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Biomedical subjects

G R John

Publications and source records attributed to G R John.

At least 19 recordsLinked to original sources
PubMed2001

Extracellular nucleotides differentially regulate interleukin-1beta signaling in primary human astrocytes: implications for inflammatory gene expression.

The cytokine interleukin-1beta (IL-1beta) is a potent activator of human astrocytes, inducing or modulating expression of multiple proinflammatory genes via activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In this study, we examined whether IL-1beta signaling is regulated in these cells by extracellular nucleotides that are released at high concentrations under inflammatory conditions and act as ligands for members of the P2 receptor family. Using reporter constructs and electromobility shift assays, we found that cotreatment of astrocyte cultures with ATP (1-100 microm) significantly potentiated IL-1beta-mediated activation of NF-kappaB and AP-1 and that ATP alone activated AP-1. These effects were blocked by the P2 receptor antagonists XAMR 0721, periodate-oxidized ATP, and suramin. A role for ATP in modulating IL-1beta-mediated inflammatory gene expression was supported further by the observation that ATP potentiated the IL-1beta-induced expression of IL-8 mRNA and protein but strongly downregulated IP-10 expression. Reverse transcription-PCR and cloning demonstrated expression of the ATP-responsive P2 receptor subtypes P2Y(1), P2Y(2), and P2X(7), as well as the ATP-insensitive receptor P2Y(4). ADP, a selective agonist for P2Y(1), produced results similar to or greater than those obtained using ATP, whereas 2'-3'-O-(4-benzoyl-benzoyl)-ATP, a selective agonist for P2X(7), was less effective than ATP. In contrast, UTP, a selective agonist for P2Y(2) and P2Y(4), was ineffective. These studies indicate that different P2 receptor subtypes play distinct roles in the modulation of IL-1beta-mediated signal transduction in human astrocytes, and that signaling via P2 receptors may fine-tune the transcription of genes involved in inflammatory responses in the human CNS.

Adenosine Triphosphate↗
PubMed2000

Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1beta in primary human fetal astrocytes.

Vertebrate tissues use multiple junctional types to establish and maintain tissue architecture, including gap junctions for cytoplasmic connectivity and tight junctions (TJs) for paracellular and/or cell polarity barriers. The integral membrane proteins of gap junctions are connexins, whereas TJs are a complex between occludin and members of a recently characterized multigene family, the claudins. In normal brain, astrocytes are coupled by gap junctions composed primarily of connexin43 (Cx43), whereas TJs have not been detected in these cells. We now show that treatment of primary human astrocytes with the cytokine interleukin-1beta (IL-1beta) causes rapid induction of claudin-1, with an expression pattern reciprocal to loss of Cx43. Treatment also led to protracted downregulation of occludin but no change in expression of zonula occludens proteins ZO-1 and -2. Immunofluorescence staining localized claudin-1 to cell membranes in IL-1beta-treated astrocytes, whereas freeze-fracture replicas showed strand-like arrays of intramembranous particles in treated cells resembling rudimentary TJ assemblies. We conclude that in human astrocytes, IL-1beta regulates expression of the claudin multigene family and that gap and tight junction proteins are inversely regulated by this proinflammatory cytokine. We suggest that in pathological conditions of the human CNS, elevated IL-1beta expression fundamentally alters astrocyte-to-astrocyte connectivity.

Astrocytes↗
PubMed2000

Modulation of interleukin-1beta and tumor necrosis factor alpha signaling by P2 purinergic receptors in human fetal astrocytes.

In human astrocytes, interleukin-1beta (IL-1beta) is a potent inducer of genes associated with inflammation. In this study, we tested the hypothesis that in primary cultures of human fetal astrocytes signaling by the P2 purinergic nucleotide receptor pathway contributes to, or modulates, cytokine-mediated signal transduction. Calcium imaging studies indicated that most cells in culture responded to ATP, whereas only a subpopulation responded to UTP. Pretreatment of astrocytes with P2 receptor antagonists, including suramin and periodate oxidized ATP (oATP), resulted in a significant downregulation of IL-1beta-stimulated expression of nitric oxide, tumor necrosis factor (TNFalpha), and IL-6 at both the protein and mRNA levels, without affecting cell viability. In cells transiently transfected with reporter constructs, IL-1beta demonstrated more potent activation of the transcription factors nuclear factor -kappaB (NF-kappaB) and activator protein-1 (AP-1) than TNFalpha. However, pretreatment with oATP downregulated activation of NF-kappaB and AP-1 by IL-1beta or TNFalpha. Electromobility shift assays using oligonucleotides containing specific NF-kappaB binding sequences confirmed that pretreatment with oATP or apyrase attenuated cytokine-mediated induction of this transcription factor. From these data, we conclude that P2 receptor-mediated signaling intersects with that of IL-1beta and TNFalpha to regulate responses to cytokines in the CNS. Because inflammation, trauma, and stress all lead to the release of high levels of extracellular nucleotides, such as ATP and UTP, signaling via P2 receptors may provide a mechanism whereby cells can sense and respond to events occurring in the extracellular environment and can fine tune the transcription of genes involved in the inflammatory response.

Adenosine Triphosphate↗
PubMed2000

Effects of 4-aminopyridine on demyelinated axons, synapses and muscle tension.

Several clinical trials have demonstrated that 4-amino-pyridine (4-AP), a potassium channel-blocking agent, improves symptoms in some patients with multiple sclerosis. The beneficial effects have typically been attributed to the restoration of conduction to demyelinated axons, since this effect was previously demonstrated experimentally. However, the clinical dose is approximately 250-1000 times lower than that used experimentally, potentially making extrapolation of the experimental findings unreliable. To examine the action(s) of 4-AP in demyelinating disorders, the drug was administered at clinical doses, both in vivo and in vitro, to rat dorsal column axons which had been experimentally demyelinated by the intraspinal injection of ethidium bromide. 4-AP had no consistent effect in restoring conduction to demyelinated axons, even to axons which were held just on the verge of conducting by adjusting the lesion temperature. However, 4-AP had prominent effects that did not involve demyelinated axons, including the potentiation of synaptic transmission and an increase in skeletal muscle twitch tension. We propose that these latter effects may be largely responsible for the beneficial action of 4-AP in multiple sclerosis patients. If so, the dominant effects of 4-AP in multiple sclerosis patients are independent of demyelination, and it follows that 4-AP may be beneficial in other neurological disorders in which function is diminished.

4-Aminopyridine↗
PubMed1999

IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.

In mammalian astrocytes, calcium waves are transmitted between cells via both a gap junction-mediated pathway and an extracellular, P2 receptor-mediated pathway, which link the cells into a syncytium. Calcium waves in astrocytes have also been shown to evoke calcium transients in neurons, and activity in neurons can elicit calcium waves in astrocytes. In this study, we show that in primary human fetal astrocytes, the P2 receptor-mediated and gap junction-mediated pathways are differentially regulated by the cytokine IL-1beta. Confocal microscopy of astrocytes loaded with Indo-1 demonstrated that intercellular calcium wave transmission in IL-1beta-treated cultures was potentiated compared with controls. However, transmission of calcium waves via the gap junction-mediated pathway was strikingly reduced. The major component of functional gap junctions in human fetal astrocytes was demonstrated to be connexin43 (Cx43), and there was a marked reduction of junctional conductance, loss of dye coupling, loss of Cx43 protein, and down-regulation of Cx43 mRNA expression after IL-1beta treatment of cultures. Conversely, transmission of calcium waves via the P2 receptor-mediated pathway was potentiated in IL-1beta-treated cultures compared with controls. This potentiation was associated with an increase in the number of cells responsive to UTP, and with a transient increase in expression of the P2Y(2) purinoceptor mRNA. Because in inflammatory conditions of the human central nervous system IL-1beta is produced both by resident glia and by invading cells of the immune system, our results suggest that inflammatory events may have a significant impact on coordination of astrocytic function and on information processing in the central nervous system.

Astrocytes↗
PubMed1999

Peripheral ulcerative keratitis--an extracutaneous neutrophilic disorder: report of a patient with rheumatoid arthritis, pustular vasculitis, pyoderma gangrenosum, and Sweet's syndrome with an excellent response to cyclosporine therapy.

The term peripheral ulcerative keratitis represents a spectrum of inflammatory diseases, characterized by cellular infiltration, corneal thinning, and ulceration. Neutrophilic dermatoses are rarely associated with peripheral ulcerative keratitis. To date, peripheral ulcerative keratitis has only been reported in patients with pyoderma gangrenosum. Separate episodes of pyoderma gangrenosum, Sweet's syndrome, and pustular vasculitis developed in a 60-year-old patient with rheumatoid arthritis over an 8-year period. Over the past 2 years, 3 episodes of peripheral ulcerative keratitis occurred. Cyclosporine (4 mg/kg/d) treatment was started on confirmation of pyoderma gangrenosum. Over the ensuing 2 years, it became evident that the activity of her ocular and skin diseases, as well as her arthritis, paralleled the administration or cessation of cyclosporine therapy. Dermatologists should be aware of the association of Sweet's syndrome, pyoderma gangrenosum, and pustular vasculitis with peripheral ulcerative keratitis. This rare ocular manifestation and the serious sequelae when left untreated make recognition crucial. Cyclosporine proved to be a very effective treatment for all of our patient's diseases.

Arthritis, Rheumatoid↗
PubMed1998

Videokeratographic abnormalities in a family with posterior polymorphous dystrophy.

PURPOSE: To describe a unique videokeratographic pattern in one family with the vesicular form of posterior polymorphous dystrophy (PPD). METHODS: Descriptive case series of five patients with PPD including keratometry, videokeratography, axial length, and pachymetry. RESULTS: All affected family members with PPD demonstrated videokeratographic steep corneas with keratometry greater than three standard deviations over reported mean. CONCLUSIONS: Videokeratographic corneal steepening appears to be associated with PPD in one family with the autosomal dominant vesicular form of disease.

Adolescent↗
PubMed1992

Rotation of posterior chamber intraocular lenses for management of lens-associated recurring hyphemas.

Management of recurring hyphemas associated with posterior chamber intraocular lenses may include a combination of medical, laser, and surgical modalities. Miotic and laser therapies have often failed, and surgical treatment has primarily relied on removal of the offending lens. We describe herein a method for rotation of the intraocular lens that provides an effective means of preventing one form of recurrent intraocular hemorrhage.

Aged↗
PubMed1989

Eye injury: prevalence and prognosis by setting.

Although an estimated 1 million Americans suffer ocular injuries each year, the setting of injury and its prognostic implications have not been closely examined. Using data compiled by the Eye Injury Registry of Alabama (EIRA) from 514 cases of serious eye injury, we examined the demographics and prognosis of ocular injury by setting of injury. Work-related injuries accounted for only 28% of total injuries, and injuries occurring at home accounted for 27%, followed by situations related to recreation (25%), assault (11%), travel (5%), and "other" (school, unknown, etc) (4%). The poorest initial vision, poorest final vision, and highest rate of enucleation occurred in patients injured by assault, whereas the lowest rate of enucleation and loss of light perception was found in patients who had work-related injuries. Patients in the "other" category had the highest rate of return to 20/100 or better vision.

Accidents, Home↗
PubMed1985

Increased activity of Ca2+-dependent enzymes of membrane lipid metabolism in synaptosomal preparations from ethanol-dependent rats.

In synaptosomal fractions of rat brain the activities of phospholipase A2 and the phospholipid base-exchange enzymes are highly dependent on external Ca2+ concentrations. Their activity is inhibited by the presence of 50 mM ethanol in vitro. Administration of ethanol to rats by inhalation causes a progressive increase in the activity of these enzymes in synaptosomal preparations at all Ca2+ concentrations studied. The increased activity of these enzymes persists in preparations from rats undergoing a physical syndrome of withdrawal from ethanol. The addition of ethanol in vitro to preparations from animals that had received ethanol in vivo had no significant effect on enzyme activity. The results are discussed in relation to the possible roles of membrane lipid metabolism and synaptic Ca2+ sensitivity in ethanol tolerance and physical dependence.

Alcoholism↗
PubMed1980

The rapid onset of functional tolerance to ethanol--role of different neurotransmitters and synaptosomal membrane lipids.

The rapid onset of functional tolerance to ethanol was studied in young adult mice of the TO Swiss and C57 Black strains. Drugs known to alter the metabolism of catecholamines and 5-hydroxytryptamine in mouse brain were without effect on the development of tolerance to ethanol. Amino-oxyacetic acid, which potentiates gamma-aminobutyric acid (GABA), slightly inhibited the development of tolerance, whereas the GABA antagonist, picrotoxin, produced a small increase in tolerance. The effects of treatments which altered synaptosomal membrane lipid composition were also investigated. Prolonged treatment of mice with diazacholesterol, an inhibitor of cholesterol synthesis, reduced the cholesterol content of synaptosomal membranes and, while not altering the initial sensitivity of mice to ethanol, prevented the subsequent development of tolerance. In contrast, feeding mice a diet rich in saturated fats throughout their lives reduced the unsaturated fat content of synaptosomal membranes and appeared to reduce the initial sensitivity of mice to ethanol. These results are consistent with our previous hypothesis that a reduction in unsaturation of synaptosomal membrane lipids underlies the rapid onset of ethanol tolerance in mice. They also provide further evidence for the suggestion that GABAnergic synapses may be particularly important in this respect.

Animals↗