Search

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease., Heller et al analyze transcriptomic and epigenetic datasets from human PSCs differentiating into pancreatic progenitors to elucidate the regulatory mechanisms behind pancreatic development. The authors report that the transcription factor ONECUT1 is expressed specifically in pancreatic development and associates with other key TFs (such as FOXA2, GATA6, PDX1) during endocrine specification.

Summary Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies., Highlights • The retina on chip (RoC) combines iPSC-organoid and organ-on-chip technology • We characterized seven adeno-associated viruses (AAVs) in terms of transduction efficacy and cell tropism • The RoC is a novel translational tool for AAV vector characterization • The presented workflow could be a blueprint for future drug development, Loskill and colleagues demonstrate that the retina on chip combining hiPSC-organoid and organ-on-chip technologies can serve as a novel translational tool for characterization and validation of AAV vectors by mimicking clinically relevant application routes. Seven AAVs were characterized in terms of transduction efficacy, cell tropism, and pharmacokinetics, showcasing a workflow that could serve as blueprint for future drug and gene therapy development.

Background Neurotrophic growth factors can stabilize the intestinal barrier by preventing the apoptosis of enteric glial cells (EGCs) and enterocytes. We reasoned that a selective 5-HT1A receptor agonist may have neuroprotective properties in the gut and that topical application of SR57746A might be an effective treatment strategy in inflammatory bowel disease (IBD). Methods The therapeutic potential of 5-HT1A receptor agonist SR57746A in IBD was evaluated in vitro (nontransformed NCM460 colonic epithelial cells, SW480 colorectal carcinoma cells) and in vivo (murine dextran sulfate sodium [DSS] colitis and CD4-T-cell transfer colitis). In vitro, we analyzed the effect of SR57746A on apoptosis in intestinal epithelial cells (IECs) and EGCs, and upon proliferation, migration, and intracellular signaling in IECs. In vivo, the effect of topical application of SR57746 on disease activity and on histological and endoscopic findings was compared with intraperitoneal infliximab and placebo, respectively. Results The SR57746A activates PI3-K/AKT- and ERK-signaling in IECs. Depending on ERK- and AKT activation, SR57746A potently prevents apoptosis of IECs without inducing proliferation or migration in these cells. Moreover, SR57746A prevented apoptosis in EGCs in vitro. Topical SR57746A treatment significantly reduced mucosal injury in 2 experimental murine colitis models and was as effective as intraperitoneal infliximab treatment. Conclusions Treatment with SR57746A prevents inflammatory cell damage and apoptosis in IECs and EGCs, similar to the neurotrophic effects of EGCs on IECs. Topical treatment with SR57746A could be a candidate for clinical evaluation in the treatment of IBD.

Zusammenfassung SARS-CoV-2 ist eine neuartige humanpathogene Coronavirus-Variante, deren Prädilektion für den Atemwegstrakt zu einer raschen pandemischen Verbreitung durch viruspartikelhaltige Aerosole geführt hat. Die individuelle Organsuszeptibilität wird maßgeblich durch die Besatzdichte mit dem membranständigen Rezeptormolekül ACE2 bestimmt, das als zentraler Interaktionspartner für das virale Spike-Protein den Adhäsions- und Fusionsprozess vermittelt und somit die Grundvoraussetzung zur Aufnahme des Virusgenoms in die Wirtszelle schafft. Ausgehend von einem umfangreichen Datensatz klinischer Studien und Fallberichte, gilt es mittlerweile als gesichert, dass auch bestimmte Zellpopulationen des Verdauungstrakts sowie des funktionell angegliederten olfaktorisch-gustatorischen Systems über die erforderliche Rezeptorenausstattung verfügen und somit durch SARS-CoV-2 „angreifbar“ sind. Zahlreiche Berichte über gastrointestinale Beschwerden und Laborabnormalitäten sind als Indizien für relevante Organdysfunktionen zu werten und untermauern die klinische Bedeutsamkeit einer Mitbeteiligung des Verdauungstrakts im Rahmen einer SARS-CoV-2-Infektion. Organoide sind dreidimensional wachsende In-vitro-Replikate von Organgeweben und nehmen insbesondere dank der organtypisch komplexen zellulären Zusammensetzung und Imitation der physiologischen Funktionsweise von Primärzellen einen hohen Stellenwert für die infektiologische Grundlagenforschung ein. Diese Übersichtsarbeit befasst sich thematisch mit den pathophysiologischen Aspekten der Infektion verdauungsrelevanter Organe mit SARS-CoV-2 unter besonderer Würdigung existierender organoid- oder primärzellkulturbasierter Infektionsmodelle und der daraus hervorgegangenen Erkenntnisse.

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

Background Uncertainty about the optimal respiratory support strategies in critically ill COVID-19 patients is widespread. While the risks and benefits of noninvasive techniques versus early invasive mechanical ventilation (IMV) are intensely debated, actual evidence is lacking. We sought to assess the risks and benefits of different respiratory support strategies, employed in intensive care units during the first months of the COVID-19 pandemic on intubation and intensive care unit (ICU) mortality rates. Methods Subanalysis of a prospective, multinational registry of critically ill COVID-19 patients. Patients were subclassified into standard oxygen therapy ≥10 L/min (SOT), high-flow oxygen therapy (HFNC), noninvasive positive-pressure ventilation (NIV), and early IMV, according to the respiratory support strategy employed at the day of admission to ICU. Propensity score matching was performed to ensure comparability between groups. Results Initially, 1421 patients were assessed for possible study inclusion. Of these, 351 patients (85 SOT, 87 HFNC, 87 NIV, and 92 IMV) remained eligible for full analysis after propensity score matching. 55% of patients initially receiving noninvasive respiratory support required IMV. The intubation rate was lower in patients initially ventilated with HFNC and NIV compared to those who received SOT (SOT: 64%, HFNC: 52%, NIV: 49%, p = 0.025). Compared to the other respiratory support strategies, NIV was associated with a higher overall ICU mortality (SOT: 18%, HFNC: 20%, NIV: 37%, IMV: 25%, p = 0.016). Conclusion In this cohort of critically ill patients with COVID-19, a trial of HFNC appeared to be the most balanced initial respiratory support strategy, given the reduced intubation rate and comparable ICU mortality rate. Nonetheless, considering the uncertainty and stress associated with the COVID-19 pandemic, SOT and early IMV represented safe initial respiratory support strategies. The presented findings, in agreement with classic ARDS literature, suggest that NIV should be avoided whenever possible due to the elevated ICU mortality risk.

Background : Accurate risk stratification of critically ill patients with coronavirus disease 2019 (COVID-19) is essential for optimizing resource allocation, delivering targeted interventions, and maximizing patient survival probability. Machine learning (ML) techniques are attracting increased interest for the development of prediction models as they excel in the analysis of complex signals in data-rich environments such as critical care. Methods : We retrieved data on patients with COVID-19 admitted to an intensive care unit (ICU) between March and October 2020 from the RIsk Stratification in COVID-19 patients in the Intensive Care Unit (RISC-19-ICU) registry. We applied the Extreme Gradient Boosting (XGBoost) algorithm to the data to predict as a binary outcome the increase or decrease in patients’ Sequential Organ Failure Assessment (SOFA) score on day 5 after ICU admission. The model was iteratively cross-validated in different subsets of the study cohort. Results : The final study population consisted of 675 patients. The XGBoost model correctly predicted a decrease in SOFA score in 320/385 (83%) critically ill COVID-19 patients, and an increase in the score in 210/290 (72%) patients. The area under the mean receiver operating characteristic curve for XGBoost was significantly higher than that for the logistic regression model {0.86 vs. 0.69, P

Frulloni, Luca/0000-0001-7417-2655; Hart, Phil/0000-0003-4346-6196; Capurso, Gabriele/0000-0002-0019-8753; de-Madaria, Enrique/0000-0002-2412-9541; Lohr, Matthias/0000-0002-7647-198X; Frokjaer, Jens Brondum/0000-0001-8722-0070 WOS:000542363500001 PubMed: 32552502 The overall objective of these guidelines is to provide evidence-based recommendations for the diagnosis and management of immunoglobulin G4 (IgG4)-related digestive disease in adults and children. IgG4-related digestive disease can be diagnosed only with a comprehensive work-up that includes histology, organ morphology at imaging, serology, search for other organ involvement, and response to glucocorticoid treatment. Indications for treatment are symptomatic patients with obstructive jaundice, abdominal pain, posterior pancreatic pain, and involvement of extra-pancreatic digestive organs, including IgG4-related cholangitis. Treatment with glucocorticoids should be weight-based and initiated at a dose of 0.6-0.8 mg/kg body weight/day orally (typical starting dose 30-40 mg/day prednisone equivalent) for 1 month to induce remission and then be tapered within two additional months. Response to initial treatment should be assessed at week 2-4 with clinical, biochemical and morphological markers. Maintenance treatment with glucocorticoids should be considered in multi-organ disease or history of relapse. If there is no change in disease activity and burden within 3 months, the diagnosis should be reconsidered. If the disease relapsed during the 3 months of treatment, immunosuppressive drugs should be added. National Societies Committee of the United European Gastroenterology (UEG) We gratefully acknowledge the support from the National Societies Committee of the United European Gastroenterology (UEG) for the conduct of these guidelines independent from other sources. No other funding was received.

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses., The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, but its spike protein doesn’t efficiently bind human ACE2. Here, the authors show that exchange of spike residue 403 between RaTG13 and SARS-CoV-2 spike proteins affects binding to human ACE2 and entry of pseudotyped viruses.

Porous materials are relevant for a broad range of technologies from catalysis and filtration, to tissue engineering and lightweight structures. Controlling the porosity of these materials over multiple length scales often leads to enticing new functionalities and higher efficiency but has been limited by manufacturing challenges and the poor understanding of the properties of hierarchical structures. Here, we report an experimental platform for the design and manufacturing of hierarchical porous materials via the stereolithographic printing of stable photo-curable Pickering emulsions. In the printing process, the micron-sized droplets of the emulsified resins work as soft templates for the incorporation of microscale porosity within sequentially photo-polymerized layers. The light patterns used to polymerize each layer on the building stage further generate controlled pores with bespoke three-dimensional geometries at the millimetre scale. Using this combined fabrication approach, we create architectured lattices with mechanical properties tuneable over several orders of magnitude and large complex-shaped inorganic objects with unprecedented porous designs., Scientific Reports, 11 (1), ISSN:2045-2322

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and associated clinical sequelae requires well-coordinated metabolic and immune responses that limit viral spread and promote recovery of damaged systems. In order to understand potential mechanisms and interactions that influence coronavirus disease 2019 (COVID-19) outcomes, we performed a multi-omics analysis on hospitalised COVID-19 patients and compared those with the most severe outcome (i.e. death) to those with severe non-fatal disease, or mild/moderate disease, that recovered. A distinct subset of 8 cytokines and 140 metabolites in sera identified those with a fatal outcome to infection. In addition, elevated levels of multiple pathobionts and lower levels of protective or anti-inflammatory microbes were observed in the faecal microbiome of those with the poorest clinical outcomes. Weighted gene correlation network analysis (WGCNA) identified modules that associated severity-associated cytokines with tryptophan metabolism, coagulation-linked fibrinopeptides, and bile acids with multiple pathobionts. In contrast, less severe clinical outcomes associated with clusters of anti-inflammatory microbes such as Bifidobacterium or Ruminococcus, short chain fatty acids (SCFAs) and IL-17A. Our study uncovered distinct mechanistic modules that link host and microbiome processes with fatal outcomes to SARS-CoV-2 infection. These features may be useful to identify at risk individuals, but also highlight a role for the microbiome in modifying hyperinflammatory responses to SARS-CoV-2 and other infectious agents.

Age-related macular degeneration (AMD) is among the most common causes of irreversible vision loss. Disease progression is strongly associated with age-related pathological changes of retinal pigment epithelial (RPE) cells, such as accumulation of intracellular lipid-containing cell debris, extracellular lipid-rich deposits (drusen) and collagen-rich basal laminar deposits. Current AMD models provide a limited understanding of the complex pathomechanisms, revealing the lack of adequate physiological human AMD models. In this study, we developed anin vitromodel applicable for the exploration of AMD pathomechanisms and risk factors for AMD progression and drusen formation. Advanced 3D culturing technologies allow long-term cultivation of hiPSC-derived RPE organoids (RPEorg) for up to 360 days, which is the time frame necessary for the development of an AMD-like phenotype. Aged RPEorg exhibit hallmarks of AMD and age-related alterations such as increased autofluorescence, accumulation of lipid droplets, calcification, and the formation of extracellular clusters of the drusen-associated proteins such as apolipoprotein E (APOE) and tissue inhibitor of metalloproteinases 3 (TIMP3). Electron microscopy further reveals drusen-like extracellular deposits mimicking the signs of late drusen formation and AMD progression. In summary, our results demonstrate that hiPSC-derived 3D RPEorg provide a promising model to study age-associated RPE pathology and drusen formation. We show here that RPEorg are applicable for disease modelling studies and early stages of drug development and provide the opportunity to uncover inter-individual genetic and epigenetic factors that alter the course of the disease.

Simple Summary The mutational landscape of pancreatic ductal adenocarcinoma has been delineated in large clinical studies. Such retrospective characterization, however, is limited to late stages of the disease, and exploring the complexity of carcinogenic routes is in its infancy. Here, we provide an in vitro and in vivo model system to directly investigate human PDAC development after the induction of specific oncogenic insults in a cancer-prone ancestry. We reprogrammed plucked human hair-derived keratinocytes from two siblings harboring a pathogenic CDKN2A variant putatively predisposing for PDAC to induced pluripotent stem cells (iPSCs) and additionally introduced an inducible KRASG12D piggyBac expression cassette. Upon KRASG12D induction in differentiated pancreatic duct-like organoids, structural and molecular changes were observed. After orthotopic transplantation either a high-grade precancer lesion or PDAC-like tumor developed. Hereby, we provide a hereditary human pancreatic cancer model which enables further dissection of tumor initiation and early development starting from patient-specific CDKN2A-mutated pluripotent stem cells. Abstract Patient-derived induced pluripotent stem cells (iPSCs) provide a unique platform to study hereditary disorders and predisposition syndromes by resembling germline mutations of affected individuals and by their potential to differentiate into nearly every cell type of the human body. We employed plucked human hair from two siblings with a family history of cancer carrying a pathogenic CDKN2A variant, P16-p.G101W/P14-p.R115L, to generate patient-specific iPSCs in a cancer-prone ancestry for downstream analytics. The differentiation capacity to pancreatic progenitors and to pancreatic duct-like organoids (PDLOs) according to a recently developed protocol remained unaffected. Upon inducible expression of KRASG12D using a piggyBac transposon system in CDKN2A-mutated PDLOs, we revealed structural and molecular changes in vitro, including disturbed polarity and epithelial-to-mesenchymal (EMT) transition. CDKN2A-mutated KRASG12D PDLO xenotransplants formed either a high-grade precancer lesion or a partially dedifferentiated PDAC-like tumor. Intriguingly, P14/P53/P21 and P16/RB cell-cycle checkpoint controls have been only partly overcome in these grafts, thereby still restricting the tumorous growth. Hereby, we provide a model for hereditary human pancreatic cancer that enables dissection of tumor initiation and early development starting from patient-specific CDKN2A-mutated pluripotent stem cells., publishedVersion

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

Background: Symptoms caused by chronic pancreatitis (CP) are common but often elusive hampering therapeutic decisions. Though correlations of morphologic findings in imaging and clinical appearance remain vague. We aimed in investigating whether a distinct combination of clinical parameters can better define the extent of pancreatic insufficiency and disease burden. Methods: Data from 350 CP patients were evaluated retrospectively from a single center data base following predefined criteria: (i) confirmed CP, (ii) endoscopic ultrasound (EUS) plus (iii) fecal elastase-1 testing, (iv) age ≥18 years, and (v) Cambridge Score ≥1 on EUS evaluation. Results: In total, 182 patients (137 male, 45 female) fulfilled criteria. Median age was 52 years (range 19–88 years). Etiology distributed as follows: idiopathic 50%, alcohol 42.3%, autoimmune 7.7%. Totally, 56.6% of patients suffered from chronic pain that was significantly associated with male sex and younger age. Stool elastase-1 activity discriminated exocrine pancreatic function in Cambridge IV significantly better than in lower stages. Similarly, the endocrine function was significantly more reduced in Cambridge IV CP. Multinominal regression analysis revealed (i) presence of diabetes, (ii) presence of complications, and (iii) extent of Cambridge score as main determinants for exocrine impairment. Conclusion: A high disease burden is linked to extensive morphological alterations in EUS, while pain is more frequent in younger and male patients. The etiology of CP predicts the course of disease in terms of complications.

Pancreatic stellate cells (PSCs) are resident cells in the exocrine pancreas which contribute to pancreatic fibrogenesis and inflammation. Studies on NF-κB in pancreatitis so far focused mainly on the parenchymal and myeloid compartments. Its function in PSCs during chronic pancreatitis has not been investigated. Here we show a protective immunomodulatory function of NF-κB in PSCs. Conditional deletion of NEMO(IKKγ) in PSCs leads to spontaneous pancreatitis with elevated circulating IgM, IgG and antinuclear autoantibodies (ANA) within 18 weeks. When further challenged with experimental chronic pancreatitis, NEMOΔCol1a2 mice show an exacerbated autoimmune phenotype characterized by increased infiltration of eosinophils, B and T lymphocytes with extremely reduced latency period. These mice fail to recover even 18 weeks after the induction of pancreatitis but show sustained fibrosis and elevated eosinophil infiltration. Transcriptomic profiling shows that NEMOΔCol1a2 mice display molecular signatures resembling autoimmune pancreatitis patients. Mechanistically, we show that PSCΔNEMO cells have higher fibrogenic activities (upregulated Col1a1 and Col3a1) and produce high levels of eotaxin-2 (CCL24) which contributes to eosinophil recruitment. Corticosteroid treatment strongly attenuates the autoimmune phenotype in NEMOΔCol1a2 mice. Our findings uncover an important and unexpected immunomodulatory role specifically of NF-κB in PSCs and its deregulation may be a trigger of autoimmune pancreatitis.

Impaired DNA damage repair (DDR) is increasingly recognised as a hallmark in pancreatic ductal adenocarcinoma (PDAC). It is estimated that around 14% of human PDACs harbour mutations in genes involved in DDR, including, amongst others, BRCA1/2, PALB2, ATM, MSH2, MSH6 and MLH1. Recently, DDR intervention by PARP inhibitor therapy has demonstrated effectiveness in germline BRCA1/2-mutated PDAC. Extending this outcome to the significant proportion of human PDACs with somatic or germline mutations in DDR genes beyond BRCA1/2 might be beneficial, but there is a lack of data, and consequently, no clear recommendations are provided in the field. Therefore, an expert panel was invited by the European Society of Digestive Oncology (ESDO) to assess the current knowledge and significance of DDR as a target in PDAC treatment. The aim of this virtual, international expert meeting was to elaborate a set of consensus recommendations on testing, diagnosis and treatment of PDAC patients with alterations in DDR pathways. Ahead of the meeting, experts completed a 27-question survey evaluating the key issues. The final recommendations herein should aid in facilitating clinical practice decisions on the management of DDR-deficient PDAC., SCOPUS: ar.j, info:eu-repo/semantics/published

Einleitung: Für rekonstruktive Verfahren hätten neue und innovative Methoden zur in vitro-Differenzierung von Urothel aus patienten-eigenen induzierten pluripotenten Stammzellen (iPSCs) eine hohe Relevanz. Sie bieten das Potential zur Generierung eines differenzierten, Barriere-bildenden Uro-Epithels.[zum vollständigen Text gelangen Sie über die oben angegebene URL], 61. Jahrestagung der Südwestdeutschen Gesellschaft für Urologie e.V.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

BACKGROUND & AIMS: Hypophosphatemia (HypoP) is associated with organ dysfunction and mortality. Despite its potential severe consequences, HypoP remains poorly characterized in terms of real prevalence and timing of onset. The primary objective was to determine the prevalence of HypoP defined as blood phosphate

© Author(s) (or their employer(s)) 2020., [Objective]: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC)., [Design]: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy., [Results]: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance., [Conclusion]: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition., Main funding is provided by the German Cancer Aid grant to AK (111879). Additional funding came from the Deutsche Forschungsgemeinschaft (DFG) ’Sachbeihilfe’ (KL 2544/1–1, 1–2, 5–1, 7-1) and ’Heisenberg-Programm’ (KL 2544/6–1), the Baden-Württemberg-Foundation ExPoChip and the INDIMEDVerbund PancChip. AK, FA, MI, SB, LW and TS are either Principal Investigators or students of HEIST RTG funded by the DFG GRK 2254/1. AK is an Else-KrönerFresenius Excellence fellow. LP received funds by the Bausteinprogramm of Ulm University. PCH is supported by a Max Eder Fellowship of the German Cancer Aid (111746), a German Cancer Aid Priority Program ’Translational Oncology’ 70112505 and by a Collaborative Research Centre grant (316249678 – SFB 1279) of the German Research Foundation. EH received funding from the German Cancer Aid (PiPAC, 70112505) and the Volkswagenstiftung/Ministry for Science and Culture in Lower Saxony (ZN3222). This work was also supported by the Deutsche Forschungsgemeinschaft (AZ.96/1–3) to NA, by the Deutsche Krebshilfe (111264) to AL and by the German Cancer Aid Priority Program Translational Oncology (70112504) to LW.

Objectives: IgG4-related disease (IgG4-RD) is a chronic fibro-inflammatory disorder affecting virtually any organ. Type 1 autoimmune (type 1 AIP) is its pancreatic manifestation. To date, steroids are considered the first-line pancreatitis treatment. The CD20-binding antibody rituximab (RTX) appears a promising steroid-sparing therapy, although long-term data are lacking. We aimed to bridge this gap with a cohort of IgG4-RD patients treated with RTX and to assess the potential value of the Responder Index (RI) as a discriminatory score for disease activity. Methods: We retrospectively evaluated 46 patients from a tertiary referral centre who were diagnosed with IgG4-RD and/or type 1 AIP according to the International Consensus Diagnostic Criteria or Unifying-AIP criteria between June 2006 and August 2019. Results: Patients resembled previous cohorts in terms of characteristics, diagnosis, and therapeutic response. Thirteen of the 46 patients with IgG4-RD/type 1 AIP were treated with RTX pulse therapy due to relapse, adverse reactions to steroids, or high-risk constellations predicting a severe course of disease with multi-organ involvement. Median follow-up after diagnosis was 52 months for all subjects, and 71 months in IgG4-RD patients treated with RTX. While patients in the RTX group showed no significant response to an initial steroid pulse, clinical activity as measured by the RI significantly decreased in the short-term after RTX induction. Within 16 months, 61% of patients relapsed in the RTX group but responded well to re-induction. Clinical and laboratory parameters improved equally in response to RTX. Conclusion: RTX therapy in patients with IgG4-RD is an effective and safe treatment to induce treatment response and possible long-term remission. Repeated RTX administration after 6–9 months may be of value in reducing the risk of relapse. The RI appears to be a reasonable index to assess disease activity and to identify patients with IgG4-related disease who may benefit from B-cell-depleting therapy., publishedVersion

SummaryGene therapies using adeno-associated viruses (AAVs) are amongst the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-Chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed Retina-on-chip that merges organoid and OoC-technology, we analyzed the efficacy, kinetics and cell tropism of seven first and second generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.

A previously healthy 30-year-old woman developed severe ARDS at the beginning of the COVID-19 pandemic. SARS-CoV-2 infection was suspected, but testing was negative. Mycoplasma pneumoniae was detected by PCR in bronchoalveolar lavage fluid and blood. This case illustrates that M. pneumoniae infection can progress to septicemia and ARDS with severe respiratory failure in young healthy adults.

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic., Graphical abstract, Comparison of the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.1 shows that inhibitors under clinical evaluation are still effective in blocking entry, though the B.1.351 and P.1 variants evade antibody responses induced upon infection as well as vaccination and evade certain therapeutic antibodies.

Central airway obstruction is a life-threatening disorder causing a high physical and psychological burden to patients. Standard-of-care airway stents are silicone tubes, which provide immediate relief but are prone to migration. Thus, they require additional surgeries to be removed, which may cause tissue damage. Customized bioresorbable airway stents produced by 3D printing would be highly needed in the management of this disorder. However, biocompatible and biodegradable materials for 3D printing of elastic medical implants are still lacking. Here, we report dual-polymer photoinks for digital light 3D printing of customized and bioresorbable airway stents. These stents exhibit tunable elastomeric properties with suitable biodegradability. In vivo study in healthy rabbits confirmed biocompatibility and showed that the stents stayed in place for 7 weeks after which they became radiographically invisible. This work opens promising perspectives for the rapid manufacturing of the customized medical devices for which high precision, elasticity, and degradability are sought., Science Advances, 7 (6), ISSN:2375-2548

Personalized invitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype invitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable invitro and invivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Real-time isolation, propagation, and pharmacotyping of patient-derived pancreatic cancer organoids (PDOs) may enable treatment response prediction and personalization of pancreatic cancer (PC) therapy. In our methodology, PDOs are isolated from 54 patients with suspected or confirmed PC in the framework of a prospective feasibility trial. The drug response of single agents is determined by a viability assay. Areas under the curves (AUC) are clustered for each drug, and a prediction score is developed for combined regimens. Pharmacotyping profiles are obtained from 28 PDOs (efficacy 63.6%) after a median of 53 days (range 21–126 days). PDOs exhibit heterogeneous responses to the standard-of-care drugs, and are classified into high, intermediate, or low responder categories. Our developed prediction model allows a successful response prediction in treatment-naïve patients with an accuracy of 91.1% for first-line and 80.0% for second-line regimens, respectively. The power of prediction declines in pretreated patients (accuracy 40.0%), particularly with more than one prior line of chemotherapy. Progression-free survival (PFS) is significantly longer in previously treatment-naïve patients receiving a predicted tumor sensitive compared to a predicted tumor resistant regimen (mPFS 141 vs. 46 days; p = 0.0048). In conclusion, generation and pharmacotyping of PDOs is feasible in clinical routine and may provide substantial benefit., publishedVersion

Pancreatic ductal adenocarcinoma remains a major challenge in cancer medicine. Given the increase in incidence and mortality, interdisciplinary research is necessary to translate basic knowledge into therapeutic strategies improving the outcome of patients. On the 4th and 5th of February 2021, three German pancreatic cancer research centers, the Clinical Research Unit 5002 from Gottingen, the Collaborative Research Center 1321 from Munich, and Clinical Research Unit 325 from Marburg organized the 1st Virtual Gottingen-Munich-Marburg Pancreatic Cancer Meeting in order to foster scientific exchange. This report summarizes current research and proceedings presented during that meeting.

To date, it is still unclear how cancer stem cells (CSCs) regulate their stemness properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. In this study, we investigate how telomerase regulation affects cancer stem cell biology in pancreatic ductal adenocarcinoma (PDAC), and delineate the mechanisms by which telomerase activity and CSC properties are linked. Using primary patient-derived pancreatic cancer cells, we show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic TERT-knockdown or pharmacological inhibitor (BIBR1532) resulted in CSC marker depletion in vitro, and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (KLF4, SOX2, OCT3/4, NANOG) and telomerase, which is essential for the self-renewal of pancreatic CSCs. Disruption the balance between telomerase activity and stemness factors, eliminates CSCs via induction of DNA damage and apoptosis, opening future perspectives to avoid CSC driven therapy resistance and tumor relapse in PDAC patients.

Preexisting diabetes increases the risk of a severe course of the pandemic coronavirus disease 2019 (COVID-19). Vice versa, exacerbations of a preexisting diabetes as well as new-onset diabetes have been reported upon SARS-CoV-2 infection. Thus, there is an imperative need to clarify whether human pancreatic endocrine cells organized within an islet of Langerhans are permissive for and affected by SARS-CoV-2 infection, and to elucidate the mechanisms underlying the development of diabetes upon COVID-19. Here, we (i) defined ACE2 and TMPRSS2 expression patterns in human pancreatic endocrine and exocrine cell types, (ii) employed human pancreatic islet cultures to demonstrate susceptibility to SARS-CoV-2 infection and to viral replication in β-cells, (iii) showed that SARS-CoV-2 attenuates glucose-stimulated insulin secretion, and (iv) tested remdesivir as eventually effective to prevent β-cell failure. In addition, we (v) visualized viral particles replicating in endocrine pancreatic cells and define their subcellular localization patterns via transmission electron microscopy, and finally (vi) present examples of cell type specific pancreatic infection patterns of COVID-19 deceased patients. Overall, our data demonstrate that SARS-CoV-2 can infect both the exocrine and endocrine compartments of the pancreas and can perturb β-cell integrity, which might lead to an increased risk for diabetes.

Central airway obstruction is a life-threatening disorder causing a high physical and psychological burden to patients due to severe breathlessness and impaired quality of life. Standard-of-care airway stents are silicone tubes, which cause immediate relief, but are prone to migration, especially in growing patients, and require additional surgeries to be removed, which may cause further tissue damage. Customized airway stents with tailorable bioresorbability that can be produced in a reasonable time frame would be highly needed in the management of this disorder. Here, we report poly(D,L-lactide-co-ε-caprolactone) methacrylate blends-based biomedical inks and their use for the rapid fabrication of customized and bioresorbable airway stents. The 3D printed materials are cytocompatible and exhibit silicone-like mechanical properties with suitable biodegradability. In vivo studies in healthy rabbits confirmed biocompatibility and showed that the stents stayed in place for 7 weeks after which they became radiographically invisible. The developed biomedical inks open promising perspectives for the rapid manufacturing of the customized medical devices for which high precision, tuneable elasticity and predictable degradation are sought-after.

Exocrine pancreatic damage is a common complication of cystic fibrosis (CF), which can significantly debilitate the quality of life and life expectancy of CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) has a major role in pancreatic ductal ion secretion, however, it presumably has an influence on intracellular signaling as well. Here we describe in multiple model systems, including iPSC-derived human pancreatic organoids from CF patients, that the activity of PMCA4 is impaired by the decreased expression of CFTR in ductal cells. The regulation of PMCA4, which colocalizes and physically interacts with CFTR on the apical membrane of the ductal cells, is dependent on the calmodulin binding ability of CFTR. Moreover, CFTR seems to be involved in the process of the apical recruitment of calmodulin, which enhances its role in calcium signaling and homeostasis. Sustained intracellular Ca2+ elevation in CFTR KO cells undermined the mitochondrial function and increased apoptosis. Based on these, the prevention of sustained intracellular Ca2+ overload may improve the exocrine pancreatic function and may have a potential therapeutic aspect in CF.

Personalized medicine in treating pancreatic ductal adenocarcinoma (PDAC) is still in its infancy, albeit PDAC-related deaths are projected to rise over the next decade. Only recently, maintenance therapy with the PARP inhibitor olaparib showed improved progression-free survival in germline BRCA1/2-mutated PDAC patients after platinum-based induction for the first time. Transferability of such a concept to other DNA damage response (DDR) genes remains unclear. Here, we conducted a placebo-controlled, three-armed preclinical trial to evaluate the efficacy of multi-DDR interference (mDDRi) as maintenance therapy vs. continuous FOLFIRINOX treatment, implemented with orthotopically transplanted ATM-deficient PDAC cell lines. Kaplan–Meier analysis, cross-sectional imaging, histology, and in vitro analysis served as analytical readouts. Median overall survival was significantly longer in the mDDRi maintenance arm compared to the maintained FOLFIRINOX treatment. This survival benefit was mirrored in the highest DNA-damage load, accompanied by superior disease control and reduced metastatic burden. In vitro analysis suggests FOLFIRINOX-driven selection of invasive subclones, erased by subsequent mDDRi treatment. Collectively, this preclinical trial substantiates mDDRi in a maintenance setting as a novel therapeutic option and extends the concept to non-germline BRCA1/2-mutant PDAC., publishedVersion