<i>In vitro</i> stress treatments change the pattern of MICA/B expression. A. Induction of TIA-1⁺ granules in Caco-2 cells.

<p>Confocal microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range temperature showing redistribution of TIA-1 (white) into stress granules. Nuclei (blue). (scan zoom 0,7, magnification 100×). <b>B.</b>- <b>Redistribution of MICA/B in treated Caco-2 cells</b>. Confocal microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range temperature showing redistribution of MICA/B (red) in cytoplasmic aggregates. Nuclei (blue). (scan zoom 0,7, magnification 100×). <b>C.</b>- <b>Distribution of MICA/B and TIA-1 in Caco-2 treated cells.</b> Confocal microscopy of Caco-2 cells treated with thapsigargin (ER stress) or sodium arsenite (oxidative stress) for 1 hour, showing MICA/B (red) and TIA-1 (white) (magnification 100×). In both cases, MICA/B⁺ structures were not associated to stress TIA-1⁺ granules. (scan zoom 0,7, magnification 100×).</p

BiP expression in duodenal mucosa.

<p>Immunofluorescent confocal analysis on duodenal biopsy samples of a healthy control (A) and a severe enteropathy of a CD patient (B) showing BiP expression (green) and nuclei (red, propidium iodide) (scan zoom 1,7, magnification 63×). Healthy control (C) and severe enteropathy of a CD patient (D) showing BiP (green) and CD138 (red) expression. (scan zoom 4.2 and 3.5, respectively, magnification 63×).</p

TIA-1⁺ granules indicate stress in the intestinal mucosa in active CD.

<p>MICA/B cytoplasmic expression colocalized with TIA-1⁺ granules. (A) MICA/B (red) and TIA-1 (green) in different cell populations in a severe enteropathy of a CD patient. Epithelium was delimited in the picture with a thin line (scan zoom 0.7, magnification 100×). (B) Enlarged picture of (A).</p

MICA expression in intestinal mucosa of CD patients.

<p><b>A.</b>- Representative immunoperoxidase staining of MICA/B in intestinal biopsy sections from pediatric CD patients with different degrees of lesion (mild, moderate and severe enteropathy; magnification 20×). <b>B.</b>- Immunohistochemical analysis for MICA/B expression in sections of intestinal biopsies from 27 pediatric patients. An arbitrary score of intensity of staining was used (from 0 to 4). The IC control antibody was defined as score zero. Each dot corresponds to the score obtained for each sample. * <i>p</i>≤0,05; ** <i>p</i>≤0,01 (Non parametric Kruskal wallis test followed by the Dunns multiple-comparison posttest).<b>C.</b>- Pattern of MICA/B expression along the epithelium on a mild enteropathy sample. Isotype Control (IC) is shown (magnification 40×).</p

Confocal immunofluorescent analysis showing MICA/B staining.

<p><b>A.</b>- sample from an untreated CD pediatric patient with mild enteropathy showing the MICA/B expression (red) in enterocytes. SYTO® 13 (Green Fluorescent Nucleic Acid Stain) was used to stain nuclei. <b>B.</b>- MICA/B staining in an intestinal section from the same patient after two years on a gluten-free diet. <b>C.</b>- healthy non-celiac control patient. <b>D.</b> IC incubated in a section corresponding to sample A. (Magnification 63×). <b>E.</b>- Flow cytometric analysis for surface and intracellular expression of MICA/B in epithelial CD3⁻ cells of a representative paediatric patient. <b>F.</b>- Flow cytometric analysis for surface and intracellular expression of MICA/B in epithelial CD3⁻ cells of duodenal samples from adult CD patients.</p

MICA/B⁺ cells in the <i>lamina propria.</i>

<p><b>A.</b>- Immunofluorescent confocal microscopic analysis was performed in paraffin embedded sections from tissues with severe enteropathy (i, iii, iv, v, vi, vii, viii) and mild enteropathy (ii). Sections were stained as follows: MICA/B (red), Nuclei (blue). i. CD3⁺ cells (green). ii and iii. CD7⁺ cells (green).. iv. CD20⁺ (green). v. CD138⁺ cells (green). vi. HAM-56⁺ cells (green). vii. CD11c⁺ cells (green). viii. IC antibody (all cell lineage markers in green). (scan zoom 0.7, magnification 100×). <b>B.</b>- Expression of MICA/B in CD7⁺ cells in sections of small intestine samples of 6 healthy controls, 8 mild enteropathy samples and 4 severe enteropathy samples. Percentage of MICA/B⁺ cells in the CD7⁺ population (left panel) and total number of <i>lamina propria</i> CD7⁺ cells per unit of m.m. (right panel) were plotted. * <i>p</i>≤0,05; (Non parametric Kruskal wallis test followed by the Dunns multiple-comparison posttest). <b>C.</b>- Expression of MICA/B in CD138⁺ cells in sections of small intestine samples of 13 healthy controls, 7 mild enteropathy and 5 severe enteropathy. Percentage of MICA/B⁺ cells on the CD138⁺ population (left panel) and total number of <i>lamina propria</i> CD138⁺ cells per unit of m.m (right panel). ** p≤0.01, (Non parametric Kruskal wallis test followed by the Dunns multiple-comparison posttest).</p