Usually, duodenal barriers are investigated using intestinal cell lines like Caco‐2, which in contrast to native tissue are limited in cell‐type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three‐dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid‐derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco‐2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco‐2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco‐2. Transcriptomic data showed that Caco‐2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco‐2 cells differ mostly in transcellular properties, ODMs reflect trans‐ and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue‐specific investigations.

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15^−/− cells and (using A549-UBA7^−/− cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α–pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control. Key Points Cell culture model of ISG15 deficiency replicates findings in ISG15^−/− patient cells. Cause of resistance in ISG15^−/− cells differs depending on duration of IFN treatment. ISG15^−/− patients without serious viral disease do not prove ISGylation is unimportant.

Animal models for cystic fibrosis (CF) have enhanced our understanding of the pathology and contributed to the development of new treatments. In the field of CF, many animal models have been developed and described. To our knowledge, thus far, none of the reviews of CF animal models has used a systematic methodology. A systematic approach to creating model overviews can lead to an objective, evidence-based choice of an animal model for new research questions. We searched Pubmed and Embase for the currently available animal models for CF. Two independent reviewers screened the results. We included all primary studies describing an animal model for CF. After duplicate removal, 12,304 publications were left. Because of the large number of models, in the current paper, only the genetic models are presented. A total of 636 publications were identified describing genetic animal models for CF in mice, pigs, ferrets, rats and zebrafish. Most of these models have an altered Cftr gene. An overview of basic model characteristics and outcome measures for these genetic models is provided, together with advice on using these data. As far as the authors are aware, this is one of the largest systematic mapping reviews on genetic animal models for CF. It can aid in selecting a suitable model and outcome measures. In general, the reporting quality of the included publications was poor. Further systematic reviews are warranted to determine the quality and translational value of these models further.

Reconstructive surgery following breast cancer ablation is a surgical gold standard, but current options comprising autologous fatty tissue transfer and artificial soft tissue implants are inferior. With the advent of powerful biofabrication technologies, researchers for the first time have the tools to engineer life-like tissues with the ultimate goal of clinical application. Here, we apply multi-material stereolithographic bioprinting together with a novel sacrificial biomaterial system to engineer complex fatty tissue constructs. Biomaterials, cellular composition and cultivation conditions of these constructs were designed to enable in vitro creation of vascularised fatty tissue. Cells within the constructs showed an overall good survival (>93%), indicated by live-dead cell staining, over the entire cultivation period of 27 d. Adipose-derived stem cells were successfully differentiated in situ, forming fat vesicles and expressing adipocyte markers PPARγ, FAPB4 and S100B. Additionally, secretion of adipokines leptin and adiponectin into culture supernatants increased significantly. Endothelial cells vascularised the constructs, creating macro- and microvascular structures within the printed channels and extending beyond with culture time. Moreover, cells invaded into the surrounding hydrogel. The engineered fatty tissue constructs could serve as a base to develop patient-specific tissue building blocks with the final goal to achieve an all-natural reconstruction of the breast.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with poor 5-year overall survival. This can be mainly explained by late diagnosis mostly at advanced stages, excluding primary tumor resection as putative curative treatment, and a profound therapy resistance. L1 cell adhesion molecule (L1CAM) is upregulated in PDAC cells and associated with different hallmarks of cancer contributing to tumor progression and therapy resistance. Using L1CAM as tumor associated antigen, two antibody drug conjugates (ADC) targeting L1CAM were developed with different toxins as payload. Both L1CAM-ADC led to a L1CAM specific reduction in tumor cell growth of up to 60% in 2D PDAC cell models. A maximum therapeutic effect was observed when at least 50% of the PDAC cells expressed L1CAM. In 3D PDAC spheroids, MMAE conjugated L1CAM-ADC still showed strong therapeutic effects, while efficacy of SG3199 conjugated L1CAM-ADC was diminished. Co-culture of PDAC spheroids with pancreatic myofibroblasts did almost not impair L1CAM-ADC anti-tumor effects, while macrophages enhanced anti-tumor responses. Application of L1CAM-ADC as adjuvant or palliative treatment in clinically adapted PDAC mouse models exerted potent anti-tumor effects even with enhanced tumor burden. Adjuvantly applied, MMAE conjugated L1CAM-ADC efficiently reduced pancreatic relapses and peritoneal metastases, whereas SG3199 conjugated L1CAM-ADC less impacted pancreatic relapses, but almost completely eliminated liver and peritoneal metastases. In the palliative setting, both L1CAM-ADC reduced number and size of liver and peritoneal metastases. Overall, our results demonstrate strong anti-tumor effects of L1CAM-ADC underscoring their potential as immunotherapeutic approach in therapy of even advanced PDAC.

Background and aims: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed to overcome these limitations and to recapitulate the authentic series of events in the primary human duodenal tissue by using the human organoid system. Methods: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis -induced barrier breakdown by functional analysis of transcriptional, electrophysiological and tight junction components. Results: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of epithelial barrier upon G. duodenalis infection. Transcriptomic analysis suggested major expression changes in genes contributing to ion transport and tight junction structure. SLC12A2/NKCC1- and CFTR-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Conclusion: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection ignited by alterations of cellular ion transport followed by breakdown of the tight junctional complex and loss of epithelial integrity. The newly established organoid-derived model to study G. duodenalis infection will help enable further molecular dissection of the disease mechanism and, thus, can help to find new options treating disease and infection, in particular relevant for chronic cases of giardiasis.