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 - Wallstabe, Julia A1 - Bussemer, Lydia A1 - Groeber-Becker, Florian A1 - Freund, Lukas A1 - Alb, Mirian A1 - Dragan, Mariola A1 - Waaga-Gasser, Ana Maria A1 - Jakubietz, Rafael A1 - Kneitz, Hermann A1 - Rosenwald, Andreas A1 - Rebhan, Silke A1 - Walles, Heike A1 - Mielke, Stephan T1 - Inflammation-Induced Tissue Damage Mimicking GvHD in Human Skin Models as Test Platform for Immunotherapeutics JF - ALTEX N2 - Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies. KW - inflammation-induced tissue demage KW - immunotherapeutics Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229974 VL - 37 IS - 3 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 - Kühnemundt, Johanna A1 - Leifeld, Heidi A1 - Scherg, Florian A1 - Schmitt, Matthias A1 - Nelke, Lena C. A1 - Schmitt, Tina A1 - Bauer, Florentin A1 - Göttlich, Claudia A1 - Fuchs, Maximilian A1 - Kunz, Meik A1 - Peindl, Matthias A1 - Brähler, Caroline A1 - Kronenthaler, Corinna A1 - Wischhusen, Jörg A1 - Prelog, Martina A1 - Walles, Heike A1 - Dandekar, Thomas A1 - Dandekar, Gudrun A1 - Nietzer, Sarah L. T1 - Modular micro-physiological human tumor/tissue models based on decellularized tissue for improved preclinical testing JF - ALTEX N2 - High attrition-rates entailed by drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia, as well as toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments. KW - modular tumor tissue models KW - invasiveness KW - bioreactor culture KW - combinatorial drug predictions KW - immunotherapies Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231465 VL - 38 ER - TY - JOUR A1 - Bittorf, Patrick A1 - Bergmann, Thorsten A1 - Merlin, Simone A1 - Olgasi, Chistina A1 - Pullig, Oliver A1 - Sanzenbacher, Ralf A1 - Zierau, Martin A1 - Walles, Heike A1 - Follenzi, Antonia A1 - Braspenning, Joris T1 - Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells JF - Molecular Therapy - Methods & Clinical Development N2 - The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 x 10\(^6\) intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 x 10\(^3\) to 1:1 x 10\(^6\) human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies. KW - outgrowth endothelial cells KW - real time PCR KW - in vivo KW - gene therapy KW - factor-VIII KW - murine KW - quantification KW - establishment KW - phenotype KW - xenotransplantation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230284 VL - 18 ER - TY - JOUR A1 - Schmitz, Tobias A1 - Jannasch, Maren A1 - Weigel, Tobias A1 - Moseke, Claus A1 - Gbureck, Uwe A1 - Groll, Jürgen A1 - Walles, Heike A1 - Hansmann, Jan T1 - Nanotopographical Coatings Induce an Early Phenotype-Specific Response of Primary Material-Resident M1 and M2 Macrophages JF - Materials N2 - Implants elicit an immunological response after implantation that results in the worst case in a complete implant rejection. This biomaterial-induced inflammation is modulated by macrophages and can be influenced by nanotopographical surface structures such as titania nanotubes or fractal titanium nitride (TiN) surfaces. However, their specific impact on a distinct macrophage phenotype has not been identified. By using two different levels of nanostructures and smooth samples as controls, the influence of tubular TiO2 and fractal TiN nanostructures on primary human macrophages with M1 or M2-phenotype was investigated. Therefore, nanotopographical coatings were either, directly generated by physical vapor deposition (PVD) or by electrochemical anodization of titanium PVD coatings. The cellular response of macrophages was quantitatively assessed to demonstrate a difference in biocompatibility of nanotubes in respect to human M1 and M2-macrophages. Depending on the tube diameter of the nanotubular surfaces, low cell numbers and impaired cellular activity, was detected for M2-macrophages, whereas the impact of nanotubes on M1-polarized macrophages was negligible. Importantly, we could confirm this phenotypic response on the fractal TiN surfaces. The results indicate that the investigated topographies specifically impact the macrophage M2-subtype that modulates the formation of the fibrotic capsule and the long-term response to an implant. KW - nanotopographical surfaces KW - combination of physical vapor deposition and electrochemical etching KW - defined humanized test system KW - inflammatory response Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203378 SN - 1996-1944 VL - 13 IS - 5 ER -