Confluent monolayers grown from primary endometrial epithelial cells were either mock-treated or exposed to HIV-1 (ADA strain, 10⁶ infectious viral units/ml, p24 280ng/ml) for 8 hours.

<p>Total RNA was extracted and cDNA was synthesized. Quantitative Real-time RT-PCR was conducted for tight junction gene expression by measuring mRNA for Claudin 1–5, Occludin and ZO-1. GAPDH, a house keeping gene, was measured for internal control (A). * p<0.01; ** p<0.001; *** p<0.0001). Immunofluorescent staining of tight junction proteins following HIV-1 exposure compared to mock-treated epithelial monolayers. Representative staining is shown for claudin-2 (B), Occludin (C), and ZO-1 (D) at 24 hours post-exposure. Magnification: 1260×. Data shown is representative of 3 separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. For RNA extraction, 6–8 replicate cultures were pooled.</p

Figure 7

<p>(A) Epithelial monolayers were treated with medium alone, HIV-1 (IIIB, 10⁶ infectious viral particles/ml), HIV-1 in combination with gp120 neutralizing antibody (35µg/ml) or isotype control antibody (35µg/ml), gp120 or isotype antibody alone. TER measurements were taken as a measure of change in permeability and presented as percent of pre-treatment TER. p<0.01. (B) Confluent T84 epithelial cell cultures were mock infected or exposed to NL4-3 (p24, 79 ng/ml) or NL4-3 Env⁻ mutant (p24, 79 ng/ml) and TER measurements were taken prior to and 24 hours post-exposure. P<0.001 (C) ZO-1 localization after exposure to wildtype HIV-1 NL4-3 or Env⁻ NL4-3 mutant. Data shown is representative of four separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.</p

Primary genital epithelial monolayers were exposed to HIV-ADA (R5 strain, 10⁶ infectious viral units/ml, p24 280ng/ml ) for 2h, 4h, 6h, 8h, 16h, 24h and 48h.

<p>To measure barrier functions, TER was measured prior to and post exposure, ZO-1 staining was done and Dextran Blue dye leakage was measured across the monolayers at all time points. (A) TER values. p<0.001. (B) Paracellular permeability measured by addition of Blue Dextran dye on the apical side of monolayers. At different time intervals post-exposure, basolateral supernatants were sampled and absorbance was measured and compared to apical absorbance at initial time point (Time “0”). Blue Dextran leakage was calculated as a percentage of apical values. Data shown is representative of 3–4 separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.</p

Primers used for Real Time PCR for different tight junction genes.

<p>Primers used for Real Time PCR for different tight junction genes.</p

Primary endometrial epithelial monolayers were exposed to TNF-α or HIV-1 alone; TNF-α or HIV-1 (ADA,10⁶ infectious viral units/ml, p24 280ng/ml) in combination with anti-TNF-α neutralizing antibody; TNF-α or HIV-1 in combination with normal mouse serum for 24 hours.

<p>TER measurements were taken as a measure of change in permeability and presented as percentage of pre-treatment TER. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.</p

Primary endometrial EC and T84 intestinal monolayers were exposed to HIV-1 (ADA, 10⁶ infectious viral units/ml, p24 280ng/ml) and apical and basolateral supernatants were collected 24 hours post-exposure and assayed by Luminex multi-analyte kit for the following cytokines: (A) TNF-α (B) IL-6, (C) IL-8, (D) MCP-1, (E) IL-10, (F) IL-1β.

<p>*p<0.01, **p<0.001. Data shown is representative of three separate experiments from different tissues, each experiment had 3–5 replicate cultures for each experimental condition.</p

Bacterial and viral translocation across mucosal epithelial monolayers following HIV-1 exposure.

<p>(A) Bacterial translocation was measured in T84 intestinal monolayers. Confluent monolayers were left untreated or treated for 6 hours with TNF-α (20ng/ml), <i>E. coli</i> (10⁸ CFU/ml) , TNF-α (20ng/ml) +<i>E. coli</i> (10⁸ CFU/ml), HIV-1 or HIV-1 (6 or 24 hours)+<i>E. coli</i> (10⁸ CFU/ml). (A) TER measurements following various treatments in the presence or absence of E. Coli. * p<0.001. (B) Basolateral supernatants were collected and bacterial counts were done. (C) Viral translocation was determined in endometrial EC monolayers exposed to HIV-1 (ADA, 10⁶ infectious viral units/ml, p24 280ng/ml) on the apical side. Basolateral supernatants were collected after different time intervals infectious and viral counts were done on TZM/b-l indicator cell line. Viral counts are depicted as percentage of inoculum added to the apical compartment of monolayers. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.</p

Primary endometrial EC monolayers were exposed to different concentrations of HIV-1 (ADA) for 24 hours.

<p>TER values were measured, starting at viral concentration of 10³ up to 10⁷ infectious viral units/ml (equivalent to p24 values of 0.2ng–2800 ng/ml) (A).* p<0.001 **p<0.05. ZO-1 staining following exposure to different concentration of virus (B). Data shown is representative of three separate experiments, each experiment had 3–5 replicate cultures for each experimental condition.</p

Induction of apoptosis in the RIP-iCasp-3 transgenic model leads to antigen specific T cell proliferation.

<p>(a) The inducible caspase-3 construct (iCasp-3) includes a human pro-caspase-3 DNA fused with a myristoylation sequence (Myr), two modified FK506 binding domains (Fv) and a hemagglutinin antigen (HA). (b) Schematic representation of how apoptosis is induced. The addition of the dimerizing agent AP20187 induces dimerization of the iCasp-3 construct and activation of caspase-3 with the subsequent induction of apoptosis. The rat insulin promoter (RIP) was used to express the iCasp-3 construct and transgenic mice were generated that express iCasp-3 in the β-islet cells of the pancreas. (c) Expression was examined by histology using an antibody specific for human caspase-3. (d) 2 mg/kg of A20187 was given by i.p. to RIP-iCasp3.4 and RIP-iCasp3.8 mice (3 times, 24 hours apart) and blood glucose levels were measured. (e) The pancreas sections from RIP-iCasp3.4 mice treated with 2 mg/kg AP20187 were analyzed by TUNEL staining at the indicated time points. (f) Purified CD45.1+ P14 CD8 were labeled with CFSE and transferred to either RIP-iCasp-3.4/gp or RIP-gp mice which were injected with 2 mg/kg AP20187 the following day. 5 days after drug injection, proliferation of P14 cells was analyzed in the pancreatic draining (PDLN) or inguinal (ILN) lymph node. (g) RIP-iCasp-3.4/gp/CD11c-DTR mice were treated with diphtheria toxin to deplete CD11c+ cells prior to AP20187 injection. Dot plots depict the proliferation of CFSE-labeled P14 cells 5 days post injection with AP20187. Data are representative of at least 5 mice per condition from independent experiments. Error bars in bar graph show S.E.M. (**<i>p</i> < 0.01, *<i>p</i> < 0.05).</p

APC maturation signals promote T cell activation and inflammation, but are unable to induce diabetes.

<p>(a) and (b) CFSE-labeled P14/CD45.1+ T cells were transferred to RIP-iCasp-3.4/gp mice and treated with AP20187. Anti-CD40 antibody was injected 2 days later. After 3 days, cells from the PDLN were analyzed for division by CFSE dilution and IFN−γ production. (c) and (d) CD8 T cell infiltration in the pancreas was evaluated by histology sections which were stained with anti-CD8. (e) Infiltrating P14 T cells and CD8+ T cells in the pancreas were quantified. (f) P14 T cells were transferred into RIP-iCasp-3.4/gp mice. After 24 hours, AP20187 was given i.p., followed by anti-CD40 antibody i.v. 2 days later. Blood glucose levels were monitored every other day for 60 days. At day 49, mice were infected with LCMV-Armstrong. Data are representative of at least 5 mice per condition from independent experiments and error bars in bar graph show S.E.M. (*<i>p</i> < 0.05).</p