Relative CCL3 (A), CCL4 (B) and CCL5 (C) expression levels in GALT tissues at different stages of SHIV_SF162P4 infection (n = 5).

<p>PID 0 was used as the calibrator (value = 1).</p

Rhesus macaque-specific primers for real-time PCR

<p>Rhesus macaque-specific primers for real-time PCR</p

Phenotyping colon and jejunum epithelial cells (ECs) by confocal microscopy.

<p>Essentially all colon (<b>A–E</b>) ECs (cytokeratin positive) were negative for (<b>A</b>) Ham56 (macrophage marker), and (<b>B</b>) CD11c (dendritic cell marker). Very few colon ECs were double positive for (<b>C</b>) both CD45 (leukocyte marker) and cytokeratin expression. A major population of colon ECs was positive for (<b>D</b>) HLA-DR expression. However, very few colon ECs were positive for (<b>E</b>) IL-10R expression which was evident in apical regions. Jejunum ECs were also negative for (<b>F</b>) CD54 (ICAM-1, cell adhesion marker). White arrow denotes the presence of double positive cells (cytokeratin and CD45/HLA-DR/IL-10R) for the specified sample and fluorochrome.</p

SHIV_SF162P4 loads in plasma (A), CD4+ T cell counts in peripheral blood (B), relative CCL3 (C), CCL4 (D) and CCL5 (E) gene expression levels in PBMCs at PID 0-180 with SHIV_SF162P4.

<p>Each point on the graph represents the mean fold change in gene expression relative to pre-infection level±SE (n = 5).</p

SIV infection induces apoptosis and upregulation of CD54 expression on intestinal epithelial cells (ECs).

<p>(<b>A</b>) Epithelial cells apoptosis was detected in jejunum by multi-labeled immunofluorescent confocal microscopy. Note that increased apoptosis of jejunum ECs was detected during acute (AV85; 21 days post infection) and chronic (HG58; 288 days post infection) SIV infection (as indicated by the white arrows). (<b>B</b>) Mean frequencies (± standard deviation) of surface CD54, CD80, CD86 and HLA-DR expression are shown for both jejunum ECs and CD45+ leukocytes from normal (n = 6) and chronically SIV-infected (n = 4) macaques. Note that a significant increase in CD54 expression on jejunum ECs was detected in SIV-infected macaques. However, increased expression of CD54, CD80 and HLA-DR on CD45+ leukocytes was observed in SIV-infected RMs. Statistically significant differences between each group of cells are shown.</p

Comparison of SHIV_SF162P4and SHIV_Ku1 primary infection in peripheral blood.

<p>Viral loads in plasma (A), CD4+ T cell counts (B), Fold-changes of CCL3, CCL4 and CCL5 gene expression levels in SHIV_SF162P4- and SHIV_Ku1–infected macaques at PID 0 vs. PID 14 (C) (n = 5 for each group). Negative sample cut-off for viral load measurements in plasma was <30 copies/ml e.g. PID 0 value.</p

Schematic representation of epithelial cell and leukocyte isolation protocols from intestinal tissues after different enzymatic treatments.

<p>Note that this protocol explains cell isolation procedures with initial DTT treatment.</p

Absolute (counts) and relative (%) levels of CCL4+ cells in MLN following SHIV_SF162P4 inoculation

a<p>Number of cells/0.5 mm² section.</p

Phenotyping epithelial cells of normal rhesus macaques.

<p>(<b>A</b>) Representative expression of cytokeratin (CK; epithelial cell marker) and CD45 (leukocyte marker) in jejunum intestinal cells isolated from lower layer (“lymphocyte enriched”) of percoll gradient after treating with DTT and EDTA solution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>; Step IX). Note that 2.8% of cells were double positive for CK and CD45 receptors. Fractions of double negative CK−CD45− cells were also evident from isolated cells. Live cells were gated first from all acquired cells and plotted based on CK and CD45 markers. Visualization of epithelial cells both in (<b>B</b>) colon at 20× and (<b>C</b>) jejunum at 5× resolution were detected by immunohistochemistry staining using anti-CK monoclonal antibody with hematoxylin counterstain.</p

The proportions of peripheral blood CD3+CD4+CCL4+ and CD3+CD8+CCL4+ T lymphocytes were compared at PID 0 and PID 14 with SHIV_SF162P4 by flow cytometry (A).

<p>Gating was performed via CD3+ lymphocyte population. Statistically significant differences between CD3+CD4+CCL4+ and CD3+CD8+CCL4+ cells over CD3+ lymphocytes at PID 0 vs. PID 14 (n = 5) are shown (B).</p