Exosomes from uninfected cells activate transcription of latent HIV-1.

HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.

Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/µl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-ß, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

Gonadal mosaicism and familial adenomatous polyposis.

De novo mutations in the adenomatous polyposis coli (APC) gene are estimated to constitute approximately 25% of familial adenomatous polyposis (FAP) cases. A small percentage of these arise in the mosaic form, affecting only a subset of cells in the affected individual. A family is described here whereby an unaffected mother with no detectible mutation in APC, transmitted the identical APC c.4729G>T (p.Glu1577X) mutation to two children. A third child, with the same APC allelic haplotype received a normal APC allele, suggesting that the mutation originated in the gonadal tissues of the mother. These results underscore the utility of mutation-specific genetic testing for the parents and siblings of a proband of an adult-onset disease, even if the proband appears to have a de novo mutation. Parents who test negative for the mutation should be counseled about the possibility of having another affected child due to gonadal mosaicism.

Corrigendum: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.

[This corrects the article on p. 1765 in vol. 7, PMID: 27872619.].

Biodegradable Nanoparticles for Delivery of Therapeutics in CNS Infection.

Despite the significant advances in neurological medicine, it remains difficult to treat ailments directly involving the brain. The blood brain barrier (BBB) is a tightly regulated, selectively permeable barrier that restricts access from the blood into the brain extracellular fluid (BEF). Many conditions such as tumors or infections in the brain are difficult to treat due to the fact that drugs and other therapeutic agents are unable to easily pass through this relatively impermeable barrier. Human Immunodeficiency Virus (HIV) presents a particular problem as it is able to remain dormant in the brain for years protected from antiretroviral drugs by the BBB. The development of nanoscale carriers over the past few decades has made possible the delivery of therapies with the potential to overcome membrane barriers and provide specific, targeted delivery. This review seeks to provide a comprehensive overview of the various aspects of nanoparticle formulation and their applications in improving the delivery efficiency of drugs, specifically antiretroviral therapeutics to the brain to treat HIV.

HIV-1 Transcription Inhibitors Increase the Synthesis of Viral Non-Coding RNA that Contribute to Latency.

BACKGROUND: HIV-1 can be preserved in long-lived resting CD4+ T- and myeloid cells, forming a viral reservoir in tissues of the infected individuals. Infected patients primarily receive cART, which, to date, is the most efficient treatment against HIV/AIDS. However, the major problem in the eradication of HIV-1 from patients is the lack of therapeutic approaches to recognize the latent HIV-1 provirus and to eliminate latently infected cells. RESULTS: In the current review, we describe the effect of HIV-1 transcriptional inhibitors CR8#13 and F07#13 using a series of in vitro and in vivo assays. We found that both of these compounds regulate p-TEFb in infected cells, and terminate transcription at two sites, either at the LTR or early gag regions. The resulting short transcripts are termed TAR and TAR-gag, respectively. These nascent RNAs are capable of binding to SWI/SNF components, including mSin3A/HDAC-1 complex and potentially serve as a scaffolding RNA. Both TAR and TAR-gag are detected as large complexes from treated infected cells when using chromatography. Both transcripts are non-coding in T-cells and monocytes, and potentially recruit suppressive factors along with RNAbinding proteins to the DNA resulting in Transcriptional Gene Silencing (TGS). Finally, these compounds suppress activated virus when using a latent humanized mouse model. CONCLUSION: Collectively, these data implicate transcription inhibitors as regulators of the viral promoter through short non-coding RNAs and chromatin remodeling factors. These RNAs give specificity toward either viral DNA and/or nascent mRNA when functioning as TGS.

Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80-90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation and eventual destruction of the T-cell and myeloid arms of the immune system (bystander lymphocyte apoptosis), allowing the virus to replicate to high titers in the immunocompromised host. Moreover, our results suggest that the use of drugs such as Oxytetracycline to modulate the levels of exosomes exiting EBOV-infected cells may be able to prevent the devastation of the adaptive immune system and allow for an improved rate of survival.

Extracellular vesicles from infected cells: potential for direct pathogenesis.

Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.

Evaluating Lynch syndrome in very early onset colorectal cancer probands without apparent polyposis.

To characterize the frequency of germline mutations associated with Lynch syndrome and review the potential expanded differential diagnoses in very early onset colorectal cancer (CRC) cases without apparent polyposis. Retrospectively reviewed medical records of 96 probands with CRC diagnosed prior to age 36 from three cancer centers. Determined the frequency of germline mutations in probands meeting different clinical criteria used to identify Lynch syndrome. Three of 46 (6.5%) single case indicators (probands without additional personal or family history suspicious for Lynch syndrome) were identified to carry a deleterious or suspected deleterious mismatch repair (MMR) mutation compared with 10 of 19 (52.6%) in the cases meeting at least one additional revised Bethesda guideline, and 11 of 15 (73.3%) in the cases meeting Amsterdam criteria. Two families without MMR mutations were documented to have a germline APC or TP53 mutation after additional clinical features were identified. Our results suggest that single cases of CRC (those without additional personal or family history suspicious of Lynch syndrome) diagnosed prior to age 36 infrequently have identifiable MMR mutations, especially when compared to cases meeting additional criteria. Careful attention to evolving or additional clinical features is warranted and may lead to an alternate genetic diagnosis in families with early onset CRC.

Population-based risk assessment for other cancers in relatives of hereditary prostate cancer (HPC) cases.

BACKGROUND: To identify associations of other cancers with hereditary prostate cancer (HPC) we estimated relative risks (RRs) of 36 different cancers in relatives of prostate cancer cases in the Utah Population Data Base (UPDB), which combines genealogical and cancer data for Utah. METHODS: We utilized known genetic relationships between prostate cancer cases and their relatives with cancer, combined with age- and sex-specific cancer rates calculated internally from the UPDB, to estimate RRs for cancer in relatives of prostate cancer cases. RESULTS: Multiple other cancers were observed in excess in both first- and second-degree relatives of HPC cases including colon cancer, non-Hodgkins lymphoma, multiple myeloma, rectal cancer, cancer of the gallbladder, and melanoma (skin). CONCLUSIONS: This analysis supports the existence of heritable prostate cancer syndromes that include other cancers. We hypothesize that the study of homogeneous pedigrees co-segregating prostate cancer and another cancer could allow more straightforward localization and identification of the gene(s) responsible.