Significance: Exosomes have been a subject of great interest in recent years, especially in the context of the microRNAs (miRNAs) that they contain. Exosome-mediated miRNA transfer between cells has been proposed to be a mechanism for intercellular signaling and exosome-associated miRNAs in biofluids have been suggested as potential minimally invasive biomarkers for multiple human disease states. Remarkably, we show here that most exosomes derived from standard preparations do not harbor many copies of miRNA molecules. These findings suggest a reevaluation of current models of the mechanism of exosome-mediated miRNA communication and indicate that stoichiometric analysis will be valuable for the study of other populations of extracellular vesicles and their associated RNAs as well.

Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20-40 and 40-100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20-40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of ∼2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5'-ends of 18-19 or 30-34 nts in length; such tRNA fragments repress translation. Thus, SE could potentially deliver regulatory signals to the recipient mucosa via transfer of small RNA molecules.

Understanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4(+) T cells. HIV-1 entered CD4(+) T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a(+) Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.

Cytotoxic CD8+ T cells play a critical role in controlling herpes simplex virus (HSV) infection and reactivation. However, little is known about the spatiotemporal dynamics of CD8+ T cells during HSV lesion evolution or about their involvement in immune surveillance after lesion resolution. Using quantum dot–conjugated peptide–major histocompatibility complex multimers, we investigated the in vivo localization of HSV-2–specific CD8+ T cells in sequential biopsies of human genital skin during acute, resolving, and healed stages of HSV-2 reactivation. Our studies revealed that functionally active CD8+ T cells selectively infiltrated to the site of viral reactivation. After lesion healing in concert with complete reepithelialization and loss of HSV DNA from skin biopsies, HSV-2–specific CD8+ T cells persisted for more than two months at the dermal–epidermal junction, adjacent to peripheral nerve endings. In two out of the six sequentially studied individuals, HSV-2 DNA reappeared in clinically and histologically normal–appearing skin. Detection of viral DNA was accompanied by increased numbers of both HSV-specific and total CD8+ T cells in the dermis. These findings indicate that the frequency and clinical course of HSV-2 reactivation in humans is influenced by virus-specific CD8+ T cells that persist in peripheral mucosa and genital skin after resolution of herpes lesions.

Infection with HSV-2 increases the likelihood of HIV acquisition, but suppression of HSV-2 reactivation with antiviral drugs does not seem to reduce the acquisition of HIV. Laurence Corey and colleagues provide a potential mechanism underlying this observation, showing that even after acyclovir treatment for the HSV-2 infection, many inflammatory and immune cells possessing the receptors required for HIV infection persist in the mucosa, making the initial 'spark' of infection more likely. To explore the mechanism by which herpes simplex virus (HSV)-2 infection is related to HIV-1 acquisition, we conducted in situ analysis of the cellular infiltrate from sequential biopsies of HSV-2 lesions from patients on and off antiviral therapy. CD4+ and CD8+ T cells and a mixed population of plasmacytoid and myeloid dendritic cells (DCs), including cells expressing the C-type lectin receptor DC-SIGN, persisted at sites of HSV-2 reactivation for months after healing, even with daily antiviral therapy. The CD4+ T cells that persisted reacted to HSV-2 antigen, were enriched for expression of the chemokine receptor CCR5, and were contiguous to DCs expressing the interleukin-3 receptor CD123 or DC-SIGN. Ex vivo infection with a CCR5-tropic strain of HIV-1 revealed greater concentrations of integrated HIV-1 DNA in cells derived from healed genital lesion biopsies than in cells from control skin biopsies. The persistence and enrichment of HIV receptor–positive inflammatory cells in the genitalia help explain the inability of anti–HSV-2 therapy to reduce HIV acquisition.