Replication In Peripheral Blood Mononuclear Cells Research Articles

Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection.

The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication.

Primary HIV-1 and Infectious Molecular Clones Are Differentially Susceptible to Broadly Neutralizing Antibodies.

To prevent the spread of HIV-1, a vaccine should elicit antibodies that block viral entry for all cell types. Recently, we have developed a virus capture assay to quantitatively examine early time points of infection. Here we present data on the ability of bNAbs to inhibit capture (1 h) or replication (48 h) of purified primary acute or chronic HIV or infectious molecular clones (IMCs) in human peripheral blood mononuclear cells (PBMCs) as quantified by qRT-PCR. Although bNAbs significantly inhibited HIV-1 replication in PBMCs in a virus subtype and in a PBMC-donor specific manner, they did not inhibit virus capture of primary viruses. In contrast, IMC capture and replication in PBMCs and purified CD4+ T cells were significantly inhibited by bNAbs, thus indicating that unlike IMCs, primary HIV-1 may initially bind to other cell surface molecules, which leads to virus capture even in the presence of bNAbs. Our results demonstrate that the initial interactions and some aspects of infectivity of primary HIV-1 and IMCs are inherently different, which underscores the importance of studying virus transmission using primary viruses in in vitro studies, an issue that could impact HIV-1 vaccine design strategies.

In Vitro Replication of HCV RNA in Peripheral Blood Mononuclear Cells Isolated from Patients Undergoing Treatment for Hepatitis C Virus Infection

Background: Although the liver is the main site for the Hepatitis C Virus (HCV) replication, there is still an essential debate about extrahepatic HCV reservoirs. Objectives: It has been proposed that Peripheral Blood Mononuclear Cells (PBMCs) could be the possible virus replication sites. Therefore, PBMCs may be candidates for recurrent HCV infection after achieving Sustained Virologic Response (SVR). In this study, we designed a lymphocyte culture to explore more about virus replication in PBMCs collected from patients with chronic hepatitis C. Methods: Plasma and PBMC samples were collected from 16 randomly selected seropositive patients for the anti-HCV antibody. Four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. PBMCs were isolated from whole blood. Between 106-107 cells were cultured with optimized concentrations of IL-2 (10 mg/ml) and phytohemagglutinin A (5 mg/ml). Total RNA was extracted from the first collected sera and harvested lymphocytes. Constructed plasmids containing the NCR coding region were used to plot the standard curve for the relative quantification of SYBR green real-time PCR. The sensitivity and specificity of the detection were established by using plasmids containing cDNA. Results: With this plasmid containing the NCR coding region, the Limit of Detection (LOD) of in-house-developed real-time RT-PCR sensitivity was 2×101 copies. Using primers for the NCR region, 10 out of 16 (62.5 %) PBMCs were positive for negative-strand HCV RNA. Among the four samples collected from patients with SVR, negative-strand HCV RNA was found in two patient samples. Conclusions: Our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with SVR, in the presence of IL-2 and PHA, markedly enhanced the detection of HCV RNA replica-tive strands. Therefore, PBMCs may be reservoirs for recurrent hepatitis infection after SVR and antiviral treatment. However, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study.

Existence of Occult Hepatitis C virus Infection in Local Haemodialysis Setting

Introduction: Among chronic hepatitis C patients, the viral agent is well known to replicate in the hepatocytes and extrahepatic cells. Previous studies have demonstrated a new form of hepatitis C called 'Occult HCV Infection' (OCI). It is defined as the presence of HCV RNA in the hepatocytes but undetectable HCV RNA in the serum or plasma. The exact natural history, epidemiology, pathogenesis and clinical importance of OCI are unknown. Several studies have been carried out worldwide, but none from Malaysia. This study investigated OCI among local patients undergoing routine hemodialysis at a referral hospital in Pahang, Malaysia. Materials and Methods: Blood was collected from both anti-HCV positive (n=10) and anti-HCV negative (n=18) patients. Inclusion criteria for the seronegative patients included elevated liver enzymes. Results: 10 chronic hepatitis C patients were recruited in this study. All had viral RNA detected in both serum and peripheral blood mononuclear cells (PBMC). Viral quasispecies was common in this group of patients. Meanwhile, out of 18 seronegative patients, 3 patients (16%) showed active viral replication in PBMC. Conclusion: This study proved the existence of OCI in local haemodialysis setting. Further investigation is needed to get the actual prevalence of OCI.

Silencing HCV Replication in Its Reservoir.

BACKGROUND:HCV infection and its complications are among the leading public health challenges; the emergence of drug-resistant variants are expected to be a major problem. A novel combinatorial small interfering RNA (siRNA) could be a novel triple therapy that could be suitable for genotype 4. Although HCV is a hepatotropic virus, there is reliable evidence about its replication in peripheral blood mononuclear cells (PBMC) of chronically infected patients; these cells act as an extra-hepatic reservoir for viral recurrence and persistence. The patients with HCV-RNA in PBMC showed a significantly lower response to therapy that supports to be one of the factors influencing therapeutic response. Almost all regions of HCV show potential for siRNA target with relative efficiencies of individual siRNA sequences.AIM:This study aims to test the efficacy of siRNA against HCV-4 replication in PBMC in vitro, to introduce an alternative therapeutic option for HCV-4 suitable to eradicate it from both hepatic and extra-hepatic reservoirs.METHODS:Efficacy of synthesised siRNA molecule that targets 5/UTR of domain IIIC within IRES of HCV RNA to eradicate HCV intra-PBMC in vitro was tested and compared with IFN/RBV in vitro, by using both qRT-PCR and western blot. Sixty genotype-4 chronic HCV patients who are naïve for any HCV treatment were enrolled and tested for the presence of HCV intra-PBMC using qRT-PCR before and after siRNA treatment in vitro.RESULTS:Real-time PCR analysis showed a significant reduction of HCV RNA levels after 24hr post-HCV-positive-PBMCs treatment by siRNA with cell vitality reached up to 98%. Besides a complete inhibition of NS5A viral protein expression, that is functionally essential for viral assembly, replication and egress.CONCLUSION:So, Targeting HCV infection using RNA interference technology might be a reliable therapeutic option for chronic HCV patients with HCV minus strand within PBMCs.

TLR7 Agonist GS-9620 Is a Potent Inhibitor of Acute HIV-1 Infection in Human Peripheral Blood Mononuclear Cells.

ABSTRACTGS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4+ T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4+ T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans. GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal.

Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS.

HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either β-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5-fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.

New evidence of replication of hepatitis C virus in short-term peripheral blood mononuclear cell cultures

BackgroundEven though hepatocytes are the main site for hepatitis C virus (HCV) replication, peripheral blood mononuclear cells (PBMC) have also been proposed as a suitable site for HCV replication. However, this issue still remains under discussion. We have previously developed an innovative system where HCV-RNA can be recovered during PBMC culture from HCV infected patients. Thus, the aim of this work was to use this novel approach in order to observe the evolution and replication of HCV genotype 1b in the PBMC of an HIV-HCV coinfected patient. MethodsHCV-RNA was extracted from serum, uncultured PBMC and PBMC culture at day 6, 20 and 33. The evolutionary analysis was performed using the direct sequences of three viral regions: 5′UTR, E2 and NS5A. Additionally, E2 region was cloned in order to extend the evolutive analysis. ResultsIn the present work, the molecular characterization of HCV along the culture showed a clear dynamic evolving process with the appearance of several nucleotide or amino acid changes in the three regions analyzed. Furthermore, the population analysis of E2 clones showed emerging and loss of lineages which indicate the fast evolutive dynamics of this system. ConclusionsSince evolution can take place only if the virus is replicating in the culture, this finding constitutes an important evidence of viral replication in PBMC. Moreover, this extrahepatic compartment could be very important due to the presence of distinctive variants that could be responsible for resistance to treatment, viral pathogenesis and other clinical implications.

Human Interferon Regulatory Factor 2 Gene Expression is Induced in Chronic Hepatitis C Virus Infection—A Possible Mode of Viral Persistence

The interferon regulatory factors (IRFs) are a family of transcription factors known to be involved in the modulation of cellular responses to interferons (IFNs) and viral infection. While IRF-1 acts as a positive regulator, IRF-2 is known to repress IFN-mediated gene expression. The increase in the IRF-1/IRF-2 ratio is considered as an important event in the transcriptional activation of IFN-α gene toward development of the cellular antiviral response. This study was performed to assess the expression of IRF mRNAs along with the expression level of IFN-α, its receptor (IFNAR-1), and the signal transduction factor (STAT-1) in treatment naive hepatitis C virus (HCV)-infected subjects. Thirty-five chronically infected (CHC) patients and 39 voluntary blood donors as controls were included in the study. Quantification of HCV-RNA (ribonucleic acid) and genotyping were done by real-time polymerase chain reaction (PCR) and hybridization assays, respectively, using patient's serum/plasma. In both controls and patients, the serum level of IFN-α and IFN-α was measured by flow cytometry. Target gene expressions were studied by retro-transcription of respective mRNAs extracted from peripheral blood mononuclear cells (PBMCs) followed by PCR amplification and densitometry. Minus-strand HCV-RNA as a marker of viral replication in PBMCs was detected by an inhouse PCR assay. Both IRF-1 and IRF-2 genes were significantly enhanced in CHC than in control subjects (P < 0.001). A significant positive correlation (r (2) = 0.386, P <0.01) was obtained between higher IRF-2 gene expression and increasing level of HCV-RNA. Chronically infected subjects (13%) harboring replicating HCV in PBMCs showed no significant differences in gene expressions than the subjects without HCV in PBMCs. Our findings indicate that HCV modulates host immunity by inducing IRF-2 gene to counteract IRF-1-mediated IFN-α gene expression. Since the IRF-2 gene is known to encode oncogenic protein, the role of IRF-2 in CHC patients developing hepatocellular carcinoma warrants further studies.

Coordinated increase of miRNA-155 and miRNA-196b expression correlates with the detection of the antigenomic strand of hepatitis C virus in peripheral blood mononuclear cells

A tight relationship has been revealed between cellular microRNAs (miRNAs) and the course of hepatitis C virus (HCV) replication in human hepatoma cells. Although the detection of the antigenomic HCV RNA strand in peripheral blood mononuclear cells (PBMCs) has provided evidence for viral replication in PBMCs, no reports have shown how miRNAs are affected upon HCV RNA synthesis in PBMCs. The aim of the present study was to assess if and how the expression levels of miRNA-155 and miRNA-196b in PBMCs are related to HCV replication in PBMCs of chronic hepatitis C (CHC) patients. Supporting analyses were performed to evaluate the expression of precursor pri-miR-155 (BIC) and Dicer protein. The genomic and antigenomic HCV RNA strands in PBMCs were detected by strand-specific qRT-PCR. The expression levels of miRNAs, BIC RNA and Dicer protein were assayed on PBMCs by qRT-PCR and Western blotting, respectively. miRNA-155 and miRNA-196b were detected in all studied PBMC samples, but their levels varied according to the presence of the antigenomic HCV RNA strand in PBMCs. Increased expression levels of miRNA-155 and miRNA-196b were associated with the presence of the antigenomic HCV RNA strand in PBMCs. In this group of patients higher frequency of BIC RNA and Dicer protein detection was also found. This study demonstrates that HCV RNA replication in PBMCs of CHC patients is connected with the increased and coordinated expression of miRNA-155 and miRNA-196b.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

BackgroundThe major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.MethodsProviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.ResultsReplication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.ConclusionsThe improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

Viral RNA but no evidence of replication can be detected in the peripheral blood mononuclear cells of hepatitis E virus-infected patients

Hepatitis E virus (HEV) infection is an important cause of acute viral hepatitis in several developing countries but has recently been shown to cause chronic hepatitis in immunosuppressed persons. Other hepatotropic viruses that cause chronic infection have been shown to infect peripheral blood mononuclear cells (PBMCs) and to persist in those cells. We therefore decided to look for evidence of replication of HEV in PBMCs obtained from patients with acute hepatitis E, using strand-specific assays for positive and negative HEV RNA. Of the 44 patients with acute hepatitis E during an outbreak in India, including 27 with detectable IgM anti-HEV and 19 with detectable serum HEV RNA, 11 had detectable HEV RNA in their PBMCs. However, of the six PBMC specimens with strong HEV RNA signal, none had detectable negative-strand HEV RNA, a marker of viral replication. These findings indicate the presence of HEV RNA but the absence of its replication in PBMCs from patients with acute hepatitis E.

Elevated levels of macrophage migration inhibitory factor (MIF) in the plasma of HIV-1-infected patients and in HIV-1-infected cell cultures: A relevant role on viral replication

The cytokine macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of inflammatory and infectious diseases, however its role in HIV-1 infection is unknown. Here we show that HIV-1-infected patients present elevated plasma levels of MIF, that HIV-1-infected peripheral blood mononuclear cells (PBMCs) release a greater amount of MIF, and that the HIV-1 envelope glycoprotein gp120 induces MIF secretion from uninfected PBMCs. The HIV-1 replication in PBMCs declines when these cells are treated with anti-MIF antibodies, and exposure of HIV-1-infected cells to the ABC-transporter inhibitor probenecid results in inhibition of MIF secretion. The addition of recombinant MIF (rhMIF) to HIV-1-infected PBMCs enhances viral replication of CCR5- or CXCR4-tropic HIV-1 isolates. Using a T CD4(+) cell lineage containing an HIV long terminal repeats (LTR)-Luciferase construct, we detected that rhMIF promotes transcription from HIV-1 LTR. Our results show that HIV-1 induces MIF secretion and suggest that MIF influences the HIV-1 biology through activation of HIV-1 LTR.

CXCL16 in HIV infection – a link between inflammation and viral replication

While some chemokines are thought to be protective in HIV-infected individuals by their ability to block HIV entry into T cells and macrophages, chemokines could also have harmful effects in HIV infection through their ability to promote inflammation. Here, we examined the regulation and the effects of CXCL16, a newly discovered chemokine of the CXC family, in HIV-infected patients. We examined serum levels of CXCL16 in clinically well-defined subgroups of HIV-infected individuals both before (n = 62) and during HAART (n = 40) as well as in age- and sex-matched healthy controls (n = 30). We also examined the effects of CXCL16 on inflammatory and anti-inflammatory cytokines and HIV replication in peripheral blood mononuclear cells (PBMC). Our main and novel findings were: (i) HIV-infected patients had significant raised CXCL16 levels according to disease severity and progression. (ii) During HAART, the immunological improvement was accompanied by a modest increase in CXCL16 level. (iii) While soluble CXCL16 promoted an anti-inflammatory response in PBMC from those on successful HAART, it induced an inflammatory response and enhanced HIV replication in PBMC from those with high viral load irrespectively of ongoing HAART. (iv) Recombinant HIV-tat protein significantly increased CXCL16 release in THP-1 macrophages. Our findings suggest a complex interaction between CXCL16 and HIV, promoting both inflammatory and anti-inflammatory effects as well as HIV replication, partly dependent on accompanying HIV replication.

Effect of porcine circovirus type 2 infection and replication on activated porcine peripheral blood mononuclear cells in vitro

The goal of this study was to examine the effect of porcine circovirus type 2 (PCV2) infection and replication on peripheral blood mononuclear cells (PBMCs) in the presence of mitogens, concanavalin A (ConA) or pokeweed mitogen (PWM) in vitro. The level of PCV2 replication and the impact of infection on PBMC proliferation, viability and the level of apoptosis in the presence or absence of mitogen stimulation were assessed. Mitogen stimulation increased viral replication in PBMCs as measured by the amount of spliced capsid mRNA (Cap mRNA). However, cell proliferation alone had no significant impact on PCV2 replication as the level of Cap mRNA in ConA or PWM stimulated PBMCs was not increased in proliferating cells compared to non-proliferating cells. No significant differences were observed in the level of PCV2 replication product in PBMCs stimulated with ConA for 12, 36, and 72 h prior to infection. Infection with PCV2 did not affect the ability of PBMCs to proliferate in response to ConA or PWM stimulation in vitro. Increased apoptosis was associated with PCV2 infection in PWM stimulated PBMCs. Interestingly, a significantly lower apoptotic index was observed in PCV2 infected PBMCs compared to mock-infected cells in the absence of mitogens. This study determined that the rate of PCV2 replication increases with cell stimulation and apoptosis is increased following PCV2 infection under certain stimulation conditions. These results further suggest that PCV2 requires a specific stimulation or trigger for increased viral replication, which is independent of cell proliferation.

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-γ), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-γ. In an in vitro study, ConA but not IFN-γ enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4 + (40%), CD8 + (54%) and IgM + (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-γ 12 h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15 dpi in non-treated and IFN-γ-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-γ-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo.

Estradiol and Progesterone Regulate HIV Type 1 Replication in Peripheral Blood Cells

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.

Active β-Catenin Signaling Is an Inhibitory Pathway for Human Immunodeficiency Virus Replication in Peripheral Blood Mononuclear Cells

The Wnt/beta-catenin pathway is involved in cell functions governing development and disease. In modeling postentry restriction of human immunodeficiency virus (HIV) replication in astrocytes, we reported that part of this natural resistance to productive replication of HIV in astrocytes involved expression of proteins of the Wnt/beta-catenin signaling pathway. We determined here whether induction of beta-catenin signaling in peripheral blood mononuclear cells (PBMCs) can modulate HIV replication. Given that lithium is an inducer of beta-catenin signaling, we used it as a tool to determine the impact of beta-catenin signaling on HIV replication in PBMCs. We demonstrated that lithium inhibited the replication of T-tropic and primary isolates of HIV by >90% and did so in noncytotoxic/noncytostatic concentrations and in a beta-catenin-dependent manner. Specifically, inhibiting beta-catenin signaling by transfection of dominant-negative mutant constructs to either T-cell factor 4, the downstream effector of Wnt signaling, or beta-catenin, the central mediator of this pathway, abrogated the ability of lithium to inhibit HIV replication. Moreover, when Wnt/beta-catenin signaling was inhibited, the level of HIV replication was enhanced by fourfold. To confirm the in vivo relevance of the beta-catenin pathway in repressing HIV replication, we evaluated HIV-positive antiretroviral therapy-naive patients who were on lithium therapy. These patients demonstrated a reduction in viral load, which increased as the dose of lithium was reduced. Collectively, these data indicate that beta-catenin signaling is an intrinsic molecular pathway restricting HIV replication in PBMCs.

Antiviral and immunomodulatory effect of a lyophilized extract of Capparis spinosa L. buds

Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are common human pathogens that in particular cases can also cause severe problems especially in immunodeficient patients. The present paper reports the antiviral and immunomodulatory properties of a methanolic extract of C. spinosa buds (CAP), rich in flavonoids, including several quercetin and kaempferol glycosides. In particular we have investigated whether the in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to CAP might inhibit the replication of HSV-2 and modulate the induction kinetics of IL-12, TNF-alpha IFN-gamma. Our findings have shown that CAP treatment interferes with HSV-2 replication in PBMCs inhibiting the extracellular virus release upregulating their production of IL-12, IFN-gamma and TNF-alpha. One could speculate that CAP may contribute in improving immune surveillance of PBMCs toward virus infection by up-regulating expression of peculiar proinflammatory cytokines; it should thus be successfully employed for treatment of HSV-2 infections in immunocompromised hosts.

Noninfectious papilloma virus–like particles inhibit HIV-1 replication: implications for immune control of HIV-1 infection by IL-27

AbstractHuman papilloma virus (HPV)–like particles (VLPs) have been used as a vaccine to prevent HPV infection. Recent studies demonstrate that VLPs bind to dendritic cells and induce the expression of antiviral cytokines such as interferon-α (IFN-α), interleukin-10 (IL-10) and IFN-γ. In the present study, we evaluated the effect of VLPs on HIV-1 replication in peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and macrophages. Here, we show that VLPs suppress the replication of both X4 and R5 HIV-1 without affecting the expression of CD4, CXCR4, and CCR5. Soluble factor(s) released by PBMCs and macrophages on VLPs treatment inhibited HIV-1 replication. To determine the inhibitory factors, DNA microarray analysis was performed using VLP-treated PBMCs and macrophages. VLPs induced the genes associated with IFN induction, immune responses, and antiviral responses, among with the recently described cytokine IL-27. Subsequently, IL-27 was found to be a potent inhibitor of HIV-1 replication in PBMCs, CD4+ T cells, and macrophages. Taken together, our studies identify a novel role of IL-27 in restricting HIV-1 replication and suggest that further examination of the inhibitory property of IL-27 may pave the way for a novel therapy for HIV-1 infection.