Research group leader: Péter Gálfi
Pharmaceutical Sciences, Clinical Pharmacology, Pharmaceutical chemistry, Animal pharmacology, Animal feed additives, Veterinary pharmacology, Prevention of oxidative stress, Performance enhancement.
HAS Chemical Research Center, Hungary
Analkrom Ltd., Hungary
University of Innsbruck, Austria
insert propagated swine jejunum cells used for modeling the oxidative stress
Interleukins and TNF-alpha expression measurement
measuring of anti-inflammarory effect of some agents
Contact: Gálfi, Péter
The effect of medicines, their active ingredients and feed components on the activity of cytochrom-enzymes, Examination of their potential interactions in the point of view of the altered efficacy and toxicity,
Interspecies differences in tissue distribution of medicines in different bird species,
The effect of species and living conditions on altered sensitivity to xenobiotics in birds
Experiments with veterinary medicines and their active ingredients:
- Pharmacokinetic examinations:
- Plasma kinetics
- Bioequivalence testing
- Tissue elimination and residue investigations
- Toxicology examinations
- Acute toxicity tests on laboratory species
- Subacute and chronic toxicity tests
- Target animal safety examinations
- Clinical efficacy tests
Investigations with cell co-culture model systems are needed to understand the complex interrelationships between such processes as intestinal absorption, bioavailability and hepatic biotransformation in the animal organism. However, at present, suitable animal co-culture models mimicking the in vivo enterohepatic cooperation of non-transformed intestinal and hepatic cells are not available. The aim of our research is to establish a new porcine cell co-culture model constituted from differentiated intestinal epithelial cells (IPEC-J2) and primary hepatocytes, and to determine the changes in the expression levels of the xenobiotic transforming cytochrome P450 in porcine liver cells, as caused by pro-inflammatory mediator molecules produced by the intestinal epithelial cells, in response to microbial lipopolysaccharide (LPS) and reactive oxygen species. The expression of pro-inflammatory cytokines and cytochrome P450 iso-enzymes are determined at the level of transcription (qRT-PCR) and translation (ELISA and Western blot analysis). Thirdly, the potential anti-inflammatory activity of total spent culture supernatant of probiotic microbes (Lactobacillus and other bacterium species) and that of their single microbial products (such as, for instance, sodium-n-butyrate) will be investigated in the co-culture model.
Flavonoids demonstrate various beneficial effects on our health - they have anti-cancer, anti-inflammatory and anti-bacterial activity. Many of these biological actions have been attributed to their antioxidant properties. There is an emerging view that flavonoids do not act as conventional hydrogen-donating antioxidants but may exert modulatory actions in cells through actions at protein kinase signalling pathways.
In the other hand, flavonoids show great potential as anti-inflammatory and cancer chemopreventive agents in cell culture studies. This does not translate well into in vivo activity, because of their extensive metabolism in intestine and liver. Methoxyflavones also show increased cancer chemopreventive properties and they have been of particular interest due to their anti-inflammatory properties too.
Our aim is to establish an in vitro intestinal model mimicking oxidative stress triggered of chemicals and pathogen bacteria. It could be achieved by hydrogen-peroxide and LPS-treatment of a non-tumoric intestinal epithelial cell culture (using cell line IPEC-J2 derived from pig). Inflammation parameters such as proinflammatory cytokines and heat-shock proteins should be followed up (at the level of transciption and translation using qPCR and ELISA method, respectively). Furthermore, we plan to test effect of methoxyflavones, which are very promising but not extensively-studied compounds to prevent and cure inflammation diseases of the intestine.
In case of bacteria, biofilm production is one representation of the so called quorum sensing, also known as local density sensing. When the size of the population exceeds a threshold value, gene expression changes, and bacteria and fungi will produce biofilms, that can protect the population from environmental effects (e.g. antibiotics, antifungal agents).
In our studies, primarily Pseudomonas aeruginosa and Malassezia pachydermatis strains are investigated. Their susceptibility is compared between their biofilm producing and planctonic (non-biofilm producing) forms. The studies are conducted with the catheter adhesion methodology, taking into account the CLSI (Clinical Laboratory Standards Institute) microdilution method. In our studies, the simple, unaided eye examinations are complemented with vital staining and their photometric evaluation.
Pseudomonas aeruginosa is a frequently isolated bacterium in the veterinary practice, causing mainly dermatological, urinary, respiratory and systemic infections. The bacterium is frequently multiresistant and can develop resistance even during therapy, thereby the effective treatment of these infections frequently proves difficult. One possibility is to use synergistic combinations against these bacteria, involving aminoglycosides, fluoroquinolones, polymixins and antipseudomonal beta-lactams. In our studies the checkerboard microdilution method is utilized to describe interactions (addition, antagonism and synergy) between the investigated antibiotics. In addition, 8-day serial passages are conducted to investigate the possible resistance-delaying effect of certain combinations.
Oncothermia (OTM) is an alternative method for treatment of tumors, which utilizes electromagnetic energy for destroying the tumor. During the treatment the tumor is mild heated, not more than 42 oC core temperature. In opposite to the classic hyperthermia, this method does not use heat or IR radiation to warm up the tumor, the energy it transferred to the treated part of the body by capacitance coupled high frequency electromagnetic energy. Utilizing this method, considerable target selectivity is elucidable, since the tumor tissue contains significantly more electrically conductive metabolites than the healthy tissues. This property makes the method self focusing.
In our investigations we are looking for appropriate combination treatment possibilities for the oncothermia which can rise the efficacy of it and also can make the local effect of the treatment systemic. For to reach this goal we are testing several possible conventional and alternative medicine component.
The incidence of malicious poisonings of predator birds has increased markedly during the past years, which particularly endangers the scarce, protected eagles (Imperial eagle, White-tailed eagle). It is shown very well by the increased number of fatal toxicoses in the previous years: e.g. the poisoning cases were higher in 2007 than in the last 10 years, and its number was exceeded in the first quarter of 2008 than in 2007. The toxic baits are used improperly or illegally to control the unwanted pests such as red fox, blackbilled magpie, hooded crow, however, protected and other huntable species such as pheasants, mallards, rabbits, wild boar, deer, follow-deer, red deer cam also ingest these baits. The poisoned animals may have fodd safety importance by consumption of them or can lead to secondary toxicosis of other animals. Experimental studies are performed on broiler chickens and other model animals to investigate the potential poisonous effect and the harmful sources of sublethal amount of pesticides to highly protected and protected birds and other animals, and to human consumer. Aspects of environmental protection and public health of a carbofuran-containing product (Chinofur 40 FW) were studied in our previous experiment. Broiler chickens were treated orally by tube with the sublethal amount of carbofuran (2.5 mg/kg bw). Then the clinical signs were observed and the concentration of carbofuran was measured by gas chromatographic method in the blood, liver and muscle tissue samples, and stomach content of the treated animals after 30 and 90 minutes of the treatment. It can be stated that the sublethal concentration of carbofuran can induce residue of it above the food-toxicological level in the breast muscle at 30-90 minutes without severe clinical signs in the poisoned birds. The detected levels of carbofuran in the breast muscle and stomach content can pose human health hazards and possible ecotoxicological risk for predators as secondary poisonous source
During the further experiments other pesticides will be investigated on chickens and other model animal species to protect the protected animals and human consumers.
Investigations performed on laboratory animals are useful as background information to the target animal safety studies and they draw attention to potential target organs and functional alterations but they do not replace them. During the target animal safety studies the test item has to be the same to the product intended to be marketed including chemical structure, purity, composition, particle size and formulation. Similarly, the route of administration has to be the same proposed by the label. The aims of the target animals safety studies are to evaluate the tolerance level of the recommended and multiple dose levels of the product applied by the recommended administration route(s) and to determine the margin of safety for the test item which cannot induce undesribale side effects including clinical signs, haematological and clinical-chemistry changes. Tolerance studies are generally required if the product contains new active substance or even new salt, or if the product marketed will be useful on a new target animal species, or if the product will be applied dermally but its active substance absorbes from the treated surface inducing systemic effect.
Persistent heavy metals (e.g. Hg, Pb, Cd) in the environment can accumulate during the food-chain in those higher animal species which were not directly exposed by the xenobiotics. Considerable concentration and accumulation of the environmental pollutants in the organisms may be realised due to the biomagnification. Heavy metal concentration of a geographical area can be characterised by the results from bird’s tissues because they are found at higher level of the food-chain, they capture the food from large area and they also live in more contaminated region such as watery habitat. To determination of heavy metal burden of birds feathers will be basically collected. Initially, the investigation will be performed at the region of Balaton by the collection of feathers from living animals and/or tissue samples (liver, kidney, muscle) from dead birds. The species, age and body weight of the animals will be determined. The measurement of heavy metal content of the biological samples will be analysed by atomic absorption spectrometry or other suitable analytical methods. Based on the results, conclusions will be drawn between the influencing effect of body size, geographical location and position in food-chain of birds, and the heavy metal content. During the health risk assessment, the sublethal harmful effect of heavy metal content of bird’s tissues will be evaluated which can modify the behaviour of birds and can induce reproductive and other problems.
During the further investigations, other bird species and their environment will be monitored at other region of Hungary.
The Erythromycin is one of the most frequently used macrolid type antibiotic in the veterinary practice, which metabolises intensively in the liver. In our in vitro experiments we investigated the changes of Erythromycin concentrations in different liver cell culture samples using different amounts of Sodium butyrate. In our in vivo experiments we determined the Erythromycin concentrations in chicken plasma samples to define the pharmacokinetic parameters.
The aim of these investigations was to develop a selective, sensitive and routinely used HPLC method, wich is usable to determine the Erythromycin concentrations in liver cell culture and chicken plasma samples.
We had to use a derivatization procedure - the reagent was fluorenylmethyl chloroformiate - with fluorescence detection for these investigations. During the sample preparation we used liquid-liquid extraction for recovering Erythromycin from liver cell culture and chicken plasma samples. We applied a reversed phase liquid chromatographic system for the measurment of Erythromycin using RP18 type stationary phase and fluorescence detection.
During the development of the HPLC method we optimized the sample preparation - the ratio of the organic and the water solution volume - , the derivatization procedure - the reaction time and temperature -, and the parameters of the HPLC measurment - type of the RP18 column, wavelength of detection, composition of the mobile phase.
Before using this HPLC method routinely, we validated it on the basis of some parameters: specificity, linearity, recovery, reproducibilty and limit of quatification.
The regulation of paracellular transport via tight junction modulators has not been fully revealed yet. Inhibition of transmembrane serine protease, matriptase found on the epithelial cell surface and in carcinogenic cells leads to decreased resistance of the epithelium presumably via the altered operation of tight junction assembly. The alterations in barrier integrity can be attributed to active interplay between extracellular and intracellular Ca2+ level. The resistance of cell monolayer can be influenced by the effect of low Ca2+ level after application of Ca2+ chelators on tight junctional Ca2+- dependent protein components. The malfunction of this delicately controlled coordination over Ca2+ homeostasis can trigger the sequences of intracellular events such as activation of protein kinases contributing to changed phosphorylation patterns of tight and adherent junctional proteins. The aim of this study is to develop a non-transformed porcine intestinal epithelial cell model cultured on polyester membrane inserts, which can be applied for in vitro investigation of permeability through IPEC-J2 cell monolayer based on the rate of transport processes for paracellular marker, fluorescently labelled dextran between apical and basolateral compartments. The effect of Ca2+ withdrawal and the consequence of modulation of matriptase activity on permeability and barrier integrity of epithelial cell monolayer is of key importance for in-depth understanding the altered functions of inflammed mucosa in vivo.
In recent years the use of low-dose dietary antibiotics has been banned in the context of growth promotion of livestock, because of the antibiotic resistance of some pathogenic bacteria. For this reason nowadays there is a growing interest to replace the use of various antibiotics by different natural alternatives, such as probiotics. Probiotics are defined as live microbial food/feed ingredients that have a beneficial effect on the host health and well-being. Probiotics have proven effects in the prevention and/or treatment of several intestinal infections, disorders and diseases but the underlying mechanisms remain to be elucidated.
In this study the role of certain probiotic bacterial strains and their metabolic products will be investigated in the prevention of LPS (lipopolysaccharide)- induced (sub)cellular changes. To monitor the complex interaction among host epithelium, inflammatory stimuli and protective agents, IPEC-J2 cell line derived from porcine jejunal epithelium will be used as model system. IL-6, IL-8, TNF-α expression will be measured at the level of transcription (determination of mRNA by real-time PCR) and translation (protein assay by ELISA). The effects of LPS treatment on IPEC-J2 monolayer integrity will be followed by measurement of transepithelial electrical resistance and via fluorometric detection of FITC-dextran 4kDa paracellular marker. The protective agent(s) - bacteria or metabolites - will be determined and attempts will be made to purify and identify the active compound(s) present among the metabolic products. The in vitro observations will be tested in in vivo animal models (rodents and/or swine).