Investigation of varicella-zoster virus DNA in lymphocyte subpopulations by quantitative PCR assay

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real-time quantitative polymerase chain reaction assay. Varicella-zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.     

Roles of macrophages in measles virus infection of genetically modified mice

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.     

Detection of Hepatitis B Virus (HBV) Genomes and HBV Drug Resistant Variants by Deep Sequencing Analysis of HBV Genomes in Immune Cell Subsets of HBV Mono-Infected and/or Human Immunodeficiency Virus Type-1 (HIV-1) and HBV Co-Infected Individuals

The hepatitis B virus (HBV) and the human immunodeficiency virus type 1 (HIV-1) can infect cells of the lymphatic system. It is unknown whether HIV-1 co-infection impacts infection of peripheral blood mononuclear cell (PBMC) subsets by the HBV. Aims To compare the detection of HBV genomes and HBV sequences in unsorted PBMCs and subsets (i.e., CD4+ T, CD8+ T, CD14+ monocytes, CD19+ B, CD56+ NK cells) in HBV mono-infected vs. HBV/HIV-1 co-infected individuals. Methods Total PBMC and subsets isolated from 14 HBV mono-infected (4/14 before and after anti-HBV therapy) and 6 HBV/HIV-1 co-infected individuals (5/6 consistently on dual active anti-HBV/HIV therapy) were tested for HBV genomes, including replication indicative HBV covalently closed circular (ccc)-DNA, by nested PCR/nucleic hybridization and/or quantitative PCR. In CD4+, and/or CD56+ subsets from two HBV monoinfected cases, the HBV polymerase/overlapping surface region was analyzed by next generation sequencing. Results All analyzed whole PBMC from HBV monoinfected and HBV/HIV coinfected individuals were HBV genome positive. Similarly, HBV DNA was detected in all target PBMC subsets regardless of antiviral therapy, but was absent from the CD4+ T cell subset from all HBV/HIV-1 positive cases (P<0.04). In the CD4+ and CD56+ subset of 2 HBV monoinfected cases on tenofovir therapy, mutations at residues associated with drug resistance and/or immune escape (i.e., G145R) were detected in a minor percentage of the population. Summary HBV genomes and drug resistant variants were detectable in PBMC subsets from HBV mono-infected individuals. The HBV replicates in PBMC subsets of HBV/HIV-1 patients except the CD4+ T cell subpopulation.     

Nipah virus infects specific subsets of porcine peripheral blood mononuclear cells

Nipah virus (NiV), a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC) in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166) highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.     

SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutières syndrome are highly susceptible to HIV-1 infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.     

Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.

The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.     

Induction of IFN-alpha in peripheral blood mononuclear cells by HIV-infected monocytes. Restricted antiviral activity of the HIV-induced IFN.

PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV-infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN-alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20-fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease.     

Abrogation of in vitro suppression of human immunodeficiency virus type 1 (HIV-1) replication mediated by CD8+ T lymphocytes of asymptomatic HIV-1 carriers by staphylococcal enterotoxin B and phorbol esters through induction of tumor necrosis factor alpha.

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (AC) suppress HIV-1 replication in vitro. Failure of host defense mechanisms and increased virus proliferation are associated with disease progression. The exact mechanisms inducing these changes at the advanced stage of the disease are still obscure. In this study, we searched for experimental conditions favoring the abrogation of the suppression of viral replication in peripheral blood mononuclear cells (PBMC) of AC by using various pharmacological and biological probes modifying cell activation. Among such agents, staphylococcal enterotoxin B (SEB) and phorbol 12-myristate 13-acetate (PMA) markedly increased otherwise low levels of HIV-1 replication in cultures of phytohemagglutinin-stimulated AC PBMC following in vitro HIV-1 LAI infection. A similar but less pronounced virus induction was also observed in macrophage-tropic HIV-1. Individual pretreatment of CD4+ and CD8+ PBMC fractions with these agents caused a reduction in CD8+ cell proliferation and enhanced HIV-1 replication in CD4+ cells. SEB- and PMA-mediated augmentation of HIV-1 replication in AC PBMC was significantly blocked by neutralizing antibody to tumor necrosis factor-alpha (TNF-alpha), although recombinant TNF-alpha alone failed to reproduce the effects of SEB or PMA. Our results suggest that the induction of TNF-alpha may be one of the mechanisms that overcomes the CD8+-induced suppression of HIV-1 replication in AC and that it may induce HIV-1 replication.     

Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status

Background

Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

Method

The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

Results

Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

Conclusions

B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

An initial in vitro investigation into the potential therapeutic use of SupT1 cells to prevent AIDS in HIV-seropositive individuals

HIV infection usually leads to a progressive decline in number and functionality of CD4+ T lymphocytes, resulting in AIDS development. In this study, I investigated the strategy of using inoculated SupT1 cells to move infection from HIV-1 X4 strains toward the inoculated cells, which should theoretically prevent infection and depletion of normal CD4+ T cells, preventing the development of AIDS-related pathologies. Interestingly, the persistent in vitro replication in SupT1 cells renders the virus less cytopathic and more sensitive to antibody-mediated neutralization, suggesting that replication of the virus in the inoculated SupT1 cells may have a vaccination effect in the long run. In order to mimic the scenario of a therapy in which SupT1 cells are inoculated in an HIV-seropositive patient, I used infected SupT1/PBMC cocultures and a series of control experiments. Infections were done with equal amounts of the wild type HIV-1 LAI virus. The SupT1 CD4+CD8+ T cell population was distinguished from the PBMC CD4+CD8- T cell population by FACS analysis. The results of this study show that the virus-mediated killing of primary CD4+ T cells in the SupT1/PBMC cocultures was significantly delayed, suggesting that the preferential infection of SupT1 cells can induce the virus to spare primary CD4+ T cells from infection and depletion. The preferential infection of SupT1 cells can be explained by the higher viral tropism for the SupT1 cell line. In conclusion, this study demonstrates that it's possible in an in vitro system to use SupT1 cells to prevent HIV infection of primary CD4+ T cells, suggesting that further exploration of the SupT1 cell line as a cell-based therapy against HIV-1 may prove worthwhile.     

Simian immunodeficiency virus is restricted to a subset of blood CD4+ lymphocytes that includes memory cells

HIV and the related simian immunodeficiency virus (SIV), which causes AIDS in macaques, infect only a small percentage of CD4+ lymphocytes at any point during the disease. We have identified three distinct cellular phenotypes within the CD4+ subpopulation in macaques, based on cell surface expression of CD44 and CD45R, which putatively represent successive stages of postthymic proliferation and functional maturation. Two of these subsets, CD44hi CD45R+, which contained virtually all circulating cells in cycle, and CD44hi CD45R-, which was noncycling and has been linked to immunologic memory, were selectively depleted in SIV-infected animals at an asymptomatic stage of disease. To test whether SIV infection was restricted to cells with this phenotype in vivo, we used the polymerase chain reaction to sensitively detect SIV DNA in purified subpopulations of CD4+ lymphocytes. We found that SIV exclusively infected blood lymphocytes expressing high levels of CD44. Within this subset infection occurred not only in the fraction containing actively proliferating cells (CD45R+), but also in resting, putative memory cells (CD45R-). These data directly demonstrate that cellular maturation stages of normal postthymic T lymphocyte differentiation are important factors in permitting lentivirus infection in vivo, and that noncycling, memory T cells may be a reservoir for SIV.     

Simian immunodeficiency virus infection of CD8+ lymphocytes in vivo.

To determine the lymphoid target cells of simian immunodeficiency virus (SIV) in vivo, peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) were positively selected (>97% purity) for surface expression of CD4, CD8, or CD20 and then analyzed for SIV provirus using semiquantitative DNA amplification. We found provirus in CD4+ and CD8+ lymphocytes but none in CD20+ lymphocytes. During acute SIV infection (< or = 214 days postinoculation), the percentage of PBL and LNL CD4+ cells containing proviral DNA ranged from 0.2 to 20% and from 0.2 to 2%, respectively. Proviral burden in the CD8+ population of either PBL or LNL ranged from 0.01 to 0.2%. Virus isolation by cocultivation was positive for both CD4+ and CD8+ purified populations. No difference in proviral burden was observed between PBL and LNL subsets during acute SIV infection. Up to 19.4% of positively selected CD8+ cells also expressed CD4, and thus the provirus may reside within a dual-positive population. This dual-positive population may represent activated lymphocytes that are particularly susceptible to infection and may provide an opportunity for virus entry into the CD8+ CD4- lymphocytes in vivo.     

The abnormal apoptosis of T cell subsets and possible involvement of IL-10 in systemic lupus erythematosus

To study the apoptosis of lymphocyte subpopulations in systemic lupus erythematosus (SLE) patients and the possible role of IL-10 in this apoptosis involved in the pathogenesis of SLE, three color fluorescence and flow cytometry were used to investigate the early apoptosis of lymphocyte subsets from freshly separated or cultured peripheral blood mononuclear cells (PBMCs). ELISA was employed to detect the levels of IL-10 in serum and the levels of sFas and sFasL in cultured PBMC supernatants, and the results of sFas and sFasL were confirmed by real-time PCR of Fas and FasL mRNA. The results showed that in cells from SLE patients, the apoptosis of CD3+, CD4+, and CD8+ T cells was distinctly increased, and the percentage of CD4+ cells and the CD4/CD8 ratio was significantly decreased, as compared with normal controls. The apoptosis of T lymphocytes cultured with SLE serum was markedly higher than that of cells cultured with control's serum. Blockade of interleukin-10 (IL-10) activation by an anti-IL-10 antibody reduced the SLE serum induced apoptosis of CD4+ and CD8+ T cells. The levels of sFas and sFasL in the culture supernatant and Fas and FasL mRNA expressions in cultured cells were significantly higher in the SLE serum-cultured groups, but decreased evidently in the presence of the anti-IL-10 antibody. Above findings suggested that SLE cells showed abnormally high apoptosis of T lymphocytes, especially of the CD4+ subpopulation, resulting in a decreased CD4/CD8 ratio. The high percentage of apoptotic T cells in SLE patients may be related to the high levels of IL-10 in SLE serum, as IL-10 may induce the abnormally activated T cells to trigger apoptosis via the Fas-FasL pathway.     

Differential cell tropism of feline immunodeficiency virus molecular clones in vivo

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4(+) and CD8(+) lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4(+) lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4(+) and CD8(+) lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

Expression of CD154 by a simian immunodeficiency virus vector induces only transitory changes in rhesus macaques

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154.     

Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.     

Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.