Comparing two ways of mixing PBMCs and washing medium by viability (A) and absolute count of live PBMCs (B).

<p>Paired samples 20 donors included. Ref.: reference group. NS: non-significant. **: p<0.01.</p

Gating strategy to identify live/dead CD45+ leukocytes.

<p>A) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of dead cells. B) Doublet exclusion C) Identification of CD45 positive cells D) Dead cells identified as 7-AAD positive events. Compensation for spectral overlap was not required.</p

Centrifugation time and force and PBMC viability (A) and absolute live PBMC count (B).

<p>Paired samples from 18 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

Temperature of centrifuge by PBMC viability (A) and absolute live PBMC recovery (B).

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

PBMC viability (A) and absolute recovery (B) as a result of varying thawing time in 37°C heated water bath.

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

Impact of washing-medium temperature on viability (A) and PBMC recovery (B).

<p>20 donors included. Ref.: reference group. **: p<0.01.</p

PBMC viability (A) and absolute live PBMC recovery (B) by incubation.

<p>Samples were stored in a 37°C incubator with 5% CO₂. Paired samples from 20 donors were included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

Oral transmission of cell-associated HIV in humanized BLT mice.

<p>(A) BLT mice (n = 7) were exposed orally to HIV-1_JR-CSF infected PBMCs. Transmission was monitored weekly by measuring the plasma viral load and percentage of human CD4⁺ T cells in peripheral blood. A two-tailed Mann-Whitney U test was used to compare the percentages of CD4⁺ T cells in peripheral blood pre-exposure (week 0) and post-exposure (p values<0.05 are indicated with an asterisk). (B) Saliva and peripheral blood (PB) were harvested from five BLT mice following oral HIV exposure with cell-associated HIV-1_JR-CSF. Saliva and peripheral blood were collected from BLT mice on the same day and the corresponding saliva and peripheral blood viral loads for each mouse are shown with the same color and shape. (C) HIV transmission of cell associated virus administered via gavage into the stomach of BLT mice. Shown is the viral load in the peripheral blood of BLT mice (n = 4) receiving a single dose of HIV-1_JR-CSF infected PBMCs directly into the stomach by gavage.</p

Description of BLT mice used to evaluate the effect of human breast milk on oral transmission of cell-free HIV-1.

*<p>Real-time PCR results representative of DNA extracted from 5×10⁴–1.6×10⁶ cells or 15–50 ul blood. The assay limit of detection is 10 copies. SPL = spleen, LN = lymph node, BM = bone marrow, TO = thymic organoid, LIV = liver, LNG = lung and PB = peripheral blood. The results are indicated as follows: (+) positive for HIV DNA, (−) negative for HIV DNA, (nd) not determined.</p

The upper GI tract of humanized BLT mice is repopulated with HIV target cells.

<p>The esophagus, stomach and duodenum were harvested from BLT mice for IHC analysis to determine if these potential sites for HIV transmission following an oral exposure possess HIV target cells. The tissues harvested were stained with the appropriate antibodies to verify the presence of human leukocytes (CD45⁺) including dendritic cells (CD11c⁺), macrophages (CD68⁺) and T cells (CD3⁺), specifically, CD4⁺ T cells (CD4⁺). Positive cells appear brown. Scale bars = 100 µm.</p