PHOTON IRRADATION AND CISPLATIN ENRICH CANCER STEM CELLS IN OVARIAN CANCER

High Grade Serous Ovarian Cancer (HGSOC) has a 5-year survival rate of less than 50%. Ovarian cancer is one of the deadliest gynecological diseases and the 7th most common female cancer worldwide. Ovarian cancer patients generally have a poor prognosis despite the relatively successful treatments. When conventional cancer treatments, such as cisplatin chemotherapy and photon irradiation, are administered, residual cancer stem-like cells (CSCs) can survive, leading to CSC enrichment. CSCs are a small population of cancer cells that exhibit stem-like characteristics: quiescence (slowing of the cell cycle), differentiation, proliferation, and self-renewal to regenerate new CSCs. We hypothesized that providing cancer treatments such as irradiation and chemotherapies enriches the CSC population. We aimed to determine the induction of CSC activity after conventional treatment in 2 ovarian cell lines: OVASHO and PDX4 (patient derived xenograft) at a 72-hour time point after treatment. Our project used a GFP reporter system, SORE-6, which fluoresces in the presence of SOX2 and OCT4, core pluripotency factors to determine levels of stemness. Here, we observed a dose-dependent increase in stemness in PDX4 after cisplatin and photon irradiation treatment. In response to photon irradiation, OVASHO exhibited a dose-dependent response and an overall increase in GFP-expression in response to cisplatin. We also observed notable changes in mRNA gene expression of OCT4 and SOX2 in response to cisplatin treatment. These results indicate that cancer cell stemness is enriched after treatment in association to CSC persistence and/or cancer stem cell reprograming

Predisposing effect of the mycotoxins deoxynivalenol and fumonisins on necrotic enteritis in broiler chickens

Mycotoxins are secondary fungal metabolites, which frequently contaminate feed and food raw materials. The Fusarium mycotoxins deoxynivalenol (DON) and fumonisins (FBs) are the most frequently detected mycotoxins on a worldwide basis. The majority of feed samples however comply with European Union (EU) maximum guidance levels. The EU maximum guidance level for poultry feed is set at 5 mg DON/kg feed and 20 mg fumonisin B1 (FB1) + fumonisin B2 (FB2)/kg feed. Although these low to moderate contamination levels do not result in obvious clinical symptoms of intoxication, they may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host pathogen interactions and thus a different outcome of eventual infections. Necrotic enteritis (NE) is one of the economically most important enteric diseases in poultry production. NE in broiler chickens may arise when changes in the gut microbial homeostasis allow colonization of the virulent Clostridium perfringens strains expressing the NetB toxin gene. The presence of predisposing factors such as coccidiosis and fishmeal is required in the pathogenesis of NE, allowing the proliferation of C. perfringens by either providing nutrients and/or favorable ecological niches. The general aim of this thesis was to examine whether DON and FBs, at concentrations in the feed approaching EU maximum guidance levels, predispose for NE in broiler chickens; and to gain insights in the mechanisms responsible for this interaction. Chapter 1 describes an experimental C. perfringens infection study in order to evaluate the predisposing effect of DON exposure to the development of NE in broiler chickens. After C. perfringens challenge, DON significantly increased the percentage of birds developing subclinical NE compared to the control group (47±3.0% vs. 20±2.6%). A negative impact of DON was demonstrated on intestinal morphology and the intrinsic component of the intestinal barrier, which is composed of the epithelial cell layer and the tight junctions that interconnect these epithelial cells. Consequently, this intestinal damage led to an increased luminal protein content by leakage of plasma proteins or altered absorption of dietary proteins, which stimulate clostridial proliferation and subsequently the development of NE. In Chapter 2 the impact of feeding a FBs contaminated diet on the intestinal morphology and microbiota composition was studied. The uptake of FBs contaminated feed also induced a negative impact on the intrinsic component of the intestinal barrier, and on the intestinal microbiota as well. A reduced abundance of the immunomodulating bacteria Candidatus Savagella and Lactobacillus spp. such as L. johnsonii, and an increased number of C. perfringens were observed in the ileum of birds fed a FBs contaminated diet. Subsequently, an experimental C. perfringens infection study was performed investigating the predisposing effect of FBs exposure to the development of NE in broiler chickens. After C. perfringens challenge, a significant higher percentage of birds developed subclinical NE in the group fed a FBs contaminated diet compared to the control group (45±2.2% vs. 30±5.5%). In Chapter 3, the impact of DON and FBs on the intestinal barrier was further examined for selected functional and structural extrinsic components of the intestinal barrier, to gain insights in the associated consequences for intestinal colonization and nutrient availability for C. perfringens. In the first part (Chapter 3.1), a negative impact of feeding broiler chickens a DON and/or FBs contaminated diet on intestinal mucus layer integrity was observed and induction of oxidative stress in intestinal epithelial cells. The last study (Chapter 3.2) investigated whether DON-induced intestinal damage influences the intestinal absorption of FBs, which may lead to an altered systemic exposure and increased toxic effects of the latter mycotoxin in broiler chickens. Although, no differences in toxicokinetic parameters of FB1 could be demonstrated between chickens fed a DON contaminated diet compared to birds fed a control diet. This doctoral thesis demonstrates for the first time that feeding a DON or FBs contaminated diet is a predisposing factor for the development of C. perfringens induced NE in broiler chickens. This coincides with negative effects on selected components of the intrinsic and extrinsic intestinal barrier of the chicken host, i.e. villus height, tight junctions, mucus, oxidative stress, and microbiota homeostasis. Consequently, exposure to DON and/or FBs at concentrations approaching the EU maximum guidance level in feed provides both nutrients and a favorable ecological niche for clostridial proliferation. Therefore, this doctoral thesis demonstrates that in addition to the best-known predisposing factors, DON and/or FBs contaminated feed should be included as major risk factor for the development of C. perfringens induced NE in broiler chickens

Fluorescence in situ hybridisation study of micronuclei in C3A cells following exposure to ELF-magnetic fields

Human C3A cells were exposed to extremely low frequency (50 Hz) magnetic fields (ELF-MF's) up to 500 mu T. They were subjected to the micronucleus assay using a Fluorescence In Situ Hybridization (FISH) technique with an in-house pan-centromere probe. We found no increased frequency in micronucleated cells and no change in the proportion of centromere positive over centromere negative micronuclei compared to the unexposed control cells. These results are in accordance with some, but in contradiction with other previously published investigations underlining that effects of environmental ELF-EMF's on cellular DNA may be very subtle and that small changes or environmental influences may determine the outcome of a (geno)toxicity study. Interestingly, a low-level (5 mu T) exposure resulted in less than the background micronucleus frequency

Chronic dietary intake of enniatin B in broiler chickens has low impact on intestinal morphometry and hepatic histology, and shows limited transfer to liver tissue

The Fusarium mycotoxin enniatin B (ENN B) is a so-called emerging mycotoxin frequently contaminating poultry feed. To investigate the impact of chronic ENN B exposure on animal health, broiler chickens were fed either a diet naturally contaminated with ENN B (2352 mu g/kg) or a control diet (135 mu g/kg) for 2, 7, 14, or 21 days. ENN B concentrations were determined in plasma and liver using a validated ultra-high performance liquid chromatographytandem mass spectrometry UHPLC-MS/MS method. Liver was evaluated histologically, and the villus length and crypt depth of the duodenum, jejunum, and ileum were measured. Histopathology of the livers did not reveal major abnormalities. Feeding an ENN B-contaminated diet could possibly inhibit the proliferation of enterocytes in the duodenal crypts, but did not affect villus length, crypt depth, or villus length-crypt depth ratio of the jejunum and ileum. ENN B levels in plasma and liver were significantly higher in the ENN B-fed group and ranged between <25-264 pg/mL and <0.05-0.85 ng/g, respectively. ENN B carry-over rates from feed to liver tissue were 0.005-0.014% and 0.034-0.109% in the ENN B and control group, respectively. Carry-over rates were low and indicated a limited contribution of poultry tissue-derived products to the total dietary ENN B intake for humans. The above results support the opinion of the European Food Safety Authority stating that adverse health effects from ENN B in broiler chickens are unlikely