Hemolysis of blood specimens transported from ED to laboratory by pneumatic tube.
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Blood Specimen Collection↗
Biomedical subjects
D J Fitzgerald
Publications and source records attributed to D J Fitzgerald.
A modified collection and rapid infusion system for shed whole blood autotransfusion during aortic aneurysm surgery.
We describe our experience in 10 patients (5 males) undergoing resection of a descending thoracic aortic aneurysm or a thoracoabdominal aortic aneurysm in which a modified shed whole blood collection and autotransfusion system was used. This modification allows several options for the processing and autotransfusion of shed blood: use of the cell saving device or the ultrafiltration of collected blood, and the autotransfusion of unprocessed shed whole blood. Either low dose heparin or sodium citrate was used for anticoagulation. All 10 patients underwent autotransfusion and volume resuscitation with the modified rapid infusion device. Total autotransfusion ranged from 1400 ml to 7843 ml. Ultrafiltration volumes ranged from 600 ml to 1100 ml. There were no intraoperative deaths and no patient reoperations for bleeding. Arterial blood gases, potassium, and platelet counts were all within the normal laboratory ranges. This modification enables the clinician to process poor quality shed blood and reinfuse whole blood, in an attempt to decrease the need for homologous blood products.
Aged↗
Sequence determinants of DNA bending in the ilvlH promoter and regulatory region of Escherichia coli.
Previous studies have shown that the promoter/regulatory region of the ilvlH operon displays intrinsic curvature, with the bend center located at position -120 relative to the transcription start site. In this report, a 57 bp sequence spanning the bend center was mutagenized in vitro in order to study the relationship between nucleotide sequence and curvature measured by electrophoresis. The strategy used for analyzing the results consisted of determining the strengths of the relationships between electrophoretic anomaly and predicted curvature calculated by computer programs that differ in wedge angle composition. The results revealed that programs which assume that bending occurs only at AA/TT display good predictive value, with correlation coefficients between electrophoretic anomaly and predicted curvature as high as 0.93. In contrast, a program which assumes that bending occurs at all 16 dinucleotide steps exhibited lower predictive value, while there were no significant relationships between the experimental data and curvature calculated by a program that was based on all non-AA/TT wedge values. These results show that the complete wedge model which incorporates values for all dinucleotide steps does not adequately describe the electrophoretic data in this report.
Acetolactate Synthase↗
Selection of Pseudomonas exotoxin-resistant cells with altered expression of alpha 2MR/LRP.
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ADP Ribose Transferases↗
Conserved patterns of bending in satellite and nucleosome positioning DNA.
Intrinsic DNA curvature has previously been implicated in the condensation of satellite DNA in chromatin. In this article, electrophoretic methods and computer programs for predicting DNA structure from nucleotide sequence were used to determine if curvature is a conserved feature of satellite DNA. The results revealed that satellite sequences display wide variation in magnitude of intrinsic bending. Less than half of the satellites examined were strongly bent when compared with control DNA. However, a conserved pattern of bending was seen in all 57 satellite sequences that were studied. The pattern consisted of repeating units of two 50-60-base pair bending elements which were separated by a 20-30-base pair region of low curvature. This pattern resembles qualitatively the bending of DNA in the nucleosome where the helix is folded approximately two turns around the histone octomer with the turns interrupted by a less bent segment in the center of the particle. The distance between successive pairs of bending elements was also similar to the average spacing of nucleosomes in chromatin. Thus, the conserved structures could play some role in the positioning of nucleosomes along satellite chromatin. In order to strengthen the correlation between DNA structure and nucleosome positioning, sequences were examined which have been shown to position nucleosomes at single major sites. This analysis revealed a conserved pattern of DNA structure resembling the two peak units seen in satellites. In addition, the nucleotide sequence patterns responsible for the conserved patterns of bending were similar in both satellite and nucleosome positioning DNA. Likewise, nucleotide sequence patterns that are thought to direct the rotational orientation of DNA in the nucleosome were similar in both sequence sets. These results are considered in terms of a general model for the role of DNA bending and nucleotide sequence in the control of nucleosome positioning and chromatin condensation in eukaryotes.
Animals↗
Warfarin and aspirin after heart-valve replacement.
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Administration, Cutaneous↗
Expression and function of connexin in normal and transformed human keratinocytes in culture.
We have studied the gap junctional intercellular communication (GJIC) of immortalized and tumourigenic human keratinocyte cell lines and of a spontaneously immortalized non-tumourigenic and a highly differentiating keratinocyte cell line (HaCaT) as the control. In homologous cultures, the GJIC capacity of five squamous cell carcinoma-derived cell lines was 1-27% that of the HaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenic potential and an SV40 DNA-immortalized cell line had markedly reduced GJIC capacities. Northern analysis and immunohistochemistry showed that connexin (Cx) 43 is the major gap junction protein expressed in the communicating cells. They do not express Cx 26 or 32. The low or absent communication observed in certain cell lines was due in some to a lack of Cx 43 gene expression, but in others to aberrant localization of the gap junction protein. GJIC of these cell lines, as well as that of primary normal human epidermal keratinocytes, was susceptible to 12-O-tetradecanoylphorbol-13-acetate-mediated inhibition. Moreover, GJIC of HaCaT cells and their tumourigenic derivatives is Ca(2+)-dependent. These results, when compared with those previously obtained for mouse keratinocyte cell lines, reveal that GJIC of human keratinocytes was correlated to the degree of differentiation and is controlled in a similar way to that of murine keratinocytes. Aberrant GJIC seems to be a common feature of human and murine skin carcinogenesis.
Carcinoma, Squamous Cell↗
Gap junctional intercellular communication and connexin expression in normal and SV 40-transformed human liver cells in vitro.
The gap junction intercellular communication (GJIC) capacity of two normal human liver-derived epithelial cell strains and their SV40 large T oncogene-transformed counterparts was examined. In homologous cultures the GJIC capacity of the transformed cells was considerably less than the parental cells. In heterologous cultures, transformed cells appeared to be able to form GJIC channels with normal cells. Only non-transformed cells expressed connexin (cx) 43 gene and cx 26 or cx 32 transcripts were not detectable in any cell strains tested. When GJIC was assayed in the presence of the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13- acetate (TPA), all four strains showed a marked sensitivity to TPA in inhibitory activity at 1-10 ng/ml. In contrast to a rat liver epithelial cell line, this effect of TPA did not appear to become refractory even after 24 h exposure. These studies demonstrate that GJIC of human liver cells in culture can be decreased by viral oncogene and tumour promoter action. Such disturbance may be an important component of the carcinogenic activity of these agents.
Adult↗
Femoral veno-arterial extracorporeal life support with minimal or no heparin.
Recent technological advances in cardiopulmonary bypass circuits achieving surface bonding with heparin have permitted prolonged extracorporeal life support (ECLS) in experimental studies without the use of systemic anticoagulation. Excessive bleeding and the need for massive transfusions after extended ECLS with systemic heparinization have often led to the development of sepsis and multisystem organ failure. The Carmeda bioactive surface circuit, along with a Bio-Medicus centrifugal pump (Bio-Pump) and the femoral veno-arterial route, were used successfully in 3 patients requiring ECLS between April 1992 and December 1992. In 2 patients the need for ECLS was acute allograft dysfunction after orthotopic cardiac transplantation with no evidence of cellular rejection. Both patients were receiving multiple inotropes and intraaortic balloon counter-pulsation; their condition continued to deteriorate rapidly before the initiation of ECLS. The third patient failed to be weaned from cardiopulmonary bypass after myocardial revascularization for ischemic cardiomyopathy. Time on ECLS ranged from 57 to 128 hours. No systemic anticoagulation was used. One patient received no heparin, and the other 2 patients received intermittent heparin infusion to maintain an average activated clotting time of 195 and 214 seconds. Multisystem organ dysfunction present before initiation of ECLS was rapidly reversed, and all patients were weaned successfully without any immediate major complications. The ability to conduct prolonged ECLS without systemic anticoagulation and without repeat sternotomy opens new avenues for the use of this procedure in profound heart failure.
Blood Coagulation↗
Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells.
We examined gap junctional intercellular communication (GJIC) of primary human mesothelial cells and cell lines of asbestos-associated human pleural mesotheliomas, and the effect of asbestos and other mineral fibres on these cells. In homologous cultures, the GJIC capacity of six out of seven tumour cell lines was markedly less than for primary mesothelial cells. This defect in GJIC appeared not to be at the expression level of mRNA and protein of the gene encoding the 43 kDa gap junction protein. In heterologous cocultures of tumour cells and primary mesothelial cells, however, 80-90% of the tumour cell/normal cell contacts were functional. Exposure of primary mesothelial cells to TPA, a phorbol ester tumour promoter, resulted in marked inhibition of GJIC, being an action common to numerous tumour promoters. Such an effect though was not observed with the carcinogenic mesothelioma-inducing mineral fibres chrysotile and amosite, neither with glass wool. These results suggest that a permanent defect in GJIC capacity is a common feature of human mesothelioma cells, but how mineral fibres are involved in the process of mesotheliomagenesis is still unclear.
Asbestos↗
Transdermal modification of platelet function. A dermal aspirin preparation selectively inhibits platelet cyclooxygenase and preserves prostacyclin biosynthesis.
BACKGROUND: Even low doses of oral aspirin inhibit prostacyclin (prostaglandin [PG] I2) formation and cause gastrointestinal toxicity. We examined the skin as a novel route for continuous low-dose aspirin administration and selective inhibition of platelet cyclooxygenase in humans. METHODS AND RESULTS: Aspirin 250 or 750 mg/d for 10 days induced a dose-dependent inhibition of serum thromboxane (TX) B2. At the highest dose, five of six subjects responded, with a mean reduction in serum TXB2 of 95 +/- 3% (P = .003). Urinary 2,3-dinor TXB2, an index of in vivo TXA2 formation, decreased by 68 +/- 7% and recovered slowly, consistent with inhibition of platelet cyclooxygenase in vivo. In contrast, PGI2 biosynthesis, determined as excretion of 2,3-dinor-6-keto PGF1 alpha, was 81 +/- 5% of baseline at 10 days. Intravenous bradykinin increased PGI2 biosynthesis 5.1 +/- 1.6-fold (n = 4) before aspirin treatment. Oral aspirin 75 mg/d for 14 days abolished bradykinin-induced PGI2 formation, whereas dermal aspirin 750 mg/d had no effect despite similar inhibition of TXA2 biosynthesis. In five subjects, plasma aspirin and salicylate were determined after a single application of 750 mg. Aspirin was absorbed slowly, with peak levels of 0.24 +/- 0.11 micrograms/mL at 3 hours. Salicylate levels peaked at 6 to 12 hours, with plasma levels of 0.79 +/- 0.14 micrograms/mL. CONCLUSIONS: Thus, it is possible to achieve selective inhibition of platelet cyclooxygenase by aspirin applied to the skin. This approach may be applicable to other antiplatelet agents and be useful in patients at risk for gastrointestinal bleeding or toxicity.
Administration, Cutaneous↗
Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.
Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.
ADP Ribose Transferases↗
Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.
Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t-PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet-induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.
Blood Platelets↗
Metsovo-tremolite asbestos fibres: in vitro effects on mutation, chromosome aberration, cell transformation and intercellular communication.
Samples of Metsovo-tremolite asbestos, previously found to be the causative agent of endemic pleural calcification and an increased level of malignant pleural mesothelioma in a rural area of north-western Greece (Metsovo area), were tested in various in vitro toxicity test systems. It was found that asbestos fibres of this type were strong inducers of micronuclei and numerical chromosomal abnormalities while they induced low levels of chromosomal aberrations in mammalian cells in culture. Furthermore, this type of asbestos can induce a low level of in vitro transformation of Syrian hamster embryo cells. The fibres had no effect on gap-junctional cell-cell communication (followed by the dye-transfer method) and did not induce any mutations in the Salmonella typhimurium strain TA102 which is known to be sensitive to the action of various oxidative agents. These results support the hypothesis generated from studies on other types of asbestos that such fibres induce tumours by causing chromosomal mutations.
Aneuploidy↗
Pharmacokinetics of tissue-type plasminogen activator during acute myocardial infarction in men. Effect of a prostacyclin analogue.
BACKGROUND: Coronary reocclusion complicates the thrombolytic therapy of acute myocardial infarction despite the routine use of aspirin. This is consistent with experimental studies demonstrating that multiple agonists, in addition to thromboxane A2, mediate the platelet activation underlying reocclusion. Consequently, a more potent antiplatelet therapy with a broader spectrum of activity than aspirin may be required in this setting. Prostacyclin and its more stable analogue, iloprost, inhibit platelet aggregation to all known agonists and exert an additional effect over aspirin alone. Experiments in animal models have demonstrated, however, that iloprost increases the clearance of tissue-type plasminogen activator (t-PA) and impairs thrombolysis in vivo. This study examines whether a similar interaction occurs in humans. METHODS AND RESULTS: Twelve patients with acute myocardial infarction received t-PA intravenously, 60 mg in the first hour and a maintenance infusion of 13.3 mg/hr for 3 hours. Patients were assigned in a double-blind fashion to iloprost (2 ng/kg/min) or placebo following the initial 90 minutes of the maintenance infusion of t-PA. Iloprost decreased mean arterial blood pressure (-10 +/- 2.9 mm Hg, p less than 0.05) but did not alter heart rate. Steady-state plasma iloprost concentration was 591 +/- 64 pmol/l. At this concentration, iloprost markedly inhibited platelet aggregation in vitro, particularly in the presence of aspirin. Steady-state clearance of t-PA was unchanged by iloprost (454 +/- 65 versus 443 +/- 136 ml/min in controls, p = NS). Furthermore, neither elimination kinetics nor plasma protein binding of t-PA was altered by iloprost. CONCLUSIONS: At plasma levels that exert a potent antiplatelet effect, iloprost did not alter the pharmacokinetics of t-PA in men. Prostacyclin analogues may prove useful as an adjunct to plasminogen activators, particularly in patients at high risk for thrombotic reocclusion.
Acute Disease↗
Subcutaneous heparin during coronary thrombolysis. Too little, too late.
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Heparin↗
Platelet activation in the pathogenesis of unstable angina: importance in determining the response to plasminogen activators.
Unstable angina is a clinical syndrome of recurrent myocardial ischemia. In some cases, this reflects episodic platelet activation and coronary thrombosis. Thus, the biosynthesis of thromboxane A2, which is largely derived from activated platelets, is increased, often coincident with chest pain. The major role of platelets in unstable angina may influence the response to plasminogen activators. Platelets increase the resistance of thrombi to lysis, by inducing clot retraction and cross-linking and by releasing inhibitors. Thus, coronary thrombi in unstable angina may be resistant to lysis. Furthermore, both t-PA and streptokinase cause platelet activation and thrombin formation in vivo, possibly via plasmin. Plasmin can activate platelets and factor V directly. These prothrombotic effects of plasminogen activators may limit their activity in unstable angina. At the very least, their therapeutic efficacy may be highly dependent on the coadministration of potent antiplatelet agents and anticoagulants.
Angina, Unstable↗
Heterogeneity of prostaglandin H2/thromboxane A2 receptors: distinct subtypes mediate vascular smooth muscle contraction and platelet aggregation.
Studies of the hierarchies of agonist and antagonist affinity for the prostaglandin (PG)H2/thromboxane (Tx)A2 receptor have been performed to establish whether distinct receptor subtypes exist in platelets and vascular smooth muscle cells (VSMC). They have yielded conflicting results. The pattern of homologous desensitization of phospholipase C activation and [Ca++i] increase induced by the PGH2/TxA2 agonist U46619 in rat aortic SMC was similar to that previously observed in human platelets: rapid desensitization of both responses followed by a delayed loss of binding sites from the cell membrane. Recently, the pattern of receptor inactivation by the antagonist ligand, GR 32191, has identified two subtypes in platelets. GR 32191 binds reversibly (GRr) to a site that mediates platelet shape change and an increase [Ca++i] and irreversibly (GRirr) to a site linked to phospholipase C activation and aggregation. In contrast to platelets, studies of ligand dissociation only identified GRr sites in rat aortic SMC and GR 32191 failed to inactivate PGH2/TxA2 receptors as detected by the PGH2/TxA2 receptor antagonist, [3H]SQ 29548. Inhibition of U46619-induced contraction of both rat aortic and human saphenous vein was competitive, consistent with the absence of GRirr sites in VSMC. Platelet activating factor, which heterologously desensitizes U46619-evoked phospholipase C activation in platelets, had no such effect in VSMC. The biochemical events attendant to PGH2/TxA2 receptor desensitization are similar in SMC and platelets. However, both the pattern of receptor inactivation by GR 32191 and of heterologous desensitization by PAF, suggest that VSMC lack the receptor subtype that transduces aggregation of platelets.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗