Therapeutic and prophylactic thalidomide in TNBS-induced colitis: Synergistic effects on TNF-α, IL-12 and VEGF production

AIM: To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor a (TNF-α), IL-12, and vascular endothelial growth factor (VEGF), hallmarks of intestinal inflammation in Crohn's disease (CD). METHODS: Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4+ T cells, macrophages, and VEGF+ cells were detected by immunohistochemistry. TNF-a and IL-12 were quantified in the supernatant of organ cultures by ELISA. RESULTS: Significant reduction in the inflammatory score and in the percentage of VEGF+ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-αand IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide. TNF-αlevels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-αand IL-12 were significantly correlated with the inflammatory score and the number of VEGF+ cells. CONCLUSION: Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-α, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD.

The full length <i>PtSRP</i>(<i>Pisolithus tinctorius</i>symbiosis related protein) fungal mRNA encodes a potential marker of ectomycorrhiza formation

The Pisolithus tinctorius symbiosis related protein expressed sequence tag (EST PtSRP) was previously identified in the first hours of the interaction between the fungus Pisolithus tinctorius and sweet chestnut Castanea sativa, and partially characterized as a fungal marker gene of ectomycorrhizal symbiosis formation. We used the 5’ rapid amplification of cDNA ends (RACE) to obtain the PtSRP mRNA 5’ region, and together with our previously reported 3’ mRNA region, the full mRNA sequence was assembled by use of bioinformatics tools and deposited to GenBank (Accession: GU733439). The full-length mRNA sequence (636 bp) revealed the locations of the 5’ and 3’ untranslated regions (UTRs) and contained the Kozak sequence (ccc aag ATG A) in the 5’ UTR. The in silico translated PtSRP open reading frame (ORF) codes for a 127 amino acid protein and contained four putative post-translational modification sites (two N-glycosylation and two phosphorylation). The protein secondary structure is postulated to be composed of one N-terminal hydrophobic transmembrane alpha helix and at least six hydrophilic beta-strands spread across the protein. Sub-cellular localization prediction suggests that the protein is involved in cellular secretory pathway, supported by the presence of a cleavage site motif close to the membrane anchor. The data presented herein indicate the role of PtSRP as a fungal membrane secreted protein involved in early stages of ectomycorrhizal formation, with application as a possible marker for nascent ectomy-corrhiza fungal development.