Lymphatic transport and organ uptake of gelatin and hyaluronan injected into the rat mesentery

The metabolic pathways of denatured collagen (gelatin) and hyaluronan were studied by injecting labelled macromolecules into the mesentery of rats. The label, [¹²⁵I]tyraminecellobiose is trapped intracellularly after endocytosis, allowing localization of the site of uptake. Mesenteric and thoracic lymph was sampled for 6 h in anaesthetized rats. Separate rats were investigated after an awake period of 6 or 24 h. About 30% of the gelatin remained at the site of injection and of the remaining activity 1.7% was recovered in lymph, 11 yo in the liver and 15% in the kidneys, whereas 3 h after an intravenous injection of gelatin > 70% was recovered in the liver. The change in preferable site of uptake from the liver to the kidney was attributed to local degradation in the mesentery as confirmed by chromatography of tissue extracts and lymph. Following hyaluronan injection and 6 h lymph sampling approximately 30% was left at the site of injection and of the remaining activity 5.7% was recovered in lymph. After an awake period of 6 or 24 h, 30% was regained in the liver. The recoveries in other organs were negligible and mesenteric lymph nodes seem quantitatively unimportant in the uptake of hyaluronan or gelatin from lymph or blood. The liver has a central role in intestinal hyaluronan metabolism, while denatured collagen is more prone to local degradation with remote uptake shared between the liver and the kidney.     

Synthesis and migration of glycoproteins in cells of the rat thymus,as shown by radioautography after 3H-fucose injection

Young male rats received a single intravenous injection of ³H-fucose and were killed after various time-intervals. Light- and electron-microscopic radioautographic studies of the thymus in animals killed shortly after injection showed that all of the different cell types present incorporated ³H-fucose label. The heaviest uptake occurred in macrophages and in hypertrophic epithelial cells located near the cortico-medullary border. Somewhat lighter incorporation was observed in medullary and cortical stellate epithelial cells and in cells designated as special cells, while the lightest reaction appeared over lymphocytes. In all cells the label was localized initially to the Golgi apparatus, where, presumably, it was incorporated into glycoproteins. With time, some of the labeled putative glycoproteins in all cell types migrated to the plasma membrane. In macrophages, much of the label migrated to lysosomal bodies, while in the special cells the label migrated to dense bodies which may also be of lysosomal nature. In stellate and hypertrophic epithelial cells much of the label migrated to characteristic vacuoles. The possible relationship between the observed glycoprotein synthesis in these cells and hormone production is discussed.     

The morphology of release of vitamin A-containing lipid droplets by hepatocytes in rat liver

Vitamin A-containing lipid droplets in the hepatocytes of rat liver were found to be exocytotically released from the cells in the form of a "lipid droplet--retinol-binding protein (RBP)--immunoreactive complex" following intraportal injection of retinol (17, 33, 67, or 100 micrograms). Evidence that the lipid droplets contain vitamin A was obtained by fluorescence microscopy of vitamin A. Intraportal injection of retinol produced varied numbers and sizes of vacuoles in the hepatocytes. The substance within the vacuoles exhibited a meshwork-like configuration in sections from slices incubated in a medium for revealing acid phosphatase activity or the corresponding control medium and was RBP-immunoreactive and proteinaceous in nature. The occurrence and number of the vacuoles depended on the dosage of injected retinol, being greatest at a dosage of 100 micrograms of retinol and becoming progressively less at dosages of 67, 33, and 17 micrograms. The vacuoles were formed by vacuolization of cisternae of the rough endoplasmic reticulum. The formation of vacuoles reached a maximum 30 min after intraportal injection of 100 micrograms retinol, and the vacuoles and lipid droplets had almost disappeared from the hepatocytes after 90 min. Little or no esterase activity was found in lipid droplets in the hepatocytes before intraportal injection of retinol, but after the injection, lipid droplets that had fused with the vacuoles become strongly positive for this enzyme activity. This suggests that hydrolysis of retinyl esters may occur in the process of complex formation in rat hepatocytes.     

Localization and induced expression of fusion genes in the rat lung.

Liposome-mediated gene transfer is useful for DNA transfection into cells in culture. We wondered whether this method could be used to introduce new DNA into the intact lung. Fusion genes containing either the Rous sarcoma virus (RSV) promoter or the mouse mammary tumor virus (MMTV) promoter (which contains glucocorticoid response elements) were linked to the bacterial gene chloramphenicol acetyltransferase (CAT), an enzyme not present in mammalian cells. Plasmids containing the RSV-CAT fusion gene were mixed with cationic liposomes (Lipofectin; BRL, Inc., Grand Island, NY), and single doses were instilled into the cervical trachea of anesthetized rats. Control rats received either liposomes or plasmid. After 24, 48, and 72 h, lungs were perfused free of blood, homogenized, and analyzed for CAT enzyme activity. Liver and kidney tissue were also obtained. We found that rats given either intratracheal liposomes or plasmid had no detectable CAT activity. By contrast, 24 h after instillation of lipid:DNA complexes, lung CAT expression remained elevated for the next 48 h but was barely detectable in liver or kidney. In another group of rats, MMTV-CAT:liposome complexes were instilled intratracheally and then the rats were injected with either dexamethasone or saline. We found that the dexamethasone-treated rats had a 5- to 10-fold higher level of lung CAT expression at 24 and 48 h than the saline-treated controls had; liver and kidney CAT levels were negligible in both groups. Dexamethasone treatment did not increase RSV-CAT expression, indicating that the dexamethasone effect on MMTV-CAT expression was related to the presence of the MMTV promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth retardation and cell death in mouse embryos following exposure to the teratogen bromodeoxyuridine

Within 24 hr after injection of mice on day 10 of pregnancy, embryos exposed to a teratogenic dose of ³H-bromodeoxyuridine (³H-BrdU) have smaller wet weights than control embryos exposed to a trace dose of ³H-thymidine (³H-TdR) or a trace dose of ³H-BrdU. This difference increased by 48 hr after injection. Measurements of total embryo DNA content indicate that these lighter embryos contain fewer cells than controls. If cell death plays a role in the observed growth retardation following administration of ³H-BrdU, one would expect label to be lost from the embryo DNA fraction. Following injection of a trace dose of ³H-TdR or ³H-BrdU, total embryo DNA label rapidly reached peak levels and remained at those levels for at least 48 hr after injection. However, within 48 hr of administering a teratogenic dose of ³H-BrdU, total embryo DNA label had decreased to 25% of peak levels. Histological evidence of cell death was found in the embryos, although it is not yet clear whether cell death was sufficiently extensive to account for the amount of label lost from the DNA fraction. Since none of these BrdU-induced cytotoxic effects were found in the maternal liver, spleen, kidney, or placenta, it is concluded that BrdU probably exerts its teratogenic effects directly on the embryo.     

Hepatic lysosomal enzymes activity and liver morphology after short-time omeprazole administration

The aim of the study was to establish the influence of short-time omeprazole administration on liver function and morphology. Omeprazole was administered intraperitoneally, twice daily, for 3 days to male Wistar rats in two doses: 0.571 mg/kg and 5.71 mg/kg. Control animals were treated with physiological saline. Half of the animals were sacrificed 12 hours after the last injection. The remaining rats were raised for another 6 weeks, without any xenobiotics, and sacrificed on the 47th day of the experiment. The activity of free and bound fractions of hepatic acid phosphatase, beta-galactosidase, beta-N-acetyl-glucosaminidase, cathepsin B, D and L, lipase, and sulphatase were determined spectrophotometrically in homogenates of the liver. The liver sections were examined by light microscopy with hematoxylin-eosin, azan, and periodic acid-Schiff stains. Marginally significant (p < 0.1) differences in activity of free sulphatase fraction, and free and bound fractions of beta-galactosidase were found in animals exposed to the higher dose of omeprazole and sacrificed 12 hours after the last injection. Enzymatic profiles were normalised during the next 6 weeks. Histological evaluation revealed small degenerative and adaptive changes in all examined groups. It could be concluded that observed differences of hepatic lysosomal enzyme activities were the result of accompanied chemical-induced peritonitis as previously reported, and not a direct drug-toxic effect.     

Quantitative and ultrastructural studies on the uptake of drug loaded liposomes by mononuclear phagocytes infected with Leishmania donovani

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.     

Pharmacokinetics of an immunomodulator peptidoglycan monomer in mice after intravenous administration

A ¹⁴C labeled low molecular weight immunomodulator, peptidoglycan monomer (¹⁴C-PGM), was injected intravenously (i.v.) into mice. At various time intervals thereafter (15 min – 6 h), radioactivity in the urine, whole blood, plasma, kidneys, liver, spleen, lungs, intestines and the brain of the mice was determined. Shortly after injection, ¹⁴C-PGM was very rapidly excreted from the organism, so that 1 h following administration, 80% of the radioactivity was found in the urine (62% as unchanged PGM and the rest as the metabolites pentapeptide and disaccharide). At the same time, around 2% of the injected material was found in the blood. Six hours after injection, equal quantities were found in the intestines, liver and blood (0.5%), slightly less in the kidneys, lungs and spleen (0.2–0.3%) and the least quantity in the brain (0.04%). However, the dynamics of retention in the organs was evidently different. In the kidneys, lungs and spleen, radioactivity steadily decreased over the studied period. In the liver following an initial decrease, radioactivity remained the same 3 and 6 h after injection. On the other hand, in the intestines and brain PGM seemed to accumulate rather than disapper following i.v. administration. This fact should be considered when explaining different biological activities of low molecular weight bacterial peptidoglycans.     

The antigenicity of aggregated and aggregate-free human gamma-globulin for rabbits

Rabbits were injected with either aggregated or aggregate-free HGG. It was found that rabbits injected with aggregated HGG produced large amounts of precipitating anti-HGG antibodies, whereas those injected with aggregate-free HGG produced no detectable antibodies, and became immunologically unresponsive to HGG.

Some rabbits were injected with ¹³¹I-labelled HGG. In these animals it was observed that aggregated HGG disappeared from the blood much faster than aggregate-free HGG. A considerable amount of the label was found in the spleen of the rabbits given aggregated HGG, but none in that of the ones given aggregate-free HGG. In the spleen the radioactivity was bound to protein. No significant protein-bound radioactivity was detected in other organs. An important amount of free ¹³¹I was found in the thyroid gland of these rabbits as early as 4 hours after the injection.

Severely hypocomplementaemic rabbits have been found sporadically. Nine such animals were included in these experiments. It was observed that their immune response to aggregated HGG was entirely normal. They also cleared the labelled protein from the blood in a normal fashion, but they retained significantly larger amounts of it in the spleen, while only small amounts of free label appeared in their thyroid glands.

The possible implications of these findings are discussed.

     

Attempts to study the localization of liposomes and liposome entrapped antigen in the spleen.

Attempts were made to study the localization of intravenously injected liposomes and enclosed labelled antigens in the spleen of mice, using autoradiography. A more than hundred fold increased uptake of antigen by the spleen was obtained when liposome entrapped antigen was compared with free antigen or antigen-antibody complexes which have been used in earlier studies. No marked accumulation of the labelled antigen was found in the follicles of the spleen, although specific antibodies to the injected antigen have been injected simultaneously with the liposome entrapped antigen. It is argued that the bulk of the labelled antigen in the spleen 0.5 to 2 h after injection was still enclosed in the liposomes, whereas part of the label, arranged in patches in the red pulp and marginal zone, corresponds with labelled antigen in macrophages, released after digestion of the liposomal membranes. The present techniques did not enable the detection of the liposomes themselves.     

Comparative study of the distribution of 7,12-dimethylbenz(a)anthracene in pregnant rats and their fetuses

On the 21st day of pregnancy rats were given an intravenous injection of 7,12-dimethylbenz(a)anthracene (DMBA) in a dose of 15 mg/kg, and its concentration in the organs of the mothers (liver, kidneys, lungs, brain, spleen, and placenta) and of their fetuses (liver, kidneys, lungs, brain, intestine, carcass, and whole fetus) was determined by a fluorescence-spectral method 30, 60, and 180 min later. The highest concentration of DMBA in the mothers at all times was found in the lungs. With an increase in the time of injection the DMBA concentration fell in all organs of the pregnant rats, except in the brain, in which it rose. In the fetuses an irregular distribution of the carcinogen in the organs was found only 60 min after injection of DNBA, when the highest concentration was detected in the liver. In all fetal organs the maximal concentration was found after 60 min. Accumulation of DMBA in the various fetal organs did not correlate with the observed highest frequency of tumors in the kidney and nervous system of rat fetuses following transplacental exposure to DMBA in the same dose.Laboratory of Biophysics and Laboratory of Experimental Tumors, N. N. Petrov Research Institute of Oncology, Ministry of Health of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 334–335, March, 1980.     

Distribution and localization of endotoxin in the reticulo-endothelial system (RES) and in the main vessels of the rat during shock

The fate of endotoxin was followed with immunohistochemistry and radio-labelled lipopolysaccharide (LPS) in organs of the reticulo-endothelial system (RES), in the great vessels and the thoracic duct of rats during a 14 day period after the injection of a shock-inducing amount of endotoxin. The immunohistochemical detectability of LPS in most tissues increased continuously during the first 48 hours, showing the strongest LPS staining in the liver and adrenal gland. Macrophages were found to be the most important cells of primary LPS uptake in all organs except the adrenal glands, where endotoxin was mainly present in phagocytic vacuoles of the cortical epithelium. The comparison of results obtained with the immunoperoxidase method and radioactivity measurements revealed that at a later stage of the experiment the persisting LPS in liver and spleen looses its antigenic activity. The correlation between the appearance of LPS positive macrophages and histological signs of tissue injury during endotoxin shock is striking.     

Interferon Induction by Poly(I):Poly(C) Enclosed in Phospholipid Particles

Liposomes were prepared with phospholipids (sphingomyelin, lecithin, and phosphatidylethanolamine) in combination with cholesterol and charged lipids (dicetyl phosphate and stearylamine) and contained either poly(I):poly(C) or poly(I). Neutral and positively charged liposomes attached much better to L-929 cells in tissue culture than did negatively charged particles. Liposomes were toxic to L cells at relatively low concentrations, making the determination of antiviral activity induced by particles containing poly(I):poly(C) difficult to measure by the plaque reduction assay. When injected into mice, all of the liposomes containing poly(I):poly(C), except phosphatidylethanolamine liposomes, greatly potentiated and extended the serum interferon response of poly(I):poly(C). Lecithin and sphingomyelin liposomes given intravenously were ten times more effective than free poly(I):poly(C) in stimulating production of serum interferon. Sphingomyelin liposomes containing [(14)C]poly(I):poly(C) were 88% cleared from the bloodstream of mice by 3 min after intravenous injection. Most of the radioactivity (70%) was captured by the liver and remained there for at least 4 h. By 2 h, 7% of the radioactivity could be found in the spleen. Five percent of the radioactivity was found in the lungs at 30 min, with decreasing amounts thereafter. Small amounts of radioactivity were found in the muscle and kidneys. The spleen was shown to contain appreciable levels of interferon at 4 h, and low levels were found in the liver. Radioactivity accumulated slowly in the liver following an intraperitoneal injection of sphingomyelin liposomes containing [(14)C]poly(I):poly(C). By 4 h, 26% of the dose was recovered from the liver and 4.9% from the spleen, with small amounts in the lung, kidney, and omentum.     

The incorporation of phenylalanine-C14 into the proteins of sarcoma 45 and of the normal organs of rats

Summary Studies of the incorporation of carbon-labelled phenylalanine-1-C¹⁴ into the proteins of sarcoma 45 and various organs of the rat were made. It was shown that the label was taken up rather intensively by the tumor and the organs under study (liver, kidneys, intestinal wall and spleen). In the whole period of study the proteins of the kidney contained the highest amount of radioactivity. The specific activity of the proteins of the tumour (sarcoma 45) was near to that of the liver and intestinal wall. However, the label in the tumour maintained on a relatively constant level without lowering in the period of study (24 hours) whereas the radioactivity in the liver was descending steeply. It was interesting to note that there was some similarity between the incorporation of phenylalanine into the proteins of sarcoma 45 and various organs and the distribution of sarcolysin (p-di-2-chloro-ethyl-amino-DL-phenylalanine) in the proteins of these organs and the tumour.(Presented by Active Member, AMS, USSR, N. N. Blokhin) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 52, No. 9, pp. 66–69, September, 1961     

Comparison of 3H-galactose and 3H-glucose as precursors of hepatic glycogen in control-fed rats

Labeling of hepatic glycogen derived from 3H-galactose and 3H-glucose was compared shortly after intravenous injection in control-fed rats. The rats were allowed to accumulate 5-8% glycogen prior to receiving label. Fifteen minutes to 2 hours after labeling, liver was excised and processed for routine light (LM) and electron microscopic (EM) radioautography (RAG) or biochemical analysis. After injection of 3H-galactose, LM-RAGs revealed that the percentage of heavily labeled hepatocytes increased from 37% after 15 minutes to 68% after 1 hour but showed no further increase after 2 hours. alpha-Amylase treatment removed most glycogen and incorporated label; thus few silver grains were observed, indicating little incorporation of label except into glycogen. EM-RAGs demonstrated that most label occurred where glycogen was located. Biochemical analysis showed initially a high blood level of label that rapidly plateaued at a reduced level by 5 minutes. Concomitantly, glycogen labeling determined by liquid scintillation counting reflected the increases observed in the RAGs. After injection of 3H-glucose, LM-RAGs revealed that only 12% of the hepatocytes were heavily labeled at 1 hour and 20% at 2 hours. In tissue treated with alpha-amylase, glycogen was depleted and label was close to background level at each interval observed. EM-RAGs showed most grains associated with glycogen deposits. Biochemically, blood levels of label persisted at a high level for 30 minutes and tissue levels increased slowly over the 2-hour period. This study shows that incorporation from 3H-galactose was more rapid than incorporation of 3H-glucose; however, label derived from both carbohydrates appeared to be incorporated mainly into glycogen.     

Liposomal chemotherapy in visceral leishmaniasis: An ultrastructural study of an intracellular pathway

The intracellular fate of liposomes administered intracardially was examined in the liver and spleen of hamsters experimentally infected withLeishmania donovani. Separate groups of animals were treated with liposomes containing either an antileishmanial agent, a colloidal gold marker, or saline. Ultrastructural examinations of lysosomal interactions with the parasitophorous vacuole and with phagocytized liposomes were made. Lysosomes readily fused with the parasitophorous vacuoles but appeared to have little effect on the parasite, possibly due to the production of enzyme inhibitors. Liposomes rapidly became localized in lysosomes subsequent to endocytosis by macrophages. Morphologic evidence suggested that secondary lysosomes containing liposomal residues then fused with the parasitophorous vacuole. Aspects of one possible pathway are discussed which may account for the greatly enhanced effectiveness of liposomal chemotherapy for experimental visceral leishmaniasis.     

Effects of preservation of rat lungs in a hypothermic medium on alveolar morphology.

The increasing use of organs such as liver, lung, heart, pancreas, kidney and small intestine for transplantation purposes necessitates the development of optimum preservation techniques. The aim of our study was to investigate time-related morphological changes in alveoli during preservation of rat lungs in hypothermic Euro-Collins solution. Lungs were perfused via the pulmonary arteries with Euro-Collins solution at a temp of 19 degrees C. Totally perfused lungs were placed in Euro-Collins solution and stored for 6, 12 and 24 h at 4 degrees C. Biopsies were taken and prepared for examination at the light and electron microscopical level. Light microscopic examination revealed good preservation of the alveoli after storage for 6 h and moderate damage of alveolar architecture after 12 h of preservation. Severe degeneration of alveoli was found after 24 h of storage. The main ultrastructural changes were observed in lungs stored for 12 h and 24 h. After 6 h of storage, tissue damage was not found. Pneumocytes type II lost their apical microvilli and lamellar bodies were electron-lucent, indicating lamellar degeneration after 12 and 24 h of storage. Pneumocytes type I were also damaged. Their cytoplasm contained many vacuoles. Endothelial lining of the capillaries was contracted. Endothelial cells also showed many vacuoles. Edema around the capillaries was observed. We conclude on the basis of our morphological study, that Euro-Collins solution at low temperature is a good preservative for a short period of time only, but serious tissue damage occurs after periods of preservation longer than 12 h.     

The fate of vacuolation in liver cells with special reference to hyaline globules.

This study was undertaken in rats to clarify the mechanisms and time necessary for recovery from vacuolation in liver cells. Vacuoles were produced by congestion of the liver due to constriction of the inferior vena cava just below the diaphragm, and changes in vacuoles were examined quantitatively and qualitatively until 24 h after release of the constriction. Vacuoles in liver cells decreased in number by half within 5 min after recovery from congestion. The remaining vacuoles metamorphosed to hyaline globules by condensation of the contents. The number of hyaline globules increased with a peak occurring at 3-6 h after recovery from congestion, although the number of vacuoles decreased gradually. Only a few small vacuoles and hyaline globules were found in liver cells in pericentral areas at 24 h after recovery from congestion. These data indicate that vacuoles may be discharged promptly from the liver cell cytoplasm after recovery from congestion, and the remaining vacuoles may metamorphose to hyaline globules by condensation of the contents and finally fade into the cytoplasm.     

Protective effect of phosphatidylcholine liposomes in cats with hemorrhagic shock

Experiments on cats indicated that the use of phosphatidylcholine liposomes in hemorrhagic shock may reduce the intensity of free-radical processes in the liver, stabilize the phospholipid bilayer of plasma membranes in hepatocytes, decrease the severity of pathomorphological changes in the target organs, and raise systemic arterial pressure with its stabilization at a subnormal level. The use of phosphatidylcholine liposomes in cats with hemorrhagic shock resulted in a considerable prolongation of their survival. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 5, pp. 480–484, May, 1995     

The Fate of Intravenously Administered Radioactive Cholesterol Dispersion in the Rat

The present work was aimed at studying the first stages of disappearance of particulate cholesterol from the circulation. The distribution of radioactivity in blood, liver, spleen, lungs and kidneys was determined at different time intervals after i.v. injections of aqueous-ethanolic dispersions of radioactive cholesterol to rats. More than 95% of the dose disappeared from the circulation in 10 min. From 71 to 93% of the dose was found in the liver 5 min-2 h after the injection. According to autoradiography, most of the particulate cholesterol was immediately taken up by the liver parenchymal cells. There were only few grains over the Kupffer cells. 2 min to 6 h after the injection the highest specific radioactivity was in the free cholesterol fraction of the liver. The specific activity of liver esterified cholesterol was 54 % of that of the free cholesterol at 1 h. They both declined gradually and reached the same level in 1–2 days. There was a gradual rise of the specific activities of plasma free and esterified cholesterol starting 30 min after the injection. Both reached a maximum in 6–16 h, when the curve of plasma esterified cholesterol intersected that of liver esterified cholesterol. The radioactivity reappearing in plasma was associated with lipoproteins and red cells; the cholesterol of the high density lipoproteins had the highest specific activity. The results indicate that particulate cholesterol is taken up chiefly by the parenchymal cells of the liver and is subsequently incorporated into the plasma lipoproteins, most rapidly into the high density lipoproteins. A large fraction of plasma esterified cholesterol originates in the liver.