidates
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The book Critical Care for Potential Liver Transplant Candidates
(D. Bezinover and F. Saner [Eds.]) focuses on patients with end-stage-liver disease (ESLD) who could possibly qualify for liver transplant. This patient cohort raises many problems: who should be treated and also, when is it too late for transplant? The authors are all dedicated experts in the field of ESLD/liver transplantation, but from different disciplines with different views of the problem.
In the past 15 years many things have changed in the treatment for these patients: cardiac assessment, treatment of porto-pulmonary hypertension, hemodynamics, coagulation assessment and management, diagnosis of kidney failure, and the timing of dialysis. These issues are comprehensively discussed in this book, in order to provide physicians starting in the field of transplantation an overview of different areas of concern.
This book is aimed at specialists and trainees in critical care, hepatology, anesthesia, surgery, and nephrology.

N. Raschzok, K.H. Hillebrandt and I.M. Sauer contributed with the chapter "Liver Assist Systems for Bridging to Transplantation: Devices and Concepts".

More information via this link.
Read More
Matters of Activity. Image Space Material
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Prof. I.M. Sauer and Prof. J. Pratschke became principal investigators in the new Cluster of Exzellence Matters of Activity. Image Space Material. This Cluster will explore materials’ own inner activity, which can be discovered as a new source of innovative strategies and mechanisms for rethinking the relationship between the analog and the digital and for designing more sustainable and energy-efficient technologies.
The project’s central vision is to develop images, spaces, and materials as active structures of a new physical and symbolic reality, in which nature and culture intertwine in a novel way. In this context, interdisciplinary research and development of sustainable processes and structures is a key priority in all areas of visual-material character, such as wearables, materials technology, medical technology, logistics, architecture, and robotics. More than 40 disciplines are systematically investigating design strategies for materials and structures that adapt to specific requirements and the environment. The cluster relies on a new role for design within the context of growing diversity and the continuous improvement of materials and forms of visualization in all disciplines.
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Composite tissue allotransplantation: opportunities and challenges
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"Composite tissue allotransplantation: opportunities and challenges" is available in Cellular & Molecular Immunology (Cell Mol Immunol. 2019 Mar 6. doi: 10.1038/s41423-019-0215-3. [Epub ahead of print]). Authors are J. Iske, Y. Nian, R. Maenosono, M. Maurer, I.M. Sauer & S.G. Tullius.

Vascularized composite allotransplants (VCAs) have unique properties because of diverse tissue components transplanted en mass as a single unit. In addition to surgery, this type of transplant also faces enormous immunological challenges that demand a detailed analysis of all aspects of alloimmune responses, organ preservation, and injury, as well as the immunogenicity of various tissues within the VCA grafts to further improve graft and patient outcomes. Moreover, the side effects of long-term immunosuppression for VCA patients need to be carefully balanced with the potential benefit of a non-life-saving procedure. In this review article, we provide a comprehensive update on limb and face transplantation, with a specific emphasis on the alloimmune responses to VCA, established and novel immunosuppressive treatments, and patient outcomes.
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Human stem cells promote liver regeneration after partial hepatectomy
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"Human Stem Cells Promote Liver Regeneration After Partial Hepatectomy in BALB/C Nude Mice" will be published in J Surg Res. 2019 (Mar 4;239:191-200. doi: 10.1016/j.jss.2019.02.010. [Epub ahead of print]).
Authors are S. Wabitsch, Ch. Benzing, F. Krenzien, K. Splith, P.K. Haber, A. Arnold, M. Nösser, C. Kamalia, F. Hermann, Ch. Günther, D. Hirsch, I.M. Sauer, J. Pratschke, and M. Schmelzle.

Mesenchymal stem cells (MSCs) have been suggested to augment liver regeneration after surgically and pharmacologically induced liver failure. To further investigate this we processed human bone marrow-derived MSC according to good manufacturing practice (GMP) and tested those cells for their modulatory capacities of metabolic alterations and liver regeneration after partial hepatectomy in BALB/c nude mice.

Human bone marrow-derived MSC attenuate metabolic alterations and improve liver regeneration after partial hepatectomy in BALB/c nude mice. Obtained results using GMP-processed human MSC suggest functional links between fat accumulation and hepatocyte proliferation, without any evidence for cellular homing. This study using GMP-proceeded MSC has important regulatory implications for an urgently needed translation into a clinical trial.
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