Search

Plasma cytokine levels were quantified among 30 persons with HIV (PWH) identified as elite controllers (15 transient controllers [studied a median of 1.38 years before losing viral control] and 15 persistent controllers). Thirty antiretroviral therapy (ART)-naive PWH, 30 ART-treated PWH with undetectable viremia, and 30 HIV-uninfected controls also were studied. Higher levels of cytokines were recognized among PWH than among controls, with EC displaying the highest levels. Elevated levels of IP-10 and MIG were identified among transient controllers as predictors of the loss of viral control. These findings offer feasible biomarkers for predicting virologic outcome and loss of control in EC.

Abstract Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle‐producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV‐related co‐morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef‐containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV‐associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV‐associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef‐containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV‐associated co‐morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV‐associated co‐morbidities.

Abstract Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-hsACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1LAI.04. This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro. These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.

HIV vaccine development can be aided by knowledge of correlates of protection. Here the authors identify engagement and reprogramming of tolerogenic CD14+ myeloid cells mediating a spatiotemporal balance of pro- and anti-inflammatory responses, as correlates of efficacy in female macaques vaccinated with the DNA/ALVAC/gp120/Alum platform.

Abstract Psychoneurological symptom clusters are co-occurring and interrelated physiological symptoms that may include cancer-related fatigue, pain, depressive symptoms, cognitive disturbances, and sleep disturbances. These symptoms are hypothesized to share a common systemic proinflammatory etiology. Thus, an investigation of systemic immune biomarkers is an important approach to test this hypothesis. Here, we investigated the associations between extracellular vesicle (EV)-associated and soluble cytokines with immune markers and symptom clusters in men with non-metastatic prostate cancer. This observational study included 40 men with non-metastatic prostate cancer at the start (T1) of external beam radiation therapy (EBRT) and 3 months post treatment (T2), as well as 20 men with non-metastatic prostate cancer on active surveillance (AS) seen at one time point. Collected questionnaires assessed patient-reported fatigue, sleep disturbances, depressive symptoms, and cognitive fatigue. In total, 45 soluble and EV-associated biomarkers in plasma were determined by multiplex assays. Principal component analysis (PCA) was used to identify psychoneurological symptom clusters for each study group and their time points. Bivariate correlation analysis was run for each identified PCA cluster with the concentrations of EV-associated and soluble cytokines and immune markers. Both EV-associated and soluble forms of RANTES significantly correlated with the symptom cluster for EBRT at T1, whereas, at T2, soluble IFNα2, IL-9, and IL-17 correlated with the corresponding symptom cluster. For the AS group, soluble survivin correlated with psychoneurological symptoms. Linking specific inflammatory cytokines with psychoneurological symptom clusters in men receiving prostate cancer treatment can enhance understanding of the underlying mechanisms of this phenomenon and aid in developing targeted interventions.

Because extracellular vesicle (EV)-associated cytokines, both encapsulated and surface bound, have been associated with symptom severity, and may vary over the lifespan, they may be potential biomarkers to uncover underlying mechanisms of various conditions. This study evaluated the associations of soluble and EV-associated cytokine concentrations with distinct symptom profiles reported by 290 women with breast cancer prior to surgery. Patients were classified into older (≥60 years, n = 93) and younger (< 60 years, n = 197) cohorts within two previously identified distinct symptom severity profiles, that included pain, depressive symptoms, sleep disturbance, and fatigue (i.e., High Fatigue Low Pain and All Low). EVs were extracted using ExoQuick. Cytokine concentrations were determined using Luminex multiplex assay. Mann Whitney U test evaluated the differences in EV and soluble cytokine levels between symptom classes and between and within the older and younger cohorts adjusting for Karnofsky Performance Status (KPS) score, body mass index (BMI), and stage of disease. Partial correlation analyses were run between symptom severity scores and cytokine concentrations. Results of this study suggest that levels of cytokine concentrations differ between EV and soluble fractions. Several EV and soluble pro-inflammatory cytokines had positive associations with depressive symptoms and fatigue within both age cohorts and symptom profiles. In addition, in the older cohort with High Fatigue Low Pain symptom profile, EV GM-CSF concentrations were higher compared to the All Low symptom profile (p < 0.05). Albeit limited by a small sample size, these exploratory analyses provide new information on the association between cytokines and symptom profiles of older and younger cohorts. Of note, unique EV-associated cytokines were found in older patients and in specific symptom classes. These results suggest that EVs may be potential biomarker discovery tools. Understanding the mechanisms that underlie distinct symptom class profiles categorized by age may inform intervention trials and offer precision medicine approaches.

BackgroundThe level of systemic inflammation correlates with the severity of the clinical course of acute myocardial infarction (AMI). It has been shown that circulating cytokines and endothelial dysfunction play an important role in the process of clot formation. The aim of our study was to assess the concentration of various circulating cytokines, endothelial function and blood clotting in AMI patients depending on the blood flow through the infarction-related artery (IRA).MethodsWe included 75 patients with AMI. 58 presented with ST-elevation myocardial infarction (STEMI) and 17 had non-ST-elevation myocardial infarction (non-STEMI). A flow-mediated dilation test (FMD test), thrombodynamics and rotational thromboelastometry as well as assessment of 14 serum cytokines using xMAP technology were performed.FindingsNon-STEMI-patients were characterized by higher levels of MDC, MIP-1β, TNF-α. Moreover, we observed that patients with impaired blood flow through the IRA (TIMI flow 0-1) had higher average and initial clot growth rates, earlier onset of spontaneous clots, C-reactive protein (CRP) and IL-10 compared to patients with preserved blood flow through the IRA (TIMI flow 2-3). Patients with TIMI 2-3 blood flow had higher level of IP-10. IL-10 correlated with CRP and pro-inflammatory cytokines levels, initial clot growth rate and clot lysis time in TIMI 0-1 patients. All these differences were statistically significant.InterpretationWe demonstrated that concentrations of the inflammatory cytokines correlate not only with the form of myocardial infarction (STEMI or non-STEMI), but also with the blood flow through the infarct-related artery. Inflammatory response, functional state of endothelium, and clot formation are closely linked with each other. A combination of these parameters affects the patency of the infarct-related artery.

Vaginal microbiota dominated by lactobacilli protects women from sexually transmitted infection, in particular HIV-1. This protection is, in part, mediated by Lactobacillus-released extracellular vesicles (EVs). Here, we investigated whether EVs derived from other Gram-positive bacteria also present in healthy vaginas, in particular Staphylococcus aureus, Gardnerella vaginalis, Enterococcus faecium, and Enterococcus faecalis, can affect vaginal HIV-1 infection. We found that EVs released by these bacteria protect human cervico-vaginal tissues ex vivo and isolated cells from HIV-1 infection by inhibiting HIV-1-cell receptor interactions. This inhibition was associated with a diminished exposure of viral Env by steric hindrance of gp120 or gp120 modification evidenced by the failure of EV-treated virions to bind to nanoparticle-coupled anti-Env antibodies. Furthermore, we found that protein components associated with EV’s outer surface are critical for EV-mediated protection from HIV-1 infection since treatment of bacteria-released EVs with proteinase K abolished their anti-HIV-1 effect. We identified numerous EV-associated proteins that may be involved in this protection. The identification of EVs with specific proteins that suppress HIV-1 may lead to the development of novel strategies for the prevention of HIV-1 transmission.

Lactobacillus associates with vaginal protection from HIV-1 infection. Here, the authors show that lactobacilli extracellular vesicles contain bacterial proteins and metabolites that inhibit HIV-1 infection in T cells and in human cervico-vaginal and tonsillar tissues ex vivo via altering viral Env proteins.

Background: Congenital cytomegalovirus (cCMV) infection is frequent and potentially severe. The immunobiology of cCMV infection is poorly understood, involving cytokines that could be carried within or on the surface of extracellular vesicles (EV). We investigated intra-amniotic cytokines, mediated or not by EV, in cCMV infection. Methods: Forty infected fetuses following early maternal primary infection and forty negative controls were included. Infected fetuses were classified according to severity at birth: asymptomatic, moderately or severely symptomatic. Following the capture of EV in amniotic fluid (AF), the concentrations of 38 cytokines were quantified. The association with infection and its severity was determined using univariate and multivariate analysis. A prediction analysis based on principal component analysis was conducted. Results: cCMV infection was nominally associated with an increase in six cytokines, mainly soluble (IP-10, IL-18, ITAC, and TRAIL). EV-associated IP-10 was also increased in cases of fetal infection. Severity of fetal infection was nominally associated with an increase in twelve cytokines, including five also associated with fetal infection. A pattern of specific increase in six proteins fitted severely symptomatic infection, including IL-18soluble, TRAILsoluble, CRPsoluble, TRAILsurface, MIGinternal, and RANTESinternal. Conclusion: Fetal infection and its severity are associated with an increase in pro-inflammatory cytokines involved in Th1 immune response.

Both gram-negative and gram-positive bacteria release extracellular vesicles (EVs) that contain components from their mother cells. Bacterial EVs are similar in size to mammalian-derived EVs and are thought to mediate bacteria-host communications by transporting diverse bioactive molecules including proteins, nucleic acids, lipids, and metabolites. Bacterial EVs have been implicated in bacteria-bacteria and bacteria-host interactions, promoting health or causing various pathologies. Although the science of bacterial EVs is less developed than that of eukaryotic EVs, the number of studies on bacterial EVs is continuously increasing. This review highlights the current state of knowledge in the rapidly evolving field of bacterial EV science, focusing on their discovery, isolation, biogenesis, and more specifically on their role in microbiota-host communications. Knowledge of these mechanisms may be translated into new therapeutics and diagnostics based on bacterial EVs.

Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely.

Objective: CMV coinfection contributes to sustained immune activation in people with chronic HIV. In particular, asymptomatic CMV shedding in semen has been associated with increased local and systemic immune activation, even during suppressive antiretroviral therapy (ART). However, the effect of seminal CMV shedding in people with HIV in the earliest phase of HIV infection is not known. Methods: Using Luminex, we measured the concentration of 34 cytokines in the blood plasma of sixty-nine men who had sex with men with or without HIV and in subgroups of CMV shedders vs. non-shedders. Differences in blood plasma cytokines between groups were investigated using the multivariate supervised partial least squares discriminant analysis method. Results: Independently of CMV, we found that concentrations of IP-10, MIG, MCP-1, I-TAC 10, IL-16, and MIP-1β were modulated in the earliest phase of HIV infection compared with control individuals without HIV. In people with HIV, there was no difference in blood cytokines among CMV shedders vs. non-shedders. Conclusion: In early/acute HIV infection, asymptomatic CMV shedding in semen does not drive additional cytokine changes in blood. Early ART initiation should remain the priority, while the added benefit of CMV suppression during the various stages of HIV infection needs to be further investigated.

Summary: The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4β7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Background: Cancer Related Fatigue (CRF) is one of the most prevalent and distressing symptoms associated with cancer treatments. The exact etiology of CRF and its mechanisms are poorly understood. Cytokine dysregulation was hypothesized to be one of these mechanisms. Here, we explored the associations of soluble and extracellular vesicle (EV)-associated markers that include cytokines, heat shock proteins (hsp27, hsp70, hsp90), and neurotrophic factors (BDNF) with CRF. Methods: Plasma was collected from men (n ​= ​40) with non-metastatic prostate cancer receiving external beam radiation therapy (EBRT) at the start of the treatment, and three months after EBRT. CRF was assessed using the Functional Assessment of Cancer Therapy - Fatigue (FACT-F) from all participants. EVs were characterized via Nanoparticle Tracking Analysis, electron microscopy, and Western blot. Concentrations of EV-associated and soluble markers were measured with a multiplexed immunoassay system. Bivariate correlation analyses and independent T tests analyzed the relationships of CRF with the markers. Findings: As CRF worsened, concentrations of EV-associated markers were upregulated. EV-associated fold changes of Eotaxin, hsp27, IP-10, MIP-3α, were significantly higher in fatigued participants compared to non-fatigued EBRT participants three months after treatment. This was not observed in soluble markers. Concentrations of EV-associated CRP and MCP-1, soluble survivin, IFNα2, IL-8, IL-12p70, and MCP-1 significantly correlated with lower (worsening) CRF scores at the start of and three months after treatment. Interpretation: Concentrations of EV-associated markers increased in fatigued men with prostate cancer three months after EBRT. Both EV-associated and soluble markers correlated with worsening CRF. EV-associated markers, which have not been previously studied in depth, may provide additional insights and serve as potential biomarkers for CRF.

Atherosclerotic cardiovascular disease (ASCVD) remains an important cause of morbidity in the general population and risk for ASCVD is increased approximately 2-fold in persons living with HIV infection (PLWH). This risk is linked to elevated CD8 T cell counts that are abundant in atherosclerotic plaques and have been implicated in disease pathogenesis yet the mechanisms driving T cell recruitment to and activation within plaques are poorly defined. Here we investigated the role of CD8 T cells in atherosclerosis in a non-human primate model of HIV infection and in the HIV-uninfected elderly; we sought to identify factors that promote the activation, function, and recruitment to endothelium of CX3CR1+ CD8 T cells. We measured elevated expression of CX3CL1 and IL-15, and increased CD8 T cell numbers in the aortas of rhesus macaques infected with SIV or SHIV, and demonstrated similar findings in atherosclerotic vessels of HIV-uninfected humans. We found that recombinant TNF enhanced the production and release of CX3CL1 and bioactive IL-15 from aortic endothelial cells, but not from aortic smooth muscle cells. IL-15 in turn promoted CX3CR1 surface expression on and TNF synthesis by CD8 T cells, and IL-15-treated CD8 T cells exhibited enhanced CX3CL1-dependent chemoattraction toward endothelial cells in vitro. Finally, we show that CD8 T cells in human atherosclerotic plaques have an activated, resident phenotype consistent with in vivo IL-15 and CX3CL1 exposure. In this report, we define a novel model of CD8 T cell involvement in atherosclerosis whereby CX3CL1 and IL-15 operate in tandem within the vascular endothelium to promote infiltration by activated CX3CR1+ memory CD8 T cells that drive further endothelial activation via TNF. We propose that these interactions are prevalent in aging and in PLWH, populations where circulating activated CX3CR1+ CD8 T cell numbers are often expanded.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

The association between the use of the injectable contraceptive depot medroxyprogesterone acetate and HIV-1 susceptibility has been addressed mainly in respect to the changes occurring in the female genital mucosa and blood. However, one of the main sites of HIV-1 pathogenesis is lymphoid organs. To investigate the immunoregulatory effect of medroxyprogesterone acetate (MPA) at this site, human tonsillar tissue explants were infected ex vivo with either a CCR5 (BaL) or CXCR4 (LAI) HIV-1 variant and the release of p24gag and cytokines was measured in culture supernatant. The response to MPA was compared with that elicited by treatment with progesterone (P4) and dexamethasone (DEX), which selectively binds the glucocorticoid receptor, in donor-matched explant cultures. MPA treatment reduced the replication of both tested HIV-1 strains as well as the production of the mediators of inflammation IL-1β, IL-17A and CCL5, but not CCL20, in a similar way to DEX, whereas P4 had no effect on HIV-1 replication. The magnitude of both MPA and DEX-mediated responses was proportional to the length of exposure and/or administered dose. Blockage of the progesterone and glucocorticoid receptors with mifepristone abolished all observed changes in HIV-1 and cytokine production, and was associated with increased IL-22 levels in HIV-infected explants. Our data indicate that elevated doses of MPA may affect the immune responses in lymphoid tissue in a glucocorticoid-like fashion with an immediate impact on local HIV-1 replication.

Abstract Cells productively infected with HIV-1 release virions along with extracellular vesicles (EVs) whose biogenesis, size, and physical properties resemble those of retroviruses. Here, we found that a significant number of EVs (exosomes) released by HIV-1 infected cells carry gp120 (Env), a viral protein that mediates virus attachment and fusion to target cells, and also facilitates HIV infection in various indirect ways. Depletion of viral preparations of EVs, in particular of those that carry gp120, decreases viral infection of human lymphoid tissue ex vivo. Thus, EVs that carry Env identified in our work seem to facilitate HIV infection and therefore may constitute a new therapeutic target for antiviral strategy.

Abstract HIV-1 envelope proteins (Envs) play a critical role in HIV infection. In a correct trimeric conformation, Env mediates virus–cell binding and fusion. Malfunctioning of this machinery renders virions incapable of infecting cells. Each HIV-1 virion carries 10–14 Envs, and therefore a defective Env may not necessarily render a HIV virion non-infectious, since other Env on the same virion may still be functional. Alternatively, it is possible that on a given virion either all the spikes are defective or all are functional. Here, we investigate Env conformations on individual virions using our new nanotechnology, “flow virometry”, and a panel of antibodies that discriminate between various Env conformations. We found that the majority of HIV-1 virions carry either only trimeric (“functional”) or only defective spikes. The relatively small subfraction of virions that carry both functional and nonfunctional Envs contributes little to HIV infection of human lymphoid tissue ex vivo. The observation that the majority of virions exclusively express either functional or nonfunctional forms of Env has important implications for understanding the role of neutralizing and non-neutralizing antibodies in the immune control of HIV infection as well as for the development of effective prophylactic strategies.

ABSTRACT Apolipoprotein A-I binding protein (AIBP) is a protein involved in regulation of lipid rafts and cholesterol efflux. AIBP has been suggested to function as a protective factor under several sets of pathological conditions associated with increased abundance of lipid rafts, such as atherosclerosis and acute lung injury. Here, we show that exogenously added AIBP reduced the abundance of lipid rafts and inhibited HIV replication in vitro as well as in HIV-infected humanized mice, whereas knockdown of endogenous AIBP increased HIV replication. Endogenous AIBP was much more abundant in activated T cells than in monocyte-derived macrophages (MDMs), and exogenous AIBP was much less effective in T cells than in MDMs. AIBP inhibited virus-cell fusion, specifically targeting cells with lipid rafts mobilized by cell activation or Nef-containing exosomes. MDM-HIV fusion was sensitive to AIBP only in the presence of Nef provided by the virus or exosomes. Peripheral blood mononuclear cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, bound less AIBP than cells from donors with other HLA genotypes and were not protected by AIBP from rapid HIV-1 replication. These results provide the first evidence for the role of Nef exosomes in regulating HIV-cell fusion by modifying lipid rafts and suggest that AIBP is an innate factor that restricts HIV replication by targeting lipid rafts. IMPORTANCE Apolipoprotein A-I binding protein (AIBP) is a recently identified innate anti-inflammatory factor. Here, we show that AIBP inhibited HIV replication by targeting lipid rafts and reducing virus-cell fusion. Importantly, AIBP selectively reduced levels of rafts on cells stimulated by an inflammatory stimulus or treated with extracellular vesicles containing HIV-1 protein Nef without affecting rafts on nonactivated cells. Accordingly, fusion of monocyte-derived macrophages with HIV was sensitive to AIBP only in the presence of Nef. Silencing of endogenous AIBP significantly upregulated HIV-1 replication. Interestingly, HIV-1 replication in cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, was not inhibited by AIBP. These results suggest that AIBP is an innate anti-HIV factor that targets virus-cell fusion.

ObjectiveAmniotic fluid cytokines have been implicated in the mechanisms of preterm labor and birth. Cytokines can be packaged within or on the surface of extracellular vesicles. The main aim of this study was to test whether the protein abundance internal to and on the surface of extracellular vesicles changes in the presence of sterile intra-amniotic inflammation and proven intra-amniotic infection in women with preterm labor as compared to the women with preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection.Study designWomen who had an episode of preterm labor and underwent an amniocentesis for the diagnosis of intra-amniotic infection or intra-amniotic inflammation were classified into three groups: 1) preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 2) preterm labor with sterile intra-amniotic inflammation, and 3) preterm labor with intra-amniotic infection. The concentrations of 38 proteins were determined on the extracellular vesicle surface, within the vesicles, and in the soluble fraction of amniotic fluid.Results1) Intra-amniotic inflammation, regardless of detected microbes, was associated with an increased abundance of amniotic fluid cytokines on the extracellular vesicle surface, within vesicles, and in the soluble fraction. These changes were most prominent in women with proven intra-amniotic infection. 2) Cytokine changes on the surface of extracellular vesicles were correlated with those determined in the soluble fraction; yet the magnitude of the increase was significantly different between these compartments. 3) The performance of prediction models of early preterm delivery based on measurements on the extracellular vesicle surface was equivalent to those based on the soluble fraction.ConclusionsDifferential packaging of amniotic fluid cytokines in extracellular vesicles during preterm labor with sterile intra-amniotic inflammation or proven intra-amniotic infection is reported herein for the first time. The current study provides insights into the biology of the intra-amniotic fluid ad may aid in the development of biomarkers for obstetrical disease.

For many years, double-layer phospholipid membrane vesicles, released by most cells, were not considered to be of biological significance. This stance has dramatically changed with the recognition of extracellular vesicles (EVs) as carriers of biologically active molecules that can traffic to local or distant targets and execute defined biological functions. The dimensionality of the field has expanded with the appreciation of diverse types of EVs and the complexity of vesicle biogenesis, cargo loading, release pathways, targeting mechanisms, and vesicle processing. With the expanded interest in the field and the accelerated rate of publications on EV structure and function in diverse biomedical fields, it has become difficult to distinguish between well-established biological features of EV and the untested hypotheses or speculative assumptions that await experimental proof. With the growing interest despite the limited evidence, we sought in this essay to formulate a set of unsolved mysteries in the field, sort out established data from fascinating hypotheses, and formulate several challenging questions that must be answered for the field to advance.

A diverse population of small extracellular vesicles (EVs) that are released by various cells has been characterized predominantly in bulk, a procedure whereby the individual characteristics of EVs are lost. Here, we used a new nanotechnology-based flow cytometric analysis to characterize the antigenic composition of individual EVs in patients with acute coronary syndrome (ACS). Plasma EVs were captured with 15-nm magnetic nanoparticles coupled to antibodies against CD31 (predominantly an endothelial marker), CD41a (a marker for platelets), and CD63 or MHC class I (common EV markers). The total amounts of EVs were higher in the ACS patients than in the controls, predominantly due to the contribution of patients with acute myocardial infarction. For all captured fractions, the differences in the EV amounts were restricted to CD41a+ EVs. The increase in the numbers of EVs in the ACS patients, predominantly of platelet origin, probably reflects platelet activation and may indicate disease progression.

Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV) acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1) infection. We identified at least three factors that mediated this suppression: (i) Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl) to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM) diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii) Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii) Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink decreasing the number of free virions. In summary, we found that lactobacilli inhibit HIV-1 replication in human tissue ex vivo by multiple mechanisms. Further studies are needed to evaluate the potential of altering the spectra of vaginal microbiota as an effective strategy to enhance vaginal health. Human tissues ex vivo may serve as a test system for these strategies.

Extracellular vesicles (EVs) are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. Investigation of EVs’ structure and function requires their isolation and precise quantification. However, in the current literature, there are significant discrepancies in the estimated numbers of EVs in different body fluids. In part, this discrepancy is due to the difference in EVs isolation protocols used by different investigators. A common protocol that includes ExoQuick ™ is often used to isolate EVs from body fluids and culture medium. Here, we show that in the case of isolation of EVs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many EVs thus leading to the underestimation of their numbers.

BackgroundAlthough an association between human herpesvirus (HHV) infection and atherosclerosis has been suggested, the data supporting such an association are controversial and, in most cases, are based on serological evidence or on the presence of cell‐associated HHV DNA, which do not report about actual viral replication. We quantified the DNA of all 8 types of HHVs in plasma, in which their presence is evidence of viral replication. Methods and ResultsUsing quantitative real‐time polymerase chain reaction, we evaluated the presence of HHV DNA in blood samples obtained at the time of hospitalization from 71 patients with acute coronary syndrome, 26 patients with stable coronary artery disease, and 53 healthy volunteers and in atherosclerotic plaques of 22 patients with peripheral artery disease who underwent endarterectomy. HHV‐5 (cytomegalovirus [CMV]) was the only HHV with a level that was higher in acute coronary syndrome patients than in the control group and that correlated with the level of high‐sensitivity C‐reactive protein. The numbers of effector memory T cells positively correlated with the numbers of CMV genome copies in carotid arteries plaques, whereas the numbers of central memory T cells negatively correlated with CMV copy numbers. ConclusionsOf all HHV levels, only CMV was higher in patients with stable coronary artery disease and acute coronary syndrome than in the healthy group, and its load correlated with the level of high‐sensitivity C‐reactive protein. The level of CMV in atherosclerotic plaques correlated with the state of immunoactivation of lymphocytes in plaques, suggesting that the reactivation of CMV may contribute to the immune activation associated with the progression of atherosclerosis.

Sexually transmitted infections (STI) are common among HIV-infected men who have sex with men (MSM). While behavioral factors are important in STI acquisition, other biological factors such as immune modulation due to chronic viral infection may further predispose to STI acquisition.Post Hoc analysis including data collected over 12 months of follow-up from 131 HIV-infected MSM receiving antiretroviral therapy and screened for incident bacterial STI every 3 months.Genital secretions collected at baseline were used to measure herpesvirus replication and inflammatory cytokines. Baseline predictors of STI were determined using survival analysis of time to incident STI.All participants were seropositive for cytomegalovirus (CMV), and 52% had detectable genital CMV at baseline. Thirty-five individuals acquired STI during follow-up, sometimes with multiple pathogen (17 syphilis, 21 gonorrhea, 14 chlamydia). Syphilis acquisition was associated with genital CMV replication at baseline (19.1% CMV-shedders versus 4.8% non-shedders, p=0.03) and younger age (p=0.02). Lower seminal MCP-1 was associated with higher seminal CMV levels and with syphilis acquisition (p

Human tissue explants are a valuable tool to study the interactions between host and infectious agents. They reliably mimic many important aspects of tissue cytoarchitecture and functions and allow us the investigation of the mechanisms of microbial pathogenesis under controlled laboratory conditions. One of the advantages of this system is that, unlike isolated cells, infection of tissue blocks with HIV-1 does not require exogenous stimulation with mitogens or activating factors. Here we describe a protocol to infect with HIV-1 human lymphoid tissue from tonsils and cervico-vaginal tissue and maintain them in culture in a non-polarized setting. These ex vivo infected tissues can be used as fruitful models to study HIV-1 pathogenesis and HIV-1 vaginal transmission, respectively, as well as an efficient platform for testing anti-HIV therapeutic and preventative strategies.

The majority of HIV-1 infections in women occur through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines, that may affect HIV-1 transmission. The concentration of interleukin (IL)-7, one of the most prominent cytokines in semen of healthy individuals, is further increased in semen of HIV-1-infected men. Here, we investigated the potential role of IL-7 in HIV-1 vaginal transmission in an ex vivo system of human cervico-vaginal tissue. We simulated an in vivo situation by depositing HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue. Similarly, we observed an enhancement of HIV-1 replication in lymphoid tissue explants. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4⁺ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl)-2. Also, IL-7 increased the fraction of cycling CD4⁺ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection.

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4⁺ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).

The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs). Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs) 5 and 9, we examined their effect on human immunodeficiency virus (HIV)-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist) treatment enhanced replication of CC chemokine receptor 5 (CCR 5)-tropic and CXC chemokine receptor 4 (CXCR4)-tropic HIV-1, treatment with oligodeoxynucleotide (ODN) M362 (TLR9 agonist) suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD) 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA)-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention.