Duration of sample storage dramatically alters expression of the human immunodeficiency virus coreceptors CXCR4 and CCR5.

Expression of the chemokine receptors CXCR4 and CCR5 was monitored using EDTA-anticoagulated whole blood held for different time periods prior to fluorescent-antibody staining. When left overnight CXCR4 expression on leukocytes was substantially increased, whereas CCR5 expression was reduced. The results were similar when heparin and acid-citrate-dextrose were used as anticoagulants.

Distribution of the human immunodeficiency virus coreceptors CXCR4 and CCR5 on leukocytes of persons with human immunodeficiency virus type 1 infection and pulmonary tuberculosis: implications for pathogenesis.

Expression of CXCR4 was significantly reduced from normal on all cell subsets of persons with pulmonary tuberculosis (TB group), with HIV-1 infection (HIV group), and those with both infections (HIV/TB group), except for on monocytes in the HIV group. The reductions were most notable in the two TB groups. Interestingly, the duration of antituberculosis treatment was significantly negatively correlated with the expression of CXCR4 on CD4+ and CD8+CD45RO+ cells, monocytes and NK cells, viral load, and proportions of CD38-expressing CD8+ lymphocytes, in HIV/TB patients. By contrast, CCR5 expression on most cell subsets analyzed was increased in all the disease groups, except for on monocytes in the two TB groups. There was no change in CCR5 expression on CD4+ cells when based on the disease groupings. However, higher proportions of CD4+CD45RA+ and CD8+ lymphocytes as well as B cells expressing CCR5 correlated with advancing HIV-1 disease, as did decreased proportions of CXCR4-expressing CD4+CD45RA+ cells.

Effects of anticoagulants and temperature on expression of activation markers CD11b and HLA-DR on human leukocytes.

A whole-blood model was used to evaluate the effects of temperature and anticoagulant on the expression of activation markers HLA-DR and CD11b on peripheral leukocytes. Venous blood, anticoagulated with either EDTA or heparin, was obtained from six healthy blood donors and 13 hospitalized patients (8 human immunodeficiency virus type 1-seropositive individuals with concurrent pulmonary tuberculosis and 5 patients with pneumonia). A preliminary evaluation was carried out with whole blood from two of the normal donors, and cells were stained immediately for HLA-DR and CD11b markers or stained after incubation at room temperature or 37 degreesC for 18 h with or without the addition of the cytokines gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-gamma plus GM-CSF, tumor necrosis factor beta, or interleukin-6. Of the cytokines tested, the combination of IFN-gamma and GM-CSF had the most pronounced modulation of marker expression on polymorphonuclear neutrophils (PMN), in particular, HLA-DR expression, which required induction for its detection. These cytokines were therefore used in further evaluations that considered the above-mentioned effects in the presence of disease. Results indicated that the expression of activation markers on PMN and lymphocytes in whole blood are influenced by the temperature of incubation and the choice of anticoagulant and the effects noted were dependent on (i) the particular cell surface marker, (ii) the cell type being studied, and (iii) the presence or absence of disease. It is therefore recommended that ex vivo whole-blood models for evaluating phenotype or immune function be carefully evaluated for the above-mentioned effects.

Depressed phagocytosis and oxidative burst in polymorphonuclear leukocytes from individuals with pulmonary tuberculosis with or without human immunodeficiency virus type 1 infection.

Phagocytosis and oxidative burst in whole-blood granulocytes were assessed by flow cytometry with Phagotest and Bursttest kits, respectively. Seventy individuals were included in this study: 15 healthy, normal donors, 18 human immunodeficiency virus (HIV) type 1 (HIV-1)-seropositive patients, 19 patients with pulmonary tuberculosis (TB), and 18 patients co-infected with Mycobacterium tuberculosis and HIV-1 (TB-HIV). Granulocyte phagocytosis was assessed by incubating whole blood with fluorescence-labelled Escherichia coli and measuring the proportion of granulocytes with ingested bacteria and the capacity (fluorescence intensity) of each cell to phagocytose E. coli. The percentage of granulocytes converting nonfluorescent dihydrorhodamine to fluorescent rhodamine 123 on production of reactive oxygen intermediates (ROIs) and the mean channel shift were assessed as a measure of oxidative burst. No differences in the proportion of granulocytes that were capable of phagocytosing or producing ROIs in response to E. coli were observed between any of the study groups. Phagocytosis was significantly enhanced in granulocytes from HIV-1-infected individuals. On the other hand, granulocytes from individuals infected with M. tuberculosis alone or in combination with HIV-1 had a significantly reduced capacity to phagocytose E. coli and to produce ROIs in response to E. coli as an agonist. These results provide evidence that granulocytes from individuals with pulmonary TB with or without concomitant infection with HIV-1 have an impaired ability to phagocytose and to undergo oxidative burst, possibly contributing to the enhanced susceptibility to opportunistic infections in these patients.

Identification of cell subsets expressing intracytoplasmic cytokines within HIV-1-infected lymph nodes.

OBJECTIVE: To describe the endogenous cytokine profile of HIV-1-infected lymph nodes (LN) and to identify the phenotype of individual cells expressing intracytoplasmic cytokines. DESIGN AND METHODS: Whole LN biopsies were collected from three HIV-seronegative controls and four HIV-1-positive individuals with persistent generalized lymphadenopathy. Three had established infection (Centers for Disease Control and Prevention 1993 criteria, stages A2, C1 and C3) and one was undergoing seroconversion illness. A combination of three methods was used to assess the impact of HIV-1 on LN architecture and endogenous cytokine expression. Immunocytochemistry was used to locate follicular dendritic cells (FDC), interdigitating cells and T and B cells. Reverse transcriptase-polymerase chain reaction was used to assess mRNA for interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma in collagenase-digested LN cells. Three-colour flow cytometry was used to identify intracytoplasmic cytokine expression within cell subsets. RESULTS: Germinal center (GC) hyperplasia was observed in LN from two patients with established HIV-1 infection, and the third, coinfected with Mycobacterium tuberculosis, showed extensive necrosis. In the patient undergoing seroconversion, there was an extensive FDC network within the expanded and confluent GC which covered expansive areas of the LN. There was varied expression of IL-1 beta, IL-4, IL-6, IL-10 and TNF-alpha mRNA from the four HIV-1-infected LN and the patient undergoing seroconversion showed evidence for a mixed cytokine profile, which also included IL-2 and IFN-gamma. Flow cytometry revealed intracytoplasmic IL-1 beta protein restricted to cells expressing CD19, CD21 and CD38 antigens. CONCLUSIONS: Cytokines were detected in freshly isolated HIV-1-infected LN cells without requiring an exogenous stimulus. Seroconversion was associated with an expanded FDC network within enlarged GC, bounded by defined mantle zones containing B cells. There was diverse cytokine mRNA expression and IL-1 beta protein was restricted to cells expressing B-cell-associated antigens.

CD4+ lymphocyte count in African patients co-infected with HIV and tuberculosis.

The objectives of this study were (a) to compare the CD4+ lymphocyte profiles over time of two groups of patients hospitalized for tuberculosis (TB) treatment [a group of patients with TB only (TB group) and a group dually infected by HIV and TB (HIV/TB group)] and (b) to assess the usefulness of the total lymphocyte count (TLC) as a surrogate of the CD4+ lymphocyte count in the HIV/TB group. A total of 345 patients were enrolled in the study of whom 104 (29.8%) were HIV seropositive (HIV/TB). On admission, the CD4+ lymphocyte counts of the HIV/TB cohort were significantly lower than the TB group with medians of 230 (interquartile range, 90-475) and 630 (500-865), respectively (p < 0.0001). The CD4+ lymphocyte count increased significantly in both cohorts on routine TB treatment. A TLC of 1,300-1,500 cells/mm3 was found to be predictive of a CD4+ lymphocyte count of < or = 200 cells/mm3 both on admission and after 1 month of TB therapy. We conclude from this study that the positive influence of TB therapy on the CD4+ lymphocyte count strongly suggests an additional avenue of influence on the course of HIV infection, whereas the usefulness of the TLC as a surrogate estimation of CD4+ lymphocyte count in HIV/TB patients has important implications for the developing world.