HIV infection: diagnosis and disease progression evaluation.
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Antibodies, Monoclonal↗
Biomedical subjects
A Landay
Publications and source records attributed to A Landay.
Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry.
Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA-1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.
Antibodies, Monoclonal↗
Prediction of CD4 count from CD4 percentage: experience from three laboratories.
OBJECTIVE: CD4 counts have been used to monitor progression of disease in HIV infection as criteria for initiation of therapy, and to stratify and follow patients in clinical trials. Recently, the Centers for Disease Control and Prevention (CDC) has made CD4 counts part of the classification of HIV disease. Because a CD4 percentage may be the only laboratory information available, this study was initiated to determine whether the correlation between CD4 percentages and CD4 counts is sufficiently high to enable these measures to be substituted for each other. DESIGN, SETTING AND PATIENTS: One thousand consecutive CD4 measurements from the University of Washington (UW) were used to create a model that was tested using datasets of 1000 CD4 measurements each from Maryland Medical Laboratories (MML) and Rush-Presbyterian-St Luke's Medical Center (Rush). The patients were not selected for age, sex, risk group or treatment. All patients from MML and Rush were known to be HIV-positive, while the HIV status of all UW patients was unknown. RESULTS: The model predicted that a patient with a CD4 percentage > or = 14% would have a CD4 count > or = 200 x 10(6)/l(if CD4 percentage of 14% was used, 9% of patients would have a CD4 count > or = 200 x 10(6)/l), and a patient with a CD4 percentage > or = 27% would have a CD4 count > or = 500 x 10(6)/l(if CD4 percentage of 27% was used, 17% of patients would have a CD4 count > or = 500 x 10(6)/l). CONCLUSIONS: These CD4 percentage correlations may be useful when a white blood cell and lymphocyte count are not available to calculate the CD4 count.
CD4-Positive T-Lymphocytes↗
A rapid manual method for CD4+ T-cell quantitation for use in developing countries.
OBJECTIVE: To evaluate a manual method (Cytosphere) for quantifying CD4+ T-cell numbers. DESIGN: Cross-sectional study of HIV-1-seronegative and HIV-1-seropositive individuals evaluated for absolute CD4 counts by both standardized flow cytometric measurements and manual Cytosphere technology using a hemacytometer. SETTING: University research hospitals in both the United States and Africa. PATIENTS, PARTICIPANTS: Blood specimens from 382 patients were evaluated. These were broken down into 294 samples obtained from HIV-1-seropositive patients and 88 samples obtained from HIV-1-seronegative patients. INTERVENTIONS: None. OUTCOME MEASURED: Absolute CD4 cell number. RESULTS: Evaluation of samples obtained from HIV-1 patients in both the United States and Africa demonstrated an overall correlation of the Cytosphere assay with flow cytometry of 0.912 (95% confidence interval, 0.895-0.928; P < 0.001). When samples were stratified based on CD4+ T-cell counts determined by flow cytometry, the Cytosphere assay had a 96% predictive value for correctly identifying individuals with CD4 T-cell counts > 200 x 10(6)/l and a 92% predictive value for correctly identifying individuals with CD4 T-cell counts < 200 x 10(6)/l. CONCLUSIONS: This assay appears to have the potential for the quantitation of CD4 cells in the limited laboratory facilities in developing countries and to have a strong correlation with standard flow cytometric technology.
Adult↗
NIH conference. Chronic fatigue syndrome research. Definition and medical outcome assessment.
A workshop was held 18 to 19 March 1991 at the National Institutes of Health to address critical issues in research concerning the chronic fatigue syndrome (CFS). Case definition, confounding diagnoses, and medical outcome assessment by laboratory and other means were considered from the perspectives of key medical specialties involved in CFS research. It was recommended that published Centers for Disease Control (CDC) case-definition criteria be modified to exclude fewer patients from analysis because of a history of psychiatric disorder. Specific recommendations were made concerning the inclusion or exclusion of other major confounding diagnoses, and a standard panel of laboratory tests was specified for initial patient evaluation. The workshop emphasized the importance of recognizing other conditions that could explain the patient's symptoms and that may be treatable. It was viewed as essential for the investigator to screen for psychiatric disorder using a combination of self-report instruments followed by at least one structured interview to identify patients who should be excluded from studies or considered as a separate subgroup in data analysis. Because CFS is not a homogeneous abnormality and because there is no single pathogenic mechanism, research progress may depend upon delineation of these and other patient subgroups for separate data analysis. Despite preliminary data, no physical finding or laboratory test was deemed confirmatory of the diagnosis of CFS. For assessment of clinical status, investigators must rely on the use of standardized instruments for patient self-reporting of fatigue, mood disturbance, functional status, sleep disorder, global well-being, and pain. Further research is needed to develop better instruments for quantifying these domains in patients with CFS.
Centers for Disease Control and Prevention, U.S.↗
Gamma/delta T cell receptor positive T cells in the inflammatory joint: lack of association with response to soluble antigens.
In patients with inflammatory synovitis, the proliferative response by lymphocytes from synovial fluid to soluble mycobacterial antigens is enhanced relative to those from peripheral blood. Earlier studies suggested that gamma/delta T cell receptor positive (TCR+) T lymphocytes may significantly contribute to the mycobacterial-specific synovial fluid response. We therefore examined the relationship of the T cell proliferative response to Mycobacterium tuberculosis antigens and the presence of gamma/delta TCR+ T cells employing several monoclonal antibodies. No consistent increase of gamma/delta TCR+ T cells was noted in inflammatory synovial fluids or tissues. Nonetheless, lymphocytes from the majority of the synovial fluids proliferated vigorously in response to water-soluble M. tuberculosis antigens. There was no relationship between the percentage of gamma/delta TCR+ T lymphocytes and the intensity of the proliferative response. In contrast, stimulation with whole mycobacterial organisms was capable of enriching the gamma/delta TCR+ cell population obtained from the peripheral blood of tuberculosis skin test positive normal controls and from some inflammatory synovial fluids. These observations do not support a role for mycobacteria reactive gamma/delta TCR+ synovial T lymphocytes in response to soluble mycobacterial antigens or in the local pathogenesis of inflammatory synovitis.
Antigens, Bacterial↗
Absolute number versus percentage of T-helper lymphocytes in human immunodeficiency virus infection.
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Adult↗
Isolation of HIV-1 from monocytes of individuals negative by conventional culture.
To improve isolation of human immunodeficiency virus (HIV) from purified monocytic cell populations, a differential culture technique was applied to blood from HIV-seropositive individuals, culture-negative for HIV by routine culture. When 206 individuals were grouped by percentage of CD4+ lymphocytes, increases in viral isolation rates were significantly associated with declines in percentage of CD4+ cells (P less than .0001). Of 158 asymptomatic individuals, 78% had greater than 400 CD4+ lymphocytes/mm3. Only 19% were culture-positive using routine methods. Separation of peripheral blood mononuclear cells (PBMC) into purified lymphocyte and monocyte subpopulations for 12 asymptomatic patients and subsequent HIV culture of each purified subpopulation using three different indicator cell lines resulted in 100% virus recovery from purified monocytes cultured in the U-937 promonocytic cell line. Culture of purified lymphocytes in U-937 cells did not increase the isolation rate, whereas culture of patient PBMC in U-937 indicator cells and in allogeneic monocytes resulted in a 50% increase in HIV isolation. A monocytic indicator cell line added to routine culture methods may improve virus recovery from asymptomatic individuals.
Acquired Immunodeficiency Syndrome↗
Circulating human immunodeficiency virus (HIV) p24 antigen-positive lymphocytes: a flow cytometric measure of HIV infection.
Asymptomatic individuals seropositive for human immunodeficiency virus (HIV) progress in a heterogeneous fashion toward AIDS. To facilitate monitoring of disease progression and response to therapy, a rapid, new flow cytometric assay (FCA) lymphocyte p24-FCA, has been devised to quantify peripheral blood lymphocytes expressing cell-associated HIV-1 p24 antigen. Results from 55 asymptomatic, HIV-1-seropositive, serum p24 antigen-negative individuals ranged from undetectable (less than 0.1%) to 13.6% p24+ lymphocytes (mean, 2.0%). Mean values for three other groups studied were 0.1% for seronegative, viral culture-negative laboratory workers (n = 24); 4.2% for untreated patients with AIDS (n = 16); and 0.3% for AIDS patients receiving zidovudine (n = 11). Lymphocyte p24-FCA values were inversely related to the number of days to positive viral cultures and to levels of CD4+ lymphocytes. The ratio of p24+ lymphocytes to CD4+ lymphocytes may reflect the fraction of infected CD4+ lymphocytes. Lymphocyte p24-FCA determination may provide a method for monitoring response to antiretroviral therapy regardless of serum p24 antigen status.
CD4-Positive T-Lymphocytes↗
Application of flow cytometry to the study of HIV infection.
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Acquired Immunodeficiency Syndrome↗
Human herpesvirus 6 inhibits human immunodeficiency virus type 1 replication in cell culture.
The SF strain of human herpesvirus 6 (HHV-6SF) isolated from the saliva of a human immunodeficiency virus (HIV)-infected individual was shown to inhibit HIV type 1 (HIV-1) replication in both peripheral blood mononuclear cells and purified CD4+ lymphocytes. This suppression of HIV-1 replication led to decreased cytopathic effects of HIV-1 and prolonged survival of CD4+ cells in culture. Even low levels of HHV-6 added to peripheral blood mononuclear cells showed an inhibitory effect on HIV-1 replication. These results differ from those previously reported showing enhanced HIV-1 production following infection with another strain of HHV-6.
Cells, Cultured↗
Results of the flow cytometry ACTG quality control program: analysis and findings.
The AIDS Clinical Trial Group's (ACTG) Immunology Committee was charged with initiating a quality control program for all laboratories participating in the ACTG program reporting flow cytometry data. Forty-one laboratories were evaluated. This report defines the goals of this program and the subsequent findings after 19 send-outs were made. Both HIV positive volunteer donors and normal age-matched donors were used. Sample sets included both heparin and EDTA anticoagulated bloods. Laboratories were asked to report hematologic parameters as well as flow cytometry data both in percentages and absolute numbers. Results were evaluated using nonparametric statistical analysis. Robust CVs and interquartile ranges were used to define the performance of individual laboratories for each CD subset analyzed. Intralaboratory reproducibility was analyzed by paired sample sets. All laboratories were found to be able to define normal samples as normal. Seventy-five percent of the laboratories were able to define abnormal samples as abnormal. Twenty-five percent could not identify two abnormal samples as abnormal. Forty percent of the labs were found unable to reproduce paired samples within an absolute of +/- 5%. EDTA was found slightly superior to heparin in bloods evaluated by flow cytometry within 30 hr of collection. The analysis of specific histograms, questionnaires, and data analysis led to a specific set of recommendations for performance of flow cytometry studies.
Acquired Immunodeficiency Syndrome↗
Lineage and stage specificity of isotype switching in humans.
The lineage and stage specificity of human isotype switch recombination was investigated by examining the IgH gene configuration in 61 hemopoietic malignancies representing different stages of B and T cell development. An unexpectedly high frequency (20%) of IgM-producing B cell leukemias and lymphomas had undergone CH gene rearrangements and deletions consistent with attempted switch recombination. These CH gene alterations were found on productive, non-productive, and 14q+ chromosomes. These data support the concept of a non-specific (common) switch recombinase activity that is often ineffective. No evidence of such switch recombination was found in 25 mu- or mu+ pre-B cell leukemias with the single exception of a mu- pre-B leukemia in which subsets of the cells were producing gamma- or alpha-H chains. The switch recombinase activity gamma- or alpha-H chains. The switch recombinase activity may be restricted to the B cell lineage, inasmuch as CH gene deletions were not observed in T lineage malignancies.
Cell Transformation, Neoplastic↗
Monocyte functional studies in asymptomatic, human immunodeficiency disease virus (HIV)-infected individuals.
Various aspects of monocyte-associated function were evaluated in the peripheral blood mononuclear cells of male homosexuals who were infected with the human immunodeficiency disease virus (HIV). The functional assessments included indomethacin-sensitive regulation of blastogenesis and lymphokine-activated killer (LAK)-cell induction, chemiluminescent responses of mononuclear leukocytes to opsonized zymosan, and the expression of HLA-DR antigen on CD-14-positive monocytes. The results obtained demonstrate that each of these functions is abnormal in asymptomatic individuals who have HIV core antigen (p24) in their circulation. These results suggest that monocyte abnormalities which could contribute to immune dysfunction in HIV-infected patients can be detected early during the course of HIV infection and are associated with the expression of serum HIV antigen.
HIV Core Protein p24↗
Diagnosis of human immunodeficiency virus infection in seronegative homosexuals presenting with an acute viral syndrome.
Early diagnosis of acute human immunodeficiency virus (HIV) infection is difficult because patients may be seronegative for HIV at the time of presentation. We have used a serum HIV antigen (HIV-Ag) enzyme immunoassay (EIA) to diagnose acute HIV infection in four high-risk patients. The clinical syndrome in these four patients was characterized by fever (four), rash (three), myalgias-arthralgias (three), and pharyngitis (two). All patients had spontaneous resolution of their symptoms within eight to 12 days. Serum HIV antibody, as measured by a commercially available screening EIA and by Western blot analysis, was negative in all patients at time of presentation and all seroconverted on subsequent testing. Human immunodeficiency virus was isolated from two of two patients during the acute illness. Initial serum samples from all four patients were positive for HIV-Ag. Serum samples of three of four patients became negative for HIV-Ag and positive for HIV antibody. These data suggest that serum HIV-Ag detection by EIA may be useful in the diagnosis of the acute syndrome caused by HIV infection.
Acquired Immunodeficiency Syndrome↗
Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination.
The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.
B-Lymphocytes↗
HIV isolation from plasma of HIV-infected persons.
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Acquired Immunodeficiency Syndrome↗
CD16+ NK lymphoproliferative disorders: cellular and molecular characterization.
We studied the mononuclear cells obtained from 2 patients with CD16+ lymphoproliferative disorders. In both subjects, over 80% of the circulating peripheral blood mononuclear cells were CD16+, CD2+, CD7+, CD3-, CD4-, and CD8-. In 1 patient, greater than 60% of the cells expressed HLA-DR and HLA-DQ gene products. Functional analysis of the natural killer (NK) cell activity of cells from this patient demonstrated 76% killing of K562 targets at ratios as low as 1:1 effector:targets. Karyotype analysis demonstrated a deletion on the long arm of chromosome 6, supporting the contention that the lymphocytosis in this patient was due to a clonally expanded population of cells. In additional studies, Southern blot analysis of DNA extracted from cells of this patient revealed that the beta-chain of the T cell receptor was of germ line configuration. This information supports the hypothesis that the clonally expanded NK population in this patient is of a lineage distinct from T cells and represents a true NK leukemia.
Aged↗