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Find video protocols related to scientific articles indexed in Pubmed.
Quantitative and stoichiometric analysis of the microRNA content of exosomes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-29-2014
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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.
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Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
Nucleic Acids Res.
PUBLISHED: 05-16-2014
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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.
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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.
PLoS ONE
PUBLISHED: 01-01-2014
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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.
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Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.
PLoS ONE
PUBLISHED: 01-01-2014
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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.
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Use of human mucosal tissue to study HIV-1 pathogenesis and evaluate HIV-1 prevention modalities.
Curr HIV/AIDS Rep
PUBLISHED: 07-17-2013
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The use of human mucosal tissue models is an important tool advancing our understanding of the specific mechanisms of sexual HIV transmission. Despite 30 years of study, major gaps remain, including how HIV-1 transverses the epithelium and the identity of the early immune targets (gate keepers). Because defining HIV-1 transmission in vivo is difficult, mucosal tissue is being used ex vivo to identify key steps in HIV-1 entry and early dissemination. Elucidating early events of HIV-1 infection will help us develop more potent and specific HIV-1 preventatives such as microbicides and vaccines. Mucosal tissue has been incorporated into testing regimens for antiretroviral drugs and monoclonal antibodies. The use of mucosal tissue recapitulates the epithelium and immune cells that would be exposed in vivo to virus and drug. This review will discuss the use of mucosal tissue to better understand HIV-1 pathogenesis and prevention modalities.
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A phase 1 randomized, double blind, placebo controlled rectal safety and acceptability study of tenofovir 1% gel (MTN-007).
PLoS ONE
PUBLISHED: 02-21-2013
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Rectal microbicides are needed to reduce the risk of HIV acquisition associated with unprotected receptive anal intercourse. The MTN-007 study was designed to assess the safety (general and mucosal), adherence, and acceptability of a new reduced glycerin formulation of tenofovir 1% gel.
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Comprehensive assessment of HIV target cells in the distal human gut suggests increasing HIV susceptibility toward the anus.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 02-09-2013
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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.
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Vaginal langerhans cells nonproductively transporting HIV-1 mediate infection of T cells.
J. Virol.
PUBLISHED: 10-05-2011
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Although implied by other models, proof that Langerhans cells (LCs) in the human vagina participate in dissemination of infectious human immunodeficiency virus type 1 (HIV-1) has been lacking. Here, we show that LCs migrate from HIV-1-exposed vaginal epithelia and pass infectious virus to CD4+ T cells without being productively infected themselves, and we point to a pathway that might enable HIV-1 to avoid degradation in vaginal LCs. Transport by migratory LCs to local lymphatics in a nonproductive but infectious form may aid HIV-1 in evasion of topical microbicides that target its intracellular productive life cycle.
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Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators.
PLoS ONE
PUBLISHED: 03-30-2011
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Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods.
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Preventing mucosal HIV transmission with topical microbicides: challenges and opportunities.
Antiviral Res.
PUBLISHED: 08-21-2010
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A combination of prevention and treatment modalities will be needed to successfully control the global spread of HIV. Microbicides, drug products topically applied to mucosal surfaces to prevent HIV infection, are one of these biomedical interventions that hold great promise. In order to be efficacious, microbicides must overcome several challenges imposed by the mucosal microenvironment they intend to protect and the mischievous human immunodeficiency virus with its enormous capacity to adapt. Recent data, however, supports the establishment of the primo-infection by only a small number of founder viruses, which are highly vulnerable to microbicidal intervention at the initial stages of mucosal invasion. The biological foundation of these challenges and opportunities in microbicide development is explored in this review. This article forms part of a special supplement on presentations covering HIV transmission and microbicides, based on the symposium "Trends in Microbicide Formulations", held on 25 and 26 January 2010, Arlington, VA.
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Ex vivo comparison of microbicide efficacies for preventing HIV-1 genomic integration in intraepithelial vaginal cells.
Antimicrob. Agents Chemother.
PUBLISHED: 11-30-2009
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Vaginally applied microbicides hold promise as a strategy to prevent sexual HIV transmission. Several nonspecific microbicides, including the polyanion cellulose sulfate, have been evaluated in large-scale clinical trials but have failed to show significant efficacy. These findings have prompted a renewed search for preclinical testing systems that can predict negative outcomes of microbicide trials. Moreover, the pipeline of potential topical microbicides has been expanded to include antiretroviral agents, such as reverse transcriptase, fusion, and integrase inhibitors. Using a novel ex vivo model of vaginal HIV-1 infection, we compared the prophylactic potentials of two forms of the fusion inhibitor T-20, the CCR5 antagonist TAK-778, the integrase inhibitor 118-D-24, and cellulose sulfate (Ushercell). The T-20 peptide with free N- and C-terminal amino acids was the most efficacious compound, causing significantly greater inhibition of viral genomic integration in intraepithelial vaginal leukocytes, measured by an optimized real-time PCR assay, than the more water-soluble N-acetylated T-20 peptide (Fuzeon) (50% inhibitory concentration [IC50], 0.153 microM versus 51.2 microM [0.687 ng/ml versus 230 ng/ml]; P<0.0001). In contrast, no significant difference in IC50s was noted in peripheral blood cells (IC50, 13.58 microM versus 7.57 microM [61 ng/ml versus 34 ng/ml]; P=0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC50, 1.8 microg/ml). These results highlight the merit of our model for screening the mucosal efficacies of novel microbicides and their formulations and potentially rank ordering candidates for clinical evaluation.
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HIV type 1 fails to trigger innate immune factor synthesis in differentiated oral epithelium.
AIDS Res. Hum. Retroviruses
PUBLISHED: 10-22-2009
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The oral mucosa is relatively resistant to human immunodeficiency virus type 1 (HIV-1) transmission. The mechanisms contributing to this resistance remain incompletely understood, but may include HIV-induced synthesis of innate immune factors. We used fully differentiated oral epithelium as a surrogate for the oral mucosa in vivo, exposed it to X4- and R5-tropic HIV-1 in culture, and quantified mRNA expression of six innate immune factors. Neither virus increased expression of human beta defensin 2 (hBD-2) mRNA over supernatants from uninfected lymphoblast controls. HIV-1 also failed to induce mRNA of four additional innate immunity-related genes. Similar results were obtained with oral monolayer epithelial cells. Interestingly, the X4-tropic virus inhibited mRNA expression of hBD-2, and of three of the other factors, at higher dosages in the differentiated oral epithelium but not the monolayers. The failure of HIV-1 to induce innate immune factors in the differentiated epithelium was not due to a lack of tissue penetration, as we detected fluorescence-tagged virions up to 30 mum deep from the apical surface. HIV-1 does not trigger de novo innate immune factor synthesis in oral epithelium, pointing to the role of a constitutive innate immunity for protection against HIV-1 in the oral cavity.
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Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells.
J. Virol.
PUBLISHED: 08-12-2009
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Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication, but these defenses fail to suppress human immunodeficiency virus (HIV) infection, which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here, we examined antiviral defenses in CD4+ cells during virus infection and coinfection, revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression, but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells, which supported robust viral replication, whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections.
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HIV infection of the genital mucosa in women.
Curr HIV/AIDS Rep
PUBLISHED: 01-20-2009
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The vast majority of new HIV infections are acquired via the genital and rectal mucosa. Here, we provide an overview of our current knowledge of how HIV establishes local infection, with an emphasis on viral invasion through the female genital tract. Studies using human explant tissues and in vivo animal studies have improved our understanding of the cellular and molecular pathways of infection; this information could be harnessed to design effective HIV vaccines and microbicides.
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Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition.
Nat. Med.
PUBLISHED: 01-07-2009
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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.
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Vpu-deficient HIV strains stimulate innate immune signaling responses in target cells.
J. Virol.
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Acute virus infection induces a cell-intrinsic innate immune response comprising our first line of immunity to limit virus replication and spread, but viruses have developed strategies to overcome these defenses. HIV-1 is a major public health problem; however, the virus-host interactions that regulate innate immune defenses against HIV-1 are not fully defined. We have recently identified the viral protein Vpu to be a key determinant responsible for HIV-1 targeting and degradation of interferon regulatory factor 3 (IRF3), a central transcription factor driving host cell innate immunity. IRF3 plays a major role in pathogen recognition receptor (PRR) signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Here we interrogate the cellular responses to target cell infection with Vpu-deficient HIV-1 strains. Remarkably, in the absence of Vpu, HIV-1 triggers a potent intracellular innate immune response that suppresses infection. Thus, HIV-1 can be recognized by PRRs within the host cell to trigger an innate immune response, and this response is unmasked only in the absence of Vpu. Vpu modulation of IRF3 therefore prevents virus induction of specific innate defense programs that could otherwise limit infection. These observations show that HIV-1 can indeed be recognized as a pathogen in infected cells and provide a novel and effective platform for defining the native innate immune programs of target cells of HIV-1 infection.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.