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J A Wagner

Publications and source records attributed to J A Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mesenteric vasodilation mediated by endothelial anandamide receptors.

Cannabinoids, including the endogenous ligand anandamide (arachidonyl ethanolamide), elicit pronounced hypotension in rats via activation of peripherally located CB1 cannabinoid receptors, which have been also implicated in endotoxin (lipopolysaccharide [LPS])-induced hypotension. The present study was designed to test the role of vascular CB1 receptors in cannabinoid- and endotoxin-induced mesenteric vasodilation. In the isolated, buffer-perfused rat mesenteric arterial bed precontracted with phenylephrine, anandamide induced long-lasting (up to 60 minutes) dose-dependent vasodilation (ED50: 79+/-3 nmol; maximal relaxation: 77+/-2%), inhibited by 0.5 to 5.0 micromol/L of the selective CB1 receptor antagonist SR141716A. Low doses of the calcium ionophore ionomycin also caused mesenteric vasodilation inhibited by SR141716A. The metabolically stable analogue R-methanandamide elicited mesenteric vasodilation (ED50: 286+/-29 nmol), whereas the potent synthetic CB1 receptor agonists WIN 55212-2 and HU-210 caused no change in vascular tone or only a minor dilator effect not affected by SR141716A, respectively. The endogenous ligand 2-arachidonyl glycerol caused no change in vascular tone, whereas Delta9-tetrahydrocannabinol and arachidonic acid caused mesenteric vasoconstriction. After endothelial denudation, the dilator response to anandamide was slightly reduced and was no longer inhibited by SR141716A. In preparations from LPS-pretreated rats, SR141716A alone caused a significant and prolonged increase in perfusion pressure, whereas it had no such effect in control preparations perfused in vitro with or without LPS or after endothelial denudation in preparations from rats pretreated with LPS. We conclude that anandamide-induced mesenteric vasodilation is mediated by an endothelially located SR141716A-sensitive "anandamide receptor" distinct from CB1 cannabinoid receptors and that activation of such receptors by an endocannabinoid, possibly anandamide, contributes to LPS-induced mesenteric vasodilation in vivo.

Animals↗
PubMed1999

Technology evaluation: tgDCC-E1A, targeted genetics/MD Anderson.

Targeted Genetics is developing, tgDCC-E1A, a E1A tumor suppressor gene therapy formulated in a non-viral, lipid-based delivery system as a potential treatment for solid tumors which overexpress the her-2/neu oncogene [221611]. Fournier is the European development partner [244180]. Preclinical studies of E1A in mouse models for human breast and ovarian cancer demonstrated inhibition of expression of the her-2/neu oncogene, a significant reduction in tumors and increased survival rate in the treated animals [244180]. Results of a phase I study of intratumoral liposomal-E1A gene therapy in patients with recurrent/refractory breast cancer and head and neck cancer were presented at the 1998 American Society of Clinical Oncology (ASCO) meeting. Results demonstrated that intratumoral delivery of the E1A gene caused a subsequent downregulation of HER-2/neu expression and tumor response. The phase I dose-escalation study treated nine patients with recurrent breast cancer and nine patients with head and neck cancer. In 16 patients evaluable for response, nine had stable disease, five had progressive disease and two had minor responses despite tumor progression at other sites. In one of six patients who had repeat biopsies of treated tumors, no pathological evidence of tumor was found. In four of seven patients evaluated to date (May 1998), evidence of downregulation of HER-2/neu was reported [287387,290118]. The first phase I trial in patients with breast and ovarian cancer was completed by the end of 1997. The second phase I trial, enrolling 12 to 24 patients with solid tumors, was initiated in April 1997. Its aim was to determine dosing and safety of tgDCC-E1A and evaluate levels of gene transfer and tumor response. Patients were administered tgDCC-E1A by intratumoral injections. The company completed this trial by the end of 1997. In October 1998, Targeted Genetics initiated a multicenter, open-label phase II clinical trial of tgDCC-E1A in patients with recurrent head and neck squamous cell carcinoma who have exhibited disease progression despite standard therapies. The study to be conducted at six medical institutions in the US will enrole up to 60 patients with an interim analysis scheduled to be performed after the first 20 evaluable patients have been enrolled. Patients will be treated with ten direct intratumoral injections of the E1A gene over a period of 8 weeks, with follow-up performed at week 12. Patients who demonstrate a response will be eligible for continued treatment and will be monitored for 1 year [300424]. Targeted Genetics has issued three patents covering methods and compositions of use of the E1A and LTgenes (qv) to treat a variety of cancers. US-05651964 covers the use of the E1A gene, US-05643567 and US-05641484 cover the use of either the E1A gene or the LT gene as cancer therapies. These three patents provide the company with broad patent protection for tgDCC-E1A and all future products based on the use of the E1A or LT gene as tumor suppressors [259808].

Adenovirus E1A Proteins↗
PubMed1998

Platelet- and macrophage-derived endogenous cannabinoids are involved in endotoxin-induced hypotension.

Macrophages are the primary cellular targets of bacterial lipopolysaccharide (LPS), but the role of macrophage-derived cytokines in LPS-induced septic shock is uncertain. Recent evidence indicates that activation of peripheral CB1 cannabinoid receptors contributes to hemorrhagic hypotension and that macrophage-derived anandamide as well as unidentified platelet-derived substances may be contributing factors. Here we demonstrate that rat platelets contain the endogenous cannabinoid 2-arachidonyl glyceride (2-AG), as identified by reverse phase high-performance liquid chromatography, gas chromatography, and mass spectrometry, and that in vitro exposure of platelets to LPS (200 microg/ml) markedly increases 2-AG levels. LPS-stimulated, but not control, macrophages contain anandamide, which is undetectable in either control or LPS-stimulated platelets. Prolonged hypotension and tachycardia are elicited in urethane-anesthetized rats treated 1) with LPS (15 mg/kg i.v.); 2) with macrophages plus platelets isolated from 3 ml of blood from an LPS-treated donor rat; or 3) with rat macrophages or 4) platelets preincubated in vitro with LPS (200 microg/ml). In all four cases, the hypotension but not the tachycardia is prevented by pretreatment of the recipient rat with the CB1 receptor antagonist SR141716A (3 mg/kg i.v.), which also inhibits the hypotensive response to anandamide or 2-AG. The hypotension elicited by LPS-treated macrophages or platelets remains unchanged in the absence of sympathetic tone or after blockade of nitric oxide synthase. These findings indicate that platelets and macrophages generate different endogenous cannabinoids, and that both 2-AG and anandamide may be paracrine mediators of endotoxin-induced hypotension via activation of vascular CB1 receptors.

Animals↗
PubMed1998

Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein.

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

Animals↗
PubMed1998

Development of a scale to measure adherence to self-monitoring of blood glucose with latent variable measurement.

OBJECTIVE: Adherence to self-monitoring of blood glucose (SMBG) is problematic for many people with diabetes. Self-reports of adherence have been found to be unreliable, and existing paper-and-pencil measures have limitations. This study developed a brief measure of SMBG adherence with good psychometric properties and a useful factor structure that can be used in research and in practice. RESEARCH DESIGN AND METHODS: A total of 216 adults with diabetes responded to 30 items rated on a 9-point Likert scale that asked about blood monitoring habits. In part I of the study, items were evaluated and retained based on their psychometric properties. The sample was divided into exploratory and confirmatory halves. Using the exploratory half, items with acceptable psychometric properties were subjected to a principal components analysis. In part II of the study, structural equation modeling was used to confirm the component solution with the entire sample. Structural modeling was also used to test the relationship between these components. It was hypothesized that the scale would produce four correlated factors. RESULTS: Principal components analysis suggested a two-component solution, and confirmatory factor analysis confirmed this solution. The first factor measures the degree to which patients rely on others to help them test and thus was named "social influence." The second component measures the degree to which patients use physical symptoms of blood glucose levels to help them test and thus was named "physical influence." Results of the structural model show that the components are correlated and make up the higher-order latent variable adherence. CONCLUSIONS: The resulting 15-item scale provides a short, reliable way to assess patient adherence to SMBG. Despite the existence of several aspects of adherence, this study indicates that the construct consists of only two components. This scale is an improvement on previous measures of adherence because of its good psychometric properties, its interpretable factor structure, and its rigorous empirical development.

Adult↗
PubMed1997

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock.

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

Animals↗
PubMed1997

Regulation of the neural-specific gene VGF in PC12 cells. Identification of transcription factors interacting with NGF-responsive elements.

Nerve growth factor (NGF) is an important regulator of differentiation and survival in both the peripheral and central nervous systems. We have begun to analyze the mechanism by which NGF induces the expression of a neural specific gene, VGF, in PC12 cells. Using DNase I footprinting and transient transfection analysis, we identified two VGF promoter regions, V1 and V2, that are required for basal promoter expression as well as gene induction by NGF, epidermal growth factor (EGF), and cAMP. The V1 element is essential for VGF promoter function, but it is not sufficient to confer NGF responsiveness to a heterologous promoter. In contrast, the V2 element can independently stimulate the expression of a linked herpes simplex virus (HSV) thymidine kinase promoter in response to NGF. We showed that the V2 region also contains a sequence that acts as a promoter-specific negative regulator of basal VGF gene expression. As determined by gel mobility shift and Southwestern analysis, the V1 sequence is recognized by a novel PC12 nuclear protein of about 110-kDa molecular mass. Using oligonucleotide competition and antibody supershift assays, we demonstrated that the cAMP-response element (CRE) motif within the V2 element interacted specifically with proteins related to cAMP-response element binding (CREB), JunB, and JunD transcription factors. The JunB-related binding activities were transiently induced by NGF, suggesting that part of the mechanism utilized by NGF to activate VGF transcription includes increased synthesis of a V2 binding protein. Taken together, our analysis suggests that the VGF promoter is regulated by a complex mechanism, and its activation requires combinatorial action of several transcription factors interacting with multiple promoter elements.

Animals↗
PubMed1997

Toward cystic fibrosis gene therapy.

Cystic fibrosis (CF) is a common genetic disorder characterized by defective epithelial chloride transport and progressive lung disease. Although great strides have been made in the treatment of CF, it remains lethal, often by early adulthood. CF is one of the most extensively researched genetic diseases as a target for gene therapy development. It may also serve as an important model for gene therapy of other diseases. Preclinical and clinical research has lead to the rapid development of a variety of vectors designed to correct the basic defect in CF, including adenovirus, adeno-associated virus, and liposomes. Clinical studies have identified the host immune response and low vector efficiency as key impediments to effective CF gene therapy. Further research promises to refine vector technology and bring CF gene therapy to the bedside.

Adolescent↗
PubMed1997

Rac is required for growth cone function but not neurite assembly.

Recent work has suggested that rac1 and other members of the rho family of small GTP-binding proteins play an important role in the formation of neural processes. We have explored the mechanism of this effect by comparing the spontaneous, growth cone-mediated growth and experimental tension-induced growth of axons in normal PC12 cells and in mutant cells expressing a dominant negative form of rac. PC12 that have been primed by exposure to NGF, but not naive PC12 cells, initiate a microtubule-rich process de novo in response to tension applied to cell body. As in chick sensory neurons, neurite elongation rate is proportional to applied tension above a threshold. Addition of cyclic AMP, which has been shown to rapidly augment NGF-induced neurite outgrowth in PC12, causes a rapid increase in the rate of neurite elongation at a given tension level. Expression of a dominant negative form of rac1 inhibits spontaneous, growth cone-mediated neurite elongation in response to NGF, but does not substantially affect tension-induced neurite elongation. That is, rac-deficient cells show a normal linear relationship between applied tension and elongation rate and the elongations contain a normal density of axial microtubules by immunofluorescent assay. Thus, rac1 is apparently required for the mechanisms that normally generate tension in an elongating neurite, but if this tension is provided from an outside source, then axonal elongation can proceed normally in rac1-deficient cells. We conclude that rac1 is required for the adhesive and motile function of growth cones rather than the assembly of neurites per se.

Animals↗
PubMed1996

Unusual expression of the Hu paraneoplastic antigen in the visual system.

The Hu paraneoplastic antigen is an RNA binding protein important in the development and maintenance of neurons. Hu has homology with the product of Elav, a gene that is essential for Drosophila visual system development. Using immunohistochemical staining, the Hu antigen was identified in most retinal neurons but was not detected in the photoreceptors. Hu is, therefore, not essential for the maintenance of all differentiated neurons. Hu immunolabeling was consistently found in glia in the optic nerve. The presence of the Hu antigen in a subset of glial cells emphasizes the heterogeneity of glial phenotype and raises the possibility that these cells may retain properties of undifferentiated neural stem cells.

Animals↗
PubMed1996

Src, ras, and rac mediate the migratory response elicited by NGF and PMA in PC12 cells.

The combination of nerve growth factor (NGF) and phorbol 12-myristate 13-acetate (PMA) rapidly induced the locomotion of PC12 cells by sequentially stimulating lamellar spreading, ruffling with pinocytosis, and polarization by retraction from the substratum. During migration, cells acquired long processes as a result of several undisrupted cell-substratum attachment points. The effect of NGF on PC12 migration was blocked by K-252a, a selective inhibitor of the trk family of receptor tyrosine kinases. When PMA was added to cells expressing pp60v-src, the cells displayed the same morphological behavior as they did with NGF and PMA addition. Activated ras only partially substituted for the effects of NGF; but, when ras was inhibited, the number of migrating cells decreased significantly due to a defect in spreading and retraction. Expression of an activated form of rac stimulated spontaneous growth of lamellipodia and enhanced cell migration in response to PMA. Expression of a dominant negative form of rac inhibited cell spreading and motility. Also, as a later effect, rac-inhibited cells extended much shorter neurites than wild type cells in response to NGF alone. These results indicate that the cytoarchitectural changes induced by NGF and PMA in PC12 cells are mediated by src, ras, and rac. Whereas ras and rac activation affect lamellipodia extension and retraction but not pinocytotic ruffling, src activation is involved in all three events.

Animals↗
PubMed1996

Gene therapy.

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Adenoviruses, Human↗