We initially hypothesized that increased transmission in the setting of GUD is the result of immediate access to target cells in the submucosa that ulcers provide for infected cells. We tested the major prediction of this hypothesis, that ulcers are sites where infected cells from the inoculum and secondarily infected cells concentrate, by examining the anatomical location of SIV RNA+ cells following exposure to infected cells.
We inoculated animals cy0089 and cy0096 with a total of 105
and animals cy0070 and cy0147 with 2.3 ×106
and 3.0 × 106
in vitro-infected allogeneic peripheral blood monocytes, respectively. On days 2 (cy0089, cy0096, cy0147) and 3 (cy0070) after inoculation, we inspected the lower female reproductive tract for signs of inflammation by using a rigid fiber-optic scope and then euthanized the animals and processed the cervix and three evenly separated sections encompassing the entire length of the vaginal mucosa. Additionally, we collected genital, inguinal, axillary, and mesenteric LN samples. We fixed a portion of each sample with 4% paraformaldehyde and processed them for in situ hybridization to examine them for SIVmac239 RNA+
cells as previously described (26
). We tested the remaining portion of every sample for SIVmac239 vRNA burden by using a quantitative reverse transcription-PCR as detailed previously (2
First we confirmed in hematoxylin-and-eosin-stained sections that epithelial disruption was associated with those foci of ulceration that we had previously identified by visual inspection (Fig. ). We investigated the association of initial infection with these sites of epithelial disruption by focusing on the 16 female genital mucosa samples obtained from animals euthanized 2 and 3 days after a cell-associated viral challenge, excluding the one animal (cy0155) euthanized 1 day after a cell-associated viral challenge, where detection of vRNA in samples from the female genital mucosa was likely due to residual inoculum (Table ). We detected vRNA in 7 out of the 16 female genital mucosa samples obtained from these four macaques. Five of the 7 positive samples were associated with visible ulcer/inflammation versus 2 positive out of the 10 nonulcerated mucosal samples (83% versus 20%, P = 0.035 by Fisher's exact test) (Table ). As additional evidence of the association of initial infection with epithelial disruption, we found that vRNA+ cells were concentrated in an ulcer (Fig. ) in one animal (cy0121) euthanized 5 days after a cell-associated viral challenge, and the ulcer site also had the highest number of copies of vRNA (Table ).
FIG. 1. Benzalkonium-induced inflammation and ulceration. (A) Vaginal ulcer with dark-staining inflammatory infiltrate and associated denuded epithelium at an original magnification of ×10. The double-headed arrow points to the inset showing the ulcer (more ...)
Cell-associated virus inocula and subsequent vRNA burdens of tissue samples from cynomolgus macaques challenged in this experiment
FIG. 2. SIV RNA+ cells concentrated at sites of ulceration and inflammation at day 5. SIV RNA+ cells were detected by in situ hybridization (black cells). The circled area is a portion of the extensive inflammatory infiltrate and associated thinned (more ...)
samples were not invariably associated with sites of visible ulceration, as we detected SIVmac239 vRNA in two samples with an intact epithelium (Fig. ). This is consistent with our earlier observation that cell-associated virus, with less frequency, can establish a persistent infection in the apparent absence of visible ulceration (9
). It is quite possible that transmission still occurs at sites of disruption of the mucosal barrier that were not detected visually or in the sections examined, as even in the section shown with an intact epithelium (Fig. ) the epithelium is thinned and there is inflammation in the underlying epithelium.