Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection

Objective Helicobacter pylori causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. Design Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air–liquid interface culture stage to study cross-talk with H. pylori and the adjacent stroma. Results The resulting ‘mucosoid cultures’, so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness—reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with H. pylori, a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. Conclusion Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of H. pylori with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence.</p

Impact of immune parameters and immune dysfunctions on the prognosis of patients with chronic lymphocytic leukemia

SIMPLE SUMMARY: In chronic lymphocytic leukemia (CLL), immune alterations—affecting both the innate and adaptive immunity—are very common. As a clinical consequence, patients with CLL frequently present with autoimmune phenomena, increased risk of infections and second malignancies. The aim of this review article is to present available data on CLL-associated alterations of immune parameters that correlate with known prognostic markers and with clinical outcome. Also, data on the impact of immune-related clinical manifestations on the prognosis of patients with CLL will be discussed. ABSTRACT: Chronic lymphocytic leukemia (CLL) is characterized by a wide spectrum of immune alterations, affecting both the innate and adaptive immunity. These immune dysfunctions strongly impact the immune surveillance, facilitate tumor progression and eventually affect the disease course. Quantitative and functional alterations involving conventional T cells, γδ T cells, regulatory T cells, NK and NKT cells, and myeloid cells, together with hypogammaglobulinemia, aberrations in the complement pathways and altered cytokine signature have been reported in patients with CLL. Some of these immune parameters have been shown to associate with other CLL-related characteristics with a known prognostic relevance or to correlate with disease prognosis. Also, in CLL, the complex immune response dysfunctions eventually translate in clinical manifestations, including autoimmune phenomena, increased risk of infections and second malignancies. These clinical issues are overall the most common complications that affect the course and management of CLL, and they also may impact overall disease prognosis

DNA methylation in human gastric epithelial cells defines regional identity without restricting lineage plasticity

BACKGROUND: Epigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail. RESULTS: Here, we establish gastric mucosa derived primary cell cultures as a reliable source of native human epithelium. We describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach, i.e., antrum, corpus, fundus together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1 and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. By extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. We present insights into the dynamics of DNA methylation across anatomical regions of the healthy stomach and patterns of its change in disease. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13148-022-01406-4

Targeting hif-1&#945; regulatory pathways as a strategy to hamper tumor-microenvironment interactions in cll

The hypoxia-inducible factor 1 (HIF-1) and the CXCL12/CXCR4 axis regulate the interaction of chronic lymphocytic leukemia cells and the tumor microenvironment. However, the interconnections occurring between HIF-1 and the CXCL12/CXCR4 axis are not fully elucidated. Here, we demonstrate that the CXCL12/CXCR4 axis plays a pivotal role in the positive regulation of the α subunit of HIF-1 (HIF-1α) that occurs in CLL cells co-cultured with stromal cells (SC). Inhibitors acting at different levels on CXCR4 downstream signalling counteract the SC-induced HIF-1α upregulation in CLL cells, also hindering the SC-mediated pro-survival effect. HIF-1α inhibition also exerts off-tumor effects on the SC component, inducing the downregulation of target genes, including CXCL12. Consistently, our data show that pretreatment of leukemic cells and/or SC with idelalisib effectively abrogates the SC-mediated survival support. A combined on-tumor and off-tumor inhibition of HIF-1α was also observed in idelalisib-treated patients, who showed, along with a downregulation of HIF-1α target genes in leukemic cells, a significant decrease in CXCL12 serum concentration and changes in the bone marrow microenvironment. Our data demonstrate that the targeting of HIF-1α or its regulatory pathways acts at the tumor- and SC-level, and may be an appealing strategy to overcome the microenvironment-mediated protection of CLL cells

Genomic aberrations after short-term exposure to colibactin-producing E. coli transform primary colon epithelial cells

Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin’s distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC

Bacteria Moving into Focus of Human Cancer

Although bacteria have long been associated with human cancer, drawing causal relationships has been difficult. In this issue of Cell Host & Microbe, Scanu et al. (2015) provide evidence for a transforming activity of Salmonella Typhimurium on predisposed host cells, which can subsequently form tumors in a xenograft model

Generation, proliferation and expansion of epithelial cells from primary tissue into mucosoid cultures

Subject of the invention is a method for long-term culture of columnar epithelial cells and the application thereof. The cells in culture survive more than 6 months and they can be expanded due to their regenerative capacity. The epithelial cells are seeded on a thin matrix-coated solid semi-permeable filter and cultivated in an air liquid interface to form a coherent monolayer. The cultivation method allows for the regeneration and expansion of the culture by enzymatic removal of the cells from the filters and seeding on a new filter. The proliferation, longevity and regenerative capacity of the cells rely on the maintenance of the stem cell niche in vitro. The stem cells are capable of multi-lineage differentiation. The cultures are sensitive to morphogens and grow factors that are inducing proliferation and/or differentiation into the specific cell lineages typical of the healthy tissue of origin. Specialized cell lineages maintain their function of origin also in the culture of the invention. By mimicking a pathological condition in the cultures of the invention, the distribution of the cells in the specific cell lineages can be altered and other lineages that are not typical of the tissue of origin may emerge (e.g. Metaplasia, neoplasia). Cultured cells of the invention maintain features of the tissue of origin including height and polarization. Polarization segregates the apical side from the basal side wherein the basal side is adjacent to the semi-permeable surface of the filter and in contact with the culture medium. The cells of the culture of invention are producing and accumulating mucus on the apical side. The apical and the basal side of the cells represent two different locations where the epithelial cells can conduct their secretory and absorptive function. The combination of these two functions may result in an active transport of substances form the apical to the basal side or vice-versa. The cells in the culture are able to respond to a variety of stimuli. The stimulus can be applied in the culture at the apical, basal or both sides. Result of the stimulation may be the activation of a specific signaling pathway that would promote the transcription of genes involved in a specific cellular function (e.g. regulation in: inflammation, differentiation, proliferation, migration, metabolism, transport, secretion, absorption and others). The stimulus can be of various sources including physical stimulation (e.g., heat, electromagnetic radiation, contact and others) chemical stimulation (e.g. natural or synthetic molecules) or biological stimulation (e.g., whole or part of organisms such as gamete, zygotes, embryos, bacteria, viruses or parasites). Other types of non-epithelial cells can be co-cultured. For example, stromal cells isolated from the same tissue can be co-cultivated below the epithelium or below the porous filters support in the same vessel reproducing the complexity of the whole mucosa. Similarly, cells of the hematopoietic lineage can be co-cultivated within or below the epithelium or below the porous filters support. We name the epithelial culture of the invention“mucosoid culture”, in reference to the fact that they recapitulate key characteristics of the polarized, mucus-secreting epithelial layers and they can optionally include other cells from the mucosa. The cultures originate from explanted epithelial cells and columnar epithelial cells. The epithelial cells can be derived from primary cells isolated from any embryonic or adult vertebrate, preferentially mammalian or human tissue; alternatively, the cell can be derived from other types of epithelial cultures that allow maintenance of stem cells in vitro, preferentially organoids or planar feeder supported culture. Cells prior cultivation into mucosoid culture can be genetically modified (e.g., using recombinant nucleic acids). The mucosoid culture of this invention can also be derived from other types of non-epithelial cells, providing they have been transformed or re-programmed to become epithelial cells, reminiscent to the authentic cells described in this invention. For example, fibroblasts can be converted into induced pluripotent cells (iPS) and the re-differentiated to form polarized epithelial cells. While the induction of pluripotency and the process of redifferentiation are not the topics of this invention, however, the outcome of the redifferentiation process, i.e. the epithelial cells, is part of the invention providing they are used as mucosoid cultures, as described herein. The mucosoid cultures are a source of mucus, cells and substances secreted in the culture medium. Analysis or direct use of these tree components, as a result of the mucosoid cultivation or of the stimulation of the mucosoid cultures, is part of this invention. The established mucosoid cultures serve a variety of purposes, including (i) the long-term maintenance or amplification of epithelial cells used in this invention, (ii) the exploration of the behavior of the cells in response to extrinsic stimuli, (iii) the transport of substances or factors from apical to basolateral directions, or vice versa, or the translation of biological, chemical and physical signals arriving from either direction, (iv) their usage as a production tool or factory for biological factors useful for technical and medical purposes, (v) their usage as an apical platform to facilitate and reproduce various extracorporeal biological processes, (vi) their usage in more complex biological settings with additional cell populations beyond the semi-permeable filter to reproduce various intracorporeal processes, and (vii) human diagnostic or therapeutic applications

Helicobacter pylori-induced NF-κB: trailblazer for gastric pathophysiology

NF-&#x3BA;B signaling pathways, induced by a variety of triggers, play a key role in regulating the expression of genes involved in the immune response and cellular responses to stress. The human pathogen Helicobacter pylori induces classical and alternative NF-&#x3BA;B signaling pathways via its effector ADP-L-glycero-&#x3B2;-D-manno-heptose (ADP-heptose). We review H. pylori- and NF-&#x3BA;B-dependent alterations in cellular processes and associated maladaptation leading to deleterious gastric pathophysiology that have implications for the diagnosis and treatment of gastric diseases. Therapeutic options for gastric cancer (GC) include clinically relevant small molecule inhibitors of NF-&#x3BA;B and epigenetic therapy approaches. In this context, gastric organoid biobanks originated from patient material, represent a valuable platform for translational applications to predict patient responses to chemotherapy, with a view to personalized medicine