A histochemical and immunohistochemical study on the gastrointestinal tract of sparrowhawk (Accipiter nisus) / Estudio histoquímico e inmunohistoquímico del tracto gastrointestinal del gavilán (Accipiter nisus)

SUMMARY: In the present study, we aimed to determine the localization and distribution of entero-endocrine cells in the gastrointestinal tract by immunohistochemical methods and understand the structure of the glycoproteins elaborated by the epithelium the digestive tract regions by histochemical methods. The nine sparrowhawks were euthanized, and gastrointestinal tract tissues were removed and fixed in formalin. The gastrointestinal tract sections were stained with immunohistochemical and histochemical techniques to evaluate the enteroendocrine cells and histomorphometric analysis. The results showed that the numbers of somatostatin in the ventriculus, gastrin in the proventriculus, serotonin in the duodenum and jejunum immunopositivity are higher, remaining segments of the gastrointestinal tract are detected slight positivity in the glucagon, gastrin, serotonin, and somatostatin. In conclusion, some endocrine cells localization and distribution and histomorphometry, and goblet cell counts were revealed in the gastrointestinal tract of the sparrowhawks.

RESUMEN: El objetivo del presente estudio fue determinar la localización y distribución de células enteroendocrinas en el tracto gastrointestinal de gavilán, a través de métodos inmunohistoquímicos y comprender la estructura de las glicoproteínas elaboradas por el epitelio de las regiones del tracto digestivo. Se sacrificaron nueve gavilanes y los tejidos del tracto gastrointestinal se extrajeron y se fijaron en formalina. Las secciones del tracto gastrointestinal se tiñeron con técnicas inmunohistoquímicas e histoquímicas para evaluar las células enteroendocrinas y se realizó análisis histo-morfométrico. Los re- sultados indicaron que los números de inmunopositividad de somatostatina en el ventrículo, gastrina en el proventrículo, serotonina en el duodeno y yeyuno son más altos, en los segmentos restantes del tracto gastrointestinal, se detecta además una ligera positividad de glucagón, gastrina, serotonina y somatostatina. En conclusión en el tracto gastrointestinal de gavilán se observó cierta localización y distribución de células endocrinas e histomorfometría, y recuentos de células caliciformes.

A Special Network Comprised of Macrophages, Epithelial Cells, and Gut Microbiota for Gut Homeostasis.

A number of gut epithelial cells derived immunological factors such as cytokines and chemokines, which are stimulated by the gut microbiota, can regulate host immune responses to maintain a well-balance between gut microbes and host immune system. Multiple specialized immune cell populations, such as macrophages, dendritic cells (DCs), innate lymphoid cells, and T regulatory (Treg) cells, can communicate with intestinal epithelial cells (IEC) and/or the gut microbiota bi-directionally. The gut microbiota contributes to the differentiation and function of resident macrophages. Situated at the interface between the gut commensals and macrophages, the gut epithelium is crucial for gut homeostasis in microbial recognition, signaling transformation, and immune interactions, apart from being a physical barrier. Thus, three distinct but interactive components-macrophages, microbiota, and IEC-can form a network for the delicate and dynamic regulation of intestinal homeostasis. In this review, we will discuss the crucial features of gut microbiota, macrophages, and IEC. We will also summarize recent advances in understanding the cooperative and dynamic interactions among the gut microbiota, gut macrophages, and IEC, which constitute a special network for gut homeostasis.

Development of Functional Thyroid C Cell-like Cells from Human Pluripotent Cells in 2D and in 3D Scaffolds.

Medullary thyroid carcinoma contributes to about 3-4% of thyroid cancers and affects C cells rather than follicular cells. Thyroid C cell differentiation from human pluripotent stem cells has not been reported. We report the stepwise differentiation of human embryonic stem cells into thyroid C cell-like cells through definitive endoderm and anterior foregut endoderm and ultimobranchial body-like intermediates in monolayer and 3D Matrigel culture conditions. The protocol involved sequential treatment with interferon/transferrin/selenium/pyruvate, foetal bovine serum, and activin A, then IGF-1 (Insulin-like growth factor 1), on the basis of embryonic thyroid developmental sequence. As well as expressing C cell lineage relative to follicular-lineage markers by qPCR (quantitative polymerase chain reaction) and immunolabelling, these cells by ELISA (enzyme-linked immunoassay) exhibited functional properties in vitro of calcitonin storage and release of calcitonin on calcium challenge. This method will contribute to developmental studies of the human thyroid gland and facilitate in vitro modelling of medullary thyroid carcinoma and provide a valuable platform for drug screening.

Molecular and Cellular Mechanisms Modulating Trained Immunity by Various Cell Types in Response to Pathogen Encounter.

The induction of trained immunity represents an emerging concept defined as the ability of innate immune cells to acquire a memory phenotype, which is a typical hallmark of the adaptive response. Key points modulated during the establishment of trained immunity include epigenetic, metabolic and functional changes in different innate-immune and non-immune cells. Regarding to epigenetic changes, it has been described that long non-coding RNAs (LncRNAs) act as molecular scaffolds to allow the assembly of chromatin-remodeling complexes that catalyze epigenetic changes on chromatin. On the other hand, relevant metabolic changes that occur during this process include increased glycolytic rate and the accumulation of metabolites from the tricarboxylic acid (TCA) cycle, which subsequently regulate the activity of histone-modifying enzymes that ultimately drive epigenetic changes. Functional consequences of established trained immunity include enhanced cytokine production, increased antigen presentation and augmented antimicrobial responses. In this article, we will discuss the current knowledge regarding the ability of different cell subsets to acquire a trained immune phenotype and the molecular mechanisms involved in triggering such a response. This knowledge will be helpful for the development of broad-spectrum therapies against infectious diseases based on the modulation of epigenetic and metabolic cues regulating the development of trained immunity.

Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila.

The adult Drosophila intestinal epithelium must be tightly regulated to maintain regeneration and homeostasis. The dysregulation of the regenerative capacity is frequently associated with intestinal diseases such as inflammation and tumorigenesis. Here, we showed that the G protein-coupled receptor Anchor maintains Drosophila adult midgut homeostasis by restricting Jun-N-terminal kinase (JNK) and Notch pathway activity. anchor inactivation resulted in aberrant JNK pathway activation, which led to excessive enteroblast (EB) production and premature enterocyte (EC) differentiation. In addition, increased Notch levels promoted premature EC differentiation following the loss of anchor. This defect induced by the loss of anchor ultimately caused sensitivity to stress or environmental challenge in adult flies. Taken together, our results demonstrate that the activity of anchor is essential to coordinate stem cell differentiation and proliferation to maintain intestinal homeostasis.

A Versatile Human Intestinal Organoid-Derived Epithelial Monolayer Model for the Study of Enteric Pathogens.

Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with in vivo-like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under in vivo-like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with S. Typhimurium causing more destruction of the terminal ileum and S. Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development. IMPORTANCE While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of Shigella, Salmonella, and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.

SUMOylation Connects Cell Stress Responses and Inflammatory Control: Lessons From the Gut as a Model Organ.

Conjugation with the small ubiquitin-like modifier (SUMO) constitutes a key post-translational modification regulating the stability, activity, and subcellular localization of its target proteins. However, the vast numbers of identified SUMO substrates obscure a clear view on the function of SUMOylation in health and disease. This article presents a comprehensive review on the physiological relevance of SUMOylation by discussing how global SUMOylation levels-rather than specific protein SUMOylation-shapes the immune response. In particular, we highlight the growing body of work on SUMOylation in intestinal pathologies, because of the unique metabolic, infectious, and inflammatory challenges of this organ. Recent studies show that global SUMOylation can help restrain detrimental inflammation while maintaining immune defenses and tissue integrity. These results warrant further efforts to develop new therapeutic tools and strategies to control SUMOylation in infectious and inflammatory disorders.

Gastrointestinal epithelial innate immunity-regionalization and organoids as new model.

The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development.

Emerging biofabrication approaches for gastrointestinal organoids towards patient specific cancer models.

Tissue engineered organoids are simple biomodels that can emulate the structural and functional complexity of specific organs. Here, we review developments in three-dimensional (3D) artificial cell constructs to model gastrointestinal dynamics towards cancer diagnosis. We describe bottom-up approaches to fabricate close-packed cell aggregates, from the use of biochemical and physical cues to guide the self-assembly of organoids, to the use of engineering approaches, including 3D printing/additive manufacturing and external field-driven protocols. Finally, we outline the main challenges and possible risks regarding the potential translation of gastrointestinal organoids from laboratory settings to patient-specific models in clinical applications.

Postembryonic development and differentiation of the midgut in the freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca, Decapoda) larvae.

Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D-localization of the midgut inside the body cavity of larval stages of N. davidi using X-ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E-cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above-mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.

Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves.

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Gastrointestinal coronavirus disease 2019: epidemiology, clinical features, pathogenesis, prevention, and management.

INTRODUCTION: The new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 2019. Some authors reported pieces of evidence that patients with SARS-CoV-2 infection could have direct involvement of the gastrointestinal tract, and in symptomatic cases, gastrointestinal symptoms (diarrhea, nausea/vomiting, abdominal pain) could be very common. AREA COVERED: In this article, we reviewed current-published data of the gastrointestinal aspects involved in SARS-CoV-2 infection, including prevalence and incidence of specific symptoms, the presumptive biological mechanism of GI infection, prognosis, clinical management, and public health-related concerns on the possible risk of oral-fecal transmission. EXPERT OPINION: Different clues point to direct virus infection and replication in mucosal cells of the gastrointestinal tract. In vitro studies showed that SARS-CoV-2 could enter into the gastrointestinal epithelial cells by the Angiotensin-Converting enzyme two membrane receptor. These findings, coupled with the identification of viral RNA found in stools of patients, clearly suggest that direct involvement of the gastrointestinal tract is very likely. This can justify most of the gastrointestinal symptoms but also suggest a risk for an oral-fecal route for transmission, additionally or alternatively to the main respiratory route.

Interstitial cells of Cajal in Wsh/Wsh c-kit mutant mice.

The c-Kit receptor tyrosine kinase regulates the development and differentiation of several progenitor cells. In the gastrointestinal (GI) tract, the c-Kit regulates the development of the interstitial cells of Cajal (ICC) that are responsible for motility regulation of the GI musculature. W-sash (Wsh) is an inversion mutation upstream of the c-kit promoter region that affects a key regulatory element, resulting in cell-type-specific altered gene expression, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the GI tract of Wsh/Wsh mice using immunohistochemistry and electron microscopy. Although the musculature of the Wsh/Wsh mice did not show any c-Kit immunoreactivity, we detected intensive immunoreactivity for transmembrane member 16A (TMEM16A, anoctamin-1), another ICC marker. TMEM16A immunopositive cells were observed as ICC-MY in the gastric corpus-antrum and the large intestine, ICC-DMP in the small intestine, and ICC-SM in the colon. Electron microscopic analysis revealed these cells as ICC from their ultrastructural features, such as numerous mitochondria and caveolae, and their close contact with nerve terminals. In the developmental period, we examined 14.5 and 18.5 day embryos but did not observe c-Kit immunoreactivity in the Wsh/Wsh small intestine. From this study, ICC subtypes developed and maturated structurally without c-Kit expression. Wsh/Wsh mice are a new model to investigate the effects of c-Kit and unknown signaling on ICC development and function.

Intestinal stem cells and intestinal organoids.

The intestinal epithelium is one of the most rapidly renewing tissues, which is fueled by stem cells at the base of the crypts. Strategies of genetic lineage tracing and organoids, which capture major features of original tissues, are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro, respectively. The combination of intestinal organoid-culturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut. Here, we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells. We will also review the applications of organoids and genetic markers in intestinal research studies. Furthermore, we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.

Euglena Gracilis and ß-Glucan Paramylon Induce Ca2+ Signaling in Intestinal Tract Epithelial, Immune, and Neural Cells.

The intestinal tract contains over half of all immune cells and peripheral nerves and manages the beneficial interactions between food compounds and the host. Paramylon is a ß-1,3-glucan storage polysaccharide from Euglena gracilis (Euglena) that exerts immunostimulatory activities by affecting cytokine production. This study investigated the signaling mechanisms that regulate the beneficial interactions between food compounds and the intestinal tract using cell type-specific calcium (Ca2+) imaging in vivo and in vitro. We successfully visualized Euglena- and paramylon-mediated Ca2+ signaling in vivo in intestinal epithelial cells from mice ubiquitously expressing the Yellow Cameleon 3.60 (YC3.60) Ca2+ biosensor. Moreover, in vivo Ca2+ imaging demonstrated that the intraperitoneal injection of both Euglena and paramylon stimulated dendritic cells (DCs) in Peyer's patches, indicating that paramylon is an active component of Euglena that affects the immune system. In addition, in vitro Ca2+ imaging in dorsal root ganglia indicated that Euglena, but not paramylon, triggers Ca2+ signaling in the sensory nervous system innervating the intestine. Thus, this study is the first to successfully visualize the direct effect of ß-1,3-glucan on DCs in vivo and will help elucidate the mechanisms via which Euglena and paramylon exert various effects in the intestinal tract.

Immunohistochemical study of morphology and distribution of CD163+ve macrophages in the normal adult equine gastrointestinal tract.

Intestinal macrophages are the largest group of mononuclear phagocytes in the body and play a role in intestinal innate immunity, neuroimmune interactions and maintaining intestinal homeostasis. Conversely, they also are implicated in numerous pathologies of the gastrointestinal tract, such as postoperative ileus and inflammatory bowel disease. As a result, macrophages could be potential therapeutic targets. To date, there are limited studies on the morphology and distribution of macrophages in the equine gastrointestinal tract (GIT). The aim of this study was to identify the location and abundance of resident macrophages in the equine GIT using CD163 as an immunohistochemical marker. Tissue samples were obtained post-mortem from 14 sites along the gastrointestinal tracts of 10 horses free from gastrointestinal disease; sample sites extended from the stomach to the small colon. CD163+ve cells were present in all regions of the equine GIT from stomach to small colon. CD163+ve cells were also identified in all tissue layers of the intestinal wall, namely, mucosa, submucosa, muscularis externa (ME), myenteric plexus and serosa. Consistent with a proposed function in regulation of intestinal motility, CD163+ve cells were regularly distributed within the ME, with accumulations closely associated with the myenteric plexus and effector cells such as neurons and the interstitial cells of Cajal (ICC).

Lipoprotein Processing and Sorting in Helicobacter pylori.

Our current understanding of lipoprotein synthesis and localization in Gram-negative bacteria is based primarily on studies of Escherichia coli Newly synthesized E. coli prolipoproteins undergo posttranslational modifications catalyzed by three essential enzymes (Lgt, LspA, and Lnt). The mature lipoproteins are then sorted to the inner or outer membrane via the Lol system (LolABCDE). Recent studies suggested that this paradigm may not be universally applicable among different classes of proteobacteria. In this study, we conducted a systematic analysis of lipoprotein processing and sorting in Helicobacter pylori, a member of the Epsilonproteobacteria that colonizes the human stomach. We show that H. pylorilgt, lspA, and lnt homologs can complement conditionally lethal E. coli mutant strains in which expression of these genes is conditionally regulated. Mutagenesis studies and analyses of conditionally lethal H. pylori mutant strains indicate that lgt and lspA are essential for H. pylori growth but lnt is dispensable. H. pylorilolA and the single lolC (or lolE) homolog are also essential genes. We then explored the role of lipoproteins in H. pylori Cag type IV secretion system (Cag T4SS) activity. Comparative analysis of the putative VirB7 homolog CagT in wild-type and lnt mutant H. pylori strains indicates that CagT undergoes amino-terminal modifications consistent with lipidation, and we show that CagT lipidation is essential for CagT stability and Cag T4SS function. This work demonstrates that lipoprotein synthesis and localization in H. pylori diverge from the canonical pathways and that lipidation of a T4SS component is necessary for H. pylori Cag T4SS activity.IMPORTANCE Bacterial lipoproteins have diverse roles in multiple aspects of bacterial physiology, antimicrobial resistance, and pathogenesis. Dedicated pathways direct the posttranslational lipidation and localization of lipoproteins, but there is considerable variation in these pathways among the proteobacteria. In this study, we characterized the proteins responsible for lipoprotein synthesis and localization in Helicobacter pylori, a member of the Epsilonproteobacteria that contributes to stomach cancer pathogenesis. We also provide evidence suggesting that lipidation of CagT, a component of the H. pylori Cag T4SS, is required for delivery of the H. pylori CagA oncoprotein into human gastric cells. Overall, these results constitute the first systematic analysis of H. pylori lipoprotein production and localization pathways and reveal how these processes in H. pylori differ from corresponding pathways in model proteobacteria.

Stromal Cells in the Pathogenesis of Inflammatory Bowel Disease.

Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma.

Bioprinting for Human Respiratory and Gastrointestinal In Vitro Models.

Increasing ethical and biological concerns require a paradigm shift toward animal-free testing strategies for drug testing and hazard assessments. To this end, the application of bioprinting technology in the field of biomedicine is driving a rapid progress in tissue engineering. In particular, standardized and reproducible in vitro models produced by three-dimensional (3D) bioprinting technique represent a possible alternative to animal models, enabling in vitro studies relevant to in vivo conditions. The innovative approach of 3D bioprinting allows a spatially controlled deposition of cells and biomaterial in a layer-by-layer fashion providing a platform for engineering reproducible models. However, despite the promising and revolutionizing character of 3D bioprinting technology, standardized protocols providing detailed instructions are lacking. Here, we provide a protocol for the automatized printing of simple alveolar, bronchial, and intestine epithelial cell layers as the basis for more complex respiratory and gastrointestinal tissue models. Such systems will be useful for high-throughput toxicity screening and drug efficacy evaluation.