We read with interest the recent research article by Lisa Simms and her colleagues (Gut 2008; 57: 903–10). While we agree with Simms et al that the NOD2 genotype is an important variable in the study of ileal Crohn’s disease (CD), we disagree with her conclusion that a-defensin expression is related to inflammation rather than NOD2 status. This genotype is especially important in the analysis of Paneth cell antimicrobial expression, given the prominent expression of NOD2 in Paneth cells. 1 In a previous investigation, we reported a significant association of the NOD2 genotype with HD5 mRNA expression levels (Wehkamp et al, 2 fig 1D), but, importantly, the effect was found only with the 1007fs mutation (SNP13). The decrease of HD5 levels with the 1007fs NOD2 genotype was confirmed at the protein level by western blot analysis (Wehkamp et al, 2 fig 1E). The significance of the western blot data was emphasised by our observation that the same 1007fs samples had no decrease in levels of either sPLA2, lysozyme or a-1-antiprotease, three other Paneth cell proteins (Wehkamp et al, 2 fig 1G). Of special note, we saw no association of either the R702W or G908R genotypes with HD5 mRNA levels (Wehkamp et al, 2 fig 1D). In their recent paper, Simms et al did not stratify their genotype data to investigate if there was a difference in HD5 expression levels associated with any particular NOD2 genotype. Since eight patients in their cohort had a 1007fs mutation, we encourage the analysis of this relevant subgroup before concluding that the defensin decrease is independent of NOD2 genotype. In addition, the conclusion of Simms et al is somewhat incongruous with the reduced defensin expression in NOD2-deficient mice. 3

A second point made by Simms et al also contradicts the published data from our previous studies. 2 4 We were aware that tissue inflammation is an obvious potential confounding variable for any investigation of altered gene expression in CD. In our 2005 investigation, an experienced gastrointestinal pathologist (Dr Robert E Petras) evaluated histological sections of the specimens used for our Paneth cell expression analysis. To minimise any possible bias, the histological sections were labelled only with an arbitrary numerical identifier, and Dr Petras had no prior knowledge of other data on any of these samples (or data from other arms of the study). We found a significant association of histological inflammation assessed by Dr Petras with interleukin 8 (IL8) mRNA levels, our surrogate molecular marker of inflammation (Wehkamp et al, 2 fig 2A), confirming the expected link between morphology and expression of this inflammatory chemokine. In contrast, and most significantly, we observed no association between inflammation and either HD5 expression levels or antibacterial activity in these samples (Wehkamp et al, 2 figs 2B and 3). When comparing samples with little or no inflammation with those with either moderate or extensive inflammation, we found no association (Wehkamp et al, 2 fig 2B). Moreover, in a 2007 study that was not cited by Simms et al, 4 we found reduced expression of TCF-4 in ileal biopsy specimens from patients with ileal CD, and linked expression of this transcription factor to reduced HD5 mRNA (Wehkamp et al, 4 figs 1A–C and 2). The levels of HD5 mRNA were also independent of IL8 mRNA levels in these experiments (Wehkamp et al, 4 figs 4, 1C, and further data that were not shown). To examine further if HD5 expression was independent of tissue inflammation, we sought to examine inflamed intestinal mucosa not affected by CD. Here, options are rather limited, since inflamed human ileal specimens are …