Gender differences in symptom burden among people living with HIV/AIDS receiving antiretroviral therapy in Yunnan, China.

ABSTRACTLittle is known about gender differences in the symptom burden of people living with HIV/AIDS (PLWHA) on antiretroviral therapy in China. This study was conducted based on a biopsychosocial-medical model to describe gender differences in symptom burden among 1035 PLWHA in Yunnan Province, China. After propensity score matching, 798 PLWHA were included in this analysis. Feeling stressed, poor sleep, and memory loss were the most burdensome symptoms among men, while feeling stressed, memory loss, and dizziness were the most burdensome symptoms among women. Among men PLWHA, factors associated with symptom burden were being of the ethnic minority, CD4 count ≥ 500 cells/mm3, physical functioning, and social support. Among women PLWHA, factors associated with symptom burden were being an inpatient, physical functioning, psychological functioning, and social support. Our findings suggest that healthcare providers need to take into account gender differences when developing optimal prevention, treatment, and care programs that provide individualized care to reduce patients' symptom burden.

Pregnancy alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism, and oxidant release of maternal neutrophils.

Pregnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women. Cytochemical studies confirmed that the distribution of the G-6-PDase antigen is coincident with functional G-6-PDase activity. Metabolic oscillations within activated pregnancy neutrophils are higher in amplitude, though lower in frequency, than activated control neutrophils, suggesting limited HMS activity. Analysis of radioisotope-labeled carbon flux from glucose to CO(2) indicates that the HMS is intact in leukocytes from pregnant women, but its level is not enhanced by cell stimulation. Using extracellular fluorescent markers, activated pregnancy neutrophils were found to release reactive oxygen metabolites (ROMs) at a lower rate than activated control neutrophils. However, basal levels of ROM production in polarized pregnancy neutrophils were greater than in control neutrophils. Microtubule-disrupting agents reversed the observed changes in G-6-PDase trafficking, metabolic oscillations, and ROM production by maternal neutrophils. G-6-PDase trafficking appears to be one mechanism regulating ROM production by maternal neutrophils.

2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine.

D-Glucosamine has been widely reported to have immunosuppressive actions on neutrophils, lymphocytes, and other cells of the immune system. However, under conditions used in biological experiments (e.g., neutral pH, and phosphate buffers), we have found that D-glucosamine self-reacts to form 2,5-deoxyfructosazine [2-(D-arabino-tetrahydroxybutyl)-5-(D-erythro-2,3,4-trihydroxybutyl)pyrazine] (1) and 2,5-fructosazine [2,5-bis(D-arabino-tetrahydroxybutyl)pyrazine] (2). When tested for bioactivity at nontoxic concentrations, these D-glucosamine derivatives were more effective inhibitors of IL-2 release from PHA-activated T cells than d-glucosamine. Hence, fructosazines constitute a novel class of immunomodulators.

Apparent role of dynein in glucose-6-phosphate dehydrogenase trafficking in neutrophils from pregnant women.

To better understand the mechanisms of metabolic microcompartmentalization associated with neutrophil hexose monophosphate shunt activity during pregnancy, we have studied the intracellular trafficking of glucose-6-phosphate dehydrogenase (G6PDase). Microtubule motor proteins colocalize with G6PDase. Dynein inhibitors block G6PDase accumulation at the microtubule-organizing center in pregnancy cells. On this basis, we conclude that microtubule motor proteins participate in hexose monophosphate shunt enzyme transport within leukocytes.

Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion.

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca(2+)-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca(2+) channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell's periphery. HT1080 cells exhibit periodic intracellular Ca(2+) spikes. High-speed imaging revealed that the Ca(2+) spikes were composed of a single Ca(2+) wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca(2+) channel blocker mibefradil inhibited Ca(2+) spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd(3+) and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca(2+) channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca(2+) wave, thereby enhancing the efficacy of emerging therapeutic protocols.

Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes.

PURPOSE: Mutations in the gene Elongation of very long-chain fatty acids-4 (ELOVL4) have been shown to be associated with autosomal dominant Stargardt-like macular dystrophy (STGD3). ELOVL4 is expressed in photoreceptors and encodes a putative transmembrane protein of 314 amino acids with an endoplasmic reticulum (ER) retention signal. A 5 bp deletion in exon 6 of ELOVL4 observed in some STGD3 patients results in the truncation of the protein and loss of the ER retention signal. To understand the disease mechanism underlying STGD3 we studied the intracellular trafficking of the wild-type and a 5 bp deletion mutant of ELOVL4. METHODS: Wild-type and mutant ELOVL4 proteins with the N-terminal GFP/V5 tags were expressed in COS-7 cells. Expression and the intracellular localization of the wild-type and mutant proteins were characterized by immunocytochemistry and western blot analysis using tag- and organelle-specific antibodies. Interaction between the wild-type and mutant proteins was studied by two-dimensional gel electrophoresis and fluorescence resonance energy transfer (FRET) analysis. RESULTS: The mutant ELOVL4 protein exerted a dominant negative effect when the wild-type and 5 bp deletion mutant ELOVL4 proteins were co-expressed in COS-7 cells. Immunocytochemical analysis, two-dimensional gel electrophoresis and FRET revealed that the mutant ELOVL4 interacts with the wild-type protein, forming higher molecular mass complexes that accumulate in aggresomes. CONCLUSIONS: In the presence of mutant ELOVL4 protein, the wild-type protein was recruited into perinuclear cytoplasmic inclusions that resemble aggresomes. The interaction between the wild-type and mutant forms of ELOVL4 and the resultant alteration in the trafficking of the wild-type ELOVL4 protein suggest a mechanism for the pathogenicity observed in patients with autosomal dominant STGD3.

Transaldolase is part of a supramolecular complex containing glucose-6-phosphate dehydrogenase in human neutrophils that undergoes retrograde trafficking during pregnancy.

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PDase) and 6-phosphogluconate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking in cells from pregnant women, but anterograde trafficking in cells from nonpregnant individuals. Using fluorescence resonance energy transfer techniques, we now demonstrate that transaldolase (TALase), a key regulatory enzyme in the nonoxidative branch of the hexose monophosphate shunt, is in close physical proximity with G6PDase, but not with lactate dehydrogenase, thus suggesting the formation of a TALase-G6PDase complex. Moreover, immunofluorescence microscopy demonstrated that TALase undergoes anterograde trafficking in neutrophils from nonpregnant individuals, whereas retrograde trafficking is found during pregnancy. However, pregnancy did not affect lactate dehydrogenase distribution. Colchicine treatment blocked the retrograde distribution of TALase, suggesting that microtubules are involved in TALase trafficking. We suggest that TALase is part of a supramolecular hexose monophosphate shunt complex, which likely increases the efficiency of the shunt via substrate channeling. We further suggest that TALase's retrograde motion contributes to uncoupling the shunt from its source of glucose-6-phosphate at the plasma membrane, thereby blunting nicotinamide adenine dinucleotide phosphate (reduced form) production and downstream oxidant production by neutrophils.

Molecular proximity of complement receptor type 3 (CR3) and the glycosylphosphatidylinositol-linked protein GPI-80 on neutrophils: effects of cell adherence, exogenous saccharides, and lipid raft disrupting agents.

GPI-80, a novel glycosylphosphatidylinositol (GPI)-anchored protein on polymorphonuclear leukocytes, has been reported to cooperate with CR3 in several aspects of cell function including cell activation, adhesion and migration. The present study investigates the physical proximity of CR3 and GPI-80 on living cells using resonance energy transfer (RET) techniques, which gives positive results when the separation distance is < or = 7 nm. RET from donor-labeled anti-CR3 to acceptor-labeled anti-GPI-80 was detected on adherent neutrophils, but not observed for non-adherent cells. Furthermore, RET was not observed on cells treated with cell adhesion inhibitors 4-bromophenacyl bromide (BPB), N-ethylmaleimide (NEM) or cytochalasin D, suggesting dynamic interactions between CR3 and GPI-80. CR3-to-GPI-80 proximity was blocked by N-acetyl-D-glucosamine (NADG), but not by other monosaccharides such as D-mannose, fructose, fucose, glucose, sorbitol, or galactose; molecular proximity was also disrupted by the glycolipid raft depleting agents 2-OH-propyl-betaCD and MbetaCD. Thus, lipid rafts may be important for the physical and functional cooperation of CR3 and GPI-80.

Maltooligosaccharides from JEG-3 trophoblast-like cells exhibit immunoregulatory properties.

PROBLEM: to better understand the immunoregulatory properties of trophoblasts, we have searched for small immunologically active carbohydrates derived from intact trophoblast-like cells. METHOD OF STUDY: using solid phase extraction coupled with HPLC and mass spectrometry methods, we have characterized a low molecular weight carbohydrate-rich fraction associated with JEG-3 cells. We have also tested the bioactivities of selected authentic oligosaccharides found in the oligosaccharide fraction. RESULTS: the most abundant components of the low molecular weight carbohydrate-rich fraction were maltotriose and maltotetraose, with detectable amounts of maltopentaose. When authentic maltooligosaccharides were tested using lymphocytes, IL-2 inhibition was observed. This activity was dependent upon the number of saccharide subunits, stereochemistry, and concentration. To further test maltooligosaccharide properties, maltopentose was attached to glass cover slips. Although spontaneous neutrophil motility was observed on unmodified and control surfaces, it was inhibited on maltooligosaccharide-derivatized surfaces. CONCLUSION: maltooligosaccharides are associated with the trophoblast's surface where they may exhibit immunoregulatory activities.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells.

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking.

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of approximately 20 s. IFN-gamma increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-gamma. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-gamma and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion.

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.

Lipid raft compartmentalization of urokinase receptor signaling in human neutrophils.

Urokinase plasminogen activator (uPA) receptors (uPAR) can be engaged for activation signaling either by aggregation or by binding exogenous uPA. These signaling mechanisms require uPAR to associate with two distinct adhesion proteins, L-selectin and complement receptor 3 (CR3), respectively. uPAR contains a glycosylphosphatidylinositol anchor, suggesting that it is concentrated within glycosphingolipid-enriched microdomains, or "lipid rafts". This study was undertaken to determine the extent to which uPAR-mediated signaling is compartmentalized to lipid rafts. Human neutrophil uPAR was cross-linked or stimulated with uPA after pretreatment with the lipid raft-disrupting agents, methyl-beta-cyclodextrin or filipin III. Both agents suppressed increases in intracellular Ca(2+) concentrations ([Ca(2+)](i)) triggered by cross-linking, but did not affect [Ca(2+) ](i) in response to uPA. Neutrophil membranes were separated into lipid raft and non-raft fractions, revealing the presence of uPAR and L-selectin, but the virtual absence of CR3 alpha chain in lipid rafts, either constitutively or in response to uPAR aggregation. Fluorescence resonance energy transfer experiments confirmed close proximity of a lipid raft marker to both uPAR and L-selectin, but not CR3. We conclude that uPAR can engage distinct signaling pathways involving different partner proteins that are functionally and physically segregated from one another in both lipid raft and non-raft domains of the plasma membrane.