PD-L1 signaling in reactive astrocytes counteracts neuroinflammation and ameliorates neuronal damage after traumatic brain injury.

BACKGROUND: Tissue damage and cellular destruction are the major events in traumatic brain injury (TBI), which trigger sterile neuroimmune and neuroinflammatory responses in the brain. While appropriate acute and transient neuroimmune and neuroinflammatory responses facilitate the repair and adaptation of injured brain tissues, prolonged and excessive neuroimmune and neuroinflammatory responses exacerbate brain damage. The mechanisms that control the intensity and duration of neuroimmune and neuroinflammatory responses in TBI largely remain elusive. METHODS: We used the controlled cortical impact (CCI) model of TBI to study the role of immune checkpoints (ICPs), key regulators of immune homeostasis, in the regulation of neuroimmune and neuroinflammatory responses in the brain in vivo. RESULTS: We found that de novo expression of PD-L1, a potent inhibitory ICP, was robustly and transiently induced in reactive astrocytes, but not in microglia, neurons, or oligodendrocyte progenitor cells (OPCs). These PD-L1+ reactive astrocytes were highly enriched to form a dense zone around the TBI lesion. Blockade of PD-L1 signaling enlarged brain tissue cavity size, increased infiltration of inflammatory Ly-6CHigh monocytes/macrophages (M/Mɸ) but not tissue-repairing Ly-6CLowF4/80+ M/Mɸ, and worsened TBI outcomes in mice. PD-L1 gene knockout enhanced production of CCL2 that is best known for its ability to interact with its cognate receptor CCR2 on Ly-6CHigh M/Mϕ to chemotactically recruit these cells into inflammatory sites. Mechanically, PD-L1 signaling in astrocytes likely exhibits dual inhibitory activities for the prevention of excessive neuroimmune and neuroinflammatory responses to TBI through (1) the PD-1/PD-L1 axis to suppress the activity of brain-infiltrating PD-1+ immune cells, such as PD-1+ T cells, and (2) PD-L1 intrinsic signaling to regulate the timing and intensity of astrocyte reactions to TBI. CONCLUSIONS: PD-L1+ astrocytes act as a gatekeeper to the brain to control TBI-related neuroimmune and neuroinflammatory responses, thereby opening a novel avenue to study the role of ICP-neuroimmune axes in the pathophysiology of TBI and other neurological disorders.

Cell-to-cell transmission of HIV-1 from provirus-activated cells to resting naïve and memory human primary CD4 T cells is highly efficient and requires CD4 and F-actin but not chemokine receptors.

Latently infected cells harboring replication-competent proviruses represent a major barrier to HIV-1 cure. One major effort to purge these cells has focused on developing the "shock and kill" approach for forcing provirus reactivation to induce cell killing by viral cytopathic effects, host immune responses, or both. We conducted kinetic and mechanistic studies of HIV-1 protein expression, virion production, and cell-to-cell virus transmission during provirus reactivation. Provirus-activated ACH-2 cells stimulated with romidepsin (RMD) or PMA produced Nef early, and then Env and Gag in parallel with the appearance of virions. Env on the surface of provirus-activated cells and cellular F-actin were critical in the formation of virological synapses to mediate cell-to-cell transmission of HIV-1 from provirus-activated cells to uninfected cells. This HIV-1 cell-to-cell transmission was substantially more efficient than transmission seen via cell-free virus spread and required F-actin remodeling and CD4, but not chemokine receptors. Resting human primary CD4+ T cells including naïve and memory subpopulations and, especially the memory CD4+ T cells, were highly susceptible to HIV-1 infection via cell-to-cell transmission. Cell-to-cell transmission of HIV-1 from provirus-activated cells was profoundly decreased by protease inhibitors (PIs) and neutralizing antibodies (nAbs) that recognize the CD4-binding site (CD4bs) such as VRC01, but not by reverse transcriptase (RT) inhibitor Emtricitabine (FTC). Therefore, our results suggest that PIs with potent blocking abilities should be used in clinical application of the "shock and kill" approach, most likely in combination with CD4bs nAbs, to prevent new HIV-1 infections.

The Impacts of COVID-19 on Musculoskeletal Health.

PURPOSE OF REVIEW: Although COVID-19 was originally characterized as a respiratory disease, recent findings have shown lingering side effects in those who have recovered, and much is still unknown about the long-term consequences of the illness. Thus, the potential of unearthing multi-system dysfunction is high, with current data revealing significant impacts on musculoskeletal health. RECENT FINDINGS: Multiple animal models of COVID-19 infection have revealed significant post-infection bone loss at several different skeletal sites. While how this loss occurred is unknown, this current review discusses the primary bone loss studies, and examines the possible mechanisms of action including: direct infection of bone marrow macrophages or hematopoietic progenitors, a proinflammatory response as a result of the COVID-19 induced cytokine storm, and/or a result of hypoxia and oxidative stress. This review will further examine how therapeutics used to treat COVID-19 affect the skeletal system. Finally, this review will examine the possible consequence that delayed care and limited healthcare accessibility has on musculoskeletal-related patient outcomes. It is important to investigate the potential impact COVID-19 infection has on musculoskeletal health.

Excessive Matrix Metalloproteinase-1 and Hyperactivation of Endothelial Cells Occurred in COVID-19 Patients and Were Associated With the Severity of COVID-19.

BACKGROUND: Systemic vascular injury occurs in coronavirus disease 2019 (COVID-19) patients; however, the underlying mechanisms remain unknown. METHODS: To clarify the role of inflammatory factors in COVID-19 vascular injury, we used a multiplex immunoassay to profile 65 inflammatory cytokines/chemokines/growth factors in plasma samples from 24 hospitalized (severe/critical) COVID-19 patients, 14 mild/moderate cases, and 13 healthy controls (HCs). RESULTS: COVID-19 patients had significantly higher plasma levels of 20 analytes than HCs. Surprisingly, only 1 cytokine, macrophage migration inhibitory factor (MIF), was among these altered analytes, while the rest were chemokines/growth factors. Additionally, only matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor A (VEGF-A) were significantly elevated in hospitalized COVID-19 patients when compared to mild/moderate cases. We further studied MMP-1 enzymatic activity and multiple endothelial cell (EC) activation markers (soluble forms of CD146, intercellular adhesion molecule 1 [ICAM-1], and vascular cell adhesion molecule 1 [VCAM-1]) and found that they were highly dysregulated in COVID-19 patients. CONCLUSIONS: COVID-19 patients have a unique inflammatory profile, and excessive MMP-1 and hyperactivation of ECs are associated with the severity of COVID-19.

Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis.

BACKGROUND: Heavy alcohol consumption disrupts gut epithelial integrity, leading to increased permeability of the gastrointestinal tract and subsequent translocation of microbes. Regenerating islet-derived protein 3α (REG3α) and Trefoil factor 3 (TFF3) are mainly secreted to the gut lumen by Paneth and Goblet cells, respectively, and are functionally linked to gut barrier integrity. Circulating levels of REG3α and TFF3 have been identified as biomarkers for gut damage in several human diseases. We examined whether plasma levels of REG3α and TFF3 were dysregulated and correlated with conventional markers of microbial translocation (MT) and pro-inflammatory mediators in heavy drinkers with and without alcoholic hepatitis (AH). METHODS: Cross-sectional and longitudinal studies were performed to monitor plasma levels of REG3α and TFF3 in 79 AH patients, 66 heavy drinkers without liver disease (HDC), and 46 healthy controls (HC) at enrollment and at 6- and 12-month follow-ups. Spearman correlation was used to measure the relationships of REG3α and TFF3 levels with MT, disease severity, inflammation, and effects of abstinence from alcohol. RESULTS: At enrollment, AH patients had significantly higher levels of REG3α and TFF3 than HDC and HC. The elevated REG3α levels were positively correlated with the 30-day fatality rate. Plasma levels of REG3α and TFF3 in AH patients differentially correlated with conventional MT markers (sCD14, sCD163, and LBP) and several highly up-regulated inflammatory cytokines/chemokines/growth factors. At follow-ups, although REG3α and TFF3 levels were decreased in AH patients with alcohol abstinence, they did not fully return to baseline levels. CONCLUSIONS: Circulating levels of REG3α and TFF3 were highly elevated in AH patients and differentially correlated with AH disease severity, MT, and inflammation, thereby serving as potential biomarkers of MT and gut epithelial damage in AH patients.

Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection.

Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively.IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.

Elevated plasma phage load as a marker for intestinal permeability in leukemic patients.

Microbial translocation (MT) and altered gut microbiota have been described in acute leukemic patients and contribute to immune activation and inflammation. However, phage translocation has not been investigated in leukemia patients yet. We recruited 44 leukemic patients and 52 healthy adults and quantified the levels of 3 phages in peripheral blood, which were the most positive phages screened from fecal samples. The content of 16S rRNA in plasma was detected by qPCR to assess the intestinal mucosa of these patients. Spearman's rank correlation was used to analyze the relationship between phage load and the relevant clinical data. We found the most prevalent phages in fecal samples were λ phage, Wphi phage, and P22 phage, and λ phage had the highest detection rate in plasma (68%). Phage content was affected by chemotherapy and course of disease and correlated with the levels of CRP (r = 0.43, p = 0.003), sCD14 (r = 0.37, p = 0.014), and sCD163 (r = 0.44, p = 0.003). Our data indicate that plasma phage load is a promising marker for gut barrier damage and that gut phage translocation correlates with monocyte/macrophage activation and systemic inflammatory response in leukemic patients.

Persistent Hyperactivation of Endothelial Cells in Patients with Alcoholic Hepatitis.

BACKGROUND: Alcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. AH patients have intense hepatic infiltration of leukocytes. Up-regulation of cell adhesion molecules (CAMs) upon endothelial cell (EC) activation plays an important role in leukocyte transendothelial migration. CAMs can shed from EC surface and accumulate in the blood, serving as soluble markers for EC activation. In this study, we examined the impact of heavy drinking on expression of soluble forms of EC activation markers (CD146, ICAM-1, VCAM-1, and VEGF-A) and the effect of alcohol abstinence on the reversal of these abnormalities in heavy drinkers with and without AH. METHODS: ELISA and multiplex immunoassays were used to measure soluble EC activation markers in plasma samples from 79 AH patients, 66 heavy drinkers without overt liver disease (HDC), and 44 healthy controls (HC) at baseline, 31 AH patients and 30 HDC at 6-month follow-up, and 18 AH patients and 25 HDC at 12-month follow-up. RESULTS: At baseline, the 4 soluble markers were significantly up-regulated in AH patients compared with HDC and HC, whereas only sVCAM-1 was elevated in HDC relative to HC. At follow-ups, plasma levels of CD146, VCAM-1, and VEGF-A remained higher in AH patients, even for those who stopped drinking. These dysregulated markers correlated with AH disease severity, clinical parameters, and several soluble inflammatory factors. CONCLUSIONS: The levels of soluble CD146, ICAM-1, VCAM-1, and VEGF-A were highly elevated in AH patients, and alcohol abstinence did not completely reverse these abnormalities.

Investigating the changes in the levels of HbA1c, blood fat and insulin sensitivity in elder patients with type II diabetes mellitus due to combined medication of pioglitazone and melbine and single-use of pioglitazone.

This study was founded for the purpose investigate the differences in effects of combined medication of pioglitazone and melbine and single-use of pioglitazone on the levels of hba1c, blood fat and insulin sensitivity of elder patients with type II diabetes mellitus (T2DM), to provide clinical reference and guidance for the treatment of T2DM in elder patients. For this purpose, we selected a total of 120 elder patients with T2DM who visited the clinic or were admitted to this hospital between July 2016 and July 2017 and divided them into the observation group and the control group (n=60 for each group). For the control group, they only took pioglitazone for treatment, while those in the observation additionally took melbine for treatment. Then, we observed the levels of FPG, 2hPG, HbA1c, blood fat, FINS, 2hINS and HOMA-IR. Results showed that after treatment, significant decreases were seen in levels of FPG, 2hPG and HbA1c in patients of two groups compared to the levels before treatment, and the levels in the observation group decreased more evidently than those in the control group (p<0.05); besides, the levels of total glyceride, total cholesterol and LDL-C were all significantly lower than those before treatment, with an elevated HDL-C, and those levels of TG, TC and LDL-C in the observation group were significantly lower than those in the control group; the level of HDL-C was higher than the control group (p<0.05). Similar decreases were identified in the levels of FINS, 2hFINS and HOMA-IR after treatment (p<0.05), and the levels in the observation group were also significantly lower than those in the control group (p<0.05). In the observation group, the incidence rate of adverse reactions was 10% (6/60), while in the control group was 8.33% (5/60), but the difference between the two groups showed no statistical significance (p>0.05). It is concluded that combined medication of pioglitazone and melbine can effectively reduce the levels of plasma glucose, blood fat and the HOMA-IR, with an elevated sensitivity to insulin, but no severe adverse reactions, manifesting a promising safety in long-term administration. It is worthy of being promoted in clinical practice.

Revision of serum ALT upper limits of normal facilitates assessment of mild liver injury in obese children with non-alcoholic fatty liver disease.

BACKGROUND: The serum alanine aminotransferase (ALT) level is a critical parameter for evaluating liver injury in non-alcoholic fatty liver disease (NAFLD). However, the currently accepted upper limits of normal (ULN) for serum ALT (ULN-ALT) are debated, as they may be excessively high. METHODS: A total of 1638 children aged 6-16 years, comprising 507 children with normal BMI (500 healthy children and 7 children with NAFLD), 199 overweight children, and 932 obese children, were included in the analysis. We re-evaluated the ULN-ALT in 500 healthy Chinese children using the 95th percentiles of serum ALT levels as revised ULN-ALT. Fatty liver was identified by ultrasound examination. RESULTS: Significant positive correlations between serum ALT levels and body mass index (BMI) were detected in overweight boys (r = .399, P < .001), obese boys (r = .398, P < .001), and obese girls (r = .392, P < .001). The prevalence percentages of NAFLD were 93.6%, 75.8%, and 37.9% in obese boys with serum ALT levels of >50, 25-50, and ≤25 U/L and were 81.6%, 67.9%, and 20.6% in obese girls with serum ALT levels of >40, 20-40, and ≤20 U/L, respectively. CONCLUSION: Serum ALT levels significantly correlated with abnormal BMI values in children, suggesting a rigorous BMI threshold is needed to establish the cutoffs for serum ULN-ALT in children. Besides, the revised serum ULN-ALT can uncover mild liver injury in obese children with NAFLD.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis.

Intestinal dysbiosis is implicated in alcoholic hepatitis (AH). However, changes in the circulating microbiome, its association with the presence and severity of AH, and its functional relevance in AH is unknown. Qualitative and quantitative assessment of changes in the circulating microbiome were performed by sequencing bacterial DNA in subjects with moderate AH (MAH) (n = 18) or severe AH (SAH) (n = 19). These data were compared with heavy drinking controls (HDCs) without obvious liver disease (n = 19) and non-alcohol-consuming controls (NACs, n = 20). The data were related to endotoxin levels and markers of monocyte activation. Linear discriminant analysis effect size (LEfSe) analysis, inferred metagenomics, and predictive functional analysis using PICRUSt were performed. There was a significant increase in 16S copies/ng DNA both in MAH (P < 0.01) and SAH (P < 0.001) subjects. Compared with NACs, the relative abundance of phylum Bacteroidetes was significantly decreased in HDCs, MAH, and SAH (P < 0.001). In contrast, all alcohol-consuming groups had enrichment with Fusobacteria; this was greatest for HDCs and decreased progressively in MAH and SAH. Subjects with SAH had significantly higher endotoxemia (P = 0.01). Compared with alcohol-consuming groups, predictive functional metagenomics indicated an enrichment of bacteria with genes related to methanogenesis and denitrification. Furthermore, both HDCs and SAH showed activation of a type III secretion system that has been linked to gram-negative bacterial virulence. Metagenomics in SAH versus NACs predicted increased isoprenoid synthesis via mevalonate and anthranilate degradation, known modulators of gram-positive bacterial growth and biofilm production, respectively. CONCLUSION: Heavy alcohol consumption appears to be the primary driver of changes in the circulating microbiome associated with a shift in its inferred metabolic functions. (Hepatology 2018;67:1284-1302).

Alcohol abstinence ameliorates the dysregulated immune profiles in patients with alcoholic hepatitis: A prospective observational study.

Alcoholic hepatitis (AH) develops in only a small proportion of heavy drinkers. To better understand the mechanisms underlying this disparity, we conducted a study to define the relationship between AH development and dysregulated immune responses that might be ameliorated by alcohol abstinence. Sixty-eight AH patients, 65 heavy drinking controls without liver disease (HDC), and 20 healthy controls were enrolled and followed up to 12 months. At baseline, HDC and healthy controls had no significant differences in their plasma levels of 38 inflammatory cytokines/chemokines measured using multiplex immunoassays. However, compared to HDC, AH patients had higher baseline levels of 11 cytokines/chemokines (tumor necrosis factor alpha, interleukin 6 [IL-6], IL-8, interferon gamma-induced protein 10, IL-4, IL-9, IL-10, fibroblast growth factor 2, IL-7, IL-15, and transforming growth factor alpha) but lower levels of the anti-inflammatory macrophage-derived chemokine. AH patients also had more activated yet dysfunctional immune cells as monocytes, T cells, and B cells expressed higher levels of cluster of differentiation 38 (CD38) and CD69 but low levels of human leukocyte antigen DR, CD80, and CD86 at baseline. In addition, CD4 T cells produced less interferon-gamma in response to T-cell stimulation. Up-regulated IL-6, IL-8, CD38, and CD69 and down-regulated macrophage-derived chemokine, human leukocyte antigen DR, CD86, and CD80 correlated positively and negatively, respectively, with disease severity. Longitudinal analysis indicated that levels of IL-6, IL-8, CD38, and CD69 were reduced, whereas levels of macrophage-derived chemokine, human leukocyte antigen DR, CD80, and CD86 were increased in abstinent AH patients. All of the cellular immune abnormalities were reversed by day 360 in abstinent AH patients; however, plasma levels of tumor necrosis factor alpha, IL-8, IL-10, fibroblast growth factor 2, and IL-7 remained higher. CONCLUSION: AH patients were in a highly immune-dysregulated state, whereas HDC showed little evidence of immune activation; alcohol abstinence reversed most, but not all, of the immunological abnormalities. (Hepatology 2017;66:575-590).

MMPs/TIMPs imbalances in the peripheral blood and cerebrospinal fluid are associated with the pathogenesis of HIV-1-associated neurocognitive disorders.

HIV-1-associated neurocognitive disorders (HAND) continue to be a major concern in the infected population, despite the widespread use of combined antiretroviral therapy (cART). Growing evidence suggests that an imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMPs (TIMPs) contributes to the pathogenesis of HAND. In our present study, we examined protein levels and enzymatic activities of MMPs and TIMPs in both plasma and cerebrospinal fluid (CSF) samples from HIV-1 patients with or without HAND and HIV-1-negative controls. Imbalances between MMPs and TIMPs with distinct patterns were revealed in both the peripheral blood and CSF of HIV-1 patients, especially those with HAND. In the peripheral blood, the protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, and the enzymatic activities of MMP-2 and MMP-9 were increased in HIV-1 patients with or without HAND when compared with HIV-1-negative controls. The enzymatic activity of MMP-2, but not MMP-9, was further increased in plasma samples of HAND patients than that of HIV-1 patients without HAND. Notably, the ratio of MMP-2/TIMP-2 in plasma was significantly increased in HAND patients, not in patients without HAND. In the CSF, MMP-2 activity was increased, but the ratio of MMP-2/TIMP-2 was not altered. De novo induction and activation of MMP-9 in the CSF of HAND patients was particularly prominent. The imbalances between MMPs and TIMPs in the blood and CSF were related to the altered profiles of inflammatory cytokines/chemokines and monocyte activation in these individuals. In addition, plasma from HIV-1 patients directly induced integrity disruption of an in vitro blood-brain barrier (BBB) model, leading to increased BBB permeability and robust transmigration of monocytes/macrophages. These results indicate that imbalances between MMPs and TIMPs are involved in BBB disruption and are implicated in the pathogenesis of neurological disorders such as HAND in HIV-1 patients.

Blockage of CD59 Function Restores Activities of Neutralizing and Nonneutralizing Antibodies in Triggering Antibody-Dependent Complement-Mediated Lysis of HIV-1 Virions and Provirus-Activated Latently Infected Cells.

UNLABELLED: Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. IMPORTANCE: There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by not only nAbs but also non-nAbs. Our results are significant in developing novel immune-based approaches to restore the functions of nAbs and non-nAbs in the circulation of HIV-1-infected individuals to specifically target and clear HIV-1 virions and infected cells. Our data also provide new insights into the mechanisms by which HIV-1 virions and infected cells escape Ab-mediated immunity and could aid in the design and/or development of therapeutic HIV-1 vaccines. In addition, a combination of antiretroviral therapy with RCA blockage, provirus activators, and therapeutic vaccines may represent a novel approach to eliminate HIV-1 reservoirs, i.e., the infected cells harboring replication-competent proviruses and residual viremia.

Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells.

Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.

Primary human macrophages serve as vehicles for vaccinia virus replication and dissemination.

UNLABELLED: Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1ß treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE: Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.

Exosome-associated hepatitis C virus in cell cultures and patient plasma.

Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

Primary human leukocyte subsets differentially express vaccinia virus receptors enriched in lipid rafts.

Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs.