Arcobacter butzleri induces a pro-inflammatory response in THP-1 derived macrophages and has limited ability for intracellular survival.

Recent case reports have identified Arcobacter (A.) butzleri to be another emerging pathogen of the family Campylobacteraceae causing foodborne diseases. However, little is known about its interaction with the human immune system. As macrophages act as first defense against bacterial infections, we studied for the first time the impact of A. butzleri on human macrophages using THP-1 derived macrophages as an in vitro infection model. Our investigations considered the inflammatory response, intracellular survival and activation of caspases as potential virulence mechanisms employed by A. butzleri. Induction of IL-1α, IL-1ß, IL-6, IL-8, IL-12ß and TNFα demonstrated a pro-inflammatory response of infected macrophages towards A. butzleri. gentamycin protection assays revealed the ability of A. butzleri strains to survive and resist the hostile environment of phagocytic immune cells for up to 22 h. Moreover, initial activation of intitiator- (CASP8) as well as effector caspases (CASP3/7) was observed without the onset of DNA damage, suggesting a potential counter regulation. Intriguingly, we recognized distinct strain specific differences in invasion and survival capabilities. This suggests the existence of isolate dependent phenotype variations and different virulence potentials as known for other intestinal pathogens such as Salmonella enterica ssp.

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu ) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event.

Structured multicellular intestinal spheroids (SMIS) as a standardized model for infection biology.

BACKGROUND: 3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens. RESULTS: We have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs. CONCLUSION: Based on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections.

TFF3-dependent resistance of human colorectal adenocarcinoma cells HT-29/B6 to apoptosis is mediated by miR-491-5p regulation of lncRNA PRINS.

Tumour necrosis factor-α (TNF-α) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-α. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-α/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue repair but is also involved in intestinal tumour progression and scattering. We hypothesised that TFF3 rescues intestinal epithelial cancer cells from TNF-α-induced apoptosis by involving regulatory RNA networks. In silico-based expression analysis revealed TFF3-mediated regulation of selected microRNAs as well as long non-coding RNAs (lncRNAs), whereas miR-491-5p was identified to target the lncRNA 'psoriasis susceptibility-related RNA gene induced by stress' (PRINS). RNA interference-based gain- and loss-of-function experiments examined miR-491-PRINS axis to exert the TFF3-mediated phenotype. Chemical inhibition of selected pathways showed that phosphatidylinositol 3-kinase/AKT accounts for TFF3-mediated downregulation of miR-491-5p and accumulation of PRINS. Moreover, we showed that PRINS colocalises with PMAIP1 (NOXA) in nuclei of HT-29/B6 possessing inhibitory effects. Immunoprecipitation experiments proved molecular interaction of PMAIP1 with PRINS. Our study provides an insight into RNA regulatory networks that determine resistance of colorectal cancer cells to apoptosis.

Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy.

Small non-coding RNA play a major part in host response to bacterial agents. However, the role of long non-coding RNA (lncRNA) in this context remains unknown. LncRNA regulate gene expression by acting e.g. as transcriptional coactivators, RNA decoys or microRNA sponges. They control development, differentiation and cellular processes such as autophagy in disease conditions. Here, we provide an insight into the role of lncRNA in mycobacterial infections. Human macrophages were infected with Mycobacterium bovis BCG and lncRNA expression was studied early post infection. For this purpose, lncRNA with known immune related functions were preselected and a lncRNA specific RT-qPCR protocol was established. In addition to expression-based prediction of lncRNA function, we assessed strategies for thorough normalisation of lncRNA. Arrayed quantification showed infection-dependent repression of several lncRNA including MEG3. Pathway analysis linked MEG3 to mTOR and PI3K-AKT signalling pointing to regulation of autophagy. Accordingly, IFN-γ induced autophagy in infected macrophages resulted in sustained MEG3 down regulation and lack of IFN-γ allowed for counter regulation of MEG3 by viable M. bovis BCG. Knockdown of MEG3 in macrophages resulted in induction of autophagy and enhanced eradication of intracellular M. bovis BCG.

Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells.

Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features.