calcimycin and deoxynivalenol

Lactic acid bacteria (LAB) are recognized for support of host gut homeostasis but the precise mechanisms remain to be identified. LABs interact with Toll-like receptors (TLRs) which might stimulate barrier function of gut epithelial cells. We previously identified six TLR2-signalling LAB strains. As TLR2 is involved in barrier-function enhancement in gut-epithelium, the epithelial barrier-protective effect of these TLR2-signalling strains was studied by using T84 human colorectal cancer cell monolayer as an in vitro gut epithelial barrier model. The protein kinase C (PKC) dependent barrier disruptor A23187 and mitogen-activated protein kinase dependent barrier stressor deoxynivalenol were tested to determine which pathways LAB influenced. We found that exclusively the PKC dependent disruption was prevented by the selected TLR2-signalling LAB strains. This study suggests that TLR2 is a pivotal epithelial barrier modulator, and provides novel insight in the molecular mechanisms by which LAB contribute to intestinal health.

Topics: Calcimycin; Cell Line, Tumor; Electric Impedance; Humans; Intestinal Mucosa; Lactobacillales; Models, Biological; Probiotics; Protein Kinase C; Toll-Like Receptor 2; Trichothecenes

The mechanisms by which trichothecene mycotoxins cause immunological effects in leukocytes such as cytokine up-regulation, aberrant IgA production, or apoptotic cell death are not fully understood. In the present study, mRNA differential display analysis was used to evaluate changes in gene expression induced by the trichothecene vomitoxin (VT or deoxynivalenol) in a T-cell model, the murine EL-4 thymoma, that was stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION). Ten differentially expressed fragments of cDNA were isolated and sequenced and three of these were identified as the known genes GRP78/BiP, P58(IPK), and RAD17. Most notably, expression of GRP78/BiP (a 78-kDa glucose-regulated protein), a stress-response gene induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function, was found to decrease in VT-exposed cells. Competitive RT-PCR analysis revealed that 250 ng/ml VT decreased GRP78/BiP mRNA expression in both unstimulated and PMA/ION-stimulated EL-4 cells at 6 and 24 h after VT treatment. Western blotting confirmed that VT (50 to 1000 ng/ml) also significantly diminished GRP/BiP protein levels in a dose-response manner in PMA/ION-stimulated cells. GRP78/BiP has been shown to play a role in regulation of protein folding and secretion, and to protect cells from apoptosis. When PMA/ION-stimulated cells were incubated with 50 to 1000 ng/ml VT for 24 h, 200-bp DNA laddering, a hallmark of apoptosis, increased in a dose-dependent manner. In addition to GRP78, mRNA expression of the cochaperone P58(IPK), which is the 58-kDa cellular inhibitor of the double-stranded RNA-regulated protein kinase (PKR), was also shown to be suppressed by VT-treatment. GRP78 and P58(IPK) are critical for maintenance of cell homeostasis and prevention of apoptosis. The down-regulation of these molecular chaperones by VT represent a novel observation and has the potential to impact immune function at multiple levels.

Topics: Animals; Blotting, Western; Calcimycin; Carrier Proteins; DNA Fragmentation; DNA, Complementary; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Ionomycin; Ionophores; Mice; Molecular Chaperones; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin; Thymoma; Trichothecenes