Enzymatic amplification of the human immunodeficiency virus in peripheral blood mononuclear cells from pediatric patients

The presence of the human immunodeficiency virus type 1 (HIV) provirus was assessed in peripheral blood mononuclear cells (PBMCs) from 27 offspring of HIV-infected mothers, 12 of these mothers, and 4 HIV-uninfected mother-infant control pairs. Enzymatic amplification of specific conserved regions of the gag and env genes was performed directly in PBMC lysates using the polymerase chain reaction (PCR) technique. The enzymatically amplified gene products were evaluated using the oligomer hybridization detection procedure. PBMCs from infected infants (as determined by Centers for Disease Control clinical criteria) and from HIV-infected mothers manifested a characteristic HIV oligomer hybridization product. Clinically uninfected seropositive infants with declining HIV antibody titers and infants who became seronegative lacked an enzymatically amplified HIV gene product. These preliminary data indicate that PCR is a valuable diagnostic technique to detect or exclude HIV infection in young infants and children.     

Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells.

Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha-mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia.     

Impaired cytokine production by peripheral blood mononuclear cells in type 1 diabetic patients

AIMS: The objective of the present investigation was to study the production of IL-1, IL-6, IL-10, IFNgamma and TNFalpha in cultures of peripheral blood mononuclear cells (PBMC) taken from type 1 diabetic patients with inadequate metabolic control. METHODS: Seventeen type 1 diabetic patients and a gender- and age-matched group of 17 healthy individuals were studied. PBMC cultures were stimulated with phytohemagglutinin (PHA; 20 microg/ml) and lipopolysaccharide (LPS; 10 microg/ml), and enzyme immunoassay (Elisa) was used to measure IL-1, IL-6, IL-10, IFNgamma and TNFalpha in the cell-culture supernatants. RESULTS: IFNgamma levels in PHA-stimulated cultures were lower in the type 1 diabetics than in the non-diabetic controls (P<0.0001) while, in contrast, IL-10 levels were increased in the PHA-stimulated culture supernatants of the diabetics compared with the controls (P<0.0001). In addition, supernatant levels of the cytokines IL-1, IL-6 and TNFalpha released in the presence of LPS in the cell cultures from the diabetic patients were significantly lower than in the non-diabetic subjects (P<0.0001, P<0.0001 and P<0.03, respectively). CONCLUSIONS: The impaired production of IL-1, IL-6, TNFalpha and IFNgamma, and the increased production of IL-10, in PBMC cultures from type 1 diabetics with inadequate metabolic control compared with healthy subjects may be an indication of a deficiency in mononuclear cell activation and, consequently, a deficient immune cellular adaptive response that, in turn, may be the cause of the increased incidence of infections in people with type 1 diabetes.     

Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro

Because helminth infections and human immunodeficiency virus (HIV) coexist in areas where the spread of AIDS is most dramatic, their in vitro interaction was explored. Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with filarial infections (n=24) and from unexposed control subjects (n=12) were depleted of CD8 T cells and were infected with macrophage (M)- and T cell-tropic viruses. A trend toward increased HIV replication in PBMC from filaria-infected patients was observed. Furthermore, PBMC from 6 filaria-infected patients before antifilarial treatment were significantly more susceptible to replication of M-tropic virus than their posttreatment PBMC (P=.03). No intergroup differences were found in the surface expression of HLA-DR, CD25, CCR5, CXCR4, CCR3 on CD4 T cells, or monocytes before infection. PBMC from filaria-infected patients produced less RANTES (P=.02) but more intracellular interleukin-4 than those of control subjects. Thus, PBMC from persons with filarial infections appear to have enhanced susceptibility to HIV-1 infection mediated by an undetermined mechanism.     

Human immunodeficiency virus type 1 RNA in peripheral blood mononuclear cells of patients receiving prolonged highly active antiretroviral therapy.

The levels of early spliced mRNA and genomic RNA of human immunodeficiency virus (HIV) type 1 in peripheral blood mononuclear cells (PBMC) of 14 patients who were receiving highly active combination antiretroviral therapy for > or =116 weeks were determined. The level of viral genomic RNA was below the level of detection in the plasma of these patients (<50 copies/mL), but cell-associated viral tat, rev, and nef mRNA were detected in 86% (12 of 14) of the patients. Cell-associated viral genomic RNA was detected in 57% (8 of 14) of the patients. Early viral spliced mRNA was detected in the PBMC of all patients who had positive results of testing for HIV-1 genomic RNA, and the level of viral genomic RNA in these patients was 34-2214 copies per 10(6) cells.     

High production of interferon gamma but not interleukin-2 by human T-lymphotropic virus type I-infected peripheral blood mononuclear cells

The transactivator protein of human T-lymphotropic virus I (HTLV-I), Tax, has been associated with the up-regulation of several host cell genes, including interleukin 2 (IL-2), the IL-2 receptor-alpha (IL-2Ralpha) chain (CD25), interferon gamma (IFN-gamma), and tumor necrosis factor (TNF). It has been proposed that an IL-2/CD25 autocrine loop plays a part in maintaining the very high proviral loads often found in HTLV-I infection. Furthermore, abnormal production of inflammatory cytokines might contribute to the pathogenesis of the inflammatory diseases associated with HTLV-I infection. However, there has been no study of the expression of these genes in freshly isolated peripheral blood mononuclear cells (PBMCs) naturally infected with HTLV-I. In the present study, flow cytometry was used to determine which cytokines are produced by freshly isolated PBMCs that spontaneously express the HTLV-I Tax protein. Surprisingly, the results show that intracellular Tax expression is associated with rapid up-regulation of IFN-gamma but not TNF or IL-2. A proportion of HTLV-I-infected cells express both IFN-gamma and the surface markers of effector memory cells. Such cells are capable of migration through peripheral tissues and could therefore contribute to the inflammation seen in diseases such as HTLV-I-associated myelopathy/tropical spastic paraparesis. (Blood. 2001;98:721-726)     

Ganglioside GD1a enhances immunoglobulin production by human peripheral blood mononuclear cells

We previously reported that ganglioside GD1a greatly enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC) in vitro. We herein examined the mechanism for the stimulatory effect of GD1a.PBMC from healthy volunteers were cultured with GD1a. The amounts of IgG, IgM, and IgA and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assays. Proliferation was determined by [3H] thymidine uptake.GD1a at 10(-6) M increased IgG, IgM, and IgA production by PBMC 2.10-fold, 2.10-fold, and 2.23-fold above the control values, respectively. GD1a did not affect the proliferation and viability of PBMC. GD1a did not alter Ig production of B cells alone. Anti-interleukin-6 (IL-6) or anti-IL-10 antibody each partially blocked the GD1a-induced enhancement of Ig production by PBMC, and the addition of both antibodies completely blocked the enhancement. GD1a increased IL-6 and IL-10 production of monocytes without altering those of T cells or B cells. The supernatant from GD1a-treated monocytes enhanced B cell Ig production to a greater extent than that from medium-treated monocytes. The supernatant-mediated effect of GD1a was partially blocked by anti-IL-6 or anti-IL-10 antibody, and the addition of both antibodies completely blocked the GD1a effect. GD1a-induced increases of IL-6 and IL-10 production in monocytes were both blocked by Ca(2)+/calmodulin (CaM)-dependent phosphodiesterase (PDE) inhibitors, 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetin, but not by other signal-transducing enzyme inhibitors. The culture with GD1a enhanced Ca(2)+/CaM-dependent PDE activity in monocytes.These results suggest that GD1a may indirectly enhance B cell Ig production in whole PBMC by increasing IL-6 and IL-10 production of monocytes via promoting Ca(2)+/CaM-dependent PDE activity.     

Ganglioside GD1b supresses immunoglobulin production by human peripheral blood mononuclear cells.

Gangliosides are sialic acid-containing glycolipids, that have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC). GD1b was one of the inhibitory gangliosides. In this study, we further examined the mechanism for the inhibitory effect of GD1b. The inhibitory effect of GD1b was revealed at 0.1 microM, increased dose dependently, and was maximized at 10 microM, which reduced spontaneous IgG, IgM, and IgA production of human PBMC by 50.5%, 52.0%, and 48.3% compared with controls, respectively. GD1b did not affect the proliferation and viability of PBMC. GD1b did not alter Ig production of B cells alone. Interleukin 6 (IL-6) and IL-10 each partially reversed the GD1b-induced inhibition of Ig production by PBMC, and the addition of both cytokines completely reversed the inhibition. When endogenous IL-6 and IL-10 were neutralized by specific antibodies, GD1b did not reveal inhibitory effects on the Ig production. GD1b inhibited IL-6 and IL-10 production of CD4+ T cells, without affecting those of CD8+ T cells, monocytes, or B cells. When CD4+ T cells were preincubated with GD1b and washed and cultured with B cells and monocytes, Ig production was also suppressed. These results suggest that GD1b may indirectly suppress Ig production of B cells in whole PBMC by reducing IL-6 and IL-10 production of CD4+ T cells. GD1b may act as an important inhibitor of human humoral immune responses.     

Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.

Gene-expression modulator 91 (GEM91) is a 25-nt antisense oligodeoxynucleotide phosphorothioate complementary to the Gag mRNA of human immunodeficiency virus type 1 (HIV-1). Cellular uptake and intracellular distribution of GEM91 within cells suggest that this oligomer is readily available for antisense activity. GEM91 inhibited HIV-1 replication in a dose-dependent and sequence-specific manner. In a comparative study, 2 microM GEM91 was as effective as 5 microM 3'-azido-3'-deoxythymidine in blocking virus replication during the 28-day treatment of an HIV-1-infected T-cell line. GEM91 also completely inhibited (> 99%) the growth of three different HIV-1 isolates in primary lymphocytes and prevented the cytopathic effect of the virus in primary CD4+ T cells. Similarly, treatment with GEM91 for 3 weeks of HIV-1/BaL-infected primary macrophages blocked virus replication. Based on GEM91 anti-HIV-activity, safety, and pharmacokinetic profile in animals, a clinical trial was started using this compound as an antisense oligonucleotide drug for the treatment of the acquired immunodeficiency syndrome.     

Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells

OBJECTIVES: To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC). METHODS: In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone. RESULTS: Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p<0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p<0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p<0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p<0.005) and PBMC-hyaluronan binding (p<0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment. CONCLUSIONS: LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.     

Human immunodeficiency virus type 1 modulates telomerase activity in peripheral blood lymphocytes

The effect of human immunodeficiency virus type 1 (HIV-1) on telomerase activity in peripheral blood lymphocytes (PBL) was examined. Telomerase is an enzyme that is involved in mechanisms that control cell life span and replicative potential. HIV-1 reduced telomerase activity in in vitro-infected PBL and impaired enzyme activation upon cell stimulation. Telomerase activity was significantly lower in PBL from 23 HIV-1-infected patients than in PBL from healthy donors and significantly increased during highly active antiretroviral therapy (HAART) in 10 patients who had both a virological and an immunological response and in 5 and 8 patients with a virological or an immunological response, respectively. Further analyses of fractionated cells revealed that telomerase activity increased mainly in CD4(+) lymphocytes. Overall, these findings demonstrate that HIV-1 infection down-modulates telomerase activity and suggest that both the HIV-1 decline and immunorestoration in response to HAART contribute to increased telomerase activity in CD4(+) lymphocytes.     

Inhibition of human immunodeficiency virus type 1 replication in human mononuclear blood cells by the iron chelators deferoxamine, deferiprone, and bleomycin

Replication of human immunodeficiency virus type 1 (HIV-1) can be influenced by iron. Hence, decreasing the availability of iron may inhibit HIV-1 replication. Deferoxamine and deferiprone, both forming catalytically inactive iron-chelator complexes, and bleomycin, by use of which iron catalyzes oxidative nucleic acid destruction, were investigated. Expression of p24 antigen in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL) was reduced by all 3 iron chelators. In PBL, p24 reduction was mirrored by a decrease in proliferation after incubation with deferoxamine or deferiprone, suggesting that viral inhibition is closely linked to a decrease in cellular proliferation. In contrast, clinically relevant bleomycin concentrations reduced p24 levels by approximately 50% without affecting proliferation. When deferoxamine and the nucleoside analogue dideoxyinosine were used in combination, they acted synergistically in inhibiting HIV-1 replication. These observations suggest that iron chelators with different mechanisms of action could be of additional benefit in antiretroviral combination therapy.     

Thalidomide inhibits the replication of human immunodeficiency virus type 1.

Thalidomide, a selective inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, suppresses the activation of latent human immunodeficiency virus type 1 (HIV-1) in a monocytoid (U1) line. The inhibition is dose dependent and occurs after exposure of the cells to recombinant TNF-alpha, phorbol myristate acetate, lipopolysaccharide, and other cytokine combinations. Associated with HIV-1 inhibition is a reduction in agonist-induced TNF-alpha protein and mRNA production. Thalidomide inhibition of virus replication in the phorbol myristate acetate- and recombinant TNF-alpha-stimulated T-cell line ACH-2 is not observed. The presence of thalidomide also inhibits the activation of virus in the peripheral blood mononuclear cells of 16 out of 17 patients with advanced HIV-1 infection and AIDS. These results suggest the use of thalidomide in a clinical setting to inhibit both virus replication and the TNF-alpha-induced systemic toxicity of HIV-1 and opportunistic infections.     

Interleukin-4 inhibits the increased production of vascular endothelial growth factor by peripheral blood mononuclear cells in patients with inflammatory bowel disease

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF), a potent angiogenic, permeability-enhancing cytokine plays an important role in tissue repair and chronic inflammatory disorders. Peripheral blood mononuclear cells (PBMCs) and the inflamed mucosa have been demonstrated to be main sources of the recently described circulating VEGF in patients with inflammatory bowel disease (IBD). There is no current information about the influence of immunoregulatory cytokines on VEGF in IBD. The present study examines the effect of interleukin-4 on the increased VEGF production of PBMCs in patients with IBD. METHODOLOGY: Unstimulated PBMCs from 17 patients with Crohn's disease, 16 patients with ulcerative colitis and 8 healthy controls were cultured with or without IL-4. VEGF production was measured in the supernatant using an enzyme-linked immunosorbent assay. RESULTS: IL-4 led to a significant reduction of the VEGF production by PBMCs of both active Crohn's disease patients (471.7 +/- 377.5 pg/mL vs. 208.2 +/- 123.2 pg/mL, P = 0.018, n = 7) and active ulcerative colitis patients (177.1 +/- 79.4 pg/mL vs. 87.4 +/- 77.2 pg/mL, P = 0.008, n = 9). IL-4 inhibited significantly the VEGF production by PBMCs of patients with inactive Crohn's disease (179.2 +/- 133.9 pg/mL vs. 87.7 +/- 56.6 pg/mL, P = 0.005, n = 10). There was no significant difference of VEGF release by PBMCs cultured with IL-4 in patients with active Crohn's disease or active ulcerative colitis compared with PBMCs cultured without IL-4 in patients with inactive disease and healthy controls (112.6 +/- 41.9 pg/mL, n = 8). CONCLUSIONS: IL-4 has been shown to reduce the increased VEGF production of PBMCs in patients with IBD to normal levels. The known defective immunosuppressive effect of IL-4 in IBD may contribute to the pathogenic cascade leading to inflammation by VEGF mediated mechanisms.     

Apoptosis induced by in vitro infection with simian-human immunodeficiency chimeric virus in macaque and human peripheral blood mononuclear cells.

We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.     

The human immunodeficiency virus type-1 Tat protein upregulates Bcl-2 gene expression in Jurkat T-cell lines and primary peripheral blood mononuclear cells

The regulatory Tat protein of human immunodeficiency virus type-1 (HIV- 1) exerts a pleyotropic activity on the survival and proliferation of different cell types in culture. In this report, we investigated the effect of either endogenous or exogenous Tat on Bcl-2 proto-oncogene expression and cell survival in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Stable and transient transfections of Jurkat cells with the cDNA of tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) stimulated CAT activity and showed an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to tat, because Jurkat cells transfected with the cDNA of tat in antisense orientation, tat carrying a mutation in the amino acid cys22-gly22, or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells. We also observed a specific correlation between tat-induced Bcl-2 gene expression and inhibition of apoptosis induced by serum withdrawal. Our results suggest that the structural integrity of the activation domain of Tat was required for the promotion of the Bcl-2 promoter and Jurkat cell survival, because a single mutation in the aminoacid cys22 was sufficient to completely block the upregulation of Bcl-2 and inhibition of apoptosis. Moreover, picomolar concentrations of native or recombinant Tat were able to upregulate Bcl-2 expression both in Jurkat and primary peripheral blood mononuclear cells, suggesting that extracellular Tat, actively released by infected cells, may also play a significant role in suppressing apoptosis. An aberrant cell survival of lymphoid cells consequent to the upregulation of Bcl-2 may represent an additional pathogenetic mechanism that could help explain both the dysregulated immune response and the frequent occurrence of hyperplastic/neoplastic disorders in HIV- 1-seropositive individuals.