ABSTRACT
Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the beta-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the gamma-globin gene. sGC, an obligate heterodimer of alpha- and beta-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different beta-like globin genes showed that, whereas the beta-subunit is expressed at similar levels, high-level expression of the alpha-subunit is preferentially observed in erythroid cells expressing gamma-globin but not those expressing beta-globin. Also, the levels of expression of the gamma-globin gene correlate to those of the alpha-subunit. sGC activators or cGMP analogs increased expression of the gamma-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with beta-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the gamma-globin gene. Furthermore, increased expression of the gamma-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC-PKG pathway constitutes a mechanism that regulates expression of the gamma-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the beta-globin disorders.
Subject(s)
Cyclic GMP-Dependent Protein Kinases/physiology , Cyclic GMP/metabolism , Gene Expression , Globins/genetics , Guanylate Cyclase/physiology , Signal Transduction/physiology , Butyrates/pharmacology , Gene Expression/drug effects , Hemin/pharmacology , Humans , K562 Cells , Leukemia, Erythroblastic, Acute , Solubility , Tumor Cells, CulturedABSTRACT
According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients.
Subject(s)
CD28 Antigens/biosynthesis , CD8-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/immunology , Lymphocyte Activation/immunology , T-Lymphocyte Subsets/immunology , Adult , CD28 Antigens/immunology , CD8-Positive T-Lymphocytes/metabolism , Clone Cells , Female , Humans , Immunophenotyping , Interferon-gamma/metabolism , Interleukins/metabolism , Male , T-Lymphocyte Subsets/metabolismABSTRACT
The presence of Ureaplasma urealyticum was evaluated on 1912 vaginal and urethral swabs from HIV-1 seronegative (HIV-) inpatients (210) and outpatients (503) suffering from acute urethritis or vaginitis; asymptomatic HIV- outpatients (201); and asymptomatic HIV-1 seropositive (HIV+) inpatients (120). The study reported an increased frequency of Ureaplasma urealyticum isolates in asymptomatic HIV+ compared to asymptomatic HIV- subjects. As expected, the frequency of Ureaplasma urealyticum isolates increased in symptomatic HIV- subjects. Strains of Ureaplasma urealyticum resistant to ciprofloxacin, tetracycline and minocycline were more frequently isolated in HIV+ (34.1%) than HIV- (3.8%) subjects; on the other hand, only 1 out of 704 (0.1%) strains isolated from outpatients was resistant to ciprofloxacin. We found no association in HIV+ patients between Ureaplasma urealyticum infection and CD4 count or HIV-1 p24 antigenemia.
Subject(s)
HIV Infections/microbiology , HIV-1 , Ureaplasma urealyticum/isolation & purification , Urethra/microbiology , Vagina/microbiology , AIDS-Related Opportunistic Infections/drug therapy , AIDS-Related Opportunistic Infections/microbiology , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Ciprofloxacin/pharmacology , Drug Resistance, Microbial , Female , HIV Infections/virology , Humans , Male , Minocycline/pharmacology , Tetracycline/pharmacology , Ureaplasma urealyticum/drug effectsABSTRACT
The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.
