Inactivation of the tumor suppressor gene Apc synergizes with H. pylori to induce DNA damage in murine gastric stem and progenitor cells

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that H. pylori induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling

GATA3 mediates nonclassical β-catenin signaling in skeletal cell fate determination and ectopic chondrogenesis

Skeletal precursors are mesenchymal in origin and can give rise to distinct sublineages. Their lineage commitment is modulated by various signaling pathways. The importance of Wnt signaling in skeletal lineage commitment has been implicated by the study of β-catenin-deficient mouse models. Ectopic chondrogenesis caused by the loss of β-catenin leads to a long-standing belief in canonical Wnt signaling that determines skeletal cell fate. As β-catenin has other functions, it remains unclear whether skeletogenic lineage commitment is solely orchestrated by canonical Wnt signaling. The study of the Wnt secretion regulator Gpr177/Wntless also raises concerns about current knowledge. Here, we show that skeletal cell fate is determined by β-catenin but independent of LEF/TCF transcription. Genomic and bioinformatic analyses further identify GATA3 as a mediator for the alternative signaling effects. GATA3 alone is sufficient to promote ectopic cartilage formation, demonstrating its essential role in mediating nonclassical β-catenin signaling in skeletogenic lineage specification

Glycoprotein (GP)96 is essential for maintaining intestinal epithelial architecture by supporting its self-renewal capacity

BACKGROUND & AIMS Glycoprotein (GP)96 is an endoplasmic reticulum (ER)-resident master chaperone for cell surface receptors including the Wnt co-receptors LRP5/6. Intestinal epithelial cells (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche has not been studied so far. Here, we investigated how GP96-loss interferes with intestinal homeostasis by compromising viability, proliferation and differentiation of IEC. METHODS Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-Villin$^{creERT2}$ mice and intestinal organoids. With H&E- and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC-types. Real-time PCR and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by a complete eradication of the epithelial layer and death within few days. These effects were due to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of ER stress. While effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS Our work demonstrates that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and is therefore indispensable for maintaining intestinal epithelial homeostasis

Terminal differentiation of villus tip enterocytes is governed by distinct Tgfβ superfamily members

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfβ. Here, we show that individual Bmp ligands and Tgfβ drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfβ induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs

Dazap2 modulates transcription driven by the Wnt effector TCF-4

A major outcome of the canonical Wnt/β-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with β-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/β-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif

Challenges for Allergy Diagnosis in Regions with Complex Pollen Exposures

Over the past few decades, significant scientific progress has influenced clinical allergy practice. The biological standardization of extracts was followed by the massive identification and characterization of new allergens and their progressive use as diagnostic tools including allergen micro arrays that facilitate the simultaneous testing of more than 100 allergen components. Specific diagnosis is the basis of allergy practice and is always aiming to select the best therapeutic or avoidance intervention. As a consequence, redundant or irrelevant information might be adding unnecessary cost and complexity to daily clinical practice. A rational use of the different diagnostic alternatives would allow a significant improvement in the diagnosis and treatment of allergic patients, especially for those residing in complex pollen exposure areas

ARIA 2016:Care pathways implementing emerging technologies for predictive medicine in rhinitis and asthma across the life cycle

The Allergic Rhinitis and its Impact on Asthma (ARIA) initiative commenced during a World Health Organization workshop in 1999. The initial goals were (1) to propose a new allergic rhinitis classification, (2) to promote the concept of multi-morbidity in asthma and rhinitis and (3) to develop guidelines with all stakeholders that could be used globally for all countries and populations. ARIA-disseminated and implemented in over 70 countries globally-is now focusing on the implementation of emerging technologies for individualized and predictive medicine. MASK [MACVIA (Contre les Maladies Chroniques pour un Vieillissement Actif)-ARIA Sentinel NetworK] uses mobile technology to develop care pathways for the management of rhinitis and asthma by a multi-disciplinary group and by patients themselves. An app (Android and iOS) is available in 20 countries and 15 languages. It uses a visual analogue scale to assess symptom control and work productivity as well as a clinical decision support system. It is associated with an inter-operable tablet for physicians and other health care professionals. The scaling up strategy uses the recommendations of the European Innovation Partnership on Active and Healthy Ageing. The aim of the novel ARIA approach is to provide an active and healthy life to rhinitis sufferers, whatever their age, sex or socio-economic status, in order to reduce health and social inequalities incurred by the disease