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To assess thepresence of Peptidoglycan (PG) in perianal fistulas as transanal advancement flap repair (TAFR) fails in one out of three patients with a high trans-sphincteric fistula. It has been suggested that failure is caused by ongoing inflammation in the remaining fistulous tract. There is no evidence that pathogenic bacteria are responsible for this ongoing inflammation. Peptidoglycan is a major component of the bacterial cell wall, is a powerful inflammatory effector in the absence of local bacterial replication. We hypothesized that PG plays a role in maintaining the fistulous disease.A consecutive series of ten patients underwent TAFR. As part of theprocedure the fistulous track was excised as far as possible. Thepresence of PG in the excised fistulous track was assessed by immunohistochemical analysis using two monoclonal antibodies (mAb) 2E9 and anti-S. aureus PG IgG3 mAb 15704. The host response to PG was examined by mAb against peptidoglycan recognitionprotein PGLYRP1 (mAb 188C424) and PGLYRP2 (mAb AAA4).PG was detected in most fistulae (90%). In 60% of the fistulae a host response to PG was identified.Our data suggests apreviously undescribed role forproinflammatory PG in perianal fistulae.