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Development of an in vitro-model for the investigation of pathogenicity mechanisms of gut diseases caused by zoonotic pathogens

Methods:

  • Coculture of human enterocytes and goblet cells
  • Cultivation of intestinal organoids from pigs
  • Analysis after treatment with bacterial toxins with Ussing chamber, RT-qPCR, Western blot, immunofluorescence

 

Project description:
The intestine plays a decisive role in many diseases that can be transmitted from animals to humans (so-called zoonoses). Both in the establishment of an infection and for the excretion of pathogens and the potentially associated spread of an infection. While various in vitro systems based on cell cultures are available for the study of disease mechanisms in mice, rats and humans, the use of primary cell or organ cultures taken directly from the animal or even the use of animals is still necessary to study such processes in farm animals (e.g. pigs, cattle or poultry). For this reason, new approaches to develop in vitro models to study the underlying molecular mechanisms of infections in farm animals are of great interest, especially given that farm animals are often carriers or vectors of zoonotic pathogens. The purpose of the present project is therefore to develop a model of the intestine by combining for the first time the so-called "colon simulation technique" (Cositec) with the "Ussing chamber technology". In this process, intact intestinal mucosa of farm animals is clamped in Ussing chambers and incubated simultaneously with the contents of the Cositec system, so that a confrontation of the intestinal epithelium with the physiological intestinal contents can take place. The combination of these two well-established methods thus makes it possible to design an in vitro model whose conditions largely correspond to the in vivo conditions. In a first step, the vitality and functionality of the intestinal epithelium in this system is checked by morphological, biochemical and functional analyses (e.g. by histology, PCR or nutrient uptake). It is of great importance that the intestinal tissue used for all analyses is not derived from laboratory animals, but is always obtained at conventional slaughterhouses. This means that the use of laboratory animals can be completely avoided. In the following step, the intestinal epithelium is to be incubated with zoonotic pathogens (e.g. enterotoxic and enteropathogenic E. coli) in order to be able to investigate the infection mechanisms of these pathogens and their effects on the intestine in more detail. If this new system were to work, it would offer a wide range of other uses, apart from research into infectious diseases. These include, for example, physiological, toxicological and pharmacological questions. Furthermore, cell cultures of the intestine could also be used in this model, which would make the use of animal material largely superfluous. Overall, a successful establishment of this system would result in a significant reduction in the number of experimental animals.

Funding:
R2N, Ministry of Science and Culture of Lower Saxony, 2017-2021

Project lead:
Prof. Dr. Gerhard Breves (gerhard.breves@tiho-hannover.de, 0511-856-7271)
Institute for Physiology and Cell Biology
Prof. Bettina Seeger, Ph.D. (bettina.seegertiho-hannover.de, 0511-856-7602)
Institute for Food Toxicology

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