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1.
PLoS One ; 13(5): e0196949, 2018.
Article in English | MEDLINE | ID: mdl-29750804

ABSTRACT

Despite the advent of highly active anti-retroviral therapy HIV-associated neurocognitive disorders (HAND) continue to be a significant problem. Furthermore, the precise pathogenesis of this neurodegeneration is still unclear. The objective of this study was to examine the relationship between infection by the simian immunodeficiency virus (SIV) and neuronal injury in the rhesus macaque using in vivo and postmortem sampling techniques. The effect of SIV infection in 23 adult rhesus macaques was investigated using an accelerated NeuroAIDS model. Disease progression was modulated either with combination anti-retroviral therapy (cART, 4 animals) or minocycline (7 animals). Twelve animals remained untreated. Viral loads were monitored in the blood and cerebral spinal fluid, as were levels of activated monocytes in the blood. Neuronal injury was monitored in vivo using magnetic resonance spectroscopy. Viral RNA was quantified in brain tissue of each animal postmortem using reverse transcription polymerase chain reaction (RT-PCR), and neuronal injury was assessed by immunohistochemistry. Without treatment, viral RNA in plasma, cerebral spinal fluid, and brain tissue appears to reach a plateau. Neuronal injury was highly correlated both to plasma viral levels and a subset of infected/activated monocytes (CD14+CD16+), which are known to traffic the virus into the brain. Treatment with either cART or minocycline decreased brain viral levels and partially reversed alterations in in vivo and immunohistochemical markers for neuronal injury. These findings suggest there is significant turnover of replicating virus within the brain and the severity of neuronal injury is directly related to the brain viral load.


Subject(s)
Acquired Immunodeficiency Syndrome , Anti-Retroviral Agents/pharmacology , Magnetic Resonance Imaging , Neurons/virology , RNA, Viral , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/cerebrospinal fluid , Acquired Immunodeficiency Syndrome/diagnostic imaging , Acquired Immunodeficiency Syndrome/drug therapy , Animals , Disease Models, Animal , Macaca mulatta , Minocycline , RNA, Viral/blood , RNA, Viral/cerebrospinal fluid , Simian Acquired Immunodeficiency Syndrome/blood , Simian Acquired Immunodeficiency Syndrome/cerebrospinal fluid , Simian Acquired Immunodeficiency Syndrome/diagnostic imaging , Simian Acquired Immunodeficiency Syndrome/drug therapy
2.
Invest Ophthalmol Vis Sci ; 48(5): 1931-41, 2007 May.
Article in English | MEDLINE | ID: mdl-17460244

ABSTRACT

PURPOSE: Matrix metalloproteinase (MMP)-9 is induced in corneal epithelial cells stimulated with platelet-activating factor (PAF), and interferes with the normal reepithelialization of wounded cornea. Here the transcriptional regulation of MMP-9 gene expression by PAF was investigated in human corneal epithelial cells (HCECs). METHODS: DNA-binding activity of NFkappaB, Sp1, and AP-1 was determined in quiescent and PAF-stimulated HCECs by electrophoretic mobility shift assay (EMSA). A series of 5' deleted human MMP-9 promoter-luciferase reporter constructs was transiently transfected into HCECs, and luciferase activity was examined after stimulation with PAF. Mutagenesis and specific deletions of some elements in the MMP-9 promoter were also introduced and analyzed. Phosphorylation of Sp1 and MEK/ERK pathway proteins was examined by Western blot analysis. Activation of Sp1 and MMP-9 was also determined by ELISA and zymography, respectively, in the absence or presence of the MEK inhibitor PD98059. RESULTS: DNA-binding activity of NFkappaB, Sp1, and AP-1 was upregulated by PAF with a peak at 1 hour after stimulation. A region spanning -670 to -460 relative to the transcription start point was required for the induction of the MMP-9 promoter by PAF. Mutation of the -79AP-1 or -600NFkappaB motif reduced the activity of MMP-9 promoter and the induction of gene expression by PAF. In untreated HCECs, mutation of the -558Sp1 motif upregulated gene expression, but it caused a significant decrease in the promoter activity induced by PAF. Inhibition of MEK activity eliminated the PAF-induced phosphorylation and activation of Sp1 and abolished the upregulation of MMP-9 expression and activity. CONCLUSIONS: These findings demonstrate that collaboration between several regulatory elements is required for the induction of MMP-9 promoter activity by PAF and that PAF overturns the repressor effect of Sp1 through activation of the MEK/ERK signaling cascade.


Subject(s)
Epithelium, Corneal/drug effects , Gene Expression Regulation, Enzymologic , Matrix Metalloproteinase 9/genetics , Platelet Activating Factor/pharmacology , Repressor Proteins/metabolism , Sp1 Transcription Factor/metabolism , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Line , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Epithelium, Corneal/enzymology , Flavonoids/pharmacology , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Plasmids , Protein Serine-Threonine Kinases/metabolism , Transcription Factor AP-1/metabolism , Transfection , NF-kappaB-Inducing Kinase
3.
JPEN J Parenter Enteral Nutr ; 28(6): 423-30, 2004.
Article in English | MEDLINE | ID: mdl-15568289

ABSTRACT

BACKGROUND: Arginine depletion by the enzyme Arginase I, decreases expression of the TCR zeta chain preventing T-cell activation and causing T-cell dysfunction. We hypothesized that citrulline could substitute for arginine under conditions of increased arginase expression. Thus, the goal was to establish a possible mechanism of how citrulline could overcome arginine depletion caused by arginase. METHODS: Jurkat cells were cultured, with or without arginase, in media containing different amino-acid constituents: complete RPMI containing arginine (C-RPMI) (arginine), Arginine-Free-RPMI (Arg-Free RPMI) and Citrulline-containing RPMI (Cit RPMI). Incorporation of citrulline was measured via uptake of 3H-citrulline, whereas proliferation was measured via 3H-thymidine incorporation. zeta Chain was analyzed by 2-color flow cytometry. Argininosuccinate synthase (AS) and argininosuccinate lyase expression was detected using Northern blots, RT-PCR, and Western blots. RESULTS: Jurkat cells exhibited a significant decrease in proliferation and 5 chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, zeta chain expression and proliferation were maintained in the absence of arginine or in the presence of the enzyme arginase. Jurkat cells, cultured in the absence of arginine, were associated with a 5-fold increase in citrulline uptake. The absence of arginine was also associated with increased expression of AS. CONCLUSIONS: T cells exhibit the molecular capability of increasing citrulline membrane transport and up-regulating AS expression, thus exhibiting the necessary mechanisms for converting citrulline into arginine and escaping the ill effects of arginine depletion. Therefore, citrulline has the potential to be a substitute for supplemental arginine in diseases associated with arginase-mediated T cell dysfunction.


Subject(s)
Arginase/metabolism , Arginine/deficiency , CD3 Complex/metabolism , Citrulline/pharmacology , Jurkat Cells/drug effects , T-Lymphocytes/drug effects , Blotting, Northern , Blotting, Western , CD3 Complex/immunology , Cell Division/drug effects , Flow Cytometry , Humans , Jurkat Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/metabolism , T-Lymphocytes/physiology , Up-Regulation
4.
Exp Eye Res ; 76(5): 613-22, 2003 May.
Article in English | MEDLINE | ID: mdl-12697425

ABSTRACT

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.


Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/physiology , Epithelium, Corneal/drug effects , Growth Substances/pharmacology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/pharmacology , Animals , Arachidonate 12-Lipoxygenase/genetics , Arachidonate 12-Lipoxygenase/metabolism , Cell Division/physiology , Cells, Cultured , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Epithelium, Corneal/enzymology , Fibroblast Growth Factor 7 , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Hepatocyte Growth Factor/pharmacology , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Rabbits , Transcriptional Activation
5.
Exp Eye Res ; 74(3): 393-402, 2002 Mar.
Article in English | MEDLINE | ID: mdl-12014920

ABSTRACT

Previous studies in the laboratory have shown that platelet-activating factor (PAF), a potent inflammatory mediator that accumulates rapidly in the cornea after an injury, stimulates the expression of urokinase (uPA) and matrix metalloproteinase-1 (MMP-1) and -9 (MMP-9). Tissue inhibitors of MMPs (TIMPs) and plasminogen activator inhibitor (PAI-1) are produced in conjunction with these enzymes and are important regulators of their activity. Here, the authors investigated how PAF affects the expression of PAI-1, TIMP-1 and -2 relative to that of uPA, MMP-1, and -9 in rabbit corneal epithelial cells. Rabbit corneas were incubated in MEM medium containing 100 nM cPAF. To block the effects of PAF in some studies, corneas were preincubated for 1 hr in the presence of the PAF antagonist BN50730 (10 microM). At several time intervals, mRNA was extracted from epithelial cells and the levels of gene expression for the enzymes and their inhibitors were determined by real-time PCR. All quantitations were normalized to the 18s rRNA values (endogenous control) and changes in gene expression were reported as fold increase relative to untreated controls. PAF produced a 20-fold increase in the gene expression of PAI-1 at 8 hr, while similar fold increases in uPA mRNA expression occurred at 2 hr. PAF treatment also stimulated the expression of TIMP-1 and -2 genes, with a six-fold increase in TIMP-1 expression occurring at 36 hr and a four-fold increase in TIMP-2 expression at 24 hr. Maximal induction of MMP-1 and -9 mRNA, on the other hand, occurred at 4 and 8 hr, respectively. Induction of MMP-1 gene expression was similar to that of its inhibitors TIMP-1 and -2, while MMP-9 mRNA induction exceeded that of these inhibitors by 100-fold. The PAF-induced expression of PAI-1, TIMP-1 and -2 mRNAs was abolished by pre-treatment with BN50730. These data indicate that PAF activates the gene expression of TIMP-1, -2, and PAI-1 in corneal epithelium by a receptor-mediated mechanism. Furthermore, PAF induced overexpression of MMP-9 mRNA relative to that of TIMP-1 and -2, suggesting an imbalance between the expression of this proteolytic enzyme and its inhibitors, which may contribute to changes in the wound-healing process and ultimately lead to corneal ulcer development.


Subject(s)
Epithelium, Corneal/metabolism , Gene Expression Regulation/drug effects , Plasminogen Activator Inhibitor 1/genetics , Platelet Activating Factor/pharmacology , Tissue Inhibitor of Metalloproteinases/genetics , Animals , Epithelium, Corneal/enzymology , Matrix Metalloproteinase 9/genetics , Organ Culture Techniques , Polymerase Chain Reaction/methods , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-2/genetics
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