Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells.

Optimal conditions for demonstrating the presence of infectious human immunodeficiency virus in peripheral blood mononuclear cells (PMCs) from seropositive individuals involved cocultivation of infected cells with phytohemagglutinin-stimulated PMCs from seronegative donors in the presence of 2 micrograms of Polybrene per ml. The size of the culture vessel also influenced the results; smaller numbers of infected cells were detected under conditions of increased cell density. In addition, an increased normal donor/patient PMC ratio was helpful. The cocultivation approach permitted identification of human immunodeficiency virus in over 90% of seropositive individuals with different clinical conditions. Moreover, reconstruction experiments indicated that this method allows detection of one productively infected CD4+ cell in a population of over 10(6) PMCs.     

HIV genome in peripheral blood mononuclear cells of seronegative regular sexual partners of HIV-infected subjects

We have investigated the presence of the human immunodeficiency virus (HIV) by using in situ hybridization on peripheral blood mononuclear cells (PBMCs) from seronegative regular sexual partners of HIV-infected subjects. The cells were hybridized with a 9 kilobase (kb) Sstl-Sstl lambda BH 10 probe, which was able to recognize both viral mRNA and proviral cDNA. Labeling was done by chemical insertion of an antigenic sulfone group in cytosine moieties and was visualized by a double-antibody immunohistochemical reaction. In all the subjects studied, the HIV genome was present. The HIV infected cells showed morphological aspects consistent with that of lymphocytes and monocytes. Our data suggest that the anti-HIV seronegative individuals who are regular sexual partners of HIV-infected subjects may be HIV-infected.     

Quantification of human immunodeficiency virus type 1-infected mononuclear cells in peripheral blood of seropositive subjects by newly developed flow cytometry analysis of the product of an in situ PCR assay.

The presence of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells (PBMC) of three groups (group 1, more than 500 CD4+ T cells per microliter; group 2, between 200 and 499 CD4+ T cells per microliter; group 3, fewer than 200 CD4+ T cells per microliter) of HIV-1-infected patients, in different stages of the disease, was determined by using a newly developed flow cytometry analysis of the product of in situ PCR assay and compared with other markers of viral replication (HIV-1 p24 antigenemia and viral isolation). Results showed varied percentages of HIV-1-infected PBMC, ranging from 0.6 to 20%. Patients with more than 500 CD4+ T cells per microliter showed the lowest percentage of HIV-1-infected PBMC (2.1 +/- 1.7), compared with patients with CD4+ T-cell counts of between 200 and 499 per microliter (6.5% +/- 4.1%; P < 0.001) and patients with fewer than 200 CD4+ T cells per microliter (4.9% +/- 4.7%; P < 0.05). The difference in the percentage of HIV-1-infected PBMC between group 2 and group 3 patients may in part reflect the loss of CD4+ T lymphocytes in more advanced stages of the disease. However, the results clearly indicate a striking coincidence between the fall of the CD4+ T-cell count below 400/microliter and the sharp increase in PBMC virus loading and p24 antigenemia. Since the procedure is relatively easy to perform, it could be used to monitor the evolution of HIV-1 infection and may prove a useful adjunct in tailoring therapeutic strategies.     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Microculture assay for isolation of human immunodeficiency virus type 1 and for titration of infected peripheral blood mononuclear cells.

To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologic endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patients cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.     

Use of cryopreserved normal peripheral blood lymphocytes for isolation of human immunodeficiency virus from seropositive men.

The possibility was investigated of using frozen stocks of phytohemagglutinin (PHA)-stimulated normal human peripheral blood lymphocytes (PBL) in cocultivation with human immunodeficiency virus (HIV)-infected lymphocytes for the isolation of HIV. Fresh and cryopreserved PBL from eight healthy volunteers were compared for their susceptibility to HIV infection in vitro. Fresh lymphocytes, as well as lymphocytes that were stimulated with PHA before or after cryopreservation, displayed comparable susceptibilities to HIV infection in vitro. In addition, HIV was recovered in all cases when lymphocytes stimulated with PHA before or after cryopreservation were cocultured in parallel with PBL from 15 patients with acquired immune deficiency syndrome. However, the cryopreserved PBL were less efficient in isolating HIV from asymptomatic men.     

Biological characterization of human immunodeficiency virus type 1 and type 2 mutants in human peripheral blood mononuclear cells

Summary Mutants of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), which have been shown to be infectious in established cell lines, were tested for ability to replicate and induce syncytium formation in human peripheral blood mononuclear cells (PBMC). Thevpu mutant of HIV-1 showed depressed kinetics of replication in an established T cell line, as reported previously, but in PBMC, its replication was similar to that of the wild type virus. Thevpx gene of HIV-2 was required for efficient virus propagation in PBMC, but not in an established T cell line, as previously reported. However, the growth rates of thevpx mutant in PBMC preparations from two individuals were different. The results of experiments on infection of PBMC with thevif andvpr mutants of HIV-1 and HIV-2 were essentially consistent with previous results of infection of established T cell lines. No negative effect of thenef gene products of HIV-1 and HIV-2 was observed. The abilities of the wild type virus and the mutants of HIV-1 to induce syncytium formation in both PBMC and established cell lines were similar. In contrast, neither the wild type nor any of the mutants of HIV-2 induced syncytium formation in PBMC. These results suggest that the functions of some genes can be detected only in mixed populations or primary cells such as PBMC. Studies on the roles of these genes in PBMC may provide a better understanding of their functions in vivo.     

The detection of the human immunodeficiency virus antigen in the lymphoid cells of seropositive subjects]

Cells from anti-HIV-positive persons were used in experiments for virus isolation. The RT-activity, viral antigen, nucleic acids, electron microscopic morphology, and infectivity were studied. The data presented allow a conclusion that the virus was isolated. These data confirmed the previous diagnosis of HIV infection.     

Microplate hybridization for Borna disease virus RNA in human peripheral blood mononuclear cells.

We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.     

Cultivation of HIV-1 virus on mononuclear peripheral blood cells of HIV-1 seropositive individuals

Using the method of co-cultivation with phytohaemagglutinin-stimulated lymphocytes from healthy donors, the author isolated the HIV-1 virus from peripheral mononuclear blood cells of three patients with the AIDS symptomatology and one patient with the ARC symptomatology. The presence of the virus in infected cells was proved by detection of the viral antigen p 24 in enzymatic immunoassays and in the immunofluorescence test. Three of the isolated strains were adapted to sensitive continual tissue cultures, where the isolates caused chronic infection of the cells associated with the development of a cytopathic effect. In the investigated patients no relationship was proved between viraemia and antigenaemia. The author discusses the importance of virus cultivation for laboratory diagnosis, epidemiology and research of HIV infection and AIDS.     

Inhibition of varicella-zoster virus in vitro by human peripheral blood mononuclear cells.

A functional in vitro assay of cell-mediated immunity to varicella-zoster virus (VZV) is described. This procedure uses an enzyme-linked immunosorbent assay (ELISA) to measure the inhibitory effect of human peripheral blood mononuclear cells on VZV antigen production by VZV-infected cell monolayers. When mononuclear cells from VZV-immune, tetanus-immune donors were stimulated with either VZV antigen or tetanus toxoid they reduced VZV antigen production. In contrast, mononuclear cells from VZV-nonimmune, tetanus-immune donors reduced VZV antigen only when stimulated with tetanus toxoid, but not when stimulated with VZV antigen. Cell-free supernatants recovered from the VZV inhibition assays contained the anti-VZV activity. The magnitude of the anti-VZV activity of the supernatants equalled the inhibition observed when the stimulated mononuclear cells were added to the VZV-infected monolayers. Treatment of either mononuclear cells or supernatants with anti-interferon gamma antibody indicated that their VZV inhibitory capability was largely due to the production of interferon gamma by stimulated mononuclear cells.     

Characterization of human immunodeficiency virus (HIV1C) variants in peripheral blood mononuclear cells and spermatozoa

The presence of distinct viral variants in different cells and secretions of the same person influences the transmission of HIV as well as the response to the host defense and to therapy. Sperm-associated virus is also a risk factor for sexual transmission of HIV. Characterization of the C2-V3 region of HIV1C env gene by the Heteroduplex Mobility Assay (HMA) and sequencing demonstrated the presence of distinct variants in the peripheral blood mononuclear cells (PBMCs) and the sperm of the same individual (n = 6). The translated amino acid sequences of HIV variants in the PBMCs of all the study participants (n = 12) and spermatozoa of the six participants characterized showed the presence of distinct variants with different numbers of N-linked glycosylation (NLG) sites. Infectivity of PBMCs of these persons by co-culture with PBMCs from healthy individuals as detected by the p24 levels in the culture supernatant did not show a correlation with the blood plasma viral load. Interestingly, the infectivity of the sperm samples from four of the five individuals showed positive correlation with the viral load in seminal plasma. The study suggests the presence of distinct viral variants in the sperm and PBMCs of the same person with differential infectivity, and the NLG sites may be associated with the affinity of HIV to receptor/co-receptor usages as well as affinity toward neutralizing antibodies which may influence the risk of sperm associated virus in sexual transmission of HIV and transmit the virus further to distal cells.     

Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry.

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.     

Comparison of blood collected in acid-citrate-dextrose and EDTA for use in human immunodeficiency virus peripheral blood mononuclear cell cultures

Paired blood samples collected in acid-citrate-dextrose and EDTA were compared for human immunodeficiency virus (HIV) infectivity on the day of collection or after 1 day of storage at room temperature. No significant differences between the anticoagulants were observed. Culture positivity was significantly associated with HIV RNA viral loads for both anticoagulants.     

CD4+-T-cell counts, spontaneous apoptosis, and Fas expression in peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1-infected subjects.

We examined the relationships among CD4+-T-cell counts, spontaneous apoptosis, and Fas expression among peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1 (HIV-1)-infected patients. After 2 days of incubation, propidium iodide DNA staining and flow cytometry revealed that peripheral blood mononuclear cells from subjects with the lowest CD4+-cell numbers (0 to 99/microl; n = 20) showed the highest frequency of apoptosis: 22.4% +/- 2.7% (mean +/- standard error) versus 13.8% +/- 1.2% and 12.7% +/- 1.4% among peripheral blood mononuclear cells obtained from patients with 100 to 499 CD4+ cells/microl (n = 19) and >500 CD4+ cells/microl (n = 17), respectively. Each of these means differed significantly from the mean frequency of apoptosis (6.3% +/- 0.7%) of peripheral blood mononuclear cells obtained from HIV-1-seronegative controls (P < 0.001, Student's t test). After incubation, the percentage of peripheral blood mononuclear cells expressing Fas antigen was increased for the HIV-1-infected subjects, and this was most evident for patients with more advanced disease. Among patients with fewer than 100 CD4+ cells/microl, 64.4% +/- 5.4% of peripheral blood mononuclear cells were Fas+, as opposed to 25.8% +/- 3.0% and 14.5% +/- 1.7% Fas+ cells among patients with more than 100 CD4+ cells/microl and healthy controls, respectively (P < 0.05 for each group comparison). Interestingly, in all populations, most apoptotic cells did not express Fas. Thus, apoptosis and Fas expression are increased in incubated peripheral blood mononuclear cells obtained from HIV-1-infected patients and these phenomena are enhanced as disease progresses.     

Esterase staining of human peripheral blood mononuclear cells.

Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12-18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.