Volume 123, Issue 5, Pages 1468-1477 (November 2002) Dysregulated peripheral and mucosal Th1/Th2 response in Whipple's disease  Thomas Marth, Nicole Kleen, Andreas Stallmach, Sabine Ring, Sheriff Aziz, Carsten Schmidt, Warren Strober, Martin Zeitz, Thomas Schneider  Gastroenterology  Volume 123, Issue 5, Pages 1468-1477 (November 2002) DOI: 10.1053/gast.2002.36583 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 (A–E) Cytokine production and (F) cell proliferation after in vitro stimulation by PBMCs with phytohemagglutinin from WD patients and healthy donors as determined by enzyme-linked immunosorbent assay and [3H] thymidine incorporation, respectively. The baseline cytokine production and cell proliferation without cell stimulation was generally very low (<3 pg/mL for IL-4; <20 pg/mL for IL-2 and IL-5; <50 pg/mL for IFN-γ; <1000 cpm for thymidine incorporation). Data represent mean ± SEM from 20 WD patients total (as denoted in Patients and Methods section, consisting of group I, n = 5 patients; group II, n = 8 patients [except for IFN-γ and the IFN/IL-4 ratio n = 7]; and group III, n = 7 patients) or mean ± SEM from 14 healthy donors. (A) IL-2 secretion; (B) IFN-γ secretion; (C) IL-4 secretion; (D) IFN-γ /IL-4 ratio; (E) IL-5 secretion; (F) cell proliferation. *P < 0.05, +P < 0.01 vs. controls. ■, All patients; □, controls. Gastroenterology 2002 123, 1468-1477DOI: (10.1053/gast.2002.36583) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Monokine production after in vitro stimulation by PBMCs with SAC, SAC + IFN-γ, or LPS + IFN-γ from WD patients and healthy donors as determined by enzyme-linked immunosorbent assay. The number of patients and controls as well as statistics are given in the legend to Figure 1. The baseline cytokine production without stimulation was low (<0.5 pg/mL for IL-12p40; <5 pg/mL for IL-12p70; <1 pg/mL for TNF-α). Data show mean ± SEM from 20 WD patients and mean ± SEM from 14 controls. For IL-12p40 stimulated with SAC + IFN-γ and with LPS + IFN-γ n = 16 (i.e., group I, n = 3; group II, n = 6; and group III, n = 7) and for SAC stimulation n = 15 (same as earlier but group II, n = 5). (A) IL-12p40 secretion–SAC stimulation; (B) IL-12p40 secretion–SAC + IFN-γ stimulation; (C) IL-12p40 secretion–LPS + IFN-γ stimulation; (D) IL-12p70 secretion–SAC + IFN-γ stimulation; (E) IL-12p70 secretion–LPS + IFN-γ stimulation; (F) TNF-α secretion–LPS + IFN-γ stimulation. *P < 0.05, and +P < 0.01 vs. controls. ■, All patients; □, controls. Gastroenterology 2002 123, 1468-1477DOI: (10.1053/gast.2002.36583) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Subgroup analysis. Cytokine and monokine production as well as cell proliferation after in vitro stimulation by PBMCs from different WD patient groups (●, group I, n = 5 patients; ●, group II, n = 8 patients; ▩, group III, n = 7 patients; in some tests only up to 15 patients were available, see legends to Figures 1 and 2) determined by techniques as detailed in the legends to Figures 1 and 2. (A) IL-2 secretion; (B) IFN-γ secretion; (C) IL-4 secretion; (D) IFN-γ/IL-4 ratio; (E) IL-5 secretion; (F) cell proliferation; (G) IL-12p40 secretion–SAC stimulation; (H) IL-12p40 secretion–SAC + IFN-γ stimulation; (I) IL-12p40 secretion–LPS + IFN-γ stimulation; (J) IL-12p70 secretion–SAC + IFN-γ stimulation; (K) IL-12p70 secretion–LPS + IFN-γ stimulation; (L) TNF-α secretion–LPS + IFN-γ stimulation. Data show mean ± SEM from the indicated WD patient groups. Gastroenterology 2002 123, 1468-1477DOI: (10.1053/gast.2002.36583) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Reduced intestinal CD4+ T-cell numbers in WD. After the isolation of LPMCs from biopsy specimens of WD patients, from normal controls and from patients with chronic H. pylori infection, CD4 T-cell numbers were determined by flow cytometry as described in the Patients and Methods section. Shown is a box plot analysis with the median (thick line), the 75% and 25% percentile (upper and lower box limits), and the standard deviation; the group size was 6 individuals each (for WD patients 2 patients each from groups I to III). The difference of the WD group was statistically significant (P < 0.05) compared with the other groups. Gastroenterology 2002 123, 1468-1477DOI: (10.1053/gast.2002.36583) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Impaired mucosal IFN-γ secretion. The production of IFN-γ from WD LPMCs and control LPMCs stimulated with anti-CD2/CD28 was determined as described in the Patients and Methods section. The difference from WD to controls was significantly different as determined by the 2-sided Wilcoxon rank test (P < 0.05). □, Individual value; ▩, group median. Gastroenterology 2002 123, 1468-1477DOI: (10.1053/gast.2002.36583) Copyright © 2002 American Gastroenterological Association Terms and Conditions