Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

Quantitative flow cytometry enables end-to-end optimization of cross-platform extracellular vesicle studies.

Flow cytometry (FCM) is a common method for characterizing extracellular particles (EPs), including viruses and extracellular vesicles (EVs). Frameworks such as MIFlowCyt-EV exist to provide reporting guidelines for metadata, controls, and data reporting. However, tools to optimize FCM for EP analysis in a systematic and quantitative way are lacking. Here, we demonstrate a cohesive set of methods and software tools that optimize FCM settings and facilitate cross-platform comparisons for EP studies. We introduce an automated small-particle optimization (SPOT) pipeline to optimize FCM fluorescence and light scatter detector settings for EP analysis and leverage quantitative FCM (qFCM) as a tool to further enable FCM optimization of fluorophore panel selection, laser power, pulse statistics, and window extensions. Finally, we demonstrate the value of qFCM to facilitate standardized cross-platform comparisons, irrespective of instrument configuration, settings, and sensitivity, in a cross-platform standardization study utilizing a commercially available EV reference material.

Isolation of the side population from neurogenic niches enriches for endothelial cells.

In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remains untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the mouse dentate gyrus and subventricular zone (SVZ), and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The majority of the cells residing in the side populations of dentate gyrus and SVZ were characterized by their expression of CD31. Additionally, at least 90% of all CD31+ cells found in the dentate gyrus and SVZ were negative for the hematopoietic marker CD45, leading to the hypothesis that the CD31+ cells in the side population were endothelial cells. These findings, therefore, suggest that the side population analysis provides an efficient method to purify CD31-expressing endothelial cells, but not adult neural stem cells.

Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus.

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus-infected cells are well known to release soluble viral proteins, defective viruses, and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small-particle flow cytometry, is an emerging analytical method enabling high-throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here, we analyzed the basic properties of Moloney MLV (M-MLV). We show that <24% of the total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contains a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus while highlighting the benefits of flow virometry for the field of virology.IMPORTANCE Gammaretroviruses, or, more specifically, murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analyzed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90 nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here, we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

Levels of circulating cortisol and cytokines in members of the Canadian Armed Forces: associations with age, sex, and anthropometry.

This study assessed blood levels of cortisol and cytokines (inflammatory and non-inflammatory) in members of the regular Canadian Armed Forces (CAF), and examined the associations between sex, age, and adiposity and circulating levels of cortisol as well as pro- and anti-inflammatory cytokines. As part of a larger ranging project, 331 blood samples were collected from a representative population of the total CAF, which included officers and noncommissioned women and men from the Air Force, Navy, and Army. The blood samples were analyzed for levels of cortisol, C-reactive protein (CRP), adiponectin, and 20 cytokines (which included interleukins, interferons, and tumor necrosis factors). Higher levels of adiponectin were found in women compared with men (median and interquartile range; 16.71 (7.68-25.32) vs 5.81 (3.52-13.19) µg/mL), and higher levels of interleukin (IL)-18 in men compared with women (89.25 (84.03-94.48) vs 75.91 (69.70-82.13) pg/mL). An association between age and levels of stress and inflammatory cytokines was observed, with CRP, IL-18, IL-2 and adiponectin all increasing with increasing age. However, contrary to trends seen in the general population, cortisol levels decreased with increasing age. Levels of CRP and IL-18 increased with an increase in adiposity, while adiponectin levels decreased. Most importantly, at the entire cohort level, a low detection rate for most of the cytokines was observed with 17 out of 22 cytokines having a detection below 10%. IN CONCLUSION: In this CAF population, although an association between age and inflammatory cytokines was observed, both sex and adiposity had a small impact on levels of cortisol and cytokines.

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry.

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.

Activating Peripheral Innate Immunity Enables Safe and Effective Oncolytic Virotherapy in the Brain.

The oncolytic mutant vesicular stomatitis virus VSVΔ51 achieves robust efficacy in multiple extracranial tumor models. Yet for malignancies of the brain, direct intratumoral infusion of VSVΔ51 causes lethal virus-induced neuropathology. Here, we have developed a novel therapeutic regime that uses peripheral immunization with a single sub-lethal dose of VSVΔ51 to establish an acute anti-viral state that enables the safe intracranial (IC) infusion of an otherwise lethal dose of VSVΔ51 within just 6 hr. Although type I interferons alone appeared insufficient to explain this protective phenotype, serum isolated at early time points from primed animals conferred protection against an IC dose of virus. Adaptive immune populations had minimal contributions. Finally, the therapeutic utility of this novel strategy was demonstrated by peripherally priming and intracranially treating mice bearing aggressive CT2A syngeneic astrocytomas with VSVΔ51. Approximately 25% of animals achieved complete regression of established tumors, with no signs of virus-induced neurological impairment. This approach may harness an early warning system in the brain that has evolved to protect the host against otherwise lethal neurotropic viral infections. We have exploited this protective mechanism to safely and efficaciously treat brain tumors with an otherwise neurotoxic virus, potentially widening the available treatment options for oncolytic virotherapy in the brain.

Single-particle characterization of oncolytic vaccinia virus by flow virometry.

Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under various handling and storage procedures. Accordingly, we find that together the infectious titer, the total number of SSPs, the number of viral genomes and the level of particle aggregation in a sample constitute useful parameters that greatly facilitate inter-sample assessment of physical quality, and also provides a means to monitor sample deterioration over time. Additionally, we have successfully employed FV sorting to further isolate virus from other particles by identifying a lipophilic dye that preferentially stains VV over other SSPs in the sample. Overall, we demonstrate that FV is a fast and effective tool that can be used to perform quality, and consistency control assessments of oncolytic VV vaccine preparations.

Smac mimetics and innate immune stimuli synergize to promote tumor death.

Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in phase 1 clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may be efficacious only in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. Therefore, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe "cytokine storm." Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFN-ß), tumor necrosis factor alpha (TNF-α) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.

Triptolide-mediated inhibition of interferon signaling enhances vesicular stomatitis virus-based oncolysis.

Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.

Detection rates of proximal or large serrated polyps in Chinese patients undergoing screening colonoscopy.

OBJECTIVE: The aim was to determine the detection rates and characteristics of large or proximal serrated polyps in Chinese patients undergoing screening colonoscopy. METHODS: Consecutive screening colonoscopies performed between 2008 and 2011 were analyzed. Serrated polyps consisted of all hyperplastic polyps, sessile serrated adenomas and traditional serrated adenomas. Large serrated polyps were defined as serrated polyps with a diameter ≥ 10 mm. Lesions proximal to the descending colon were considered as proximal lesions. Advanced neoplasia included invasive adenocarcinomas, adenomas with high grade dysplasia, adenomas with any villous histology and tubular adenomas ≥ 10 mm. RESULTS: In total, 1282 colonoscopies were included. The detection rates for adenoma, advanced neoplasia, proximal serrated polyps and large serrated polyps were 26.1%, 10.5%, 7.2% and 2.3%, respectively. There was a significant association between synchronous advanced neoplasia and large serrated polyps (P = 0.002) or proximal serrated polyps (P = 0.013). Age ≥ 55 years (OR 1.9, 95% CI 1.2-2.8) and the presence of advanced neoplasia (OR 1.8, 95% CI 1.0-3.1) were significantly associated with the presence of large or proximal serrated polyps. Males had more advanced neoplasia (OR 2.0, 95% CI 1.4-2.9), but not more large or proximal serrated polyps, than females. CONCLUSIONS: Large and proximal serrated polyps were detected in 2.3% and 7.2% of Chinese patients undergoing screening colonoscopies, respectively. Individuals with large or proximal serrated polyps have a higher risk of synchronous advanced neoplasia.

Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine.

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV's extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.

Intravaginal infection with herpes simplex virus type-2 (HSV-2) generates a functional effector memory T cell population that persists in the murine genital tract.

Although the female genital tract is the main portal of entry for sexually transmitted infections in women, we still have limited understanding of the generation, maintenance and characteristics of memory T cells in the local tissue. Here, we utilized a mouse model of intravaginal HSV-2 infection and tetramers against the immunodominant HSV glycoprotein B epitope recognized by CD8+ T cells to examine the generation, maintenance and characteristics of anti-HSV memory T cells in the genital tract following acute infection. Our results show that the highest percentage of HSVgB-specific CD8+ T cells was found in the genital tract compared to the spleen or iliac lymphnode. Indeed, although the actual number of CD8+ T cells contracted following viral clearance, approximately one quarter of the CD8+ population that remained in the genital tissue was HSVgB-specific. Memory gB-tetramer+CD8 T cells in the genital tract were positive for CD127 and KLRG1 and negative for CD62L and CCR7, thus confirming that HSV-specific CD8 cells were effector memory T cells that lack the capacity for homing to lymphoid tissues. Functionally, both memory CD8+ and CD4+ HSV-specific populations in the genital tract produced IFNγ when stimulated in vitro and CD4+ cells also produced TNFα. Genital HSVgB-specific memory T cells expressed tissue-homing integrins CD103 (αE integrin) and CD49a (VLA-1 or α1 integrin). Our findings suggest that HSV-specific memory T cells are retained in the genital tract, poised to act as an early line of defense against future virus encounter.

Protection against genital herpes infection in mice immunized under different hormonal conditions correlates with induction of vagina-associated lymphoid tissue.

The present study was undertaken to examine the effect of the hormonal environment on immunization with an attenuated strain of herpes simplex virus type 2 (HSV-2 TK(-)) and subsequent protection against challenge. Ovariectomized mice were administered saline (S; control), estradiol (E(2)), progesterone (P(4)), or a combination of estradiol and progesterone (E+P) and immunized intravaginally (IVAG) with HSV-2 TK(-). Three weeks later, the immunized mice were challenged IVAG with wild-type HSV-2. Mice that were immunized following E treatment were not protected, whereas complete protection against the challenge was seen in mice from the S- and P(4)-treated groups. In the P(4)-treated group, 15% of mice developed chronic pathology following TK(-) immunization. Interestingly, about 40% of the E+P-treated mice were also protected. Upon examination of viral shedding in the vaginal secretions, it was clear that protection against challenge was dependent on the ability of the TK(-) virus to cause productive genital infection under different hormonal conditions. In the protected mice (the S and P groups and part of the E+P group), induced vagina-associated lymphoid tissues composed of CD11c(+) dendritic cells and CD3(+) and CD4(+) T cells were formed transiently in the vaginal lamina propria from day 2 to day 5 postchallenge. These aggregates were absent in the unprotected mice (the E group and part of the E+P group). Significant HSV-2-specific activation of lymphocytes was observed in the local draining lymph nodes of protected mice. This response was absent in the unprotected groups. High titers of gB-specific local immunoglobulin A (IgA) antibodies were present in the vaginal secretions of S- and P(4)-treated immunized mice following HSV-2 challenge. The S-treated group of mice also had high gB-specific IgG titers. These studies show that sex hormones modify the induction of protective immune responses following IVAG immunization.

Accuracy of a new near patient test for the diagnosis of Helicobacter pylori infection in Chinese.

BACKGROUND AND AIM: The performance of existing near patient tests for the diagnosis of Helicobacter pylori remains unsatisfactory. The aim of this study is to evaluate the accuracy of a new near patient test (Signify H. pylori) for the diagnosis of H. pylori and the usefulness of the Signify H. pylori test for a test and treat strategy. METHODS: Consecutive dyspeptic patients referred for upper endoscopy were recruited. Rapid urease test and histology were used as the gold standard. After endoscopy, blood was collected for the Signify H. pylori test and compared with a gold standard. RESULTS: Two hundred and forty-four patients were eligible for analysis and 121 (49.5%) were positive for H. pylori. The Signify H. pylori test showed a sensitivity, specificity, and accuracy of 84.3, 89.4%, and 86.9%, respectively, for whole blood and 79.3, 88.6, and 84.0% for serum, respectively. The sensitivity and specificity of the Signify H. pylori whole blood test was 87.5 and 92.6% for patients less than 45-years-old and the accuracy was similar between patients referred from primary care physicians or gastroenterologists. The test is easy to operate and results are available within 5 min. CONCLUSION: The Signify H. pylori test is accurate for the near patient diagnosis of H. pylori infection.