Five hybridoma cell lines producing monoclonal antibodies against human pancreatic phospholipase A2 (PLA2) were established after selection from prepared hybridomas on the basis of the affinity of the antibodies they produced. All the monoclonal antibodies (1008, 1015, 1045, 1085, and 2003) proved to have very high affinity toward human pancreatic PLA2 (Ka=1.5×1011-1.2×1012M-1), but to have no cross reactivity with porcine pancreatic PLA2, snake venom PLA2, and membrane-associated PLA2 from human spleen. Cross reactivities with human pancreatic prophospholipase A2 (proPLA2) ranged from 4 to 49%. Three of the monoclonal antibodies neutralized the enzyme activity of PLA2, while the other two had no effect on it. The three neutralizing antibodies had smaller cross reactivities with proPLA2 than the other two. On radioimmunoassay using these monoclonal antibodies, the PLA2 contents of sera from patients with acute pancreatitis were above normal in every case. These monoclonal antibodies are expected to be useful tools in the diagnosis of acute pancreatitis.
Monoclonal antibodies were developed against human pancreatic prophospholipase A2 (proPLA2). They had high affinity for proPLA2 and small cross reactivities with human pancreatic phospholipase A2 (PLA2). By S-Sepharose ion-exchange chromatography combined with radioimmunoassay for proPLA2 and that for PLA2 both using monoclonal antibodies, proPLA2 and PLA2 in human sera could be separated and measured. In healthy individuals, proPLA2 proved to be the major form of the enzyme, while two out of three patients with pancreatitis showed a larger proportion of PLA2, which may be an indication of the severity of the pancreatitis. In patients with pancreatic cancer and chronic renal failure, proPLA2 remained in larger proportion in spite of the very high radioimmunoassay level of PLA2. The concentrations of both proPLA2 and PLA2 in human sera could be estimated by a combination of the two radioimmunoassay systems, suggesting that this combination radioimmunoassay could be used to more precisely diagnose pancreatitis and other diseases exhibiting high levels of immunoreactive PLA2 than a single radioimmunoassay system.
Female mice were fed from delivery until weaning by lactating mothers consuming a vitamin A-free diet. Three groups of weanling female mice were fed diets containing different amounts of vitamin A (0mg/kg diet; 0.45mg/kg; 4.5mg/kg) for 7 weeks. A control pair-fed group (PF) was paired with the group fed the vitamin A-free diet. Seven mice received the vitamin A-free diet for 7 weeks, and then were rehabilitated (R) on the 4.5mg/kg diet for 10 days. Mice fed the 0mg vitamin A/kg diet were severely vitamin A deficient (SD), whereas those fed the 0.45mg vitamin A/kg one were moderately deficient (MD), as shown by their vitamin A levels in blood and in liver. Mice receiving the 4.5mg/kg dose of vitamin A served as the control (C). No significant differences were observed in thymus weight. Total number and concentration of thymocytes per mg of tissue were decreased in SD mice compared with the control, and number and proportion of CD4-8+ cells were lower than in C, PF, and R. No significant difference was found in subsets in lymph node. In spleen, the proportion and the total number of CD4+8- was lower in SD mice. No difference was observed in the proportion of splenic B lymphocytes.
The interaction between lipopolysaccharide (LPS) and bile acids was studied in rat erythrocyte membranes. Addition of LPS isolated from E. coli (J5 mutant) to erythrocytes resulted in a decrease in the membrane fluidity as determined by spin labeling using electron spin resonance (ESR). This was accompanied by membrane fragility. The generation of hydroxyl radicals in the LPS-treated erythrocytes was detected by ESR spin trapping. However, pretreatment of erythrocytes with taurine-conjugated bile acids, i.e., taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), and taurochenodeoxycholic acid (TUDCA), modified the membrane damage induced by LPS. TCA and TUDCA prevented the decrease in membrane fluidity induced by LPS; and as a result, the membrane integrity was maintained although no significant changes were observed in the amount of hydroxyl radicals produced by LPS addition. However, TCDCA exhibited little beneficial effect on the dynamic properties and function of the erythrocyte membrane, although the hydroxyl radical content declined markedly in erythrocytes treated with it. Therefore, our data suggest that TCA and TUDCA have a protective effect against LPS-induced membrane fragility by modulating membrane fluidity.
Using subcutaneous solid tumor produced by U251-SP human glioma cells, we studied the in vivo transfection of the cells with the human β-interferon (HuIFN-β) gene by means of novel liposomes. Since the tumor usually forms a single or multiple cysts when the tumor becomes over 15mm in diameter, we injected liposomes entrapping the gene into the cyst, and found that HuIFN-β was secreted into the cystic fluid. It reached a level of approximately 450IU/ml 48h after the transfection and remained at the same level for 8 days. The growth of the tumor was completely inhibited when the injection was carried out every second day for 10 days. The tumor did not grow at all for 30 days.
Acid lipase (AL) activity was measured in mononuclear leukocytes (MNL) isolated from 214 subjects. An inverse relation was observed between the AL activity and serum cholesterol (CHO) with age variation: The age groups above 30, which showed low AL activities in their MNL, exhibited high serum CHO; whereas the 20-to-29 age group, which had the highest AL activity among the age groups, showed the lowest CHO value. Moreover, the hypercholesterolemic group with high apo-B levels showed significantly low AL activity in both sexes when compared with the normolipidemic group. Significant negative correlations were also observed in the hypercholesterolemic group between the AL activity and both CHO and apo-B, and between the AL activity and lipid peroxides (LPO). On the other hand, while the addition of low-density lipoprotein (LDL) from patients with type II hypercholesterolemia to the AL assay system and the culture medium of MNL resulted in the inhibition of the AL activity, the inhibitory effect was prevented by preincubation of LDL with anti apo-B serum. These data suggest a certain regulatory mechanism of LDL, the physiological substrate of AL, on the AL activity; this action possibly occurs through lipid peroxides in LDL.
It has been shown in this study that the selenium (Se) content and glutathione peroxidase (GSH-Px) activity in blood and tissues of Keshan disease (KD) patients were lower than those of control subjects. Also these patients showed abnormal lipid composition of plasma and of membranes of erythrocytes and myocardium, such as a decrease in phospholipid (PL) and in cardiolipin (CL) and an increase in the molar ratio of cholesterol/PL and of sphingomyelin/phosphatidylcholine. In addition the cytochrome c oxidase (CCO) activity in their myocardial mitochondria was decreased. A guinea pig model fed cereals from Yunnan, a KD-endemic area, for 15 weeks, showed a series of changes similar to those observed in KD patients, as above, such as decreases in Se level and activity of GSH-Px and of glutathione-S-transferase, and significant increases in lipid peroxides, hydroperoxides (conjugated dienes), and fluorescent chromolipids. The lipid composition of myocardial mitochondria in the animal model was abnormal; especially, the CL was decreased. Even though the CCO content was almost unchanged, activity of the enzyme was decreased remarkably. This result coincides exactly with the changes seen in the circular dichroism spectra of myocardial mitochondria, the peak values (208 and 224nm) of which were lower significantly than those of the control.
We measured the serum apolipoprotein (Apo) levels in various hyperlipidemias and determined their correlations with the serum cholinesterase and albumin levels. The objective was to elucidate which apolipoprotein(s) commonly reflects the liver function, as shown by the albumin and cholinesterase levels, in various hyperlipidemias. The subjects consisted of 53 normal controls and 86 type IIa, 38 type IIb, 28 type IV, and 9 type V hyperlipidemias. Each apolipoprotein level generally reflected the characteristic changes of lipoproteins for each hyperlipidemia. As for the correlation of apolipoproteins with cholinesterase and albumin, Apo A-II showed a positive correlation with cholinesterase in the normal controls and in all types of hyperlipidemia, though it was not significant in types IIb and V. And Apo A-II also showed a significantly positive correlation with albumin in all types of hyperlipidemia except type V. Considering that cholinesterase and albumin are known to reflect the liver function, these results are consistent with the idea that among the various apolipoproteins, Apo A-II may best reflect the liver function in all types of hyperlipidemia.