Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.     

In vivo and in vitro ultrastructural alterations induced by human immunodeficiency virus in human lymphoid cells

The ultrastructural alterations induced by human immunodeficiency virus (HIV) in human lymphoid cells have been evaluated. Electron microscopic examination of peripheral blood mononuclear cells (PBMC) from 14 male homosexuals with confirmed acquired immunodeficiency syndrome (AIDS) or AIDS-related complex revealed that tubuloreticular inclusions were present in 5-15% of the cell sections from each case. In 5 of 14 cases, cylindrical confronting lamellae were found in 1-2% of the cell sections. No retrovirus-like particles or surface membrane alterations were detected. Neither of these structural alterations was observed in control PBMC obtained from six HIV-seronegative, hepatitis B virus surface antigen (HBsAg)-positive carriers or in 11 healthy subjects. When primary cultures of CD4+-enriched lymphocytes were infected in vitro with HIV, tubuloreticular inclusions could be detected in 3-10% of the cell sections, but no cylindrical confronting lamellae-like structures were found. In contrast, neither of these alterations were seen in uninfected or HIV-infected H9-HT continuous cell lines. These in vivo and in vitro studies indicate that there is an association between the appearance of the tubuloreticular inclusions and cytopathic HIV infection, although no correlation between cytopathic changes and active viral replication was observed at the single cell level. Further studies will be required to establish the mechanism(s) of formation of the tubuloreticular inclusions and to determine their prognostic potential.     

Effect of Shipment, Storage, Anticoagulant, and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus-Infected Patients

Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.     

Interferon alpha (IFN)-macrophage interactions in human immunodeficiency virus (HIV) infection: role of IFN in the tempo and progression of HIV disease.

Components of the host immune response that constrain virus replication and affect long-lasting antiviral immunity following HIV infection are incompletely defined. IFNs are critical participants in host antiviral processes. While IFN induces significant anti-retroviral activities, they also serve as harbingers for poor clinical outcomes. Moreover, monocytes, a major cellular source of IFN and HIV in man, are poor producer cells for IFN following HIV infection. Indeed, HIV infection of monocytes results in a diminished production and induction of IFN. IFN is only produced during cell to cell contact between HIV-infected cells and uninfected PBMC. Analysis of the biologic activity of HIV-induced IFN(s) shows that it poorly restricts HIV replication. Thus, the role of IFN in HIV disease is complex and seemingly paradoxical. The diminished capacity of HIV-infected monocytes to produce IFN and the production of defective IFNs likely reflect specific viral adaptive mechanisms for persistent infection.     

Inhibition of hepatitis C virus adsorption to peripheral blood mononuclear cells by dextran sulfate

Summary.  Human peripheral blood mononuclear cells (PBMC) can be infected in vitro with sera of hepatitis C virus (HCV)-infected patients. We have treated pools of PBMC with dextran sulfate MW 8 000, before infection with various HCV(+) sera. When the cells were treated with dextran sulfate 10⁻³M, HCV RNA was no longer detectable after infection with all HCV genotypes tested. When the cells incubated with dextran sulfate 10⁻³M and 10⁻⁴M were maintained in culture for 4 weeks, no significant HCV replication could be observed. Our data suggest that dextran sulfate inhibits the attachment of HCV to the cell surface, since the HCV RNA detection was similar to control cells when dextran sulfate was added after infection. Accepted October 15, 1997 Received August 25, 1997     

Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.     

In vitro and in vivo evaluations of sodium lauryl sulfate and dextran sulfate as microbicides against herpes simplex and human immunodeficiency viruses

The efficacy of sodium lauryl sulfate (SLS), a sulfated anionic chaotropic surfactant, and dextran sulfate (DS), a polysulfated carbohydrate, against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) infections was evaluated in cultured cells and in different murine models of HSV infection. Results showed that both SLS and DS were potent inhibitors of the infectivities of various HSV-1 and HSV-2 strains. Pretreatment of HIV-1 (strain NL4-3) with SLS also reduced its infectivity to 1G5 cells. DS prevented the binding of HSV to cell surface receptors and therefore its entry into cells. Pretreatment of HSV-1 (strain F) with 50 microM SLS resulted in a complete loss of virus infectivity to Vero cells. However, viruses were able to enter into cells and to produce in the nuclei capsid shells devoid of a DNA core. The amount of the glycoprotein D gene produced in these cells remained unchanged compared to controls, suggesting that SLS could interfere with the maturation of the virus. At a higher SLS concentration (100 microM), HSV was highly damaged by SLS pretreatment and only a few viral particles could enter into cells to produce abnormal capsids. Although DS was a more potent inhibitor of HSV infectivity in vitro, it was unable to provide any protection in murine models of HSV infection. However, SLS conferred a complete protection of animals infected cutaneously with pretreated viruses. In addition, skin pretreatment of mice with a polymer formulation containing SLS completely prevented the development of cutaneous lesions. More interestingly, intravaginal pretreatment of mice with SLS in a buffered solution also completely protected against lethal HSV-2 infection. Taken together, our results suggest that SLS could thus represent a candidate of choice as a microbicide to prevent the sexual transmission of HIV, HSV, and possibly other pathogens that cause sexually transmitted diseases.     

3例艾滋病患者不同组织来源HIV分离株异同性的研究

目的：了解艾滋病（AIDS）患者外周血和部分组织人免疫缺陷病毒（HIV）生物学性状和基因序列的特点。方法：将3例患者的PBMC、淋巴结活检组织或脑脊液接种于PHA刺激72h的正常人PBMC进行病毒分离，将分离的毒株接种于MT2细胞,观察分离株的致细胞病毒性。同时提取前病毒DNA，对外膜基因进行序列分析。结果：同一患者不同组织来源的毒株，其生物学性状和基因序列存在差异；2例来自脑脊液的毒株，其基因序列与国际标准株SF162同源性较高。结论：在同一宿主体内的不同组织、体液内有不同的病毒种群存在；中国部分HIV感染者中存在与国际标准株同源的嗜神经株。     

Anti-HLA antigen class I heavy chain monoclonal antibodies inhibit human immunodeficiency virus production by peripheral blood mononuclear cells

We have examined the capacity of monoclonal antibodies (mAb) specific for HLA class I heavy chain to interfere with the human immunodeficiency virus (HIV) replicative cycle in human T cells. Among six anti-HLA class I heavy chain-specific mAb assayed, two mAb, RL4-24-6 and W6/32, were able to delay HIV1 and HIV2 cytopathic effect on MT4 cells, a human T cell leukemia virus type I (HTLVI) immortalized T cell line, mAb RL4-24-6, chosen for further studies, also inhibited HIV1 production by peripheral blood mononuclear cells (PBMC), and this inhibition was dose dependent. However, no effect was observed when mAb treatment was performed with either the CEM or Jurkat T cell lines. Our investigation of how RL4-24-6 interferes with the HIV replicative cycle revealed that: (a) incubation of PBMC with RL4-24-6 prior to HIV exposure did not change the susceptibility of these cells to HIV infection, (b) syncytia formation between CD4+ MT4 cells and HIV chronically infected PBMC was not affected by RL4-24-6 and (c) treatment of freshly infected PBMC with RL4-24-6, however, inhibited viral production. These data, together with those we previously reported using anti-beta 2-microglobulin (beta 2m) mAb, suggest that anti-HLA class I/beta 2m complex mAb can modify an early step of the HIV replicative cycle without affecting the viral entry.     

BAY 50-4798, a novel, high-affinity receptor-specific recombinant interleukin-2 analog, induces dose-dependent increases in CD25 expression and proliferation among unstimulated, human peripheral blood mononuclear cells in vitro

Interleukin-2 administration induces CD4 T cell expansion in HIV-infected patients, however, toxicity can limit dosing. BAY 50-4798 is a recombinant IL-2 analog with >1000-fold specificity for the high-affinity IL-2 receptor. The effects of this compound on unstimulated human PBMC were evaluated. PBMC from HIV(-) and HIV(+) donors were cultured in vitro with incremental doses of BAY 50-4798 or aldesleukin. CD25 expression and proliferation were evaluated with flow cytometry. Cytokine levels were measured by ELISA in culture supernatants. BAY 50-4798 induced dose-dependent increases in CD25 expression and proliferation of T cells, NK, and B cells and showed selectivity for CD4 T cells expressing CD25. Induction of pro-inflammatory cytokines was also dose-dependent and was observed at the concentrations of BAY 50-4798 with the highest biologic activity. These data suggest that BAY 50-4798 can induce proliferation of unstimulated T cells but loss of T cell selectivity and induction of pro-inflammatory cytokines occur at concentrations exerting the highest biologic activity.     

Human immunodeficiency virus infection and levels of Dehydroepiandrosterone sulfate in plasma among Indians

The shift in cytokine profile during human immunodeficiency virus (HIV) disease progression is influenced by dehydroepiandrosterone sulfate (DHEAS) level. Radioimmunoassay was used to measure plasma DHEAS for 30 treatment-na?ve HIV-infected and 30 uninfected individuals. There was a significant negative correlation of viral load with DHEAS level (P<0.05). Further studies of the use of DHEAS levels for monitoring HIV patients economically are warranted.     

Fas antigen expression and apoptosis of lymphocytes in macaques infected with simian immunodeficiency virus strain mac

Summary.  To investigate the role of apoptosis in the pathogenesis of HIV infection we used macaques infected with simian immunodeficiency virus (SIV) as a primate model and examined the characteristics of the apoptosis of lymphocytes in SIV mac-infected macaques. In vitro apoptosis was more strongly induced in peripheral blood mononcuclear cells (PBMC) from SIV mac239- infected macaques than those from uninfected controls. We found that the frequency of Fas antigen-positive cells was higher in PBMC from SIV mac-infected macaques than from uninfected controls, and in vitro apoptosis of PBMC was suppressed by an inhibitor of the interleukin-lβ converting enzyme (ICE) family proteases. In biopsied lymph nodes, the number of apoptotic nuclei in T cell-dependent areas was higher in SIVmac-infected macaques than in unifected controls. A higher number of apoptotic nuclei in lymph nodes of SIVmac-infected macaques was observed in the stage of persisent general lymphadenopathy than in those with AIDS-related complex, while there was no significant difference in the extent of apoptosis of cultured PBMC among the SIVmac-infected macaques. These results suggest that in vitro apoptosis is mediated by the Fas/Fas ligand and ICE system and that apoptosis in lymph nodes may be more closely related to the stage of SIVmac infection than is that of cultured PBMC. Accepted November 17, 1997 Received October 2, 1997     

Quantitation of hepatitis C virus RNA in peripheral blood mononuclear cells in HCV-monoinfection and HIV/HCV-coinfection

Peripheral blood mononuclear cells (PBMCs) represent an extrahepatic hepatitis C virus (HCV) reservoir, the significance of which is unclear due to limited studies and varying test methodologies. In this study, a commercial viral load assay for measuring cell‐associated PBMC HCV RNA was evaluated. HCV RNA was extracted from PBMCs, sorted CD14+, and CD19+ cells and corresponding plasma samples using the Abbott m2000 and Real‐Time HCV assay. Test performance and influence of HIV seropositivity on plasma and PBMC HCV RNA were studied. Among 51 patients, 67 and 62 unique patient samples had detectable plasma and PBMC HCV viral load, respectively. The median PBMC viral load was 535 IU/1 M cells (range 29–5,190). CD19+ cells had significantly higher viral load than CD14+ cells (median log₁₀ HCV viral load 2.63 vs. 1.50 IU/ml; P< 0.001). Stability of PBMC viral load over time was demonstrated in untreated patients; all patients with an undetectable plasma HCV viral load after HCV treatment also demonstrated undetectable PBMC viral load. Repeated testing in nine samples yielded consistent PBMC viral load, differing by only 1.3‐fold (range 1.0–1.7‐fold). Among samples with detectable plasma HCV RNA, the correlation between PBMC and plasma viral load was moderate (r = 0.66) and was greater among HCV mono‐infected compared to HIV/HCV co‐infected subjects (r = 0.80 vs. 0.52). Measurement of cell‐associated PBMC HCV RNA using a commercial assay demonstrated promising test characteristics. Differences in PBMC HCV viral load based on HIV‐coinfection status and the significance of greater copy number in B‐cells requires further study. J. Med. Virol. 84:431–437, 2012. © 2011 Wiley Periodicals, Inc.     

Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1

We investigated the role of different CC chemokines, including regulated upon activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-lalpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), and MCP-3 on virus replication in cultures established from CD8+ T cell-depleted peripheral blood mononuclear cells (PBMC) of HIV-infected individuals that were either cocultivated with allogeneic T cell blasts (ATCB) of uninfected individuals or directly stimulated by mitogen plus interleukin-2. RANTES was the only chemokine that showed a clear-cut suppressive effect on HIV replication in both culture systems, although inhibitory effects were frequently also observed with MIP-1alpha, MCP-3, and, occasionally, with MCP-1. In contrast, MCP-1 frequently enhanced HIV production in most patients' cultures or cocultures that were characterized by secreting relatively low levels (<20 ng/mL) of MCP-1. When CD8-depleted PBMC of HIV+ individuals were cocultivated with ATCB of uninfected healthy donors, a positive correlation was observed between MCP-1 concentrations and the enhancement of HIV-1 replication occurring after depletion of CD8+ cells from donors' cells. Depletion of CD14+ cells (monocytes) from ATCB resulted in the down-regulation of virus replication during co-cultivation with CD8-depleted PBMC of infected individuals. Of interest, MCP-1 up-regulated HIV production in these CD14-depleted ATCB cocultures. Altogether these observations suggest that MCP-1 may represent an important factor enhancing HIV spreading, particularly in anatomical sites, such as the brain, where infection of macrophages and microglial cells plays a dominant role.     

Effect of polyions on Japanese encephalitis virus Difference in interaction of virus with DEAE dextran and Dextran sulfate between PS cell adapted and non-adapted virus

Summary The effect of DEAE dextran and Dextran sulfate on Japanese encephalitis virus was studied to elucidate the difference in characteristics between porcine kidney stable (PS) cell cultures-adapted and nonadapted virus. The infectivity of PS-adapted virus was markedly inhibited by the addition of either DEAE dextran or Dextran sulfate, because of interference by the polyions with virus adsorption. Furthermore, release of PS-adapted virus from cells was found to be enhanced by the addition of the polyions. Consequently, when virus was grown in the presence of the polyions, virus titers in the supernatant fluid were always higher than in the control cultures.On the other hand, DEAE dextran and Dextran sulfate did not inhibit or enhance the growth of PS-nonadapted virus. The interactions between virus and cells, such as adsorption and viral growth, were unaffected by the addition of polyions.     

Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis

Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.     

A comparative study of the effect of dextran sulfate on the fusion and the in vitro replication of influenza A and B,Semliki Forest,vesicular stomatitis,rabies, Sendai,and mumps virus

Summary The effect of dextran sulfate on the fusion of a series of enveloped viruses, bearing specifically different fusion proteins, was investigated. The fusion with model- and with biological membranes was monitored by an R18 fluorescence-dequenching fusion assay. Dextran sulfate strongly suppresses the fusion of orthomxyo- (influenza A (H₁N₁ and H₃N₂ subtypes) and influenza B), of toga- (Semliki Forest virus), and of rhabdoviruses (vesicular stomatitis and rabies virus). The fusion of the paramyxo-viruses Sendai and mumps was not significantly affected by the anionic polysaccharide. The response to dextran sulfate was virus-specific, and identical for the different members of one virusfamily, bearing the same fusion protein. It was shown that dextran sulfate attaches with high affinity to the viruses studied, but not to erythrocytes. The anionic polymer appears to attach to the fusion epitope of the viral membrane. The inhibition of virus replication in vitro shows a remarkable correlation with the observed anti-fusion effects of dextran sulfate.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

Human Immunodeficiency Virus Infection and Levels of Dehydroepiandrosterone Sulfate in Plasma among Indians

The shift in cytokine profile during human immunodeficiency virus (HIV) disease progression is influenced by dehydroepiandrosterone sulfate (DHEAS) level. Radioimmunoassay was used to measure plasma DHEAS for 30 treatment-naïve HIV-infected and 30 uninfected individuals. There was a significant negative correlation of viral load with DHEAS level (P < 0.05). Further studies of the use of DHEAS levels for monitoring HIV patients economically are warranted.     

In vivo infection of ramified microglia from adult cat central nervous system by feline immunodeficiency virus

Infection of microglial cells by the human immunodeficiency virus (HIV) is supposed to play an important role in the pathogenesis of AIDS-related central nervous system (CNS) complications. So far, however, experimental data about interactions between HIV and ramified microglia from the adult CNS were only occasionally reported, making it difficult to understand the exact nature of pathogenic events contributing to HIV-encephalopathy. Therefore, we used the animal model of feline immunodeficiency virus (FIV) infection of domestic cats to establish an experimental system which is suitable for studying the relationships between an immunodeficiency virus and the mature ramified microglia of the central nervous system. By means of density gradient centrifugation approximately 95% pure microglial cells could be isolated from adult feline brain that were characterized by their CD45(low) phenotype. Resident microglia extracted from the CNS of experimentally infected cats harbored FIV-specific DNA and cocultivation with mitogen-activated, but uninfected peripheral blood mononuclear cells (PBMC) resulted in recovery of high-titered infectious virus. Double labeling of brain cell monocultures explanted from persistently infected animals for both microglia and FIV markers disclosed less than 1% of viral antigen expressing microglial cells. This suggests that during the subclinical phase of the infection only a small number of brain-resident macrophages are productively infected. However, interaction of FIV-infected microglia and inflammatory lymphocytes may promote viral replication, thus supporting viral spread in brain tissue.