Biomedical subjects
Publications and source records attributed to M A Wells.
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The immune response to influenza infection was evaluated in mice using recently developed techniques to measure the induction of cytotoxic thymus-derived (T) lymphocytes and complement-dependent cytolytic antibody, locally and systemically, during primary and secondary immunization. Cytolytic antibody responses were compared to antibody titres measured by haemagglutination-inhibition (HI) and neutralization in the same samples. The development of these responses was also correlated with the titres of virus in the lung, in an attempt to further define the role of these host immune responses which can kill virus infected cells during recovery from influenza infection in vivo.
Cytotoxic T cells were detected in the cervical lymph nodes, lungs, spleen, and peripheral blood of mice with influenza. Lymphocytes decreased in the peripheral circulation and increased in the lung during the period of acute inflammation and pneumonia. Peak cytotoxic T-cell activity was present at the time of marked pulmonary infiltration, and it decreased with resolution of the pneumonia. The cytotoxic T cells in the lung were shown to be H-2 restricted and specific for the hemagglutinin of the infecting virus. The results indicate that hemagglutinin specific cytotoxic T cells are (a) induced during influenza infection; (b) they circulate in the blood; (c) they are present in greatest number; and (d) they have their peak cytotoxic effect when pneumonia is most marked. We interpret the results to indicate that specific cytotoxic T cells in the infected target organ are part of the immunological and pathological response to virus infection.
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The interaction of mouse macrophages with influenza virus was examined as part of a study into the defense mechanisms against influenza infection. Macrophages exposed to A/Port Chalmers/1/73 virus produced infectious foci on susceptible indicator cell monolayers. Sampling of supernatant fluids and cells from infected macrophage cultures showed release of virus adsorbed to the cell surface. Active virus replication in macrophages could not be demonstrated. Exposing macrophages to specific antibody before or after virus infection resulted in a significant decrease in the number of infectious macrophages. The results suggest that although macrophages are not the source of replicating influenza virus, they are able to spread the infection by having virus attaching to their surface. This activity is interfered with by the presence of specific antibody.
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Estimations of molecular weights from sedimentation equilibrium experiments indicate that the degree of aggregation of egg lecithin in benzene solutions increases with increasing addition of water. The molecular weight rises monotopically from about 14,000 in the anhydrous state to about 57,000 at a water/lecithin ratio of 0.31. Sedimentation coefficients were shown to behave in the same manner. Frictional coefficients obtained from the combined results fail to indicate definite differences in asymmetry; however, they seem to indicate that the aggregates in all stages are compact in shape.
The synthesis of 1-acyl-sn-glycero-3-phosphorylcholines in particular those containing short fatty acyl chains are described. The method involves the use of 1-acyl-2-tert-butyl-dimethylsilyl-sn-glycero-3-phosphorylcholines which can be readily prepared by reacting hens' egg yolk 1-acyl-sn-glycero-3-phosphorylcholines with tert-butyldimethylchlorosilane with imidazole as catalyst and dimethylformamide as solvent. Deacylation of the 1-acyl-2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholines with saturated anhydrous potassium carbonate in methanol yields the 2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholine. Reacylation of the 2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholine with fatty acyl anhydride in the presence of 4-dimethylaminopyridine in anhydrous chloroform followed by removal of the tert-butyldimethylsilyl protecting group by treatment with dry hydrogen chloride gas in anhydrous chloroform at 0 degrees yields the desired 1-acyl-sn-glycero-3-phosphorylcholine. Various facets of the reactions involved in developing the synthetic procedures in this study are discussed.
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The action of snake venom phospholipases A2 in intact human erythrocytes was investigated in detail. The basis phospholipase from Agkistrodon halys blomhifii was found to induce both hydrolysis of membrane phospholipids and total cell hemolysis under certain experimental conditions. The hydrolytic action of the basic enzyme was found to consist of two sequential events: (a) hydrolysis of 70% of the total cell ph osphatidylcholine without any evident hemolysis; and (b) complete hydrolysis of the remaining phosphatidylcholine, followed closely by extensive phosphatidylethanolamine hydrolysis and finally with onset of hemolysis, attack on the phosphatidylserine. At pH 7.4 and 10 mM Ca2+ only stage (a) occurred. However, a slight elevation of the pH of incubation to pH 8.0 and/or inclusion of 40 mM Ca2+ in the reaction mixture caused both events (a) and (b) to occur. The addition of glucose limited the action of the enzyme to stage (a) under any reaction conditions. An investigation showed that enzymically induced hemolysis occurred under conditions where the intracellular ATP levels were lowered. Data are presented which suggest that stage (b) is mediated by in influx of Ca2+ into the cell when the levels of ATP are low. Interestingly the phosphllipase from Naja naja venom (Pakistan) yielded results similar to those observed with the basic enzyme from Agkistrodon venom. However, the enzyme from Crotalus adamanteus and the acidic enzyme also present in the Agkistrodon venom produced only slight hydrolysis or hemolysis under any of the conditions studied. Other species of erythrocytes, e.g., guinea pig, monkey, pig, and rat, were tested but only those from guinea pig behaved similarly to the human cells. Pig, monkey, and rat erythrocytes underwent very limited hydrolysis and hemolysis. It is evident that the use of these phospholipases to probe the localization of phospholipds in erythrocyte membranes must be approached with caution. Certain facets of this problem are discussed.
A new, high yield, procedure for the purification of the phospholipases A2 of Crotalus adamanteus venom is described. The procedure involves precipitation of the bulk of venom proteins with 50% isopropanol, precipitation of the enzymes from the isopranol soluble material with neodynium chloride, and final purification on DEAE cellulose. An overall yield of approximately 80% is achieved.
The interactions of calcium, magnesium, and the rare earth cations, cerium, neodymium, and praseodymium, with phosphatidylcholines were studied by proton magnetic resonance and infared spectroscopy. The calcium-induced chemical shifts for the various protons of phosphatidylcholine were C alpha choline greater than C beta choline greater than N(CH3)3 greater than C3 glycerol. No significant chemical shifts were observed for the C1 and C2 glycerol protons. None of the acyl chain protons were affected by the presence of calcium. Analysis of the salt-induced chemical shifts yielded binding curves with an excellent fit with the theoretical. The vicinal coupling constants for the various protons of phosphatidylcholine did not appear to change in the presence of calcium. The lanthanide-induced isotropic shifts for the protons of phosphatidylcholines followed the order Cbeta choline greater than C3 glycerol greater than Calpha choline greater than N(CH3)3. Examination of the P=O stretching band (1150-1300 cm-1) of phosphatidylcholines by differential infrared spectroscopy showed that this band shifted to shorter wavelengths in the presence of calcium. The site of calcium binding to phosphatidylcholines as deduced from the proton magnetic resonance and infrared data is discussed in light of the high specificity for calcium in enhancing the amino-catalyzed methanolysis of phosphatidylcholines.
The level of endotoxin in influenza virus vaccine lots was reduced 10- to 20-fold after barium sulfate adsorption-elution. The amount of viral antigen lost was negligible.
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