Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract.

The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.

Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Challenges, advances, and opportunities in RNA structural biology by Cryo-EM.

RNAs are remarkably versatile molecules that can fold into intricate three-dimensional (3D) structures to perform diverse cellular and viral functions. Despite their biological importance, relatively few RNA 3D structures have been solved, and our understanding of RNA structure-function relationships remains in its infancy. This limitation partly arises from challenges posed by RNA's complex conformational landscape, characterized by structural flexibility, formation of multiple states, and a propensity to misfold. Recently, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool for the visualization of conformationally dynamic RNA-only 3D structures. However, RNA's characteristics continue to pose challenges. We discuss experimental methods developed to overcome these hurdles, including the engineering of modular modifications that facilitate the visualization of small RNAs, improve particle alignment, and validate structural models.

Semen inhibits Zika virus infection of cells and tissues from the anogenital region.

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.

Evolution of gag and gp41 in Patients Receiving Ritonavir-Boosted Protease Inhibitors.

Several groups have proposed that genotypic determinants in gag and the gp41 cytoplasmic domain (gp41-CD) reduce protease inhibitor (PI) susceptibility without PI-resistance mutations in protease. However, no gag and gp41-CD mutations definitively responsible for reduced PI susceptibility have been identified in individuals with virological failure (VF) while receiving a boosted PI (PI/r)-containing regimen. To identify gag and gp41 mutations under selective PI pressure, we sequenced gag and/or gp41 in 61 individuals with VF on a PI/r (n = 40) or NNRTI (n = 20) containing regimen. We quantified nonsynonymous and synonymous changes in both genes and identified sites exhibiting signal for directional or diversifying selection. We also used published gag and gp41 polymorphism data to highlight mutations displaying a high selection index, defined as changing from a conserved to an uncommon amino acid. Many amino acid mutations developed in gag and in gp41-CD in both the PI- and NNRTI-treated groups. However, in neither gene, were there discernable differences between the two groups in overall numbers of mutations, mutations displaying evidence of diversifying or directional selection, or mutations with a high selection index. If gag and/or gp41 encode PI-resistance mutations, they may not be confined to consistent mutations at a few sites.

A novel peptide that improves metabolic parameters without adverse central nervous system effects.

Intracellular peptides generated by limited proteolysis are likely to function inside and outside cells and could represent new possibilities for drug development. Here, we used several conformational-sensitive antibodies targeting G-protein coupled receptors to screen for novel pharmacological active peptides. We find that one of these peptides, DITADDEPLT activates cannabinoid type 1 receptors. Single amino acid modifications identified a novel peptide, DIIADDEPLT (Pep19), with slightly better inverse agonist activity at cannabinoid type 1 receptors. Pep19 induced uncoupling protein 1 expression in both white adipose tissue and 3T3-L1 differentiated adipocytes; in the latter, Pep19 activates pERK1/2 and AKT signaling pathways. Uncoupling protein 1 expression induced by Pep19 in 3T3-L1 differentiated adipocytes is blocked by AM251, a cannabinoid type 1 receptors antagonist. Oral administration of Pep19 into diet-induced obese Wistar rats significantly reduces adiposity index, whole body weight, glucose, triacylglycerol, cholesterol and blood pressure, without altering heart rate; changes in the number and size of adipocytes were also observed. Pep19 has no central nervous system effects as suggested by the lack of brain c-Fos expression, cell toxicity, induction of the cannabinoid tetrad, depressive- and anxiety-like behaviors. Therefore, Pep19 has several advantages over previously identified peripherally active cannabinoid compounds, and could have clinical applications.

Outcomes and associated risk factors for aortic valve replacement in 160 children: a competing-risks analysis.

BACKGROUND: We sought to define patient characteristics, outcomes, and associated risk factors after aortic valve replacement (AVR) in children. METHODS AND RESULTS: Clinical records from children undergoing AVR from 1974 to 2004 at our institution were reviewed. Competing-risks methodology determined the time-related prevalence of 3 mutually exclusive end states: death, repeated replacement, and survival without subsequent AVR and their associated risk factors. Longitudinal echocardiographic data were analyzed by mixed linear-regression models. Children (n=160) underwent 198 AVRs, with 33 having >1. Competing-risks analysis predicted that 10 years from the initial AVR, 19% had died without subsequent AVR, 34% underwent a second AVR, and 47% remained alive without replacement. Risk factors for death without a second AVR included lower weight (P<0.001) and younger age at AVR (P=0.04), performance of aortic arch reconstruction together with AVR (P=0.03), and nonautograft use (P=0.03). Risk factors for a second AVR included earlier operation year (P=0.04) and implantation of a bioprosthetic or homograft valve (P=0.004). Analysis of serial echocardiographic measurements showed that pulmonary autograft use was associated with slower progression of peak aortic gradient (P=0.002), smaller left ventricular dimension (P=0.04), and decreased prevalence of aortic regurgitation (P=0.04). CONCLUSIONS: Mortality and repeated valve replacement are common after initial AVR in children, especially in younger patients and those with bioprosthetic or homograft valves. Pulmonary autograft use is associated with decreased mortality, slower gradient progression, and smaller left ventricular dimension.

Maternal high-sodium intake alters the responsiveness of the renin-angiotensin system in adult offspring.

AIMS: The goal of the current study was to evaluate the impact of maternal sodium intake during gestation on the systemic and renal renin-angiotensin-aldosterone-system (RAAS) of the adult offspring. MAIN METHODS: Female Wistar rats were fed high- (HSD-8.0% NaCl) or normal-sodium diets (NSD-1.3% NaCl) from 8 weeks of age until the delivery of their first litter. After birth, the offspring received NSD. Tail-cuff blood pressure (TcBP) was measured in the offspring between 6 and 12 weeks of age. At 12 weeks of age, the offspring were subjected to either one week of HSD or low sodium diet (LSD-0.16% NaCl) feeding to evaluate RAAS responsiveness or to acute saline overload to examine sodium excretory function. Plasma (PRA) and renal renin content (RRC), serum aldosterone (ALDO) levels, and renal cortical and medullary renin mRNA expression levels were evaluated at the end of the study. KEY FINDINGS: TcBP was higher among dams fed HSD, but no TcBP differences were observed among the offspring. Male offspring, however, exhibited increased TcBP after one week of HSD feeding, and this effect was independent of maternal diet. Increased RAAS responsiveness to the HSD and LSD was also observed in male offspring. The baseline levels of PRA, ALDO, and cortical and medullary renin gene expression were lower but the RRC levels were higher among HSD-fed male offspring (HSDoff). Conversely, female HSDoff showed reduced sodium excretion 4 h after saline overload compared with female NSDoff. SIGNIFICANCE: High maternal sodium intake is associated with gender-specific changes in RAAS responsiveness among adult offspring.

Estimating the prevalence of myelodysplastic syndromes in patients with unexplained cytopenias: a retrospective study of 322 bone marrows.

Although unexplained anemia is common in the elderly, the prevalence of MDS is poorly defined. We reviewed 2267 bone marrows reviewed at our center between the years 2002 and 2005. Of these, 322 met our criteria for inclusion (14%). Seventy-three patients (22.6%) had a confirmed diagnosis of MDS and 32 (9.9%) had suspected MDS. Confirmed or suspected MDS was more likely in patients aged >65 (31.5% and 11%, respectively). Age, MCV, LDH and RDW were independently predictive of MDS. Extrapolation of our findings to the elderly anemic individuals in the community suggests MDS prevalence may be higher than previously postulated.