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, 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

Safety and uptake assessment of nanostructured silica in an advanced intestinal in vitro model

The food industry has identified the benefits of nanotechnology and exploited the unique properties of engineered nanomaterials (ENM) over the last decade. The number of products available containing ENM or nanostructured materials worldwide is expected to increase even further. Due to this presumed increase in number of available products and manufactur-ing quantities, authorities as well as consumers are concerned about potential adverse ef-fects of nano sized materials in food on public health. Materials directly added to food but also those leaking from the packaging into food might be ingested. Also nanostructured food processing agents, which are added to prevent caking, to improve flowing or to clarify and absorb, might be ingested. Considering the high oral exposure to all these food additives, a better understanding of the uptake, the accumulation and the biological effects of food rele-vant nano sized materials at the intestinal epithelium is needed. Ten differently produced synthetic amorphous silica (SAS) materials with different specific surface areas, different primary structure sizes and different surface charges have been characterised. Their biological impact has been screened in a cell line (Caco 2) representa-tive for the most common cell type in the small intestine, enterocytes. No acute impairment of viability or barrier integrity could be identified. Furthermore, the adhesion and internalization of one representative of fumed and precipitat-ed SAS have been investigated, exploiting flow cytometry, scanning electron microscope coupled with energy dispersive X ray spectroscopy, time of flight secondary ion mass spec-trometry, transmission electron microscopy micrographs, confocal, dark field and hyperspec-tral microscopy. Furthermore, also the impact of food grade titanium dioxide has been inves-tigated in the same setup. Titanium dioxide has been studied to identify if the restrictions in the detection of SAS, were due to the material or the cell environment. The SAS materials were only detected on the cell layer with scanning electron microscope coupled with energy dispersive X ray spectroscopy or the time of flight secondary ion mass spectrometry. It has been shown that for the detection of silica in cell environment a subsequent elemental analy-sis is needed to recognise the SAS materials. In the second part of this work an advanced co culture model has been established to better evaluate the impact of food grade materials in a more in vivo like setting. Caco 2 monocul-ture only presents one cell type of the very complex intestine. The newly established ad-vanced co culture model consists of Caco 2 cells and a mucus producing HT 29 cell line. The addition of B lymphocytes allowed the differentiation of one additional cell type: M cells. The exposition of the advanced co culture model to six different SAS selected due to the different production routes, specific surface areas and their different silanol content has led to no differences in the viability, barrier integrity, microvilli function and lipid uptake. Neverthe-less, the treatment has shown that the mucus production increases after the treatment with SAS materials with an aggregate size above 200 nm and which are highly negatively charged. A co effect has been found for the investigation of the iron cell type precipitated SAS with a small specific surface area decreased the iron uptake in the advanced co culture only in the ferritin uptake but not on the corresponding gene level. This newly established model also offers the possibilities to further investigated broader sci-entific questions. As one other scientific question the colloidal structural formation during milk digestion in the advanced co culture of Caco 2 and HT 29 has been compared with struc-tures formed in a cell free environment during milk digestion. The incorporation of cells has not resulted in different types of structural formations and has not increased the speed at which the colloidal structures are formed during the milk digestion compared to the setup without cells. The results show that the use of this advanced in vitro model can lead to an improved predic-tion on potential adverse outcomes of food components on the intestine. Mucus seems to be a very important protective barrier in the interaction of food components with the intestinal epithelium and should be studied in more detail. The advanced co culture model established in this thesis can be used for a first estimate of the interactions of food components with in-testinal epithelium and a further reduction of animal experiments in the future

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

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.ISSN:0340-5761ISSN:1432-073

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 mu g/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