Sean M. Hughes

Quantitative and stoichiometric analysis of the microRNA content of exosomes

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. Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions

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.

Mucosal effects of tenofovir 1% gel

Tenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against HIV transmission. Because this is a new prevention strategy, we broadly assessed its effects on the mucosa. In MTN-007, a phase-1, randomized, double-blinded rectal microbicide trial, we used systems genomics/proteomics to determine the effect of tenofovir 1% gel, nonoxynol-9 2% gel, placebo gel or no treatment on rectal biopsies (15 subjects/arm). We also treated primary vaginal epithelial cells from four healthy women with tenofovir in vitro. After seven days of administration, tenofovir 1% gel had broad-ranging effects on the rectal mucosa, which were more pronounced than, but different from, those of the detergent nonoxynol-9. Tenofovir suppressed anti-inflammatory mediators, increased T cell densities, caused mitochondrial dysfunction, altered regulatory pathways of cell differentiation and survival, and stimulated epithelial cell proliferation. The breadth of mucosal changes induced by tenofovir indicates that its safety over longer-term topical use should be carefully monitored. Clinical trial registration: NCT01232803. DOI: http://dx.doi.org/10.7554/eLife.04525.001

Comprehensive assessment of HIV target cells in the distal human gut suggests increasing HIV susceptibility toward the anus

Background:Prevention of rectal HIV transmission is a high-priority goal for vaccines and topical microbicides because a large fraction of HIV transmissions occurs rectally. Yet, little is known about the specific target-cell milieu in the human rectum other than inferences made from the colon. Methods:We conducted a comprehensive comparative in situ fluorescence study of HIV target cells (CCR5-expressing T cells, macrophages, and putative dendritic cells) at 4 and 30 cm proximal of the anal canal in 29 healthy individuals, using computerized analysis of digitized combination stains. Results:Most strikingly, we find that more than 3 times as many CD68+ macrophages express the HIV coreceptor CCR5 in the rectum than in the colon (P = 0.0001), and as such rectal macrophages seem biologically closer to the HIV-susceptible CCR5high phenotype in the vagina than the mostly HIV-resistant CCR5low phenotype in the colon. Putative CD209+ dendritic cells are generally enriched in the colon compared with the rectum (P = 0.0004), though their CCR5 expression levels are similar in both compartments. CD3+ T-cell densities and CCR5 expression levels are comparable in the colon and rectum. Conclusions:Our study establishes the target-cell environment for HIV infection in the human distal gut and demonstrates in general terms that the colon and rectum are immunologically distinct anatomical compartments. Greater expression of CCR5 on rectal macrophages suggests that the most distal sections of the gut may be especially vulnerable to HIV infection. Our findings also emphasize that caution should be exercised when extrapolating data obtained from colon tissues to the rectum.

Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study

Background Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. Methods and Findings We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor. Conclusions CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines. Methods and Findings We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women. Conclusion Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.

Properties and mechanisms of spontaneous activity in the embryonic chick hindbrain.

Spontaneous activity regulates many aspects of central nervous system development. We demonstrate that in the embryonic chick hindbrain, spontaneous activity is expressed between embryonic days (E) 6–9. Over this period the frequency of activity decreases significantly, although the events maintain a consistent rhythm on the timescale of minutes. At E6, the activity is pharmacologically dependent on serotonin, nACh, GABAA, and glycine input, but not on muscarinic, glutamatergic, or GABAB receptor activation. It also depends on gap junctions, t‐type calcium channels and TTX‐sensitive ion channels. In intact spinal cord‐hindbrain preparations, E6 spontaneous events originate in the spinal cord and propagate into lateral hindbrain tissue; midline activity follows the appearance of lateral activity. However, the spinal cord is not required for hindbrain activity. There are two invariant points of origin of activity along the midline, both within the caudal group of serotonin‐expressing cell bodies; one point is caudal to the nV exit point while the other is caudal to the nVII exit point. Additional caudal midline points of origin are seen in a minority of cases. Using immunohistochemistry, we show robust differentiation of the serotonergic raphe near the midline at E6, and extensive fiber tracts expressing GAD65/67 and the nAChR in lateral areas; this suggests that the medial activity is dependent on serotonergic neuron activation, while lateral activity requires other transmitters. Although there are differences between species, this activity is highly conserved between mouse and chick, suggesting that developmental event(s) within the hindbrain are dependent on expression of this spontaneous activity.

Anti-proliferative therapy for HIV cure: a compound interest approach

In the era of antiretroviral therapy (ART), HIV-1 infection is no longer tantamount to early death. Yet the benefits of treatment are available only to those who can access, afford, and tolerate taking daily pills. True cure is challenged by HIV latency, the ability of chromosomally integrated virus to persist within memory CD4+ T cells in a non-replicative state and activate when ART is discontinued. Using a mathematical model of HIV dynamics, we demonstrate that treatment strategies offering modest but continual enhancement of reservoir clearance rates result in faster cure than abrupt, one-time reductions in reservoir size. We frame this concept in terms of compounding interest: small changes in interest rate drastically improve returns over time. On ART, latent cell proliferation rates are orders of magnitude larger than activation and new infection rates. Contingent on subtypes of cells that may make up the reservoir and their respective proliferation rates, our model predicts that coupling clinically available, anti-proliferative therapies with ART could result in functional cure within 2–10 years rather than several decades on ART alone.

Effect of Mucosal Cytokine Administration on Selective Expansion of Vaginal Dendritic Cells to Support Nanoparticle Transport

The capacity of antigen‐carrying vaccine nanoparticles (NPs) administered vaginally to stimulate local immune responses may be limited by the relatively low numbers of antigen‐presenting cells (APCs) in the genital mucosa. Because inflammation is associated with increased susceptibility to sexually transmitted infections, we sought to increase APC numbers without causing inflammation.

Cryopreservation of Human Mucosal Leukocytes.

Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. Methods and Findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.