ABSTRACT
Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.
Subject(s)
Cell Separation/methods , Flow Cytometry/methods , CD28 Antigens/immunology , CD3 Complex/immunology , Carbocyanines , Cell Division , Cell Survival , DNA , Dactinomycin/analogs & derivatives , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique , Fluorescent Dyes , Humans , Lymphocyte Subsets/cytology , Lymphocyte Subsets/immunology , Staining and Labeling/methods , Titrimetry , Tumor Cells, CulturedABSTRACT
Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules. A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.
Subject(s)
Antigens/analysis , Flow Cytometry/methods , Cells/cytology , Cells/immunology , Fluorescent Antibody Technique , Indicators and ReagentsABSTRACT
OBJECTIVE: The pathogenesis of human disease is often multifactorial. For fatal diseases, it is common to combine survival information in relation to different aspects of pathogenesis. Here we explored a scoring system for HIV disease markers that combines measures of immunodeficiency (CD4 cell count), plasma viral burden, and immune activation (CD38 expression on CD8 T cells) DESIGN: Observational data from 252 HIV-infected individuals from the Multicenter AIDS Cohort Study (MACS) was used for model development. METHODS: A statistical model was used to develop a system that related marker values to the outcomes of clinical AIDS or death. RESULTS: Mathematical formulae were derived to calculate AIDS and death progression scores. These scores give the number of days at which there is a 50% probability that a person may develop AIDS or die. Graphs were constructed that can be used to determine the probability that a person will be AIDS-free or alive for times between 6 months and 4 years. The model was valid when tested on data from 240 additional people from the MACS cohort. CONCLUSIONS: These formulae and graphs are a convenient way for individuals and their physicians to apply existing MACS cohort data to HIV disease markers in order to estimate probabilities of progression to AIDS or death. Further investigation is needed to determine whether modifications of the formulae are required to predict outcome accurately in those patients on highly active antiretroviral therapy or in other demographic groups.
Subject(s)
Acquired Immunodeficiency Syndrome/diagnosis , HIV Infections/diagnosis , Acquired Immunodeficiency Syndrome/immunology , Acquired Immunodeficiency Syndrome/mortality , Acquired Immunodeficiency Syndrome/virology , Biomarkers/analysis , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cohort Studies , Disease Progression , Disease-Free Survival , HIV Infections/immunology , HIV Infections/mortality , HIV Infections/virology , Humans , Models, Biological , Predictive Value of Tests , Probability , Prognosis , Reproducibility of Results , Survival Rate , Time Factors , Viral Load , Viremia/diagnosis , Viremia/immunology , Viremia/pathology , Viremia/virologyABSTRACT
OBJECTIVE: To examine compartmental differences in co-receptor expression on CD4 lymphocytes between blood and gut using endoscopic biopsies. DESIGN: Mucosal and peripheral CD4 T cells from healthy controls were compared for co-receptor expression and vulnerability to infection by HIV-1. METHODS: Expression of CCR5 and CXCR4 was quantified by flow cytometry on isolated mucosal CD4 lymphocytes obtained from endoscopic biopsies and blood from healthy controls. Vulnerability to in vitro infection by both R5 and X4 strains was assessed by measuring p24. RESULTS: Biopsies yielded sufficient lymphocytes for flow cytometric characterization and infectivity studies. The percentage of mucosal CD4 T lymphocytes that expressed CCR5 and the per cell expression of CCR5 were both significantly increased compared with that in peripheral blood CD4 T lymphocytes. CXCR4 was expressed on the majority of CD4 lymphocytes in both compartments. In vitro infection of mucosal mononuclear cells supported greater viral replication of both R5 and X4 strains than peripheral blood mononuclear cells. CONCLUSIONS: Enhanced expression of CXCR4 and CCR5 on CD4 lymphocytes in normal intestinal mucosa predicts increased vulnerability to infection by both R5 and X4 HIV-1. Endoscopic biopsies provide a useful mucosal tissue sampling technique to identify compartmental immunologic differences that may be exploited by HIV-1 in establishing initial mucosal infection.
Subject(s)
HIV-1 , Intestinal Mucosa/immunology , Receptors, HIV/physiology , T-Lymphocytes/metabolism , Biopsy , CD4 Antigens/metabolism , Flow Cytometry , HIV Core Protein p24/metabolism , Humans , In Vitro Techniques , Intestinal Mucosa/virology , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Receptors, HIV/metabolism , T-Lymphocytes/virology , Time FactorsABSTRACT
RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta are human immunodeficiency virus (HIV) suppressor factors by virtue of their ability to compete with HIV for access to cell surface R5. Their ability to block HIV infection in vitro is unequivocal; however, their role as HIV suppressor factors in vivo is not firmly established. We therefore conducted a study to test the hypothesis that production of these factors in vitro was a correlate of decreased virus burden in vivo. Moreover, we asked whether higher beta chemokine production could be demonstrated with cells from people who are R5D32 heterozygotes, compared with people who are R5 wild-type homozygotes. Our data support the thesis that RANTES, MIP-1alpha, and MIP-1beta production is associated with decreased in vivo virus load. Moreover, enhanced production of these factors may be explained in part by the genetic background of the host.
Subject(s)
Chemokine CCL5/blood , HIV Infections/immunology , HIV Infections/virology , Macrophage Inflammatory Proteins/blood , Viral Load , CD4 Lymphocyte Count , CD8-Positive T-Lymphocytes/immunology , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/genetics , Cohort Studies , Enzyme-Linked Immunosorbent Assay , HIV Infections/blood , Heterozygote , Homozygote , Humans , Macrophage Inflammatory Proteins/genetics , Protein Isoforms/blood , Protein Isoforms/genetics , RNA, Viral/blood , SurvivorsABSTRACT
CONTEXT: HIV-1 RNA and lymphocyte subset levels are the principal indications for antiretroviral treatment. Past reports have differed with regard to the effect of gender and race on these measures and in measures of disease progression. OBJECTIVE: To assess racial and gender differences in HIV-1 RNA levels and CD4+ lymphocyte decline. DESIGN: A longitudinal study based in the two largest HIV natural history cohort studies conducted in 7 metropolitan areas of the United States. RESULTS: In all, 1256 adult women and 1603 adult men for whom multiple data points were available prior to initiation of antiretroviral therapy were included. Women were more likely to be nonwhite, to have a history of injection drug use, and to have HIV-associated symptoms. After adjustment for differences in measurement method, baseline CD4+ cell count, age, and clinical symptoms, HIV-1 RNA levels were 32% to 50% lower in women than in men at CD4+ counts >200 cells/mm3 (p <.001) but not at CD4+ cell counts <200 cells/mm3. HIV-1 RNA levels were also 41% lower in nonwhites than in whites (p <.001) and 21% lower in persons reporting a prior history of injection drug use (p <.001). Women had more rapid declines in CD4+ cell counts over time than men (difference in slope of 46 cells/year) and nonwhite individuals had slower decline in CD4 cell counts than whites (difference of 39 cells/year). CONCLUSIONS: Both race and gender influence the values of HIV-1 RNA and the rate of HIV-1 disease progression as indicated by decline in CD4 cell counts over time. These effects could provide clues regarding the factors that influence HIV-disease progression and may indicate that guidelines for therapy should be adjusted for demographic characteristics.
Subject(s)
HIV Infections/virology , HIV-1/isolation & purification , RNA, Viral/analysis , Acquired Immunodeficiency Syndrome/ethnology , Acquired Immunodeficiency Syndrome/virology , Adolescent , Adult , Black or African American , Aged , CD4 Lymphocyte Count , Cities , Cohort Studies , Female , HIV Infections/ethnology , HIV Infections/immunology , HIV-1/genetics , Humans , Male , Middle Aged , Multicenter Studies as Topic , Multivariate Analysis , Reverse Transcriptase Polymerase Chain Reaction , Substance Abuse, Intravenous/immunology , Substance Abuse, Intravenous/virology , United States , White PeopleABSTRACT
A homozygous 32-bp deletion in the gene encoding CCR5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1, and heterozygosity for the deletion is associated with delayed disease progression in persons infected with HIV-1. We investigated the effect of CCR5 heterozygosity on disease progression as measured by both CD4+ T-cell count decline and the occurrence of clinical AIDS symptoms. Using a unified statistical model for CD4 count progression and AIDS development, we examined whether the effect of CCR5 heterozygosity on clinical AIDS is direct or indirect through its effect on CD4 counts. Based on data from the Multicenter AIDS Cohort Study, we noted a protective effect of CCR5 heterozygosity on both CD4 cell count progression and on AIDS occurrence. Furthermore, we found that this protective effect on the occurrence of AIDS was completely mediated through an effect on the CD4 marker. Additional adjustment for the effect of an initial viral load measurement indicate that CCR5 heterozygosity did not have predictive value for either CD4 progression or the development of AIDS beyond its association with early viral load.
Subject(s)
HIV Infections/genetics , HIV Infections/physiopathology , Receptors, CCR5/genetics , Adult , Bisexuality , CD4 Lymphocyte Count , Cohort Studies , Disease Progression , Genotype , Homosexuality, Male , Humans , Male , Prospective Studies , Risk Factors , Viral LoadABSTRACT
BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.
Subject(s)
Cell Cycle , DNA/analysis , Flow Cytometry/methods , Fluorescent Antibody Technique , RNA/analysis , Antigens, CD/immunology , Dactinomycin/analogs & derivatives , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Leukocytes , Phycocyanin , T-Lymphocytes/chemistryABSTRACT
We have utilized combination antiretroviral therapy following human immunodeficiency virus type 1-induced human CD4(+) thymocyte depletion in the SCID-hu mouse to examine the immune competence of reconstituting thymocytes which appear following administration of combination therapy. These cells express a normal distribution of T-cell receptor variable gene families and are responsive to costimulatory signals. These results suggest that normal thymic function may be restored following antiretroviral treatment.
Subject(s)
HIV Infections/drug therapy , HIV Infections/immunology , HIV-1/immunology , Thymus Gland/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Didanosine/pharmacology , Disease Models, Animal , Drug Therapy, Combination , HIV Protease Inhibitors/pharmacology , Humans , Indinavir/pharmacology , Mice , Mice, SCID , Receptors, Antigen, T-Cell, alpha-beta/genetics , Reverse Transcriptase Inhibitors/pharmacology , Thymus Gland/cytology , Zidovudine/pharmacologyABSTRACT
To identify factors associated with development of AIDS at high CD4+ cell levels a nested case-control study using data from the Multicenter AIDS Cohort Study (MACS) was conducted. HIV-1-infected men who developed AIDS with > or =300/mm3 CD4+ cells (AIDS men) were compared to men who had > or =300/mm3 of CD4+ cells, but remained AIDS free for at least 2 years. The AIDS men had higher plasma HIV-1 RNA levels (mean 10(5.02) vs. 10(4.42), p<0.01) and neopterin levels (mean 18.3 vs. 11.5 units/ml, p<0.05) before the AIDS diagnosis than did the AIDS-free men. A significantly higher proportion of the AIDS men reported genital herpes within the year prior to their initial AIDS diagnosis than did the AIDS-free men (21.9 vs. 4.4%, p<0.05). The higher viral load at relatively high CD4+ cell levels in men who subsequently developed AIDS within 6 months supports the hypothesis that elevated levels of HIV precede CD4+ decline and are the major factor in determining risk of AIDS even at high levels of CD4+ cell levels.
Subject(s)
Acquired Immunodeficiency Syndrome/pathology , CD4-Positive T-Lymphocytes/pathology , HIV-1/pathogenicity , Viral Load , Acquired Immunodeficiency Syndrome/virology , CD4 Lymphocyte Count , Case-Control Studies , Herpes Genitalis/complications , Humans , Male , Multivariate Analysis , Neopterin/blood , RNA, Viral/analysis , Statistics, NonparametricABSTRACT
We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. Furthermore, light scatter properties of cells are preserved after permeabilization; this permits gating on cell populations that differ in scatter signals on the flow cytometer. Because of the low pH of the phosphate-citrate staining buffer, Alexa488, a pH-independent green-fluorescent fluorochrome is used instead of fluorescein-isothiocyanate (FITC) for cell surface staining in combination with phycoerythrin (PE) and with allophycocyanin (APC) which are both pH insensitive. Removal of 7-AAD after staining and replacing it with non-fluorescent actinomycin D (AD) retains DNA staining and allows detection of Alexa488, PE and APC cell surface immunofluorescence without interference from fluorescent 7-AAD in solution for clear identification of cell subpopulations even after prolonged stimulation in culture. Thus, using a four-color benchtop flow cytometer, measurement of Alexa488, PE and APC three-color immunofluorescence can be combined with 7-AAD DNA content analysis. Furthermore, we demonstrate that sample storage overnight without fixation for later analysis on the flow cytometer is possible without compromising results. Application of the method to the assessment of the differential proliferative responses of lymphocyte subsets of human peripheral blood mononuclear cells (PBMC) that were costimulated with CD3 and with CD28.2 is presented.
Subject(s)
DNA/isolation & purification , Flow Cytometry/methods , Fluorescent Antibody Technique , Lymphocyte Subsets/cytology , CD28 Antigens/metabolism , CD3 Complex/metabolism , CD8-Positive T-Lymphocytes/cytology , Cell Cycle , Dactinomycin/analogs & derivatives , Fluorescent Dyes , Humans , Lymphocyte Activation , Phycocyanin , Phycoerythrin , Propidium , Receptors, Transferrin/isolation & purification , Specimen Handling , Staining and Labeling/methodsABSTRACT
BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.
Subject(s)
DNA, Neoplasm/analysis , Flow Cytometry/methods , Cell Death , Cell Division , Cell Survival , Coloring Agents/analysis , Dactinomycin/analogs & derivatives , Dactinomycin/analysis , Fluorescent Antibody Technique , Fluorescent Dyes/analysis , Humans , Leukemia, T-Cell/genetics , Leukemia, T-Cell/pathology , Leukocytes, Mononuclear/cytology , Phenotype , Propidium/analysis , Pyronine/analysis , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/cytologyABSTRACT
We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.
Subject(s)
Acquired Immunodeficiency Syndrome/diagnosis , HIV-1/isolation & purification , Polymerase Chain Reaction/methods , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/virology , Anticoagulants/pharmacology , DNA, Viral/analysis , HIV Reverse Transcriptase/analysis , Humans , Microbiological Techniques , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
Reconstituting the immune response will be critical for the survival of HIV-infected individuals once viral load is brought under control. While the adult thymus was previously thought to be relatively inactive, new data suggest it may play a role in T cell reconstitution. We examined thymopoiesis in adults up to 56 years of age and found active T cell receptor (TCR) rearrangement, generating a diverse TCR Vbeta repertoire. The resulting thymocytes are functional and are capable of responding to costimulatory signals. These data demonstrate that the adult thymus remains active late in life and contributes functional T cells to the peripheral lymphoid pool.
Subject(s)
T-Lymphocytes/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Adult , CD4-Positive T-Lymphocytes/virology , Cell Division/immunology , Cells, Cultured , Gene Rearrangement, T-Lymphocyte , Genetic Variation , HIV Infections/immunology , Humans , Lymphocyte Activation , Middle Aged , Receptors, Antigen, T-Cell/genetics , T-Lymphocyte Subsets/immunologyABSTRACT
Sera from human immunodeficiency virus-1-infected participants in the Multicenter AIDS Cohort Study were tested to assess the association between serum neutralizing antibodies (NAbs) and disease progression. Each of 14 pairs, retrospectively matched for age, sex, race, and CD4+ lymphocyte numbers early in the study, consisted of a rapid progressor (RP) who developed AIDS and a long-term nonprogressor (LTNP) who remained asymptomatic. Serum samples were drawn early, when all participants were asymptomatic, and late, when the RPs had developed clinical AIDS. The LTNPs and RPs had similar levels of NAbs against primary isolates at the early time point, indicating that NAb levels are not predictive of disease progression; at the late time point, the LTNPs had significantly higher titers because of an increase in the level of serum NAbs in the LTNPs and/or a decrease in the NAbs in the RPs. The patterns of neutralizing activity over time suggest that changes in effective NAbs against different viruses do not occur in parallel.
Subject(s)
HIV Antibodies/blood , HIV Infections/immunology , HIV-1/immunology , Adult , CD4 Lymphocyte Count , Disease Progression , HIV Long-Term Survivors , Humans , Longitudinal Studies , Male , Neutralization Tests , Retrospective Studies , Single-Blind Method , Time FactorsABSTRACT
We have previously reported that circulating effector cytotoxic CD8+ T-lymphocytes (CTLs) against HIV-1 express CD38 and HLA-DR activation antigens. In this study, we performed two series of FACS sorts to phenotype and characterize precursors of CTL effectors. First we looked at memory CTL activity against HIV-1 stimulated by antigen as well as CTL activity stimulated by CD3 mAb with regard to whether the precursors expressed CD45RA and/or CD62L. We found that the precursor cells that could be stimulated with antigen to become effectors within 7 days predominated in the CD45RA CD62L subset. However. in donors with low levels of CD8+ T-cell activation as measured by CD38 antigen expression, memory cells could also be found in the CD45RA+ CD62L+ subset. Our data indicate that reversion of memory cells to the CD45RA+ CD62L+ phenotype can occur in humans, especially in donors with low levels of virus replication and minimal CD8 + T-cell activation. Next, we looked at CD28 expression with regard to antigen specific memory cells and again found that the level of virus replication and CD8+ T-cell activation influenced the subset that contained the memory cells. In donors with high levels of virus replication, our results indicated that CTL were being actively recruited from both CD28+ and CD28 subsets, while in donors with undetectable levels of viral replication, the memory cells were entirely in the CD28 compartment.
Subject(s)
CD28 Antigens/immunology , HIV Infections/immunology , HIV-1/immunology , Immunologic Memory/immunology , L-Selectin/immunology , Leukocyte Common Antigens/immunology , T-Lymphocytes, Cytotoxic/immunology , Humans , Immunophenotyping , Male , T-Lymphocytes, Cytotoxic/classificationABSTRACT
OBJECTIVES: To use follow-up on untreated HIV-positive men to assess the prognostic information provided by baseline data on plasma HIV RNA, CD4 cell count, age, and HIV-related symptom status, separately for three specific AIDS-defining illnesses: Pneumocystis carinii pneumonia (PCP), cytomegalovirus (CMV), and Mycobacterium avium complex (MAC). METHODS: The study population were 734 HIV-positive homosexual men enrolled in the Multicenter AIDS Cohort Study, with follow-up (1984-1985 through mid-1988) restricted to the antiretroviral treatment-free and prophylaxis-free era. Baseline marker values were categorized and assessed as predictor variables in separate time-to-event analyses for each of the three specific outcomes. RESULTS: A total of 138 cases of PCP, 25 cases of CMV, and 25 cases of MAC were observed. For PCP and CMV, higher categories of HIV RNA and lower categories of CD4 cell count were associated with increased risk relative to the respective reference groups. For MAC, oral candidiasis or fever and elevated HIV RNA at baseline were the primary risk factors. Further analysis highlighted the importance of monitoring HIV RNA levels in addition to CD4 cell counts when evaluating patients' risk of developing PCP. CONCLUSIONS: In the absence of treatment, plasma HIV RNA levels provide prognostic information about the risk of these three specific AIDS-defining illnesses, independently of the CD4 cell count. These data provide a useful reference as researchers investigate changing patterns in the incidence and predictors of opportunistic infections in the era of increasingly active antiretroviral therapies.
Subject(s)
AIDS-Related Opportunistic Infections/physiopathology , HIV Infections/physiopathology , RNA, Viral/blood , Viral Load , Adult , CD4 Lymphocyte Count , Cohort Studies , Cytomegalovirus Infections/physiopathology , Disease Progression , HIV/isolation & purification , Humans , Male , Mycobacterium avium Complex , Mycobacterium avium-intracellulare Infection/physiopathology , Pneumonia, Pneumocystis/physiopathology , Prognosis , Regression Analysis , Risk Factors , Viremia/virologyABSTRACT
To define predictors of survival time in late human immunodeficiency virus type 1 (HIV-1) disease, long- and short-duration survivors were studied after their CD4+ T cells fell to =50/mm3. Immune activation of CD4+ and CD8+ T cells, as measured by elevated cell surface expression of CD38 antigen, was strongly associated with shorter subsequent survival (P=.002). The naive CD45RA+CD62L+ T cell reserve was low in all subjects and did not predict survival (P=.34 for CD4+ and.08 for CD8+ cells). Higher virus burden correlated with CD8+ but not CD4+ cell activation and, after correcting for multiple comparisons, was not associated with shorter survival (P=.02). All of the patients' viruses used CCR5, CXCR4, or both, and coreceptor usage did not predict survival (P=. 27). Through mechanisms apparently unrelated to higher virus burden, immune activation is a major determinant of survival in advanced HIV-1 disease.
Subject(s)
Acquired Immunodeficiency Syndrome/mortality , Antigens, CD , HIV-1 , Lymphocyte Activation , RNA, Viral/blood , Receptors, CCR5/physiology , Receptors, CXCR4/physiology , T-Lymphocytes/immunology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Acquired Immunodeficiency Syndrome/immunology , Acquired Immunodeficiency Syndrome/virology , Adult , Antigens, Differentiation/analysis , CD4 Lymphocyte Count , HLA-DR Antigens/analysis , Humans , Male , Membrane Glycoproteins , Middle Aged , NAD+ Nucleosidase/analysisABSTRACT
The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 10(7) cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 x 10(6) (50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4(+) lymphocytes. Storing 10(7) PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals.