Volume 115, Issue 3, Pages 551-563 (September 1998) Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease  Ellen M. Nilsen, Frode L. Jahnsen, Knut E.A. Lundin, Finn–Eirik Johansen, Olav Fausa, Ludvig M. Sollid, Jørgen Jahnsen, Helge Scott, Per Brandtzaeg  Gastroenterology  Volume 115, Issue 3, Pages 551-563 (September 1998) DOI: 10.1016/S0016-5085(98)70134-9 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Agarose gel electrophoresis for semiquantitative determination of PCR-amplified cytokine mRNA in duodenal specimens from 8 control subjects with normal histology. Specimens from 2 subjects (controls [Ctr.] 1 and 2) were stimulated for 2, 4, and 6 hours with gluten or for 6 hours with medium alone (*) (control 1), and were compared with unstimulated counterparts (0). For the other subjects (controls 3–8), results are shown only for unstimulated specimens. Total RNA (500 ng) was reverse-transcribed to cDNA. Amplification was performed by gene-specific primers with 2 μL cDNA for 25 cycles (β-actin) or 35 cycles (cytokines). The indicated size (base pairs) of the amplification product matched that predicted from the position of the primer pairs. Gastroenterology 1998 115, 551-563DOI: (10.1016/S0016-5085(98)70134-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Agarose gel electrophoresis for semiquantitative determination of PCR-amplified cytokine mRNA in duodenal specimens from 4 patients with celiac disease. Specimens taken from 1 untreated patient (Pt. 23) and 3 treated patients (Pt. 37, Pt. 40, and Pt. 41) were gluten-stimulated for different times (0.5–10 hours) and compared with unstimulated counterparts (0). Amplification and display as described in the legend to Figure 1. Gastroenterology 1998 115, 551-563DOI: (10.1016/S0016-5085(98)70134-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Agarose gel electrophoresis for semiquantitative determination of PCR-amplified IL-12p40 mRNA in duodenal specimens taken from both histologically normal controls and patients with celiac disease. Specimens were stimulated with gluten for different times as indicated and compared with unstimulated counterparts (0). In addition, 2 specimens were stimulated for 6 hours with medium alone (6*). Total RNA (500 ng) was reverse-transcribed to cDNA. Amplification was performed on 2 μL cDNA for 35 cycles followed by nested PCR on 1 μL of the product (diluted 1:10, 20 cycles). The 274-bp amplification product matched that predicted from the position of the primer pairs. A 123-bp ladder was used as molecular-weight marker, and β-actin served as sample control. Gastroenterology 1998 115, 551-563DOI: (10.1016/S0016-5085(98)70134-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunohistochemical cytokine detection in duodenal specimen from a patient with untreated celiac disease and in cytospins from gluten-specific T-cell clones. (A and B) Perinuclear staining of IFN-γ in lamina propria cells (closed arrows) identifies mucosal expression of this cytokine, whereas a few other cells with a diffuse brown cytoplasm (open arrow) were deemed to be nonspecifically decorated. Note that cytokine-expressing cells were absent from the epithelium (EP) shown at the top. (C and D) Gluten-specific T-cell clones used as positive controls. After stimulation with gluten peptides and blockade of the secretory pathway by monensin, cytospins showed characteristic perinuclear staining both for (C) IFN-γ and (D) IL-4 (immunoperoxidase staining lightly counterstained with hematoxylin; original magnification 1000×). Gastroenterology 1998 115, 551-563DOI: (10.1016/S0016-5085(98)70134-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Scatter diagram comparing the density of lamina propria cells positive for IFN-γ or IL-4 in duodenal specimens from histologically normal controls (Ctr.) and patients with untreated celiac disease (Pt.). Gastroenterology 1998 115, 551-563DOI: (10.1016/S0016-5085(98)70134-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions