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4. Tools development

4.	Tools development
Within the team we have developed many tools to analyze the effects of mycotoxins in pigs: quantitative PCR and DNA chip to analyze gene expression of the immune response, intestinal explants, contamination experimental and isolation of swine cell types.

4.1 Development of quantitative PCR (qPCR) to analyze the expression of pig immune genes
The main limit of the study of the genes expression in the porcine species is the lack of reagents for proteins analysis. The knowledge on the porcine genome allows the design of primers for the study of mRNA expression. qPCR is a widely used technology that combines the DNA amplification with the immediate detection of the products. We developed the SYBR® Green I qPCR approach to quantify the initial amount of target mRNAs presents in samples obtained from exposed tissues or cells. We focus on immune-related genes like cytokines and regulatory genes or intestinal structure-related genes. Primers are obtained from our own design or from bibliography. Around 60 sets of primers are validated for the study of intestinal or immune cells gene expression. We applied this technology for the evaluation of the effect of mycotoxin exposure of immune or intestinal cells (Meissonnier et al., 2008) as well as the effects of food additives (Le Gall et al., 2009).

4.2. Development of a DNA array specific for the pig immune response
In collaboration with C. Rogel-Gaillard (INRA, UMR GABI, Jouy-en Josas) we have developed a DNA array specific for the pig immune response. This array, made with long-oligonucleotides, combines a generic set of 13297 probes with a newly designed SLA-RI set. This set comprises 3773 probes, which target all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex. This DNA array was first validated by studying the immune response of porcine peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin (Gao et al., 2010). We are currently using this array to investigate the effect of mycotoxins on pigs.

4.3. Development of experimental intoxications
Our animal facility allows us to perform in vivo experiments (maximum 40 piglets). This facility received the agreement of the Veterinary authority. People involved in the animal experiments obtained the diploma authorizing living animal experimentation. We perform experimental intoxications, either by using artificial or naturally contaminated feed, or orally treatments by gavage as well. Depending on the aim of our studies, short and long-term exposure (from 1 week to 7 weeks) can be considered.
We mainly focused on the effects of mycotoxins at the systemic and intestinal levels working with blood samples, collected from jugular vein. We also work with organs samples from euthanatized animals, flash-frozen in liquid nitrogen and stored at -80°C until processing, or stored in formalin.
Systemic parameters encompass general toxicological parameters (performance, hematology/biochemistry, organ histology), as well as specific parameters describing immune system responses. We paid particular attention on the impact of mycotoxins on the specific immune response following antigenic challenges. Indeed, vaccination or pathogenic models have been already used to evaluate the effects on the establishment of the specific response, humoral and cellular. (Pinton et al., 2008, Meissonnier et al., 2008).
Intestinal parameters encompass histopathological analysis (immunohistochemistry, histology, villi and crypt measurements), evaluation of intestinal permeability (junction proteins, cellular signaling pathways) and analysis of intestinal immunity (bacteria colonization, cytokines content) (Pinton et al., 2009).

4.4. Development of intestinal explants as an alternative technique to investigate the effect of mycotoxin on the intestine
In the context of reducing the number of experimental animals (3Rs principles), intestinal explants represent a powerful model. Organ culture of intestinal explants allows to preserve normal histological structure in vitro. In addition, large numbers of explants can be prepared from a single animal, thus reducing the number of animals required for a given study. In the context, we develop a model of jejunal explant and we used it to study the effect of mycotoxin on pig intestine (Kolf-Clauw et al., 2009).

4.5. Development of cell cultures to analyze the mechanism of action of mycotoxins
With the aim of identifying the mode of action of mycotoxins, we have developed techniques to isolate and/or differentiate porcine cells. The members of the team master the isolation of several blood cells such as lymphocytes, neutrophils or monocytes (Marin et al., 2007; Yerle-Boissou et al., 2009). We also worked on porcine alveolar macrophages and dendritic cells (Guzylack-Piriou, 2006). For intestinal epithelial cells, we used the porcine intestinal epithelial cell line IPEC-1. This cell line is able to differentiate when cultured on semi-porous filter (Bouhet et al., 2004; Pinton et al., 2010)