Dietary effects of eicosapentaenoic and docosahexaenoic acid esters on lipid metabolism and immune parameters in Sprague-Dawley rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-gamma-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTBs productivity were observed in the PEC, in response to the treatment with 5 microM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Dietary Effects of Eicosapentaenoic and Docosahexaenoic Acid Esters on Lipid Metabolism and Immune Parameters in Sprague-Dawley Rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB₄ productivity and an increase of LTB₅ productivity were observed in the PEC, in response to the treatment with 5 μM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product.

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.     

Effects of Short- and Long-Term Protriptyline Treatment on Phospholipid Metabolism in Rat Brain

Wistar rats were injected intraperitoneally with 10 mg/kg of protriptyline according to one of the following schedules: a single dose or daily for 4 days (short-term), or daily for 2 or 13 weeks (long-term). Total lipid, total phospholipid, and individual phospholipid contents in the brain were determined. Further, the incorporation of 32P into individual phospholipids in vivo and the fatty acid composition of phosphatidylethanolamine in the brains of rats treated with protriptyline for 13 weeks were studied. Three alternative phases of changes of total and individual phospholipid contents in the brain during 13 weeks of experimentation were distinguished. An increase of phospholipid contents after 4 days, a decrease after 2 weeks, and a further increase after 13 weeks of protriptyline administration were found. However, phosphatidylinositol and phosphatidic acid levels after 13 weeks of protriptyline administration were diminished. The decrease of specific radioactivity of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine and the increase of phosphatidylinositol, phosphatidic acid, and sphingomyelin in rats treated with the drug for a longer period of time were noted. No greater differences in fatty acid composition of phosphatidylethanolamine in the brains of the same group of rats were observed as compared to control. These results indicate that during long-term treatment with protriptyline the contents of lipids and phospholipids in rat brain are altered. The modification of the biological function of phospholipids in brain cell membranes is suggested.     

Effect of macrophages and antibodies on in vivo growth of Moloney sarcoma in the rat.

Brown Norway and Lewis rats were challenged with a Brown Norway Moloney sarcoma tumor, MST-1, admixed with nonimmune peritoneal exudate macrophages syngeneic to the host; or admixed with nonimmune peritoneal exudate macrophages and hyperimmune anti-MST-1 antibodies. In vivo growth of MST-1 in BN and Lewis rats was inhibited by admixing Brown Norway or Lewis macrophages, respectively, with BN anti-MST-1 antibodies. The inhibiting BN antibodies were of the IgG2 class, lacking IgG2a antibodies. Brown Norway anti-MST-1 of IgG2 class without macrophages did not affect growth of MST-1. Brown Norway and Lewis anti-MST-1 antibodies of IgG2a class enhanced tumor growth, whether admixed with macrophages or not. Anti-MST-1 antibodies of IgM and IgG1 classes did not influence tumor growth. Peritoneal exudate macrophages removed from Lewis donors 8 to 10 days after inoculation of MST-1 inhibited completely growth of the challenge tumor; macrophages of Brown Norway origin were inhibitory only when harvested from hyperimmune donors, that is, 40 or more days after inoculation of MST-1. Macrophages from hyperimmune donors were specifically cytotoxic to MST-1 and did not inhibit an unrelated syngeneic BN tumor of chemical origin.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Interactions of dietary fats and proteins on fatty acid composition of immune cells and LTB4 production by peritoneal exudate cells of rats

The interaction of dietary fats and proteins on lipid parameters of rats was studied using safflower oil (linoleic acid-rich), borage oil (gamma-linolenic acid-rich) or perilla oil (alpha-linolenic acid-rich) in combination with casein or soybean protein. The experiment was focused on the fatty acid composition of immune cells and the leukotriene B4 production by peritoneal exudate cells. Serum total cholesterol, triglyceride, and phospholipid levels were low in perilla oil-fed or soybean protein-fed rats. Fatty acid compositions of serum and liver phospholipids reflected those of dietary fats. However, feeding borage oil resulted in a marked increase in the proportion of dihomo-gamma-linolenic acid in phospholipids of peritoneal exudate cells, spleen lymphocytes, and mesenteric lymph node lymphocytes in relation to those of liver and serum. It is suggested that activities of metabolic n-6 polyunsaturated fatty acids are different between immune and other tissues. In addition, the magnitude of the reduction of the proportion of linoleic acid of perilla oil in immune cells was considerably more moderate than serum and liver, indicating a different degree of interference of alpha-linolenic acid with linoleic acid metabolism. Leukotriene release from peritoneal exudate cells was in the order of safflower oil > borage oil > perilla oil groups as reflecting the proportion of arachidonic acid, and tended to be lower in soybean protein-fed groups. These suggest an anti-inflammatory property of gamma-linolenic acid as well as alpha-linolenic acid tended to be strengthened when they were combined with soybean protein than with casein.     

The modulation of phagocyte oxygen metabolism by recombinant cytokines and a complex of natural cytokines

The data on the modulating function of cytokines on the oxygen-producing function of peritoneal exudate cells of rats are presented. As priming agents, recombinant cytokines IL1 beta and TFR beta 1, as well as the natural complex of cytokines, were used. The priming action of cytokines was studied by changing in the production of active forms of oxygen by peritoneal exudate cells of rats, stimulated with opsonized zymosan, by the method of luminol-dependent chemiluminescence. The study revealed that IL1 beta and the natural complex of cytokines primed peritoneal exudate cells for the production of active forms of oxygen. The maximum value of the prestimulation index was 1.9 +/- 0.1 and 2.95 +/- 0.27 respectively. The preincubation of peritoneal exudate cells of rats with TFR beta 1 led to the pronounced inhibition of the intensity of the chemiluminescent response of cells. The prestimulation index did not exceed 1.06 +/- 0.1. Moreover, as revealed with the use of the probe Fura-2/AM, in the process the prestimulation of phagocytes with the natural complex of cytokines the intracellular concentration of calcium increased from 0.86 +/- 0.15 to 1.86 +/- 0.2 microM/ml. The mechanism of the prestimulation of peritoneal exudate cells of rats cytokines seems to be calcium-dependent.     

Effect of orexin-A on phagocytic activity of peritoneal macrophage in starved rats

The aim of this study was to investigate the effect of fasting-induced orexin-A (OXA) on inflammation and macrophage phagocytic activity. Fifty six male wistar rats were fasted for 36 h to stimulate OXA synthesis. In 24 rats, air pouches were induced subcutaneously in the intrascapular area. After (6 h) carrageenan injection into the pouches, the contents of the air pouches were removed. The exudate volume, protein content and cell count were measured. After the determination of fasting on inflammation, the peritoneal macrophages were collected from 32 rats to investigate the effect of fasting-induced OXA on macrophage phagocytic activity. Plasma OXA levels were markedly higher in fasted rats compared with control rats. The phagocytic capability of peritoneal macrophages was obtained as a percentage of phagocytosing macrophages and number of phagocytosed particles per cell. In spite of increased blood OXA level SB-334867, selective orexin type 1 receptor antagonist (10 mg/kg) did not change phagocytic activity of peritoneal macrophages. These findings indicate that 36 h fasting-induced OXA has no significant effect to phagocytosis of peritoneal macrophages.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.     

Phospholipids of rat tissues after feeding pure phosphatidyl ethanolamine and lecithin

Pure phosphatidyl ethanolamine and lecithin from egg yolks were fed to rats in saline or in olive oil and the changes in individual phospholipids in the intestinal wall, liver, and plasma of the animals were studied. Ingestion of olive oil alone produced increased levels of all phospholipid fractions in each of the three tissues. Feeding phosphatidyl ethanolamine in saline resulted in slightly increased plasma phospholipids, but levels of liver total phospholipids were greatly reduced; when phosphatidyl ethanolamine was fed with olive oil, liver phospholipids were again reduced but this reduction was confined to the phosphatidyl ethanolamine and phosphatidic acid fractions. Feeding lecithin alone did not produce significant changes in levels of plasma or tissue phospholipids. The results suggest that liver phospholipid synthesis is depressed by feeding phosphatidyl ethanolamine; in the presence of olive oil, hepatic synthesis of phosphatidyl ethanolamine seems to be more selectively inhibited.     

Recruitment of exogenous macrophages into metastases at different stages of tumor growth

Summary The endogenous tumor-associated macrophage content and recruitment of labeled peritoneal exudate cells into experimental murine B16 melanoma metastases has been examined at different stages in the progressive growth of metastatic lesions. The recruitment of thioglycollate-elicited peritoneal exudate cells and peritoneal exudate cells activated in vitro with muramyl dipeptide was studied. Tumor-associated macrophages and labeled peritoneal exudate cells were identified in paraffin sections by specific histochemical staining and their density in individual metastases measured morphometrically. The density of tumor-associated macrophages and exogenously recruited peritoneal exudate cells was high in very small lesions but decreased rapidly as a function of enlargement of metastases, MD:Aⁿ; where MD is macrophage density, A is the cross-sectional area of the lesion and n is a negative number. No significant difference was observed in the recruitment of activated and nonactivated peritoneal exudate cells. These results suggest that decreased recrutiment of macrophages from the circulation may explain the decrease in the density of tumor-associated macrophages as metastases grow and indicate that macrophage activation is not accompanied by enhanced localization and/or uptake of macrophages into metastases.     

Studies on the effect of inflammation on the acute phase response using rat liver slices

Liver slices from control and inflamed rats were incubated in McCoy's medium and incorporation of [3H]leucine into liver and medium proteins and into albumin and alpha 1-acid glycoprotein was monitored over 48 hr. The release of the new acute phase reactant, sialyltransferase was also monitored in this system. Earlier observations in which liver slices were incubated for 6 hr showed that increased leucine incorporation into liver and medium proteins and alpha 1-acid glycoprotein, coupled with decreased incorporation into albumin, correlated with the acute phase response of these proteins. Increased incorporation of leucine into these proteins was found following 48 hr incubation in McCoy's medium showing that slices were able to express the changes characteristic of the acute phase response over this longer time period of incubation. Sialyltransferase was released into medium in a linear fashion up to 15 hr and continued to increase for 30 hr in this system; there was a substantial increase in release of enzyme activity from slices from inflamed rats when compared to controls. Monokine-conditioned medium prepared from peritoneal exudate cells isolated from rats at various times after lipopolysaccharide administration was used to induce the acute phase response by intraperitoneal injection. Slices were prepared from these rats and sialyltransferase release from slices was monitored. Monokines prepared from peritoneal exudate cells isolated from rats at about 30 hr were most effective in stimulating sialyltransferase release from liver slices.     

Diabetes and host resistance. I. Effect of alloxan diabetes upon the phagocytic and bactericidal efficiency of rat leukocytes for Pneumococcus

Briscoe, H. Frances (University Medical Center, Jackson, Miss.), and Fred Allison, Jr. Diabetes and host resistance. I. Effect of alloxan diabetes upon the phagocytic and bactericidal efficiency of rat leukocytes for pneumococcus. J. Bacteriol. 90:1537-1541. 1965.-Chronic diabetes mellitus was induced in rats with alloxan monohydrate. Glycosuria persisted for the 6 weeks of study, but ketonuria was never encountered. The cellular composition of peritoneal exudate recovered from diabetic rats after starch aleuronat administration was the same as that obtained from normal rats. The quantity of exudate recovered from the diabetic rats was thought to be less than that obtained from normal rats subjected to the same irritant. Phagocytosis was found to be essentially the same for both diabetic and normal cells when suspended in normal saline. The killing efficiency of harvested peritoneal phagocytes suspended in saline from both diabetic and normal rats for type 1 pneumococcus was compared and no difference between the groups was found.     

Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.     

Short-term diabetes increases triacylglycerol arachidonic acid content in the rat liver

Fatty acid compositions of liver phospholipid, cholesterol ester and triacylglycerol fractions obtained from streptozotocin-induced diabetic rats were compared to those from control or from simple-acidotic rats. Significant reductions of arachidonic acid proportions in phospholipid and cholesterol ester were found on the 3rd day after the streptozotocin treatment. In triacylglycerol, arachidonic acid and the other desaturation and elongation products of linoleic acid except for gamma-linolenic acid were increased in the diabetic rats. Although essential fatty acid composition in liver phospholipid and cholesterol ester of simple-acidotic rats did not differ from control rats, dihomo-gamma-linolenic acid, arachidonic acid, adrenic acid and docosapentaenoic acid (22:5(n - 6] contents in liver TG were significantly increased over those in control rats and were similar to those in diabetic rats. These results suggest that metabolic acidosis may contribute to the fatty acid abnormalities observed in diabetic animals.     

Effects of dietary selenium and tocopherol on glutathione peroxidase and superoxide dismutase activities in rat phagocytes

Glutathione peroxidase activities (GSH-Px) of peritoneal exudate polymorphonuclear neutrophils, pulmonary alveolar macrophages, and peritoneal exudate macrophages of rats depleted of dietary selenium for four to six weeks were markedly lower than the corresponding activities in rats fed the same diet supplemented with 0.5 ppm selenium as sodium selenite. GSH-Px in phagocytes from selenium-supplemented rats adequate or deficient in tocopherol status did not differ significantly. In selenium deficient animals, the residual GSH-Px of polymorphonuclear neutrophils and peritoneal macrophages, but not of alveolar macrophages were slightly higher in tocopherol-deficient rats than in tocopherol-supplemented animals. Superoxide dismutase activities of each cell type were comparable and were not significantly affected by dietary selenium or tocopherol.     

Novel protease bound with chromatins in normal and tumorous tissues of rats

A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma? Rhodamine sarcoma≥ AH 130≥ thymus> spleen? kidney≥ liver? brain.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Age-related features of cataractogenesis in salmon fry. 3. Age-related dynamics of liver lipid composition during cataractogenesis

When studying the cataract pathogenesis in salmon fry, we found changes in the content of individual; phospholipid fractions and fatty acid composition in the liver of diseased and healthy fish. The age-related changes correlated with the increased antioxidant activity and decreased liver content of malondialdehyde.