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Biomedical subjects

D J Fitzgerald

Publications and source records attributed to D J Fitzgerald.

At least 109 records · Page 6Linked to original sources
PubMed1991

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin.

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.

Animals↗
PubMed1991

Cytotoxic activity of chimeric proteins composed of acidic fibroblast growth factor and Pseudomonas exotoxin on a variety of cell types.

Chimeric proteins composed of acidic fibroblast growth factor (acidic FGF) and several forms of Pseudomonas exotoxin (PE) that cannot bind to the PE receptor have been produced in Escherichia coli by expressing chimeric genes in which DNA encoding acidic FGF is fused to various mutant forms of PE. These acidic FGF-PE fusion proteins were found to be cytotoxic to a variety of tumor cell lines including hepatocellular (PLC/PRF/5 and HEPG2), prostatic (LNCaP), colon (HT29), and breast (MCF-7) carcinomas at concentrations of 1-70 ng/ml. The cytotoxic effects of acidic FGF-PE were FGF-receptor specific as demonstrated by competition with excess acidic FGF and by showing that acidic FGF-PE bound to the FGF receptor with the same affinity as acidic FGF. Furthermore, the cell-killing activity of acidic FGF-PE was toxin-mediated, as an acidic FGF-PE mutant, which does not possess ADP-ribosylation activity, failed to kill cells. These findings demonstrate that acidic FGF-PE is a potent cytotoxic molecule that can be targeted to FGF receptor-bearing cells. Because acidic FGF is a potent angiogenic molecule, cytotoxic acidic FGF-PE chimeras may have utility as anti-angiogenic agents. These molecules could be helpful in determining the functional role of FGF receptors in cellular processes.

ADP Ribose Transferases↗
PubMed1991

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.

ADP Ribose Transferases↗
PubMed1991

Combined administration of aspirin and a specific thrombin inhibitor in man.

BACKGROUND: Heparin is of limited value as an antithrombotic drug in the presence of platelet activation and residual thrombus. Greater anticoagulant activity can be achieved in vivo with more specific thrombin inhibitors. Heparin may also increase the risk of bleeding by an effect on platelets that is independent of its thrombin inhibitory activity. METHODS AND RESULTS: The pharmacodynamic and pharmacokinetic effects of a novel thrombin inhibitor, argatroban, were examined alone and in combination with aspirin in normal male volunteers. Argatroban induced a dose-dependent prolongation of the thrombin time and the activated partial thromboplastin time (aPTT). aPTT had returned to its pretreatment value 1 hour after stopping the infusion of argatroban. Six male subjects received an infusion of 1 micrograms/kg/min argatroban after the administration of two doses of 162.5 mg aspirin or a matching placebo. At this dose, aspirin decreased serum thromboxane B2 by a mean of 99% and prolonged the bleeding time (230 +/- 52 versus 320 +/- 113 seconds, p less than 0.01). Argatroban given alone increased thrombin time by 454 +/- 18% and aPTT by 160 +/- 3%. Steady-state plasma concentrations were achieved at 1 hour and declined exponentially with an elimination half-life of 24 +/- 4 minutes. Neither the anticoagulant effects nor the plasma concentrations of argatroban were altered by aspirin. Furthermore, argatroban did not increase the bleeding time when given alone and did not further prolong the bleeding time when combined with aspirin. CONCLUSION: The combination of aspirin and argatroban may prove to be an effective therapeutic strategy in the prevention of coronary thrombosis.

Adult↗
PubMed1991

Systemic lysis protects against the effects of platelet activation during coronary thrombolysis.

Systemic lysis may protect against the platelet activation and ongoing thrombosis associated with coronary thrombolysis. To address this hypothesis, we compared urokinase and tissue-type plasminogen activator (t-PA) given intravenously in a chronic, canine model of coronary thrombosis. T-PA 10 micrograms/kg per min induced reperfusion in 55 +/- 7 min but complete reocclusion occurred in 9/10 animals. Reocclusion was prevented by combining t-PA with 7E3, an antibody to the platelet glycoprotein IIb/IIIa which abolished ex vivo platelet aggregation. A similar time to reperfusion was seen with urokinase 750-1,000 U/kg per min. In contrast to t-PA, complete reocclusion occurred in only 1/20 cases (P less than 0.001 vs. t-PA), despite evidence of continued platelet activation in vivo and platelet aggregation ex vivo. Furthermore, this did not reflect a difference in the clearance of the two plasminogen activators. However, plasma fibrinogen was undetectable after urokinase in contrast with t-PA. Furthermore, in animals treated with prourokinase 20 micrograms/kg per min, reocclusion (4/7) correlated with the degree of systemic lysis. To determine whether platelet activation modified the response to urokinase, it was combined with 7E3. 7E3 0.8 mg/kg reduced the time to reperfusion with t-PA (30 +/- 5, n = 6; P = 0.025), but not with urokinase (56 +/- 8 vs. 62 +/- 6, P = ns). Systemic lysis protects against the propensity of continued thrombosis during coronary thrombolysis to delay reperfusion and induce reocclusion. This may modify the requirement for adjunctive antiplatelet therapy.

Animals↗
PubMed1991

A prostacyclin analog impairs the response to tissue-type plasminogen activator during coronary thrombolysis: evidence for a pharmacokinetic interaction.

Inhibition of thromboxane (TX) A2 with aspirin enhances the response to coronary thrombolysis. However, experimental evidence suggests that platelet activation during coronary thrombolysis is mediated by a number of agonists, in addition to TXA2. As a consequence, greater benefit would be expected with antiplatelet agents that have a broader spectrum of activity. However, a recent clinical trial, combining tissue plasminogen activator (t-PA) with the prostacyclin analog, iloprost, did not detect such a benefit. To address the mechanism of this response, we compared the effect of iloprost, a stable analog of prostacyclin, with GR32191, a TXA2/prostaglandin endoperoxide receptor antagonist, on the response to i.v. t-PA in a closed chest, canine model of coronary thrombosis. GR32191 reduced the time to reperfusion by 47% (n = 6, P less than .05), consistent with a role for TXA2-mediated platelet activation in impairing thrombolysis. In contrast, iloprost increased the time to reperfusion by 50% (n = 5, P = NS) and in four of nine animals reperfusion failed to occur despite inhibition of platelet aggregation. In a separate series of experiments, steady-state plasma t-PA clearance increased by 38% (407 +/- 49 vs. 294 +/- 42 ml/min; n = 8, P less than .02) during infusion of iloprost and recovered after its withdrawal. This appeared to be a specific effect, as infusion of nitroglycerin at a dose which induced a similar fall in blood pressure altered neither the time to reperfusion nor plasma t-PA. Iloprost impairs the thrombolytic response to t-PA via an increase in the clearance of this agent.

Alprostadil↗
PubMed1990

IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells.

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.

ADP Ribose Transferases↗
PubMed1990

The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems.

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1990

Thromboxane A2 synthesis in pregnancy-induced hypertension.

Urinary excretion of thromboxane B2 metabolites as markers of thromboxane A2 synthesis was measured in eight patients with moderate to severe pregnancy-induced hypertension and in six normotensive pregnant women at term. Excretion of both 2,3-dinor-TxB2 (median 3919, range 683-16,680 pg/mg creatinine) and 11-dehydro-TxB2 (median 10,187, range 434-57,203 pg/mg creatinine) was significantly higher in the patients with pregnancy-induced hypertension than in the normotensive pregnant group (927[273-1343] and 774[500-2760] pg/mg creatinine, respectively). Thromboxane metabolite excretion correlated with mean arterial blood pressure, plasma lactate dehydrogenase, and platelet count which are indices of the severity of pregnancy-induced hypertension. Excretion of both metabolites fell rapidly post partum in parallel with resolution of clinical signs. Thus, increased thromboxane A2 biosynthesis correlates with disease severity and may have a pathogenetic role in pregnancy-induced hypertension. These findings provide a rationale for the use of aspirin in the treatment as well as in the prevention of this disorder.

Adolescent↗
PubMed1990

Tumor promotion: models and assay systems.

Tumor promotion is defined operationally from two-stage models of experimental carcinogenesis. It is, therefore, in a strict sense, possible to identify tumor promoters only from such models. The development and use of in vitro two-stage cell transformation assays was a logical extension toward in vitro short-term testing for tumor promoters. Another approach is to apply mechanistic knowledge of the tumor promotion process in developing end points for such assays. In this context, we have been examining the role of blocked gap-junctional intercellular communication (GJIC) in tumor promotion, using in vitro and in vivo systems. Many promoters have been shown to block GJIC in vitro; our studies support the idea that inhibition of GJIC does play an important role in the promotion stage of BALB/c 3T3 cell transformation. In animal studies, we have shown that the rat liver tumor promoter phenobarbital can decrease the level of expression of the 32 Kd gap junction protein gene specifically in liver upon systemic exposure in rats. Further examination of the role of GJIC in tumor promotion is indeed warranted. Also, deployment of in vitro GJIC and transformation assay systems should provide useful short-term tests for detecting tumor promoting activity of environmental chemicals.

Animals↗
PubMed1990

Endogenous biosynthesis of arachidonic acid epoxides in humans: increased formation in pregnancy-induced hypertension.

Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Recombinant CD4-Pseudomonas exotoxin hybrid protein displays HIV-specific cytotoxicity without affecting MHC class II-dependent functions.

The present study describes several in vitro activities of CD4(178)-PE40, a recombinant protein containing a portion of human CD4 linked to active regions of Pseudomonas aeruginosa exotoxin A. Using assays for cell viability, we demonstrate that the hybrid toxin displays highly selective cytotoxicity for HIV-infected T lymphocytes. In a latently infected human T-cell line which is inducible for HIV expression, toxin sensitivity is observed only upon virus induction. At concentrations which readily kill HIV-infected T cells, CD4(178)-PE40 has no observable cytotoxic effects on uninfected human cell lines expressing surface major histocompatibility complex (MHC) Class II molecules, and does not interfere with cellular responses known to be dependent on functional association between CD4 and MHC Class II molecules.

ADP Ribose Transferases↗
PubMed1990

Effects on intercellular communication in human keratinocytes and liver-derived cells of polychlorinated biphenyl congeners with differing in vivo promotion activities.

Several purified polychlorinated biphenyl (PCB) congeners with differing toxicity/tumor promotional activities in rat liver in vivo were tested for their effects on gap-junctional intercellular communication (GJIC) in cell strains and lines derived from human liver and skin. This in vitro assay is being developed to detect various classes of tumor promoters. The 3-methylcholanthrene (MCA)-type cytochrome P450 inducer and hepatotoxic promoter 3,3',4,4'-tetrachlorobiphenyl was inactive in this assay for all of the cells tested, suggesting this promoter acts by other mechanisms. The phenobarbital-like enzyme inducer and less toxic promoter 2,2',4,4',5,5'-hexachlorobiphenyl inhibited GJIC in both liver and skin cells, whereas the 2,2',5,5'-tetrachlorobiphenyl congener, which does not act as a promoter in rat liver, inhibited GJIC only in the skin cell types and in one of the liver cell strains thought to be of bile duct origin. 2,3,4,4',5-Pentachlorobiphenyl, a mixed (phenobarbital plus MCA) inducer of cytochrome P450, inhibited GJIC in both liver and skin cells, suggesting that it may be a promoter in vivo. The results suggest that GJIC inhibition is a property of PCB congeners with phenobarbital-like enzyme induction capabilities, and that there exist some tissue/cell type differences in sensitivity to these congeners.

Carcinogens↗
PubMed1990

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.

ADP Ribose Transferases↗
PubMed1990

Dietary fish oil accelerates the response to coronary thrombolysis with tissue-type plasminogen activator. Evidence for a modest platelet inhibitory effect in vivo.

To assess the platelet inhibitory effect of high doses of fish oils and relate it to alterations in eicosanoid synthesis, we used a canine model in which coronary thrombosis, the time to reperfusion induced by recombinant tissue-type plasminogen activator (rt-PA), and the rate of spontaneous reocclusion are sensitive to platelet inhibition. In the animals fed fish oil, the time to rt-PA induced thrombolysis was accelerated (mean, 63 vs. 27 minutes; p less than 0.003). The time to thrombotic occlusion and the rate of reocclusion were unaltered. The ratio of eicosapentaenoic acid (EPA) to arachidonic acid rose in platelet and endothelial cell membranes, whereas serum thromboxane (Tx) B levels fell a mean 86%, and basal excretion of 2,3-dinor-TxB2 (TxA2-M) declined. Basal prostaglandin (PG) I2 formation was unaltered, whereas biosynthesis of EPA-derived TxA3 and PGI3 increased. In control animals, TxA2 formation increased during thrombosis; there was a further, more marked rise during reperfusion. PGI2 formation also increased, probably as a response to platelet-vascular interactions. Stimulated production of both eicosanoids was strikingly suppressed in the animals fed fish oil. Fish oils significantly enhance the efficacy of rt-PA in vivo, albeit to a modest extent. Because the time to reperfusion is highly sensitive to Tx-dependent platelet activation, this effect is likely to reflect the demonstrated suppression of TxA2 biosynthesis by fish oils.

Animals↗
PubMed1989

Gap-junctional intercellular communication in epidermal cell lines from selected stages of SENCAR mouse skin carcinogenesis.

Homologous and heterologous gap-junctional intercellular communication (IC) was characterized in a panel of cell lines derived from selected stages of SENCAR mouse skin carcinogenesis. This panel included a "carcinogen-altered" cell line, 3PC, obtained from Ca2+-resistant primary adult keratinocytes after exposure to dimethylbenz(a)anthracene as well as cell lines obtained from early and late-stage papillomas and a squamous cell carcinoma (CA3/7) generated during standard in vivo initiation/promotion protocols (dimethylbenz(a)anthracene/12-O-tetradecanoyl-phorbol-13-acetate). Also studied was a cell line (B66BA) obtained from a metastatic lesion following benzo(a)pyrene-induced skin tumorigenesis. Intercellular communication was measured in low-calcium (0.05 mM) medium by quantitation of cell-cell transfer of microinjected fluorescent dye Lucifer Yellow CH. Homologous IC ability diminished progressively from 68 dye-coupled cells per injection for 3PC cultures, to between 21 and 54 dye-coupled cells per injection for three papilloma-derived cell lines, to six and three dye-coupled cells per injection for CA3/7 and B66BA cells, respectively. To test communication of these cells with their normal counterparts, heterologous IC was examined in cocultures with primary adult keratinocytes. Under the conditions used, normal cells established functional communication channels with each cell line tested, showing no selectivity. These results suggest that progressive loss of homologous but not heterologous IC capacity accompanies neoplastic development in mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗