Investigating the effects of differently produced synthetic amorphous silica (E 551) on the integrity and functionality of the human intestinal barrier using an advanced in vitro co-culture model.

E 551, also known as synthetic amorphous silica (SAS), is the second most produced food additive. However, according to the re-evaluation of E 551 by the European Food Safety Authority (EFSA) in 2018, the amount of available data on the oral toxicity of food grade E 551 is still insufficient for reliable risk assessment. To close this gap, this study aimed to investigate six food-grade SAS with distinct physicochemical properties on their interaction with the intestinal barrier using advanced in vitro intestinal co-cultures and to identify potential structure-activity relationships. A mucus-secreting Caco-2/HT-29/Raji co-culture model was treated with up to 50 µg/ml SAS for 48 h, which represents a dose range relevant to dietary exposure. No effects on cell viability, barrier integrity, microvilli function or the release of inflammatory cytokine were detected after acute exposure. Slight biological responses were observed for few SAS materials on iron uptake and gene expression levels of mucin 1 and G-protein coupled receptor 120 (GPR120). There was no clear correlation between SAS properties (single or combined) and the observed biological responses. Overall, this study provides novel insights into the short-term impact of food-relevant SAS with distinct characteristics on the intestinal epithelium including a range of intestine-specific functional endpoints. In addition, it highlights the importance of using advanced intestinal co-cultures embracing relevant cell types as well as a protective mucus barrier to achieve a comprehensive understanding of the biological response of food additives at the intestinal barrier in vitro.

The impact of synthetic amorphous silica (E 551) on differentiated Caco-2 cells, a model for the human intestinal epithelium.

For several decades, food-grade synthetic amorphous silica (SAS) have been used as a technological additive to reduce caking of food powders. Human exposure is thus inevitable and safety concerns are taken seriously. The toxicity of silica in general and SAS in particular has been studied extensively. Overall, there is little evidence that food-grade SAS pose any health risks to humans. However, from the available data it was often not clear which type of silica was used. Accordingly, the latest report of the European food safety authority requested additional toxicity data for well-characterised "real food-grade SAS". To close this gap, we screened a panel of ten well-defined, food-grade SAS for potential adverse effects on differentiated Caco-2 cells. Precipitated and fumed SAS with low, intermediate and high specific surface area were included to determine structure-activity relationships. In a physiological dose-range up to 50 µg/ml and 48 h of incubation, none of the materials induced adverse effects on differentiated Caco-2 cells. This held true for endpoints of acute cytotoxicity as well as epithelial specific measures of barrier integrity. These results showed that despite considerable differences in production routes and material characteristics, food-relevant SAS did not elicit acute toxicity responses in intestinal epithelial cells.

Nanostructure generation during milk digestion in presence of a cell culture model simulating the small intestine.

HYPOTHESIS: The development of advanced oral delivery systems for bioactive compounds requires the fundamental understanding of the digestion process within the gastrointestinal tract. Towards this goal, dynamic invitro digestion models, capable of characterising the molecular as well as colloidal aspects of food, together with their biological interactions with relevant invitro cell culture models, are essential. EXPERIMENTS: In this study, we demonstrate a novel digestion model that combines flow-through time resolved small angle X-ray scattering (SAXS) with an invitro Caco-2/HT-29 cell co-culture model that also contained a mucus layer. This set-up allows the dynamic insitu characterisation of colloidal structures and their transport across a viable intestinal cell layer during simulated digestion. FINDINGS: An integrated online SAXS - invitro cell co-culture model was developed and applied to study the digestion of nature's own emulsion, milk. The impact of the invitro cell culture on the digestion-triggered formation and evolution of highly ordered nanostructures in milk is demonstrated. Reported is also the crucial role of the mucus layer on top of the cell layer, protecting the cells from degradation by digestive juice components such as lipase. The novel model can open unique possibilities for the dynamic investigation of colloidal structure formation during lipid digestion and their effect on the uptake of bioactive molecules by the cells.

Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients.

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish "MGMT methylated" from "MGMT unmethylated" patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, < 10% mean methylation), 10.4 months in the low methylated group (LM, 10-20% mean methylation) and 19.83 months in the highly methylated group (HM, > 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant.

Classification of in vitro genotoxicants using a novel multiplexed biomarker assay compared to the flow cytometric micronucleus test.

Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow® assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): γH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow® micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow® assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow® assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a lab-specific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow® . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow® assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens. Environ. Mol. Mutagen. 58:662-677, 2017. © 2017 Wiley Periodicals, Inc.

Phospho-AXL is widely expressed in glioblastoma and associated with significant shorter overall survival.

Receptor tyrosine kinase AXL (RTK-AXL) is regarded as a suitable target in glioblastoma (GBM) therapy. Since AXL kinase inhibitors are about to get approval for clinical use, patients with a potential benefit from therapy targeting AXL need to be identified. We therefore assessed the expression pattern of Phospho-AXL (P-AXL), the biologically active form of AXL, in 90 patients with newly diagnosed GBM, which was found to be detectable in 67 patients (corresponding to 74%). We identified three main P-AXL expression patterns: i) exclusively in the tumor vasculature (13%), ii) in areas of hypercellularity (35%), or iii) both, in the tumor vasculature and in hypercellular areas of the tumor tissue (52%). Pattern iii) is associated with significant decrease in overall survival (Hazard ratio 2.349, 95% confidence interval 1.069 to 5.162, *p=0.03). Our data suggest that P-AXL may serve as a therapeutic target in the majority of GBM patients.