TY - JOUR A1 - Hetz, Susan A1 - Acikgoez, Ali A1 - Moll, Corinna A1 - Jahnke, Heinz-Georg A1 - Robitzki, Andrea A. A1 - Metzger, Roman A1 - Metzger, Marco T1 - Age-related gene expression analysis in enteric ganglia of human colon after laser microdissection JF - Frontiers in Aging Neuroscience N2 - The enteric nervous system (ENS) poses the intrinsic innervation of the gastrointestinal tract and plays a critical role for all stages of postnatal life. There is increasing scientific and clinical interest in acquired or age-related gastrointestinal dysfunctions that can be manifested in diseases such as gut constipation or fecal incontinence. In this study, we sought to analyze age-dependent changes in the gene expression profile of the human ENS, particularly in the myenteric plexus. Therefore, we used the laser microdissection technique which has been proven as a feasible tool to analyze distinct cell populations within heterogeneously composed tissues. Full biopsy gut samples were prepared from children (4–12 months), middle aged (48–58 years) and aged donors (70–95 years). Cryosections were histologically stained with H&E, the ganglia of the myenteric plexus identified and RNA isolated using laser microdissection technique. Quantitative PCR was performed for selected neural genes, neurotransmitters and receptors. Data were confirmed on protein level using NADPH-diaphorase staining and immunohistochemistry. As result, we demonstrate age-associated alterations in site-specific gene expression pattern of the ENS. Thus, in the adult and aged distal parts of the colon a marked decrease in relative gene expression of neural key genes like NGFR, RET, NOS1 and a concurrent increase of CHAT were observed. Further, we detected notable regional differences of RET, CHAT, TH, and S100B comparing gene expression in aged proximal and distal colon. Interestingly, markers indicating cellular senescence or oxidative stress (SNCA, CASP3, CAT, SOD2, and TERT) were largely unchanged within the ENS. For the first time, our study also describes the age-dependent expression pattern of all major sodium channels within the ENS. Our results are in line with previous studies showing spatio-temporal differences within the mammalian ENS. KW - enteric nervous system KW - myenteric plexus KW - aging KW - sodium channels KW - laser microdissection Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118308 SN - 1663-4365 VL - 6 ER - TY - JOUR A1 - Hetz, Susan A1 - Acikgoez, Ali A1 - Voss, Ulrike A1 - Nieber, Karen A1 - Holland, Heidrun A1 - Hegewald, Cindy A1 - Till, Holger A1 - Metzger, Roman A1 - Metzger, Marco T1 - In Vivo Transplantation of Neurosphere-Like Bodies Derived from the Human Postnatal and Adult Enteric Nervous System: A Pilot Study JF - PLOS ONE N2 - Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via 'bystander' mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders. KW - Hirschsprung disease KW - benzalkonium chloride KW - progenitors KW - GUT KW - rat KW - colon KW - biology KW - therapy KW - neural stem-cell KW - motility disorders Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116793 VL - 9 IS - 4 ER - TY - JOUR A1 - Rosenbaum, Corinna A1 - Schick, Martin Alexander A1 - Wollborn, Jakob A1 - Heider, Andreas A1 - Scholz, Claus-Jürgen A1 - Cecil, Alexander A1 - Niesler, Beate A1 - Hirrlinger, Johannes A1 - Walles, Heike A1 - Metzger, Marco T1 - Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo JF - PLoS One N2 - Background Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. Methods and Results In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Conclusion and Significance Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine. KW - gene expression KW - gastrointestinal tract KW - inflammatory bowel disease KW - central nervous system KW - systemic inflammatory response syndrome KW - inflammation KW - astrocytes KW - cytokines Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146544 VL - 11 IS - 3 ER - TY - JOUR A1 - Markenstein, Lisa A1 - Appelt-Menzel, Antje A1 - Metzger, Marco A1 - Wenz, Gerhard T1 - Conjugates of methylated cyclodextrin derivatives and hydroxyethyl starch (HES): Synthesis, cytotoxicity and inclusion of anaesthetic actives JF - Beilstein Journal of Organic Chemistry N2 - The mono-6-deoxy-6-azides of 2,6-di-O-methyl-beta-cyclodextrin (DIMEB) and randomly methylated-beta-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu+-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 degrees C (DIMEB-HES) and 84.5 degrees C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives. KW - midazolam KW - supermolecular carrier systems KW - beta-cyclodextrin KW - pharmaceutical applications KW - gamma-cyclodextrin KW - anaesthetics KW - complexation KW - cyclodextrin KW - LCST KW - lower critical solution temperature KW - acid dissociation KW - drug KW - rat KW - cycloaddition KW - occupancy KW - polymer KW - sevoflurane KW - solubility KW - starch Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114280 SN - 1860-5397 VL - 10 ER - TY - JOUR A1 - Gordon, Sarah A1 - Daneshian, Mardas A1 - Bouwstra, Joke A1 - Caloni, Francesca A1 - Constant, Samuel A1 - Davies, Donna E. A1 - Dandekar, Gudrun A1 - Guzman, Carlos A. A1 - Fabian, Eric A1 - Haltner, Eleonore A1 - Hartung, Thomas A1 - Hasiwa, Nina A1 - Hayden, Patrick A1 - Kandarova, Helena A1 - Khare, Sangeeta A1 - Krug, Harald F. A1 - Kneuer, Carsten A1 - Leist, Marcel A1 - Lian, Guoping A1 - Marx, Uwe A1 - Metzger, Marco A1 - Ott, Katharina A1 - Prieto, Pilar A1 - Roberts, Michael S. A1 - Roggen, Erwin L. A1 - Tralau, Tewes A1 - van den Braak, Claudia A1 - Walles, Heike A1 - Lehr, Claus-Michael T1 - Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology JF - ALTEX: Alternatives to Animal Experimentation N2 - Models of the outer epithelia of the human body namely the skin, the intestine and the lung have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report. KW - on-a-chip KW - asthmatic bronchial epithelium KW - vesicle-based barrier KW - pulmonary drug-delivery KW - epithelial cell culture KW - cytotoxicity KW - transport studies KW - permeability KW - in vitro models KW - air-liquid interface KW - respiratory syncytial virus KW - reconstructed human epidermis KW - artificial membrane-permeability KW - embryonic stem cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144275 VL - 32 IS - 4 ER - TY - JOUR A1 - Wohlfarth, Carolin A1 - Schmitteckert, Stefanie A1 - Härtle, Janina D. A1 - Houghton, Lesley A. A1 - Dweep, Harsh A1 - Fortea, Marina A1 - Assadi, Ghazaleh A1 - Braun, Alexander A1 - Mederer, Tanja A1 - Pöhner, Sarina A1 - Becker, Philip P. A1 - Fischer, Christine A1 - Granzow, Martin A1 - Mönnikes, Hubert A1 - Mayer, Emeran A. A1 - Sayuk, Gregory A1 - Boeckxstaens, Guy A1 - Wouters, Mira M. A1 - Simrén, Magnus A1 - Lindberg, Greger A1 - Ohlsson, Bodil A1 - Schmidt, Peter Thelin A1 - Dlugosz, Aldona A1 - Agreus, Lars A1 - Andreasson, Anna A1 - D'Amato, Mauro A1 - Burwinkel, Barbara A1 - Bermejo, Justo Lorenzo A1 - Röth, Ralph A1 - Lasitschka, Felix A1 - Vicario, Maria A1 - Metzger, Marco A1 - Santos, Javier A1 - Rappold, Gudrun A. A1 - Martinez, Cristina A1 - Niesler, Beate T1 - miR-16 and miR-103 impact 5-HT4 receptor signalling and correlate with symptom profile in irritable bowel syndrome JF - Scientific Reports N2 - Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT4 receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT4R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT4 receptor gene \(HTR4\) to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms \({HTR4b/i}\) and putatively impairs \(HTR4\) expression. Subsequent miRNA profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. \(In\) \(vitro\) assays confirmed expression regulation via three 3′UTR binding sites. The novel isoform \(HTR4b\_2\) lacking two of the three miRNA binding sites escapes miR-16/103/107 regulationin SNP carriers. We provide the first evidence that \(HTR4\) expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or bydiminished levels of miR-16 and miR-103 suggesting that \(HTR4\) might be involved in the development of IBS-D. KW - Medicine KW - Gene regulation KW - Irritable bowel syndrome Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173478 VL - 7 ER - TY - JOUR A1 - Burns, Alan J. A1 - Goldstein, Allan M. A1 - Newgreen, Donald F. A1 - Stamp, Lincon A1 - Schäfer, Karl-Herbert A1 - Metzger, Marco A1 - Hotta, Ryo A1 - Young, Heather M. A1 - Andrews, Peter W. A1 - Thapar, Nikhil A1 - Belkind-Gerson, Jaime A1 - Bondurand, Nadege A1 - Bornstein, Joel C. A1 - Chan, Wood Yee A1 - Cheah, Kathryn A1 - Gershon, Michael D. A1 - Heuckeroth, Robert O. A1 - Hofstra, Robert M.W. A1 - Just, Lothar A1 - Kapur, Raj P. A1 - King, Sebastian K. A1 - McCann, Conor J. A1 - Nagy, Nandor A1 - Ngan, Elly A1 - Obermayr, Florian A1 - Pachnis, Vassilis A1 - Pasricha, Pankaj J. A1 - Sham, Mai Har A1 - Tam, Paul A1 - Vanden Berghe, Pieter T1 - White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies JF - Developmental Biology N2 - Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic. KW - Neural crest cells KW - Rat mynteric plexus KW - Intestinal pseudoobstruction KW - Hypertrophic pyloric-stenosis KW - Hirschsprung disease liability KW - Slow-transit constipation KW - Oxide synthase gene KW - Term follow-up KW - Nitric-oxide KW - In-vivo KW - Enteric nervous system KW - Enteric neuropathies KW - Stem cells KW - Cell replacement therapy KW - Hirschsprung disease Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187415 VL - 417 IS - 2 ER - TY - JOUR A1 - Schulte, Leon N. A1 - Schweinlin, Matthias A1 - Westermann, Alexander J. A1 - Janga, Harshavardhan A1 - Santos, Sara C. A1 - Appenzeller, Silke A1 - Walles, Heike A1 - Vogel, Jörg A1 - Metzger, Marco T1 - An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection JF - mBio N2 - A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host. KW - Salmonella KW - gene expression KW - infectious disease Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229428 VL - 11, 2020 IS - 1 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - JOUR A1 - Appelt-Menzel, Antje A1 - Cubukova, Alevtina A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Piontek, Jörg A1 - Krause, Gerd A1 - Stüber, Tanja A1 - Walles, Heike A1 - Neuhaus, Winfried A1 - Metzger, Marco T1 - Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells JF - Stem Cell Reports N2 - In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm\(^{2}\) and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. KW - blood-brain barrier (BBB) model KW - human induced pluripotent stem cells (hiPSCs)human induced pluripotent stem cells (hiPSCs) KW - multipotent fetal neural stem cells (fNSCs) KW - neurovascular unit in vitro Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170982 VL - 8 IS - 4 ER - TY - JOUR A1 - Kress, Sebastian A1 - Baur, Johannes A1 - Otto, Christoph A1 - Burkard, Natalie A1 - Braspenning, Joris A1 - Walles, Heike A1 - Nickel, Joachim A1 - Metzger, Marco T1 - Evaluation of a miniaturized biologically vascularized scaffold in vitro and in vivo JF - Scientific Reports N2 - In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine. KW - biological models KW - translational research Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176343 VL - 8 IS - 4719 ER - TY - JOUR A1 - Alzheimer, Mona A1 - Svensson, Sarah L. A1 - König, Fabian A1 - Schweinlin, Matthias A1 - Metzger, Marco A1 - Walles, Heike A1 - Sharma, Cynthia M. T1 - A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni JF - PLoS Pathogens N2 - The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. KW - in vitro KW - stem cells KW - invasion KW - host KW - adhesion KW - epithelial cells KW - translocation KW - virulence KW - responses KW - microenvironment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229454 VL - 16 IS - 2 ER - TY - JOUR A1 - Herbert, Saskia-Laureen A1 - Fick, Andrea A1 - Heydarian, Motaharehsadat A1 - Metzger, Marco A1 - Wöckel, Achim A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera A1 - Wulff, Christine T1 - Establishment of the SIS scaffold-based 3D model of human peritoneum for studying the dissemination of ovarian cancer JF - Journal of Tissue Engineering N2 - Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer. KW - ovarian cancer KW - 3D tissue model KW - co-culture KW - peritoneal metastasis KW - cancer dissemination Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301311 SN - 2041-7314 VL - 13 ER - TY - JOUR A1 - Straßer, Marion A1 - Schrauth, Joachim H. X. A1 - Dembski, Sofia A1 - Haddad, Daniel A1 - Ahrens, Bernd A1 - Schweizer, Stefan A1 - Christ, Bastian A1 - Cubukova, Alevtina A1 - Metzger, Marco A1 - Walles, Heike A1 - Jakob, Peter M. A1 - Sextl, Gerhard T1 - Calcium fluoride based multifunctional nanoparticles for multimodal imaging JF - Beilstein Journal of Nanotechnology N2 - New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5–10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd\(^{3+}\) ions on the surface, the NPs reduce the MR T\(_{1}\) relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about r = 0.471 mL·mg\(^{−1}\)·s\(^{−1}\). Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL\(^{−1}\) was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs are suitable for medical imaging. KW - calcium fluoride nanoparticles KW - magnetic resonance imaging (MRI) KW - multifunctional nanoparticles KW - multimodal imaging KW - photoluminescence Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170657 VL - 8 ER - TY - JOUR A1 - Trujillo‐Viera, Jonathan A1 - El‐Merahbi, Rabih A1 - Schmidt, Vanessa A1 - Karwen, Till A1 - Loza‐Valdes, Angel A1 - Strohmeyer, Akim A1 - Reuter, Saskia A1 - Noh, Minhee A1 - Wit, Magdalena A1 - Hawro, Izabela A1 - Mocek, Sabine A1 - Fey, Christina A1 - Mayer, Alexander E. A1 - Löffler, Mona C. A1 - Wilhelmi, Ilka A1 - Metzger, Marco A1 - Ishikawa, Eri A1 - Yamasaki, Sho A1 - Rau, Monika A1 - Geier, Andreas A1 - Hankir, Mohammed A1 - Seyfried, Florian A1 - Klingenspor, Martin A1 - Sumara, Grzegorz T1 - Protein Kinase D2 drives chylomicron‐mediated lipid transport in the intestine and promotes obesity JF - EMBO Molecular Medicine N2 - Lipids are the most energy‐dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron‐mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high‐fat diet‐induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity. KW - chylomicron KW - fat absorption KW - intestine KW - obesity KW - protein kinase D2/PKD2/PRKD2 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239018 VL - 13 IS - 5 ER - TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Schweinlin, Matthias A1 - Schwarz, Thomas A1 - Rawal, Ravisha A1 - Walles, Heike A1 - Metzger, Marco A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera T1 - Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity JF - Journal of Tissue Engineering N2 - Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment. KW - Triple co-culture KW - biomimetic 3D tissue model KW - Neisseria gonorrhoeae KW - perfusion-based bioreactor system KW - neutrophil transmigration Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259032 VL - 12 ER - TY - JOUR A1 - Koenig, Leopold A1 - Ramme, Anja Patricia A1 - Faust, Daniel A1 - Mayer, Manuela A1 - Flötke, Tobias A1 - Gerhartl, Anna A1 - Brachner, Andreas A1 - Neuhaus, Winfried A1 - Appelt-Menzel, Antje A1 - Metzger, Marco A1 - Marx, Uwe A1 - Dehne, Eva-Maria T1 - A human stem cell-derived brain-liver chip for assessing blood-brain-barrier permeation of pharmaceutical drugs JF - Cells N2 - Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood–brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier. KW - blood-brain barrier (BBB) model KW - human induced pluripotent stem cells (hiPSCs) KW - microphysiological systems (MPS) KW - multi-organ chip KW - brain–liver chip Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290375 SN - 2073-4409 VL - 11 IS - 20 ER - TY - JOUR A1 - Däullary, Thomas A1 - Imdahl, Fabian A1 - Dietrich, Oliver A1 - Hepp, Laura A1 - Krammer, Tobias A1 - Fey, Christina A1 - Neuhaus, Winfried A1 - Metzger, Marco A1 - Vogel, Jörg A1 - Westermann, Alexander J. A1 - Saliba, Antoine-Emmanuel A1 - Zdzieblo, Daniela T1 - A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection JF - Gut Microbes N2 - Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay. KW - intestinal enteroids KW - biological scaffold KW - Salmonella Typhimurium KW - OLFM4 KW - NOTCH KW - filamentous Salmonella Typhimurium KW - bacterial migration KW - bacterial virulence KW - 3D tissue model KW - olfactomedin 4 KW - infection Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350451 VL - 15 IS - 1 ER -