1. Volume 133, Issue 4, Pages 1175-1187 (October 2007)
Evidence for the Role of Interferon-alfa Production by Dendritic Cells in the Th1 Response in Celiac Disease 
Antonio Di Sabatino, Karen M. Pickard, John N. Gordon, Virginia Salvati, Giuseppe Mazzarella, Robert M. Beattie, Anna Vossenkaemper, Laura Rovedatti, Nicholas A.B. Leakey, Nicholas M. Croft, Riccardo Troncone, Gino R. Corazza, Andrew J. Stagg, Giovanni Monteleone, Thomas T. MacDonald 
Gastroenterology 
Volume 133, Issue 4, Pages (October 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Lamina propria DCs from an untreated celiac patient (ucd), a celiac patient on a gluten-free diet (tcd), and a control subject (hc). (A) DCs were identified by multicolor flow cytometry in LPMCs (see side scatter/forward scatter dot plots) as HLA-DR–positive, lineage-negative cells. Within the DC gate, the 2 subsets of CD123-positive (plasmacytoid) cells and CD11c-positive (myeloid) cells were identified (see HLA-DR/CD123 and HLA-DR/CD11c dot plots, respectively). Numbers within the dot plots represent the percentages of CD123-positive and CD11c-positive cells in gated HLA-DR–positive/lineage-negative LPMCs. Data are representative of experiments performed in 15 untreated celiac patients, 8 treated celiac patients, and 15 controls. (B) CD123-positive DCs co-express the plasmacytoid DC marker TLR9. Data are expressed as dot plots together with the isotype control. Numbers within the dot plots represent the percentages of TLR9-positive/CD123-positive DCs and TLR9-positive/CD11c-positive DCs (upper right quadrant). (C) Frequency of DC subtypes, calculated as percentage of HLA-DR–positive/lineage-negative LPMCs in the duodenum of 15 untreated celiac patients, 8 treated celiac patients, and 15 control subjects. Horizontal bars represent median values. §P < .05 vs treated celiac patients, *P < .001 vs controls, †P < .01 vs treated celiac patients.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Flow cytometric analysis of CD31 expression in CD123-positive DCs. (A) DCs were identified by multicolor flow cytometry in LPMCs (see side scatter/forward scatter dot plots) as HLA-DR–positive, lineage-negative cells (see HLA-DR/lineage dot plots). Within the DC gate, the subset of CD123-positive (plasmacytoid) cells was identified (see HLA-DR/CD123 dot plot). (B) To exclude the possibility that CD123-positive/DR-positive/lineage-negative cells included endothelial cells, the endothelial cell marker CD31 was analyzed in gated CD123-positive DCs. Numbers within the dot plots represent the percentage of CD31-positive cells in the gate of CD123-positive DCs (upper right quadrant). Data are representative of experiments performed in 7 active celiac patients.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
CD123 and CD11c immunofluorescence by confocal microscopy in active celiac and normal duodenal mucosa. In comparison with control mucosa, in whom CD123 positivity is evident in a few mononuclear cells scattered in the lamina propria, a considerable proportion of cells are CD123-positive in untreated celiac mucosa. An increase of CD11c-positive mononuclear cells also is evident in active celiac mucosa in comparison with normal mucosa in which CD11c positivity is limited to few cells located beneath the epithelium. No staining is evident in celiac and normal sections processed with the isotype control antibody. Data are representative of staining performed in 13 celiac patients and 13 control subjects.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Activation and maturation state of DCs. (A) Within the DC gate, CD123-positive or CD11c-positive cells were identified. Activation (CD80 and CD86) and maturation (CD83) markers then were assessed. Data are representative of experiments performed in 11 untreated celiac patients, 8 treated celiac patients, and 12 controls. (B) CD80, CD86, and CD83 expression in gated DCs from 11 untreated celiac patients, 8 treated celiac patients, and 12 control subjects. Horizontal bars represent median values. *P < .001 vs treated celiac patients and controls. *P < vs treated celiac patients and controls. †P < vs treated celiac patients and controls. (C) CD80, CD86, and CD83 expression in gated CD123-positive (gray) and CD11-positive (black) DCs from 11 untreated celiac patients, 8 treated celiac patients, and 12 control subjects. Results are mean (±SD). *P < .001 vs treated celiac patients and controls. mfi, mean fluorescence intensity.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Response to p31-43 and p57-68 gliadin peptides of lamina propria DCs from patients with untreated celiac disease, treated celiac disease, and control subjects. (A) LPMCs isolated from 9 untreated celiac patients, 7 treated celiac patients, and 10 control subjects were cultured in the absence or presence of nonimmunodominant (p31-43) or immunodominant (p57-68) gliadin peptides, TNF-α, and PT-gliadin. After 24-hour culture, multicolor flow cytometry was performed to assess CD80, CD86, and CD83 expression on gated DCs and HLA-DR expression on total LPMCs. Results are mean (±SD). mfi, mean fluorescence intensity. *P < .0001, §P < .01, and †P < .05 vs cells treated with p31-43 or p or medium only. (B) Cyclooxygenase-2 protein expression in duodenal biopsy specimens from a patient with untreated celiac disease incubated in the absence or presence of p31–43 or p57–68.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Cytokine transcripts determined by quantitative RT-PCR in sorted DCs from patients with untreated celiac disease and control subjects. (A) The 2-parameter histograms of HLA-DR and markers show the cells before and after sorting in an untreated celiac patient and control subject. (B) Levels of IFN-alfa, IL-18, IL-23p19, IL-12p40, IL-12p35, IL-27, IL-4, IL-5, and TGF-β transcripts in 7 patients with untreated celiac disease and 7 control subjects. Horizontal bars represent median values. r.u., relative units.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
IL-6, IL-12p40, and IL-10 production by DCs in (A) untreated celiac mucosa and (B) control mucosa. Cytokine production by DCs was assessed by intracellular cytokine staining (see Materials and Methods section). The right panels show cytokine production in the absence (nom) or presence of monensin (+m) (see HLA-DR/lineage dot plots). In the left panels, representative intracellular staining for IL-6, IL-12p40, and IL-10 in (A) untreated celiac DCs and (B) normal DCs are shown. In each case, pairs of histograms are presented in which one histogram shows staining of cells cultured without monensin (nom) and the second histogram shows staining of DCs cultured in the presence of monensin together with the result of the subtraction analysis (mws). Here the open histogram shows staining of DCs in the presence of monensin and the shaded area represents the fraction of cytokine-positive DCs. Numeric values show the percentage of cytokine-positive DCs. Data are representative of experiments performed in 6 untreated celiac patients and 7 control subjects.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
IL-6, IL-12p40, and IL-10 production by untreated celiac and control DCs assessed by intracellular cytokine staining. Both the percentage of cytokine-positive DCs and the cytokine fluorescence intensity ratio (fir) are shown in 6 untreated celiac patients (ucd) and 7 controls (hc). Horizontal bars represent median values.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

10. Figure 9
Effect of neutralizing IFN-alfa on gluten-induced IFN-γ transcripts. Biopsy specimens from 5 treated celiac patients cultured in the presence or absence of a PT-gliadin with and without the addition of a neutralizing anti–IFN-alfa antibody. Results are mean (±SD). r.u., relative units. *P < vs biopsy specimens cultured with medium or vs biopsy specimens cultured with PT-gliadin and anti–IFN-alfa antibody. Data are representative of experiments performed in 5 untreated celiac patients. Ethical restrictions of our study concerning the maximum number of biopsy specimens that could be collected from each patient did not allow us to perform control experiments with the isotype control antibody for IFN-alfa, however, we have performed many organ culture studies using control antibodies in celiac biopsy specimens challenged with gluten and have seen no effects.5,63
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions