Hepatitis B virus DNA in mononuclear cells and analysis of cell subsets for the presence of replicative intermediates of viral DNA

To determine whether peripheral blood mononuclear cells (PBMCs) contain replicating forms of hepatitis B virus (HBV) DNA and to define which cell subset may be permissive for viral replication, we analyzed the PBMC DNA from 14 carriers positive for hepatitis B surface antigen (HBsAg) by Southern blot hybridization. HBV-related DNA, which was present exclusively in an extrachromosomal state, was found in the PBMCs of all five hepatitis B e antigen (HBeAg)-positive and three of nine HBeAg-negative carriers. Serum-associated HBV DNA was detected only in those HBsAg carriers whose PBMCs contained HBV DNA forms resembling replicative intermediates (1.0-3.2 kilobase pairs in the EcoRI digests). Analysis of PBMC subsets revealed that replicating forms of the HBV genome were present primarily in monocytes. Low levels of hybridization also were detected in B cells, whereas the T cell fraction (which contained natural killer cells) appeared to be devoid of these replicating forms.     

Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression

Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.     

Peripheral blood lymphocytes express recombination-activating genes 1 and 2 during Epstein-Barr virus-induced infectious mononucleosis

Implicit in the persistence of Epstein-Barr virus (EBV) in B lymphocytes is the successful circumvention of ongoing cell selection for competence of B cell receptors (BCRs). Because the EBV infection of B cells in vitro induces enzymatic machinery that is responsible for secondary immunoglobulin gene rearrangement, we examined the expression of the recombination-activating genes (RAGs) in peripheral blood mononuclear cells (PBMCs) from 26 patients with infectious mononucleosis (IM). RAG1 and/or RAG2 RNA was detected in PBMCs from 42% of patients with IM but not from healthy control subjects. EBV may usurp the cellular mechanism that diversifies the BCR, to guarantee a level of survival signaling sufficient for its own persistence.     

Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines.

Human immunodeficiency virus type 1 (HIV-1) isolates from various patients were divided into two major groups, rapid/high and slow/low, according to their replication properties in vitro. Rapid/high isolates grow well in cell lines and induce the formation of syncytia in peripheral blood mononuclear cells. In contrast, slow/low isolates do not replicate in cell lines and rarely induce syncytia in peripheral blood mononuclear cells. To understand the differences in replicative capacity of these isolates, a panel of indicator cell lines was used. These cell lines were generated for sensitive detection of HIV-1 isolates and show characteristics of T-lymphoid or monocytoid cells. As a result of infection, chloramphenicol acetyltransferase expression is activated. Rapid/high viruses activate chloramphenicol acetyltransferase expression in T-cell and monocytoid indicator cell lines, whereas slow/low isolates activate chloramphenicol acetyltransferase expression only in monocytoid cell lines. The block in infection of T-lymphoid cells by the slow/low isolates appears to occur early in the infection cycle, prior to the production of the virally encoded tat protein. HIV-1 isolates can thus be distinguished according to target-cell tropism. Monocyte-derived cells seem to be a more general target for the various HIV-1 isolates.     

Ability to induce p53 and caspase-mediated apoptosis in primary CD4+ T cells is variable among primary isolates of human immunodeficiency virus type 1

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with dramatic depletion of CD4(+) T cells, the major HIV-1-induced pathogenesis. Apoptosis has been suggested to play an important role for the T cell depletion and a number of mechanisms have been proposed for the apoptosis in T cells. Here, we compared the levels for apoptosis induction in primary peripheral blood mononuclear cells (PBMCs) among several laboratory strains and primary isolates of the HIV-1 subtypes B and E. The results showed that apoptosis in infected PBMCs, preferentially in CD4+ T cell population, became detectable around the time for virus production by flow cytometric terminal transferase dUTP nick end labeling (TUNEL) technique and staining with the nuclear dye Hoechst 33342. The abilities to induce apoptosis in PBMCs were highly variable in individual isolates. The increase of p53 protein in infected PBMCs, which was initiated before virus production, was observed in infected PBMCs and the levels of p53 protein were almost proportional to the rates of the isolates to induced apoptosis. The cells infected and cultured in the presence of Z-VAD-FMK had significantly decreased cell mortalities, indicating that activated caspases also played a significant role in the apoptosis. Thus, HIV-1-induced apoptosis in primary T cells was accompanied by the p53 protein and caspase activation at varied levels in primary isolates.     

Entry inhibitors SCH-C,RANTES, and T-20 block HIV type 1 replication in multiple cell types

The small-molecule CCR5 antagonist SCH-C (SCH 351125) was tested for its ability to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells, immature dendritic cells (DCs), and macrophages. Inhibition of infection of PBMCs by virus associated with mature DC in trans was also studied. For comparison, the peptide-based fusion inhibitor T-20 and the CC-chemokine RANTES were also evaluated. Although some cell type-dependent differences in potency were observed, each of the three entry inhibitors was active against the replication of three different CCR5-using primary isolates in each cell type. CCR5-dependent HIV-1 infectivity, whether DC associated or not, is thus vulnerable to inhibitors that block the virus-cell fusion process by different mechanisms. Together, these results suggest that SCH-C and other entry inhibitors should be evaluated for their clinical potential as inhibitors of HIV-1 replication in several settings, including the prevention of maternal-infant transmission and the prevention of sexual transmission by topical application as a microbicide.     

Cytotoxic factors secreted by cells infected by human immunodeficiency virus type I

Conditioned media from human immunodeficiency virus type I (HIV-1) infected cells were tested for cytotoxic cell-derived factors. The assay used a murine fibroblast cell line which is sensitive to the effects of tumor necrosis factors, but nonpermissive for HIV-1 replication. Cytotoxic activity was detected in cultures of peripheral blood mononuclear cells infected with HIV-1. However, no differences in activity were found in conditioned media from infected lymphoid or monocytoid cell lines compared to their uninfected counterparts. These data suggest that cytotoxic activities of this type are not mediators of cell killing resulting from HIV-1 infection. Thus, this cytotoxic activity is a direct or indirect result of virus replication or cytopathicity. One should consider a role for this cytotoxic factor, secreted by HIV-1 infected mononuclear cells, in various aspects of infection in vivo, such as AIDS encephalopathy or the systemic manifestations accompanying ARC.     

Human herpesvirus 8 enhances human immunodeficiency virus replication in acutely infected cells and induces reactivation in latently infected cells

Human herpesvirus 8 (HHV-8) is etiologically associated with Kaposi sarcoma (KS), the most common AIDS-associated malignancy. Previous results indicate that the HHV-8 viral transactivator ORF50 interacts synergistically with Tat protein in the transactivation of human immunodeficiency virus (HIV) long terminal repeat (LTR), leading to increased cell susceptibility to HIV infection. Here, we analyze the effect of HHV-8 infection on HIV replication in monocyte-macrophage and endothelial cells, as potential targets of coinfection. Primary or transformed monocytic and endothelial cells were infected with a cell-free HHV-8 inoculum and subsequently infected with lymphotropic or monocytotropic strains of HIV. The results show that HHV-8 coinfection markedly increases HIV replication in both cell types. HHV-8 infection induces also HIV reactivation in chronically infected cell lines and in peripheral blood mononuclear cells (PBMCs) from patients with asymptomatic HIV, suggesting the possibility that similar interactions might take place also in vivo. Furthermore, coinfection is not an essential condition, since contiguity of differently infected cells is sufficient for HIV reactivation. The results suggest that HHV-8 might be a cofactor for HIV progression and that HHV-8-infected endothelial cells might play a relevant role in transendothelial HIV spread.     

Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.     

Class I-unrestricted noncytotoxic anti-HTLV-I activity of CD8(+) T cells

Although it is widely believed that viral clearance is mediated principally by the destruction of infected cells by cytotoxic T cells, noncytolytic antiviral activity of CD8(+) T cells may play a role in preventing the progression to disease in infections with immunodeficiency viruses and hepatitis B virus. We demonstrate here that (1) replication of human T-lymphotropic virus type I (HTLV-I) is more readily detected from CD8(+) T-cell-depleted (CD8(-)) peripheral blood mononuclear cells (PBMCs) of healthy HTLV-I carriers than from unfractionated PBMCs, (2) cocultures of CD8(-) PBMCs with autologous or allogeneic CD8(+) T cells suppressed HTLV-I replication, and (3) CD8(+) T-cell anti-HTLV-I activity is not abrogated in trans-well cultures in which CD8(+) cells are separated from CD8(-) PBMCs by a permeable membrane filter. These results suggest that class I-unrestricted noncytolytic anti-HTLV-I activity is mediated, at least in part by a soluble factor(s), and may play a role in the pathogenesis of HTLV-I infection. (Blood. 2000;96:1994-1995)     

Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens

CD8(+) cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-gamma ELISPOT assay to the quantitative analysis of EBV-specific CD8(+) cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8(+) cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/10(6) CD8(+) cells). LMP2 peptide-responsive CD8(+) cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/10(6) PBMC). BMLF1 peptide-responsive CD8(+) cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/10(6) PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8(+) cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8(+) cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8(+) cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8(+) responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines. (Blood. 2000;95:241-248)     

拉米夫定治疗慢性乙型肝炎患者血清及PBMC内HBV DNA的动态变化

目的 :研究拉米夫定对慢性乙型肝炎 (慢乙肝 )患者血清及PBMC内HBVDNA的抑制作用。方法 :应用荧光定量PCR方法动态观察 72例拉米夫定治疗组和对照组患者血清及PBMC内HBVDNA的含量变化。结果 :治疗组血清HBVDNA在治疗 6周时 ( 2～ 8周 ) 2 1例转阴 ,PBMC中HBVDNA在治疗 16周 ( 8～ 2 4周 )时 17例转阴。结论 :拉米夫定对慢乙肝患者血清HBVDNA有较强的抑制作用 ,起效快 ,同时 ,随着治疗时间的延长 ,对PBMC内HBVDNA也有一定的抑制作用 ,但滞后于血清中HBVDNA的转阴     

Interaction of mannose-binding lectin with HIV type 1 is sufficient for virus opsonization but not neutralization

Mannose-binding lectin (MBL), a microbe-recognition protein in serum, binds to high mannose glycans on HIV-1 gp120 and has been reported to neutralize the cell line-adapted strain HIV(IIIB). Because HIV primary isolates (PI) are generally more resistant to neutralization by antibodies and considering that PI are produced in primary cells that could alter the number of high mannose glycans on HIV relative to cell lines, we assessed the ability to MBL to neutralize HIV PI. MBL at concentrations up to 50 microg/ml mediated relatively little neutralization (<20%) of HIV PI infection of peripheral blood mononuclear cells (PBMCs). MBL-neutralizing activity was slightly higher for cell line-adapted HIV infection of the H9 T cell line (up to 64% at 50 microg/ml). However, this effect was specific for H9 cells since MBL did not neutralize cell line-adapted virus infection of PBMCs, HIV PI infection of the GHOST cell line, or VSV pseudotyped with HIV gp160 from cell line-derived virus or PI. In contrast to its low activity in neutralization assays, MBL efficiently bound infectious HIV PI and opsonized HIV PI for uptake by monocytic cells. These results show that both PI and cell line-adapted HIV, despite binding of MBL, are relatively resistant to neutralization by levels of MBL normally present in serum. However, binding and opsonization of HIV by MBL may alter virus trafficking and viral-antigen presentation during HIV infection.     

Synthesis of human immunodeficiency virus DNA in a cell-to-cell transmission model.

Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was modelled by coculturing virus-infected cells with uninfected target cells at a ratio of 1:4. While H9 cells persistently infected with HTLV-IIIB did not contain unintegrated viral DNA detectable by Southern blotting, when cocultured with uninfected HUT-78 cells the mixed culture effectively underwent a new round one-step virus replication which began de novo synthesis of free viral DNA within 4 hours. Linear DNA was synthesized before the accumulation of circular DNA, and two seemingly distinct phases of viral DNA synthesis were involved. When both virus donor cells and recipient cells were arrested in the G0/G1 phase of the cell cycle, accumulation of circular viral DNA was inhibited. In contrast to cell-free virus infection of resting human peripheral blood mononuclear cells (PBMC), where no free viral DNA of discrete sizes could be detected by Southern blot, cell-to-cell transmission infection of resting PBMC resulted in the synthesis of full-length linear as well as circular viral DNA. The efficiency with which cell-to-cell transmission of HIV initiates virus replication underlines the importance of this mode of transmission in virus dissemination in vivo.     

Cellular factors influence the binding of HIV type 1 to cells

The goal of this study was to determine the importance of cellular factors for binding of HIV to cells. HIV primary isolates (PIs) produced in peripheral blood mononuclear cells (PBMCs) bound at relatively high levels to PBMCs but at low levels to cell lines, whereas T cell line-adapted (TCLA) virus produced in the H9 T cell line bound at high levels to both cell lines and PBMCs. Expression of CD4 in CD4-negative cells or blocking CD4 with antibody on CD4-positive cells did not affect virus binding. Blocking of gp120/gp41 with antibodies or a lack of expression of gp120/gp41 in virus particles also did not affect virus binding. However, the cell type from which virus was produced did affect virus binding. Thus, the binding pattern of TCLA virus shifted to that of a PI virus when produced in PBMCs. A PI binding pattern also occurred when a cloned TCLA virus (NL4-3) was produced in PBMCs, indicating that the virus-producing cell type has more of an effect on virus binding than the virus strain. These experiments show that both the virus-producing cell and the target cell have a major influence on HIV binding and suggest that host cell factors incorporated into virions are important for virus binding.     

CD8+ T cell depletion amplifies hepatitis C virus replication in peripheral blood mononuclear cells

We investigated the ability of CD8+ T cells to inhibit hepatitis C virus (HCV) replication in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA. Removal of CD8+ T cells from these PBMCs resulted in detection of HCV RNA, and depletion of CD8+ T cells from PBMCs that had detectable HCV RNA amplified HCV replication. Reconstitution of CD8- PBMCs with autologous CD8+ T cells led to inhibition of HCV replication. Interferon-gamma produced by CD8+ T cells was partially responsible for CD8+ T cell-mediated noncytotoxic anti-HCV activity in PBMCs. This noncytotoxic anti-HCV activity was confirmed in HCV replicon cells. Supernatants from CD8+ T cell cultures inhibited HCV RNA expression in the replicon cells. These findings may have important implications for the immunopathogenesis of HCV in both immune and hepatic cells and are relevant to the development of host innate immunity-based anti-HCV interventions.     

In vitro immune responses to hepatitis B surface antigens S and preS2 during acute infection by hepatitis B virus in humans

This study evaluated the in vitro immune responses to different components of the hepatitis B surface antigen (HBsAg) over the course of acute hepatitis B virus (HBV) infection. Early in the convalescent phase of infection, while HBsAg was present in the serum, peripheral blood mononuclear cells (PBMCs) were stimulated with preS2 peptide or hepatitis B surface protein. Specific IgG directed to different components of HBsAg was produced without a polyclonal increase in total IgG production. Stimulation with preS2 peptide produced IgG to the preS2 peptide (anti-preS2) and to the S antigen (anti-HBs). Hepatitis B surface protein stimulation produced anti-HBs but not anti-preS2. After this initial reactive phase, the PBMCs did not produce specific antibody when stimulated with either component of HBsAg; this effect lasted greater than 1 year. At some time 1-2 years after acute infection, the pattern of in vitro S antigen- and preS2 antigen-stimulated anti-HBs response reemerged in the PBMCs. Reemergence of sustained preS2 peptide-stimulated anti-preS2 response was not observed.