TLR2 and TLR4 Up-regulation and Colonization of the Ileal Mucosa by Clostridiaceae spp. in Chronic/Relapsing Pouchitis

Innate Immune Environment in Ileal Pouch Mucosa: α5 Defensin Up-regulation as Predictor of Chronic/Relapsing Pouchitis

“…16,17 Briefly, all patients with UC who had undergone restorative proctocolectomy with ileal pouch-anal anastomosis from September 2007 to January 2008 and attending our outpatient's clinic for routine endoscopic and clinical follow-up 31 were considered as eligible candidates. Patients with cuffitis (inflammation of the rectal mucosa remnant) or Crohn's disease of the pouch (with perianal fistulae or with inflammation of the afferent ileal limb) as well as patients who had received antibiotic or probiotic therapy during the previous 30 days were excluded from the study.…”

“…Primers and probes sequences and PCR conditions are summarized in Table 1 and in. 16,17 Microbiological Culture and Assay After being weighed, the biopsy samples for microbiological cultures were immediately placed on 500 μL of icecold thioglycollate medium (Oxoid Limited, Cambridge, UK) to preserve anaerobes in the samples. The specimens were then washed under agitation for 2 min at room temperature in saline solution (NaCl 0.9%, pH=7) containing dithiothreitol 0.016% (w/v) to remove bacteria loosely adherent to the ileal pouch mucous.…”

“…16,17 Briefly, after fixation in 10% neutral-buffered formalin at room temperature for 24/48 h, the mucosal biopsy specimens were dehydrated and embedded in paraffin wax. Three-micrometre-thick sections were then cut and a standard haematoxylin/eosin stain was applied.…”

“…and Streptococcaceae spp., may have a role in maintaining immunologic homeostasis within the pouch mucosa, with low levels of these bacteria possibly favouring the development of acute pouchitis. 16,17 Deregulated mucosal cell immunity toward the microbiota also seems to be implicated in the pathogenesis of pouchitis. Epithelial and immune cells within the intestinal mucosa recognize conserved microbial structures through membrane-bound and cytoplasmic receptors which, when activated, trigger intracellular signalling pathways to elicit a variety of innate immune responses.…”

“…19 In one of our recent studies, we confirmed that high mucosal TLR-2 and TLR4 mRNA levels were associated to chronic/relapsing pouchitis. 16 TLR-2 recognizes and binds to fragments of bacterial peptidoglycan (the main component of bacterial cell wall), while TLR-4 recognizes and binds to bacterial lipopolysaccharides (LPS), endotoxins found in the outer membrane of gramnegative bacteria. The binding of these conserved bacterial structures to TLRs triggers a number of intracellular signalling cascades, such as nuclear transcription factor κB (NF-κB) and protein kinases, leading to the transcription of a series of pro-inflammatory and regulatory genes which influence the function and integrity of the intestinal mucosa.…”