Volume 119, Issue 1, Pages 23-31 (July 2000) Identification of a novel bacterial sequence associated with Crohn's disease  Christopher L. Sutton, Jeong Kim, Akemi Yamane, Harnisha Dalwadi, Bo Wei, Carol Landers, Stephan R. Targan, Jonathan Braun  Gastroenterology  Volume 119, Issue 1, Pages 23-31 (July 2000) DOI: 10.1053/gast.2000.8519 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Representational difference analysis of involved and uninvolved CD mucosa. (A) Lamina propria mononuclear cells were prepared from involved and uninvolved regions of a CD colectomy specimen, and DNA from the 2 preparations underwent representational difference analysis. RDA products after 3 cycles of RDA were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. Bands I1 to I4 were subtractively enriched from involved mucosa; bands U1 and U2 were subtractively enriched from uninvolved mucosa. (B) Alignment of the I2 ORF with the closest database homologues was identified by BLAST analysis with the NIH nonredundant database. The homology of I2 to these bacterial proteins is 30%-40% similarity. C. pasteur., Clostridium pasteurinum; M. tb., Mycobacterium tuberculosis; A. aeolicus, Aquifex aeolicus. Gastroenterology 2000 119, 23-31DOI: (10.1053/gast.2000.8519) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Representational difference analysis of involved and uninvolved CD mucosa. (A) Lamina propria mononuclear cells were prepared from involved and uninvolved regions of a CD colectomy specimen, and DNA from the 2 preparations underwent representational difference analysis. RDA products after 3 cycles of RDA were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. Bands I1 to I4 were subtractively enriched from involved mucosa; bands U1 and U2 were subtractively enriched from uninvolved mucosa. (B) Alignment of the I2 ORF with the closest database homologues was identified by BLAST analysis with the NIH nonredundant database. The homology of I2 to these bacterial proteins is 30%-40% similarity. C. pasteur., Clostridium pasteurinum; M. tb., Mycobacterium tuberculosis; A. aeolicus, Aquifex aeolicus. Gastroenterology 2000 119, 23-31DOI: (10.1053/gast.2000.8519) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Serum antibodies to the I2 ORF. (A and B) ELISA wells were coated with I2-GST fusion protein, reacted with human sera at the indicated dilutions, and detected for anti-I2 binding activity with anti-IgG or anti-IgA reagents. Absorbances for nonspecific binding to control GST (typically <0.25 for IgG, 0.05 for IgA) were subtracted from raw I2-GST absorbances to obtain specific absorbances. (C) I2-GST ELISA wells were reacted with human sera at 1:200 dilution, and detected with anti-IgA. Shaded area is the mean ± 2 SD for the normal group. The 37 non-IBD inflammatory controls included 10 infectious (Campylobacter, Salmonella, Shigella, Blastocystis, Schistosomiasis), 4 collagenous colitis, 3 eosinophilic colitis, 3 peptic ulcer disease, 2 celiac disease, 2 radiation-associated proctitis, and 13 other. Gastroenterology 2000 119, 23-31DOI: (10.1053/gast.2000.8519) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Serum antibodies to the I2 ORF. (A and B) ELISA wells were coated with I2-GST fusion protein, reacted with human sera at the indicated dilutions, and detected for anti-I2 binding activity with anti-IgG or anti-IgA reagents. Absorbances for nonspecific binding to control GST (typically <0.25 for IgG, 0.05 for IgA) were subtracted from raw I2-GST absorbances to obtain specific absorbances. (C) I2-GST ELISA wells were reacted with human sera at 1:200 dilution, and detected with anti-IgA. Shaded area is the mean ± 2 SD for the normal group. The 37 non-IBD inflammatory controls included 10 infectious (Campylobacter, Salmonella, Shigella, Blastocystis, Schistosomiasis), 4 collagenous colitis, 3 eosinophilic colitis, 3 peptic ulcer disease, 2 celiac disease, 2 radiation-associated proctitis, and 13 other. Gastroenterology 2000 119, 23-31DOI: (10.1053/gast.2000.8519) Copyright © 2000 American Gastroenterological Association Terms and Conditions