Volume 139, Issue 4, Pages 1289-1300.e2 (October 2010) Acute HIV Infection Induces Mucosal Infiltration With CD4+ and CD8+ T Cells, Epithelial Apoptosis, and a Mucosal Barrier Defect  Hans–Jörg Epple, Kristina Allers, Hanno Tröger, Anja Kühl, Ulrike Erben, Michael Fromm, Martin Zeitz, Christoph Loddenkemper, Jörg– Dieter Schulzke, Thomas Schneider  Gastroenterology  Volume 139, Issue 4, Pages 1289-1300.e2 (October 2010) DOI: 10.1053/j.gastro.2010.06.065 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Representative immunohistochemical analysis of mucosal T-cell subsets in duodenal mucosa from HIV-negative controls and from patients with acute or chronic HIV infection. Mucosal T cells were detected as mononuclear cells expressing CD3, CD4, or CD8 (red). Bars, 50 μm. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Density and frequency of mucosal T-cell subsets in HIV-negative controls and in patients with acute or chronic HIV infection. (A) Density (per 10 high-power fields) of mucosal T cells (CD3+), (B) mucosal CD4+, or (C) CD8+ T cells. Relative frequency (per CD3+ cells) of (D) mucosal CD4+ or (E) CD8+ T cells. The horizontal lines denote the median values of each group. *P < .05; **P < .01; ***P < .001; ns, not significantly different. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Characterization of mucosal CD4 T cells in patients with acute HIV infection. (A) Immunofluorescence and confocal analysis identified cells expressing both CD3 (green) and CD45RO or CD45 RA (red), or both CD4 (green) and CD 40L (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (dapi) (blue). (B) Flow cytometry revealed an activated (CD38+) effector memory (CD45RO+ CD62L−) phenotype in more than 90% of mucosal CD4 T cells. (C) Immunohistochemistry showed only very few (<2%) proliferating (Ki-67+) mucosal CD4 T cells. Bars, 50 μm. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Representative analysis of mucosal perforin- and T-cell intracellular antigen-1 (tia-1)–expressing cells and apoptotic epithelial cells in patients with acute or chronic HIV infection and in HIV-negative controls. (A) Mucosal mononuclear cells expressing the TIA-1 or perforin were visualized by immunohistochemistry (upper 2 panels). Immunofluorescence and confocal analysis (third panel from top) identified cells expressing both CD8 (green) and perforin (red), resulting in a yellow overlay of double-positive cells. Apoptotic epithelial cells were detected as epithelial cells positive for activated caspase-3 by immunohistochemistry (bottom panel). (B) Apoptotic cells within the epithelial layer were characterized as apoptotic epithelial cells by immunofluorescence and confocal analysis visualizing cells positive for cleaved caspase 3 (red), cytokeratin or CD3 (green), and 4′,6-diamidino-2-phenylindole (dapi) (blue). The arrow points to an intraepithelial CD3-positive T cell immediately adjacent to 2 apoptotic epithelial cells. Bars, 50 μm. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Lamina propria and intraepithelial mononuclear cells expressing markers of cytotoxicity, and frequency of apoptotic enterocytes in patients with acute or chronic HIV infection and in HIV-negative controls. (A) Density (per 10 high-power fields) of lamina propria and (B) frequency (per 1000 enterocytes) of intraepithelial T-cell intracellular antigen-1 (tia-1)–positive mononuclear cells. (C) Density of lamina propria and (D) frequency of intraepithelial perforin-positive mononuclear cells (per 10 high-power fields or 1000 enterocytes, respectively). (E) Frequency of apoptotic (caspase-3+) epithelial cells per 1000 enterocytes. (F) Correlation of apoptotic enterocytes with mucosal cytotoxic (perforin+) T cells. The linear regression analysis was performed by minimizing the sum of deviation squares. The horizontal lines denote the median values of each group. *P < .05; **P < .01; ***P < .001; ns, not significantly different. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Density and frequency of mucosal T-cell subsets in a single patient before and during acute HIV infection. (A) Density (per 10 high-power fields) and (B) frequency (per CD3+ cells) of mucosal CD4+ T cells before (HIV−) and during acute HIV infection (HIV+). (C) Density and (D) frequency of mucosal CD8+ T cells before (HIV−) and during acute HIV infection (HIV+). (E) Density of mucosal cytotoxic T cells before (HIV−) and during acute HIV infection (HIV+), as immunohistochemically quantified by perforin (squares), T-cell intracellular antigen-1 (tia-1) (diamonds), or granzyme B (circles) stain. (F) Frequency of caspase-3+ epithelial cells per 1000 enterocytes. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Duodenal barrier function in HIV-negative controls and in patients with acute or chronic HIV infection. Representative impedance locus plots of duodenal specimens obtained by endoscopic forceps biopsy from (A) an HIV-negative control, (B) a patient with acute HIV infection, and (C) a patient with chronic HIV infection. zreal gives the Ohmic component and zimaginary gives the reactive component of the complex impedance resulting from the capacitive properties of the epithelial cell membranes. Intersections between fitted semicircle and the x-axis at low and high frequencies represent transmural resistance (Rt) and subepithelial resistance (Rsub), respectively. The epithelial resistance (Re) results from Rt minus Rsub. (D) Quantification of the epithelial resistance (Re) by impedance spectroscopy, and (E) quantification of the paracellular mannitol permeability by mucosal-to-serosal 3H-mannitol fluxes. Values are means ± standard error of the mean. **P < .01; *P < .05; ns, not significant vs controls. Gastroenterology 2010 139, 1289-1300.e2DOI: (10.1053/j.gastro.2010.06.065) Copyright © 2010 AGA Institute Terms and Conditions