1. Abnormal intestinal intraepithelial lymphocytes in refractory sprue
Christophe Cellier, Natacha Patey, Laurent Mauvieux, Bana Jabri, Eric Delabesse, Jean–Paul Cervoni, Marie–Laure Burtin, Delphine Guy–Grand, Yoram Bouhnik, Robert Modigliani, Jean–Philippe Barbier, Elisabeth Macintyre, Nicole Brousse, Nadine Cerf–Bensussan 
Gastroenterology 
Volume 114, Issue 3, Pages (March 1998)
DOI: /S (98)70530-X
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Histology and immunohistochemistry in 1 patient with refractory sprue (UPN 1070). (A and B) H&E on paraffin sections. Approximately two thirds of the examined intestinal mucosa shows total villous atrophy (A), but some areas only exhibited partial villous atrophy (B). (C–F) Comparative immunoperoxidase staining of intestinal frozen tissue sections. The epithelium is massively infiltrated by lymphocytes (arrow), stained by (C) anti-CD3ϵ, but not by (D) anti-CD8, (E) anti-TCRαβ, and (F) anti-TCRγδ antibodies. In contrast, numerous lamina propria T cells (arrowhead) are stained by anti-TCRαβ antibody (E).
Gastroenterology  , DOI: ( /S (98)70530-X)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Medians and interquartiles of the numbers of IELs in (A) patients with refractory sprue (n = 6), (B) patients with celiac sprue sensitive to a GFD (n = 7), and (C) controls (n = 19). Numbers of IELs were estimated on serial duodenal sections by counting peroxidase-stained lymphocytes per 100 epithelial cells. Statistical differences were calculated using the Kruskal–Wallis nonparametric test. Note that the scale of the figures is different in the three panels and depends on the mean number of IELs in each studied group. In group I patients, there is a significant increase in the number of CD3+ IELs that express neither βF1 nor TcRδ1, represented as X (X being the calculated difference between the numbers of CD3+ IELs and the sum of TcRαβ+ and γδ+ IELs). *Statistically significant difference (P < 0.05) between the mean numbers of labeled IELs in patients with celiac sprue sensitive to GFD (group II) and/or with refractory sprue (group I) compared with controls (group III). †Statistically significant difference (P < 0.05) between the mean numbers of labeled IELs in the patients with celiac sprue sensitive to a GFD compared with those with refractory sprue.
Gastroenterology  , DOI: ( /S (98)70530-X)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Fluorescence-activated cell sorter (FACS) analysis of isolated intestinal lymphocytes in 2 patients with refractory sprue. Lymphocytes isolated from (A) duodenal or (B) rectal biopsy specimens of patient UPN 1286 were labeled with fluorescein isothiocyanate–conjugated anti-CD103 and PE-conjugated anti-CD3ϵ antibodies. FACS analysis indicates that the majority of CD103+ lymphocytes, presumably IELs, do not express surface CD3ϵ. Lymphocytes isolated from duodenal biopsy specimens of patient UPN 1070 were surface-labeled with (C) fluorescein isothiocyanate–conjugated anti-CD103 and PE-conjugated anti-TCRαβ, (D) anti-TCRγδ, and (E) anti-CD8 antibodies. FACS analysis indicates that the majority of CD103+ lymphocytes express neither TCRαβ, TCRγδ, nor CD8.
Gastroenterology  , DOI: ( /S (98)70530-X)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 4
(A) TCRγ multiplex PCR. Ethidium bromide–stained PAGE of TCR VγfI/Vγ10 multiplex PCR products from celiac disease and normal control biopsy specimens (upper panel) and from refractory sprue biopsy specimens (lower panel). Whereas one or two discrete bands are seen in duodenal biopsy specimens from all cases of refractory sprue, samples from celiac sprue and from controls show only polyclonal smears. Discrete bands of the same size as those in duodenal biopsy specimens are observed in two of the four rectal biopsy specimens shown (UPN 1286 and 1399). The two larger PCR products (approximately 350 bp) observed in UPN 1286 represent heteroduplex formation. D, duodenum; R, rectum; MWM, molecular weight markers; Peer 100% and 10% refer to the positive control cell line diluted or not into polyclonal lymphoid DNA. (B) High-resolution fluorescent runoff analysis of VγfI/Vγ10 multiplex PCR from sorted duodenal fractions. Vγ usage and accurate size assessment was performed by limited cycle runoff with a mix of three differently labeled internal Vγ-specific primers and electrophoresis on an ABI373 automated fragment analyzer. One of the refractory sprue patients (UPN 1286) showed two bands compatible with a Vγ3/5 rearrangement in the duodenal (top panel, right lane) and rectal (not shown) biopsy specimens. Identically sized Vγ3/5 products were observed in the surface CD3ϵ-negative intestinal lymphocytes (s-CD3ϵ−IL) (top panel, left lane), whereas the rearrangements observed in the surface CD3ϵ-positive intestinal lymphocytes (s-CD3ϵ+ IL) were polyclonal (top panel, center lane). Ethidium bromide PAGE from the same samples are shown below (bottom left), with parallel control DNA amplification of a DNA fragment from the RAG1 gene to assess DNA content (bottom right). The clonal population in the duodenal biopsy specimen was not visible by ethidium bromide staining due to limited DNA quantity (see parallel control PCR) but can be seen in A (UPN1286D).
Gastroenterology  , DOI: ( /S (98)70530-X)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 4
(A) TCRγ multiplex PCR. Ethidium bromide–stained PAGE of TCR VγfI/Vγ10 multiplex PCR products from celiac disease and normal control biopsy specimens (upper panel) and from refractory sprue biopsy specimens (lower panel). Whereas one or two discrete bands are seen in duodenal biopsy specimens from all cases of refractory sprue, samples from celiac sprue and from controls show only polyclonal smears. Discrete bands of the same size as those in duodenal biopsy specimens are observed in two of the four rectal biopsy specimens shown (UPN 1286 and 1399). The two larger PCR products (approximately 350 bp) observed in UPN 1286 represent heteroduplex formation. D, duodenum; R, rectum; MWM, molecular weight markers; Peer 100% and 10% refer to the positive control cell line diluted or not into polyclonal lymphoid DNA. (B) High-resolution fluorescent runoff analysis of VγfI/Vγ10 multiplex PCR from sorted duodenal fractions. Vγ usage and accurate size assessment was performed by limited cycle runoff with a mix of three differently labeled internal Vγ-specific primers and electrophoresis on an ABI373 automated fragment analyzer. One of the refractory sprue patients (UPN 1286) showed two bands compatible with a Vγ3/5 rearrangement in the duodenal (top panel, right lane) and rectal (not shown) biopsy specimens. Identically sized Vγ3/5 products were observed in the surface CD3ϵ-negative intestinal lymphocytes (s-CD3ϵ−IL) (top panel, left lane), whereas the rearrangements observed in the surface CD3ϵ-positive intestinal lymphocytes (s-CD3ϵ+ IL) were polyclonal (top panel, center lane). Ethidium bromide PAGE from the same samples are shown below (bottom left), with parallel control DNA amplification of a DNA fragment from the RAG1 gene to assess DNA content (bottom right). The clonal population in the duodenal biopsy specimen was not visible by ethidium bromide staining due to limited DNA quantity (see parallel control PCR) but can be seen in A (UPN1286D).
Gastroenterology  , DOI: ( /S (98)70530-X)
Copyright © 1998 American Gastroenterological Association Terms and Conditions