Biomedical subjects
Publications and source records attributed to J R Dawson.
A case is described of Streptococcus viridans endocarditis involving a left atrial myxoma. The diagnosis was made by echocardiography and the infected tumour was successfully treated by surgical removal. A review of the published literature about left atrial myxomas underlines the importance of performing echocardiography in all patients with suspected endocarditis.
Cell-mediated cytotoxicity involves a localized secretory process in which lytic agents stored in specialized granules of the effector cells are released upon contact with the appropriate target cell membrane and cause membrane damage. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibits cytotoxicity of natural killer cells, cytotoxic T-lymphocytes and lymphokine-activated killer cells. This inhibition is due to an effect of CCCP on the cytolytic cells, rather than on their targets, and is reversible. Treatment with CCCP does not inhibit the formation of effector-target conjugates, but seems to affect the programming of the effector cells for lysis. CCCP only inhibits lysis if added during a certain period of the lytic cycle: it has an effect only if added before, or within 5 minutes of the initiation of killing by a pulse of Ca++. Effector cells treated with CCCP retain their characteristic beta-glucuronidase-positive granules, but in the presence of the drug, these are no longer oriented to face the contact area with the target cell membrane.
Left ventricular cineangiograms (30 degrees right anterior oblique projection) and high fidelity left ventricular pressure were recorded at rest in 10 individuals with normal hearts and at rest and during an episode of angina provoked by rapid atrial pacing in 14 patients with obstructive coronary artery disease. Angiograms were digitised frame by frame. The ventricular perimeter was divided into 40 segments and regional ventricular function was examined by means of isometric and contour plots and by the construction of segmental pressure-wall displacement loops. In 10 patients 12 abnormal resting regions were identified. The commonest (11 regions) was delayed onset of inward endocardial motion during systole which was manifest by diagonal contour lines on the contour plot. Six regions (five with associated delay in onset of inward motion) showed resting hypokinesis. Angina was associated with the development of 19 new regions of abnormal wall motion in 12 patients. Fourteen of these regions of abnormality were thought to be primary events occurring as a consequence of ischaemia and five to be secondary events occurring in normally perfused regions of myocardium. Hypokinesis occurred in 12 regions, developing in a region with normal amplitude at rest in seven and as an extension of resting hypokinesis in five. In 10 out of 12 cases the region of hypokinesis developed in a region showing diagonal contour lines at rest. Asynchrony with delay in the timing of peak inward displacement relative to minimum volume occurred with angina in eight regions (in six cases concomitant with hypokinesis and in two cases in isolation). In contrast with hypokinesis the pattern of wall motion at rest did not permit regions developing asynchrony with angina to be identified. Pressure-displacement loops show that regional hypokinesis is associated with reduced segmental work and that regional asynchrony (delayed or premature timing of peak inward endocardial displacement) is associated with a loss of efficiency of energy transfer between the myocardium and the circulation. These observations illustrate the complex nature of wall motion abnormalities occurring with angina.
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The metabolic, hormonal and haemodynamic effects of oral pirbuterol, a new beta 2-adrenoceptor agonist, were studied acutely (n = 19) and after 3 months treatment (n = 11) in patients with severe heart failure receiving chronic frusemide therapy. In the acute study fasted patients (n = 10) showed reductions in plasma K+ (P less than 0.005) and cortisol (P less than 0.01) and increases in plasma glucose (P less than 0.005), insulin (P less than 0.01), lactate (P less than 0.005) and pyruvate (P less than 0.0025). These acute changes were less in unfasted subjects (n = 9). Maximal increase in stroke volume occurred at approximately half the plasma pirbuterol concentration required for maximal effect on plasma insulin. Treatment with pirbuterol for 3 months was associated with sustained increases in stroke volume and fasting plasma glucose and insulin, but there was loss of all other acute metabolic effects. Despite concurrent frusemide and digoxin therapy acute hypokalaemia caused no adverse effects. Hypokalaemia did not occur with chronic pirbuterol administration.
When 13 B cell lines were phenotyped with a panel of B cell, stage-specific monoclonal antibodies and ordered with respect to differentiation state; their sensitivity to natural killer (NK) cell-mediated conjugate formation and cytolysis was found to be stage dependent. Target cell lines expressing an early B cell phenotype (B1+B2-CALLA+DU-ALL1 +/-) or an intermediate phenotype (B1+B2+CALLA-DU-ALL1+) were NK resistant. Late phenotypic B cells (B1+B2-CALLA-DU-ALL1-) were highly susceptible to NK cytolysis. Individual B cell lines when induced with sodium butyrate or retinoic acid expressed altered B cell phenotype and NK susceptibility. For example, SB, an intermediate B cell line and initially NK resistant, when induced to express a later B cell phenotype became NK sensitive. BJA.B, a late B cell line and initially NK sensitive, when induced to differentiate lost most of its sensitivity to natural killing. Since loss of the B2 antigen is associated with B cell activation, we further phenotyped the B cell lines and induced B cell lines with the 4F2 and 5E9 (anti-transferrin receptor) monoclonal reagents. All cell lines tested expressed each of these antigens, but with varying intensities. While the intensity of 4F2 expression appeared to correlate well with NK sensitivity on both resting and differentiated B cell lines, the intensity of 5E9 expression did not. Peripheral blood B cells express a similar pattern of reactivity to natural killing when stimulated with pokeweed mitogen (PWM) in vitro. B cell sensitivity to NK-mediated events peaked at day 4 of incubation and correlated with the loss of the B2 antigen and acquisition of the 4F2 and 5E9 antigens; sensitivity to natural killing was diminished in the presence of the PCA-1 antigen. The expression of the NK-susceptible phenotype among B cells appears to be differentiation stage- and activation state-dependent. It is during this period of ontogeny that the NK cell may cytolytically regulate the B cell transition to a plasma cell.
We have detected formation of stable associations, or conjugates, between fluorescein diacetate-(FDA) stained human natural killer (NK) cells and Hoechst 33342-(HO342) stained tumor cells by dual laser flow cytometry. Conjugates in mixtures of effectors and targets emitted both green (FDA) and blue (HO342) fluorescence. This was confirmed by cell sorting. More than 90% of the conjugates included one target and one effector cell. Conjugate formation frequency was temperature independent between 4 and 37 degrees C, optimized by 10 min, and stable for 1 hr. Enrichment of effector populations for cells mediating lysis of standard NK targets and for cells reacting with OKM1, Leu-7, and Leu-11b monoclonal antibodies also enriched conjugate-forming cells. Lysis of either OKM1+, Leu-11b+ effector subpopulations with antibody and complement eliminated, but treatment with these antibodies alone had no effect on, conjugate formation. Effector pretreatment with Leu-4 or 3A1 and complement increased the frequency of conjugation slightly. Flow-determined frequencies of NK-conjugate formation with 14 target cell lines correlated well with data derived from standard microscopic assays. However, the flow method was more rapid, could be used when target and effector were of comparable size, and permitted isolation of conjugates by sorting.
Oxygen-reactive metabolites are not detected at the natural killer (NK)-target cell interface in quantities comparable to those seen for other effector-target cell interactions. A novel luminol-coated target cell chemiluminescence assay is described in which luminol is conjugated to the target cell surface with the bifunctional crosslinker 3,3'-dithiobis(propionic acid N-hydroxysuccinimide ester) (DSP). This modification of the basic chemiluminescence assay precludes exclusion of the detection system from the tightly occluded intercellular junction, a possible deficiency in previous investigations. Luminol conjugation does not affect NK-mediated conjugate formation or cytolysis. As NK activity is enriched by a standard series of effector fractionation procedures, chemiluminescence generated against labeled target cells diminishes. Residual chemiluminescence in the most highly NK-active effector fraction is ablated upon antibody and complement depletion of MO2+ cells. This indicates that monocyte contamination is the source of luminol-detectable oxygen-reactive metabolites.
Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.
The effects of intracoronary and intravenous infusions of amrinone were studied to distinguish the drug's direct cardiac actions from its peripheral vascular and neuroendocrine properties. Intracoronary infusions of amrinone were found to have no haemodynamic effect other than producing a slight reduction in the left ventricular ejection fraction and some suggestion of coronary vasodilatation in patients with impaired left ventricular function. They did not improve contractility, cardiac output, or filling pressures and had no significant effect on myocardial metabolism, although therapeutic concentrations of the drug were detected in coronary sinus blood. Intravenously administered amrinone reduced filling pressures and improved the cardiac index in all patients, but haemodynamic improvements were most pronounced in the patients with the worst cardiac function. These changes were accompanied by improvements in the indices of contractility only in patients in whom alterations in concentrations of free fatty acid, glycerol, and glucose suggested peripheral catecholamine release. In the patients with the best basal cardiac function intravenously administered amrinone produced a reduction in myocardial work and evidence of myocardial ischaemia, as a result of excessive reduction of coronary perfusion pressure and increased heart rate, without any appreciable increase in cardiac index. It is concluded that, at the concentrations of the drug that can be achieved in man without adverse effects, amrinone has no direct positive inotropic effect. Haemodynamic changes are predominantly the result of vasodilatation, although catecholamines may be released in some patients.
Dopexamine, a new compound with postjunctional dopamine receptor activating and beta adrenoceptor agonist properties, was given to 10 patients with chronic heart failure at diagnostic cardiac catheterisation to investigate its acute haemodynamic and metabolic effects. The drug was administered by intravenous infusion in three incremental doses and produced significant dose related increases in cardiac index, stroke volume index, and heart rate and falls in systemic vascular resistance and left ventricular end diastolic pressure; aortic and pulmonary artery pressures were unchanged. Isovolumic phase (max dP/dt and KVmax) and ejection phase (peak aortic blood velocity, maximum acceleration of blood, and maximum rate of change of power with time during ejection) indices of myocardial contractility were all increased by dopexamine but these changes were hard to interpret in the presence of an increase in heart rate. Myocardial efficiency and ejection fraction were both increased and left ventricular end diastolic and end systolic volumes fell. These largely beneficial changes were achieved without a statistically significant increase in myocardial oxygen consumption or disturbance of myocardial metabolic function. Dopexamine was well tolerated but tremor was reported by two patients at the intermediate dose and mild chest pain by two patients at the high dose.
The lysosome content of lymphocytes has been analyzed with lysosomotropic vital stains and the fluorescence-activated cell sorter (FACS). Large granular lymphocytes (LGL), which account for virtually all natural killing activity in peripheral blood, are quantitatively different from small lymphocytes (SL) in this respect. LGL obtained by Percoll gradient density centrifugation accumulate more of the lysosomotropic vital dyes than SL do, staining with either neutral red or mepacrine (quinacrine). Furthermore among the LGL-rich, low density lymphocyte population highly, granulated cells can be separated from less granulated ones by mepacrine staining and FACS. Thus, separated highly granulated LGL express very high natural killing, whereas the less granulated low density large lymphocytes do not kill.
The human T lymphoblastoid cell line CEM was subjected to immunoselection by co-culture with peripheral blood mononuclear cells (PBMC) for resistance to natural killer (NK) cell-mediated lysis. The NK susceptibility of the resulting subline, CEM.NKR, was 8.4 to 20.6% of that of CEM when PBMC or adherent cell-depleted PBMC were used as effector cells, and -7.1 to 12.1% of that of CEM when Percoll gradient-enriched large granular lymphocytes (LGL) were used. However, CEM and CEM.NKR exhibited comparable sensitivity to antibody-dependent cellular cytotoxicity. Unlabeled CEM was eight- to 32-fold more effective than unlabeled CEM.NKR in inhibiting the NK lysis of labeled CEM target cells, and CEM bound 1.9 to 3.9-fold more Percoll gradient-enriched LGL than CEM.NKR in single cell-binding assays, suggesting that the NK-resistant variant has lost the expression of NK target antigens. However, CEM.NKR was comparable to CEM in its ability to induce interferon (IFN)-alpha production by PBMC in vitro, and the NK-resistant variant maintained its susceptibility to the antiproliferative effects of IFN-alpha, indicating that these phenomena may be mediated by molecules other than NK target structures. Comparison of CEM and CEM.NKR by indirect immunofluorescence with monoclonal antibodies specific for leukocyte antigens and the transferrin receptor, and by microcytotoxicity typing for HLA-A and B specificities, revealed no major differences.
Treatment of human peripheral blood lymphocytes (PBL) with ammonium or other lysosomotropic amines eliminates natural killing (NK) activity of effector cells without affecting their viability. Inhibition of NK by these reagents is probably due to blocking of events subsequent to effector cell target cell binding but before programming for cytolysis. Treatment of K562 target cells with the same reagents has no effect on target cell sensitivity to NK. Pretreatment of effector cells with the NK inhibitory lysosomotropic agents decreases lysosomal thiol cathepsin activity without affecting the lysosomal acid phosphatase activity. Thiol-reactive agents, tosyl-L-lysine chloromethyl ketone (TLCK) and iodoacetamide also inhibit NK and thiol cathepsin activity in PBL. These data suggest a role for thiol-containing enzymes in the early stages of NK cytolysis.
Previous studies in the once-through perfused rat liver preparation have shown that the techniques of normal and retrograde delivery of substrate and computer simulation of enzyme distributions along the sinusoidal flow path in liver were useful in delineating the relative distributions of sulfation and glucuronidation activities for harmol metabolism (Pang et al., J. Pharmacol. Exp. Ther. 224: 647-653, 1983). The observed steady-state hepatic extraction ratios of harmol and the steady-state formation rates of harmol sulfate and harmol glucuronide were consistent with three enzyme-distribution models which described a proximal localization of sulfation activity and a more distal distribution of glucuronidation activities. The present study was a further refinement on the definition of the distribution of these activities. The experimental approach used included the perturbation of delivery of harmol (10 microM) at 8, 12 and 16 ml/min by normal flow, and the alternation of normal and retrograde directional flows for delivery of harmol (10 microM) at constant hepatic blood flows (8, 12 or 16 ml/min) in the single-pass perfused rat liver preparation. The observed steady-state sulfation and glucuronidation rates were compared against predicted values afforded by models previously shown to be adequate and additional enzyme-distributed models. The observed and predicted data point to a periportal distribution of sulfation activity and an even distribution of glucuronidation activity for the metabolism of harmol.
The phenolic compound, harmol, is metabolized by sulfation and glucuronidation in the rat in vivo. In the present study, various harmol infusion rates into the jugular vein were used to delineate first-order conditions whereby total body clearance was maximal and constant; at low infusion rates the steady state harmol concentration in blood varied proportionally with the infusion rate. At infusion rates of 167 nmole/min and below, the steady state clearance of harmol was 60 ml/min or 200 ml/min/kg. Because this value for total body clearance greatly exceeded the value for hepatic blood flow rate (20 ml/min for a 300 g rat), considerable extrahepatic conjugation of harmol was suggested. At higher harmol infusion rates the total clearance decreased. Since an intraportal infusion of 167 nmole/min to the rat yielded, during steady state, the same arterial harmol blood concentration as a 52 nmole/min jugular infusion, the hepatic extraction ratio of harmol in vivo was 0.7. Extrahepatic clearance, therefore, constituted about 77% of total body clearance (after taking the difference between total body clearance and hepatic clearance). Total sulfation clearance was 52 ml/min, and greatly exceed the value for hepatic clearance (14 ml/min). Extrahepatic clearance for sulfation (at least 38 ml/min) therefore accounted for a major proportion of the sulfation activity. Blood platelets did not seem to contribute to sulfation or glucuronidation in vivo.
Cyst and ascites fluid was obtained from 31 different patients with ovarian epithelial neoplasms and was analyzed for the molecular forms and quantities of IgG, IgM, and IgA. The fluids were grouped on the basis of the pathologic diagnosis of the tumor tissue for investigation of the possibility that immunoglobulin levels in fluids from ovarian neoplasms varied with the type of tumor. Immunoglobulin concentrations in most cyst fluid samples were comparable to amounts detected in normal human serum; however, significant reductions in the amounts of IgA in fluids from mucinous cystadenocarcinomas and in the amounts of both IgA and IgM in fluids from mucinous cystadenoma were demonstrated. The cyst fluid immunoglobulins were present in the same molecular forms as serum immunoglobulins, that is, pentameric IgM, predominantly monomeric IgA, and monomeric IgG. The usefulness of cyst effusions as a source of autologous antibodies in the investigation of the immune response to tumors is discussed.
Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH.