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Gastrointestinal Physiology

Prof. Dr. med. vet. Gerhard Breves

 

Development of an in vitro-model for the investigation of pathogenicity mechanisms of gut diseases caused by zoonotic pathogens (Msc Pascal Hoffmann).

The intestine plays an important role in the frame of infectious diseases that can be transmitted from animals to humans (so-called zoonoses) as well as in the establishment of an infection and its potential spreading through the excretion of pathogens. While cell culture-based in-vitro-systems are available for investigating disease mechanisms in mice, rats and humans, the analysis of such mechanisms in livestock animals (e.g., pigs, cattle or poultry) still requires the use of live animals, primary cells or organ cultures directly obtained from living animals. The purpose of the present project is therefore to develop an in-vitro-system replicating the intestine by combining the so-called “colon-simulation-technique” (cositec) with the well-established Ussing-chamber-technology.

 

Large roundworm induced malabsorption: modulation of intestinal porcine nutrient  transport (TÄ Nicole Issel)

The roundworm Ascaris suum is the most important parasite in pig farming in Europe. Infections are acquired by ingestion of embryonated eggs containing the infective third-stage larva. These larvae hatch after ingestion by the host, reach the liver via the mesenteric veins and remain within the liver tissue during a histotropic phase until they continue migration via blood stream into the lungs. Following migration through alveoli, larvae reach the oral cavity via the trachea and are subsequently swallowed. Thus, they reach the gastrointestinal tract about 4 weeks post infection, where they develop into the preadult stage and finally into sexually mature adults and complete the life cycle by producing large amounts of eggs. Infected pigs most commonly show unspecific clinical signs like decreased performance and retardation of growth. Within the host, the development of Ascaris suum has well been described; however, mechanisms of action of this parasite are mostly unknown. Few data obtained from pigs as well as studies comprising laying hens indicate an influence of Ascaris suum on intestinal nutrient transport mechanisms. Therefore, the aim of the project is the investigation of the effect of an Ascaris suum-infection in pigs on intestinal transport of glucose, amino acids and dipeptides. Additionally, the effect of excretory-secretory antigens as well as cuticle antigens of the parasite on these transport mechanisms will be evaluated as these antigens are in direct contact with the intestinal wall. Analyses will be conducted utilizing in vitro-measurements of the intact epithelium after mucosal addition of nutrients via electrophysiological response. Furthermore, measurements on nutrient flux rates employing radioactively labeled substrates as well as uptake studies into membrane vesicles of luminal and basolateral membranes of enterocytes will be conducted. Molecular approaches comprising quantitative real-time PCR, immunoblotting and immunohistochemistry to evaluate transcription and expression of transporter systems will complete the functional analyses. The expected insights will not only contribute to a better understanding of the host-parasite interactions, but will also provide the basis for new, innovative therapeutic approaches. Beside therapeutic intervention concerning malabsorption in porcine or human ascariosis, this may also include the potential identification of target molecules contributing to the development of therapeutics for the treatment of etiologically different absorption disorders or obesity. This precludes the complete functional characterization of intestinal nutrient transport systems in response to A. suum infections, which will be achieved by the planned project.


Melanie Eger, PhD

 

In vitro investigations of the rumen microbiome and metabolome under physiological and pathological conditions

The topic of our research is to characterize the ruminal bacterial community and its fermentation products in the RUSITEC in detail by using next generation sequencing and metabolomics techniques. Moreover, the effect of various physiological and pathological conditions on the ruminal fermentation and the microbial community are investigated. For example, a model to simulate a subacute ruminal acidosis in the RUSITEC is established and characterized.

 

Dr. rer. nat. Stefanie Klinger

 

Studies on effects and mechanisms of trans-resveratrol on intestinal nutrient transport in the porcine small intestines

In recent years, several health promoting effects have been described for the polyphenol resveratrol including anti-carcinogenic, anti-inflammatory and anti-hyperglycemic properties. The underlying mechanisms are of high scientific interest at the moment and a variety of intracellular pathways was found to be influenced by resveratrol. Because of the potential of resveratrol to be used as a dietary supplement, its metabolism in enterocytes has been intensively characterized with regard to its bioavailability, whereas only few studies are available on effects of resveratrol on the intestinal absorption of nutrients and electrolytes. The aim of the project is to investigate in functional studies using the Ussing chamber technique, whether the intestinal absorption of nutrients apart from the absorption of glucose is influenced by resveratrol.

 

 

Dr. rer. nat. Alexandra Muscher Banse

 

Modulation of calcitriol and IGF 1 synthesis by dietary protein in young goats

A protein reduction leads to a decline in calcitriol and IGF1 concentrations, a decrease in intestinal calcium (Ca) absorption and therefore diminished blood Ca levels in young goats. The underlying mechanisms which are involved in the modulation of hepatic IGF1 synthesis during protein reduction are unknown in young goats. Components of the somatotropic axis (growth hormone (GH)/IGF1 axis) will be investigated to identify potential factors for the reduction of IGF1 levels during protein reduction. Described investigations are likely to improve the comprehension of the effects of a protein reduced diet on the hypothalamic-pituitary axis (especially GH and IGF1) as an explanation for the already known changes of calcitriol synthesis in young goats during this feeding regime. Knowledge about changed expression of modulators of calcitriol and IGF1 synthesis can help to better understand resulting consequences such as reduced calcitriol concentrations and therefore reduced intestinal Ca absorption and diminished blood Ca levels and as a consequence alteration of further metabolic pathway like bone metabolism.

 

 

Dr. rer. nat. Susanne Riede

 

Glypho-Bak-Project

Since approximately 40 years, glyphosate-based herbicides are applied in agriculture. The active ingredient Glyphosate inhibits the enzyme 5-enolpyruvlyshikimate-3-phosphate synthase (EPSPS), which is involved in the biosynthesis of aromatic amino acids, leading to plant death. EPSPS is existent as well in bacteria and fungi which degrade the forage in the forestomach of ruminants. The present study dedicates the question whether glyphosate-based herbicides affect the rumen microbial metabolism in vitro by applying the rumen simulation technique (RUSITEC). 

 

PD Dr. med. vet. habil. Mirja Wilkens

 

Ca balance in laying hens

In collaboration with the Institute of Farm Animal Genetics, Federal Research Institute for Animal Health Mariensee we are investigating potential differences in respect to the capacity of four different genotypes of purebred laying hens (WLA: high performing white layer, R11: low performing white layer, BLA: high performing brown layer, L68: low performing brown layer) to respond to transient challenges of their Ca homeostasis. The hypothesis that selection for performance has resulted in the maintenance of production even under unfavorable conditions and despite the negative impact on the organism itself, is the key question of this project.

 

Supplementation with different vitamin D metabolites: non-classical effects

Supplementation with different vitamin D metabolites is common practice not only in humans, but also in livestock. Because first results obtained with sheep have indicated that such treatments might influence the metabolism and elimination of hormones and therapeutics, we are conducting systematic studies to address this topic in rats now.

 

Dietary interventions to stabilize Ca homeostasis in ruminants

Different studies in collaboration with the Institute of Animal Nutrition, Vetsuisse Faculty, University of Zurich, and the Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, are carried out in cattle and sheep as model animals. We are investigating to which extent the gastrointestinal absorption, renal excretion and mobilization of Ca and P from the skeleton can be stimulated or inhibited by feeding different contents of Ca and P, dietary induction of a compensated metabolic acidosis and supplementation with vitamin D metabolites. The overarching objective of these projects is the deeper understanding, the improvement and the development of the currently used strategies to stabilize the peripartum Ca homeostasis of the dairy cow.

 

 

Methods

 

Rumen simulation technique, RUSITEC

 

 

The Rumen Simulation Technique (RUSITEC) is a well-established method for simulating rumen microbial fermentation in vitro. At the beginning of an experiment the fermentation vessels are inoculated with liquid and solid rumen content from ruminants and thereafter are supplied with new feed daily and continuously infused with buffer solution similar to the ruminants’ saliva. The effluent and fermentation gas are collected. The RUSITEC can be used for testing e.g. the effect of various feedstuff or feed additives on microbial fermentation (measurement of short chain fatty acids via gas chromatography, photometric assay for measuring ammonia and lactate concentrations), gas production (CO2 and methane) or the microbial community (SSCP) over several weeks. For a more detailed investigation of the bacterial and archaeal community in the RUSITEC DNA can be isolated and send to next generation sequencing.

 

The Ussing chamber technique: An in vitro method for analyzing electrphysiological and transportphysiological processes using intact epithelia or cell culture models

 

 

 

 The absorption of nutrients ions is mediated by complex transport processes by the epithelia of the gastrointestinal tract. Various factors as the food composition, the age or the hormonal status as well as pathophysiological events like diseases or infections are involved in the regulation of intestinal nutrient and ion transport. Since an adequate nutrient supply and nutrient utilization are essential for the health of an organism, the cellular mechanisms of nutrient absorption and their regulation are of considerable interest.    

The Ussing chamber technique, was initially described by USSING and ZERAHN in 1951, allows studying epithelial transport processes using intact epithelial tissues or cell culture models under controlled in vitro conditions for several hours.

 

Molecular biological techniques

In order to provide some insight into cellular mechanisms which form the basis for effects on the level of an organ or a whole organism, different molecular biological techniques are used. While real-time PCR is used for the quantification of RNA, changes in protein expression levels are measured semi quantitatively using Western Blot analysis. Protein can also be detected in histological tissue sections. The enzyme-linked Immunosorbent Assay (ELISA) is used for the determination of Vitamin D metabolites or bone markers in plasma samples.

The DNA fingerprint method SSCP (single strand conformation polymorphism) is based on the detection of microbial 16S rRNA and is used for the characterization of the microbial community in the rumen.

 

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