Neutrophil Recruitment and Barrier Impairment in Celiac Disease: A Genomic Study  Begoña Diosdado, Harm van Bakel, Eric Strengman, Lude Franke, Erica van Oort, Chris J. Mulder, Cisca Wijmenga, Martin C. Wapenaar  Clinical Gastroenterology and Hepatology  Volume 5, Issue 5, Pages 574-581.e5 (May 2007) DOI: 10.1016/j.cgh.2006.11.014 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Profiles of differentially expressed immune-related genes during mucosal recovery. The pattern of expression of 27 immune-related genes selected by MAANOVA analysis showed a gradual change as opposed to the discrete histologic transitions during remission. The upper panel shows the relative expression of genes that either were enhanced (red) or decreased (green) during remission. Expression was normalized to the values measured in untreated Marsh III patients. The lower panels show the corresponding histology and Marsh classification of the biopsy samples taken before treatment and during remission on a gluten-free diet. Histopathologic features include villous atrophy (MIII), crypt hyperplasia (MIII–MII), and lymphocytosis (MIII–MI). Patients in complete remission (M0) are comparable with controls. miii-ut, Marsh III untreated; miii-t, Marsh III treated; mii, Marsh II; mi, Marsh I; m0, Marsh 0. The numbers of independent individuals included in each group are indicated. Clinical Gastroenterology and Hepatology 2007 5, 574-581.e5DOI: (10.1016/j.cgh.2006.11.014) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Immunohistochemical localization of HLA-E and MPO in the duodenal mucosa of celiac patients and controls. Shown is the expression of the nonclassic major histocompatibility complex class I molecule HLA-E and the neutrophil-specific marker MPO in patients (Marsh III and Marsh 0) and healthy controls. (A) HLA-E was expressed strongly in the Marsh III epithelial layer but absent in normalized patients (Marsh 0) and controls. No differences in staining were found at the lamina propria. (B) Increased number of MPO-positive cells in the lamina propria of Marsh III and Marsh 0 patients compared with controls. This indicated a higher number of neutrophils in the celiac mucosa regardless of the histologic stage or the gluten-free diet followed. Clinical Gastroenterology and Hepatology 2007 5, 574-581.e5DOI: (10.1016/j.cgh.2006.11.014) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Relative expression of (A) CYP4F2 and (B) CYP4F3 determined by qRT-PCR. Gene expression was measured with qRT-PCR on RNA from individual M0 (n = 16) and MIII (n = 13) biopsy samples. Data were averaged and normalized to that obtained from a pool of complementary DNAs (n = 16) derived from normal controls (nc). Error bars indicate SDs. Measurements were in duplicate. Clinical Gastroenterology and Hepatology 2007 5, 574-581.e5DOI: (10.1016/j.cgh.2006.11.014) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Fraction of the MPO-positive neutrophils counted in mucosal sections of celiac patients (MIII and M0) and controls. On average, a 2-fold increase in neutrophils was observed in both active and remission patients compared with healthy controls. Percentages are based on the average of approximately 1000 total cells counted (see also Supplementary Table 3). Error bars indicate the standard error. Clinical Gastroenterology and Hepatology 2007 5, 574-581.e5DOI: (10.1016/j.cgh.2006.11.014) Copyright © 2007 AGA Institute Terms and Conditions