sq-23377 and Chronic-Disease

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and/or predict disease progression relative to CD4(+) T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term non-progressors with low/undetectable viral loads, those with chronic plasma viremia, but clinically healthy, exhibited significantly lower numbers and functional impairment of CD4(+) T cells, but not CD8(+) T cells, in terms of IL-2 production by central memory subset in response to PMA and ionomycine (PMA+I) stimulation. Highly viremic animals showed impaired cytokine-production by all T cells subsets. These results suggest that functional impairment of CD4(+) T cells in general, and of central memory subset in particular, may be a potential indicator/predictor of chronic infection with immune dysfunction, which could be assayed relatively easily using non-specific PMA+I stimulation.

Topics: Animals; Biomarkers; CD4-Positive T-Lymphocytes; Chronic Disease; Cytokines; Disease Progression; Humans; Immunologic Memory; Ionomycin; Macaca mulatta; Prognosis; Simian Acquired Immunodeficiency Syndrome; Tetradecanoylphorbol Acetate; Viral Load; Viremia

Topics: CD4-Positive T-Lymphocytes; Cell Separation; Chronic Disease; Disease Progression; Enterotoxins; Flow Cytometry; HIV Infections; Humans; Interleukins; Ionomycin; Ionophores

The nitric oxide/soluble guanylyl cyclase (sGC) signal transduction pathway plays an important role in smooth muscle relaxation and phenotypic regulation. However, the transcriptional regulation of sGC gene expression is largely unknown. It has been shown that sGC expression increases in pulmonary arteries from chronic hypoxia-induced pulmonary hypertensive animals. Since the transcription factor NFATc3 is required for the upregulation of the smooth muscle hypertrophic/differentiation marker alpha-actin in pulmonary artery smooth muscle cells from chronically hypoxic mice, we hypothesized that NFATc3 is required for the regulation of sGC-alpha1 expression during chronic hypoxia. Exposure to chronic hypoxia for 2 days induced a decrease in sGC-alpha1 expression in mouse pulmonary arteries. This reduction was independent of NFATc3 but mediated by nuclear accumulation of the mRNA-stabilizing protein human antigen R (HuR). Consistent with our hypothesis, chronic hypoxia (21 days) upregulated pulmonary artery sGC-alpha1 expression, bringing it back to the level of the normoxic controls. This response was prevented in NFATc3 knockout and cyclosporin (calcineurin/NFATc inhibitor)-treated mice. Furthermore, we identified effective binding sites for NFATc in the mouse sGC-alpha1 promoter. Activation of NFATc3 increased sGC-alpha1 promoter activity in human embryonic derived kidney cells, rat aortic-derived smooth muscle cells, and human pulmonary artery smooth muscle cells. Our results suggest that NFATc3 and HuR are important regulators of sGC-alpha1 expression in pulmonary vascular smooth muscle cells during chronic hypoxia-induced pulmonary hypertension.

Topics: Animals; Antigens, Surface; Base Sequence; Binding Sites; Calcineurin; Cell Line; Cell Nucleus; Chronic Disease; ELAV Proteins; ELAV-Like Protein 1; Gene Deletion; Guanylate Cyclase; Humans; Hypertension, Pulmonary; Hypoxia; Ionomycin; Male; Mice; NFATC Transcription Factors; Point Mutation; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Pulmonary Artery; Rats; Receptors, Cytoplasmic and Nuclear; RNA-Binding Proteins; Soluble Guanylyl Cyclase; Transfection

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carbon Dioxide; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Ionophores; Male; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Polycythemia; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Chronic Disease; Clonal Anergy; Cricetinae; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Ionomycin; Ionophores; Leishmania donovani; Leishmaniasis, Visceral; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred BALB C; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by increased platelet clearance because of antiplatelet antibodies. It was recently reported that the balance of T helper 1 (Th1) and T helper 2 (Th2) subsets has been implicated in the regulation of many immune responses. In this study, the intracellular interleukin-4 and interferon-gamma production in CD4+ T-lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin was assessed via flow cytometry in order to determine the clinical significance of the Th1/Th2 ratio in 42 patients with ITP. The study cohort included 28 untreated patients, seven postprednisolone therapy patients and seven postsplenectomy patients. The mean level of the Th1/Th2 ratio in the untreated group was 36.9 (95% CI 25.8-47.9), and significantly higher than in the control group (mean 12.8, 95% CI 9.5-16.1). The mean levels of the Th1/Th2 ratio in the postprednisolone therapy and postsplenectomy groups were 20.5 (95% CI 8.4-32.6) and 16.4 (95% CI 3.1-29.7), respectively, but were no significant differences as compared with control subjects. When untreated patients were divided into two subgroups by Th1/Th2 ratio, the mean level of platelet associated IgG in the high Th1/Th2 subgroup (higher than upper limit of control group) tended to be higher than in the normal Th1/Th2 subgroup. In conclusion, the high Th1/Th2 ratio was closely related to the etiology and disease status of chronic ITP.

Topics: Adult; Aged; Case-Control Studies; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chronic Disease; Cohort Studies; Cytokines; Female; Flow Cytometry; Humans; Immunoglobulin G; Ionomycin; Ionophores; Male; Middle Aged; Prednisolone; Purpura, Thrombocytopenic, Idiopathic; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells; Time Factors

Activated CD4(+) T helper cells (Th) comprise at least two functionally distinct subsets, Th1 and Th2, which mediate different immunological effector functions. Experimental leishmaniasis is widely used to study the effector function of Th cell subsets in vivo. Healing and nonhealing Leishmania major infections have been correlated with polarized Th1 and Th2 responses, respectively. In the study presented here, a stable cell surface marker expressed on Th2 cells, T1/ST2, has been used to assess the distribution of CD4(+) T1/ST2(+) T cells in different organs of healer and nonhealer strains of mice during the course of L. major infection. The frequency of CD4(+) T cells expressing the T1/ST2 cell surface marker and Th2 cytokines in the lymphoid organs was low in both strains of infected mice; however, CD4(+) T1/ST2(+) T cells could be enriched from the lymphoid organs of infected nonhealer but not from healer strains of mice. The highest frequency of CD4(+) T1/ST2(+) T cells was detected in the footpads of mice with nonhealing disease, showing that CD4(+) T1/ST2(+) T cells home to the footpads. Since the majority of parasites persist at the local site of infection in nonhealing BALB/c mice, these results show that CD4(+) T1/ST2(+) T cells are localized at the site of active infection and inflammation in this model.

Topics: Animals; CD5 Antigens; Cell Differentiation; Chronic Disease; Disease Susceptibility; Female; Interleukin-1 Receptor-Like 1 Protein; Ionomycin; Leishmania major; Leishmaniasis, Cutaneous; Lymph Nodes; Lymphocyte Activation; Lymphoid Tissue; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Organ Specificity; Phenotype; Receptors, Interleukin; Schistosomiasis mansoni; Specific Pathogen-Free Organisms; Spleen; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Th2 Cells

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17 beta-estradiol (E(2)beta), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E(2)beta attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E(2)beta. Finally, responses to S-nitroso-N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E(2)beta on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Endothelium, Vascular; Enzyme Inhibitors; Estradiol; Female; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Ionomycin; Ionophores; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Nitrogen Oxides; Ovariectomy; Penicillamine; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Spermine; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals.. Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion-imaging system, and the spatial distribution of Ca2+ within intracellular organelles.. There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P < 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P < 0.05). Non-ER Ca2+ stores were not similarly affected.. We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity.

Topics: Arthritis, Rheumatoid; Calcium; Calcium Signaling; Chronic Disease; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Ionomycin; Ionophores; Organelles; Reference Values; Synovial Fluid; T-Lymphocytes; Thapsigargin

We previously demonstrated augmented endothelium-derived nitric oxide (EDNO)-dependent pulmonary arterial dilation and increased arterial endothelial nitric oxide synthase (eNOS) levels in chronic hypoxic (CH) and monocrotaline (nonhypoxic) models of pulmonary arterial hypertension. Therefore, we hypothesized that the long-term elevation of arterial eNOS levels associated with CH is related to pulmonary hypertension or some factor(s) associated with hypertension and not directly to hypoxia. To test this hypothesis, we examined responses to the EDNO-dependent dilator ionomycin in U-46619-constricted, isolated, saline-perfused lungs from control rats, CH (4 wk at 380 mmHg) rats, and rats previously exposed to CH but returned to normoxia for 4 days or 2 wk. Microvascular pressure was assessed by double-occlusion technique, allowing calculation of segmental resistances. In addition, vascular eNOS immunoreactivity was assessed by quantitative immunohistochemistry, and eNOS mRNA abundance was determined by RT-PCR assays. Our findings indicate that 4-day and 2-wk posthypoxic rats exhibit persistent pulmonary hypertension, likely due to maintained arterial remodeling and polycythemia associated with prior exposure to CH. Furthermore, arterial dilation to ionomycin was augmented in lungs from each experimental group compared with controls. Finally, arterial eNOS immunoreactivity and whole lung eNOS mRNA levels remained elevated in posthypoxic animals. These findings suggest that altered vascular mechanical forces or vascular remodeling contributes to enhanced EDNO-dependent arterial dilation and upregulation of arterial eNOS in various models of established pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Gases; Gene Expression Regulation; Hematocrit; Hemodynamics; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Ionomycin; Lung; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vascular Resistance

We have previously demonstrated that arterial, but not venous, vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent agonists are enhanced in lungs isolated from rats with chronic hypoxia (CH)-induced pulmonary arterial hypertension. These data suggest that CH is associated with increased endothelial nitric oxide synthase (eNOS) activity within the pulmonary arterial vasculature. In addition, the correlation of increased pulmonary arterial pressure with selectively enhanced arterial responsiveness to EDNO-mediated agonists suggests that arterial hypertension, rather than hypoxia per se, is a contributing factor in this response. Therefore, we hypothesized that 1) CH selectively upregulates eNOS within the pulmonary arterial vasculature and 2) monocrotaline (MC)-induced pulmonary arterial hypertension selectively enhances pulmonary arterial dilation to EDNO-dependent dilators and upregulates arterial eNOS. We examined the responses to the EDNO-dependent dilators arginine vasopressin and ionomycin in U-46619-constricted isolated perfused lungs from control and MC-treated rats. Microvascular pressure was assessed by the double-occlusion technique, allowing calculation of segmental resistances. Lungs from MC-treated rats exhibited augmented arterial dilation to arginine vasopressin compared with control lungs. However, the responses to ionomycin were not different between the two groups. Quantitative immunocytochemistry was used to compare pulmonary eNOS immunoreactivity in vessels from control, CH, and MC-treated rats. eNOS staining was more intense in the arteries of CH and MC-treated rats compared with those of control animals, whereas CH and MC treatment had no effect on eNOS staining in veins. We conclude that pulmonary arterial hypertension, or altered vascular mechanical forces associated with hypertension, may be responsible for the augmented EDNO-dependent arterial dilation and upregulation of arterial eNOS in lungs from CH and MC-treated rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine Vasopressin; Blood Pressure; Chronic Disease; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionomycin; Lung; Male; Monocrotaline; Nitric Oxide Synthase; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thromboxane A2; Vascular Resistance; Vasoconstrictor Agents