Next, we assessed the signs of intoxication through measurement of cell trans-epithelial electrical resistance (TEER) and cell viability, colon epithelium permeability to macromolecules, gene expression of TJ proteins, as well as the immunofluorescence (IF) analysis of the cytoskeletal structure and TJ proteins in IPEC-J2 cells. 2. in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (= 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from infections in the offspring. (syn. has been documented as an important cause of uncontrolled enteritis outbreaks in neonatal pigs [1,2]. Toxins A (TcdA) and B (TcdB) besides a binary toxin are the main infection agents leading to loss of epithelial integrity, immune response and intestinal damage [3]. Their action is related to the modulation of the intestinal epithelial cell physiology and disruption of barrier function via inactivation of Rho proteins involved in the formation of the cytoskeleton. This leads to disruption of tight junction (TJ) proteins and finally to a loss of epithelial integrity, as demonstrated in porcine and human cell lines [4,5,6,7]. Such a scenario of events may lead Zearalenone to the typical signs of infection (CDI) [8,9] with severe consequences, such as animal death or growth retardation in piglets that survived the infection [10]. However, why some piglets are asymptomatic carriers of high concentrations of spores and toxins and others are not, is not exactly clear [11]. In addition, the reasons why some piglets of the same litter get sick and others do not remain unknown so far. Maternal factors may play an important role in protection from CDI. Porcine milk is rich in bioactive components and has been shown to be beneficial for the offspring [12,13]. Porcine milk has also been demonstrated to strengthen the epithelial barrier function in the piglet gut [14] and previous evidence suggests that it could have the potential to neutralise clostridial toxins [8] and possibly prevent CDI. Based on the association between the mother sow and offspring in relation to piglet susceptibility to CDI, we hypothesised that porcine colostrum exerts protective effects against toxins Zearalenone on IPEC-J2 cells in vitro and in colon epithelium of healthy suckling and weaned piglets. We therefore treated IPEC-J2 cells and the explants of colon tissues with a culture supernatant containing toxins. In addition, IPEC-J2 cells were treated with porcine colostrum alone and together with the toxins. Next, we assessed the signs of intoxication through measurement of cell trans-epithelial electrical resistance (TEER) and cell viability, colon epithelium permeability to macromolecules, gene expression of TJ proteins, as well as the immunofluorescence (IF) analysis of the cytoskeletal structure and TJ proteins in IPEC-J2 cells. 2. Materials and Methods 2.1. Ethical Statement The institutional and national guidelines for the care and use of animals were followed and the study was approved by the State Office of Health and Social Affairs (Landesamt fr Gesundheit und Soziales Berlin, LAGeSo Reg G0255/14 (20 January 2015) and G0269/16 (16 February 2017). 2.2. C. difficile Toxins To obtain the spent culture supernatant with toxins, spores of infectious ribotype 078, known to produce toxins A and B and carrying genes encoding a binary toxin, were inoculated into BHI media supplemented with yeast extract and taurocholate and without L-cysteine [8,15,16,17]. They were then incubated at 37 C for 1C2 weeks to produce a sufficient concentration of toxins. Thereafter, the spent culture supernatant was centrifuged at 10,000 for 10 min, filtered through 0.22 m pore size MF-Millipore membrane filters (Merck, Darmstadt, Germany) and subjected to a commercial ELISA kit Zearalenone (tgcBIOMICS GmbH, Bingen, Germany) to determine the concentrations of TcdA and TcdB [11]. Previously performed tests showed that a two-hour incubation at a toxin concentration of 13 ng/mL Rabbit Polyclonal to VGF of TcdA and 8 ng/mL of TcdB was suitable to demonstrate detrimental changes in IPEC-J2 cells by lowering the TEER values Zearalenone within time but without inducing prominent cell damage or death (light microscopic evaluation), so that further manipulations such as cell washing could.
Next, we assessed the signs of intoxication through measurement of cell trans-epithelial electrical resistance (TEER) and cell viability, colon epithelium permeability to macromolecules, gene expression of TJ proteins, as well as the immunofluorescence (IF) analysis of the cytoskeletal structure and TJ proteins in IPEC-J2 cells