METABOLIC EFFECTS OF LATHYROGENIC AGENTS ON CARTILAGE IN VIVO AND IN VITRO

The effects of lathyrogenic agents in vivo and in vitro are described, in respect to some biochemical indices of cartilage metabolism. Lathyrogenic agents in vivo inhibited the incorporation of radiosulfate into rat epiphyseal cartilage and the isolated chondroitin sulfate. No significant changes in hydroxyproline or hexosamine content of epiphyseal cartilage were found, but there was a marked increase in water content. The content of chondroitin sulfate, measured as uronic acid, was decreased. The importance of taking growth rate differences between control and experimental rats into account in assessing the effects of lathyrogenic agents in vivo is emphasized. In an in vitro system, utilizing fresh calf costal cartilage slices, the presence of low concentrations of lathyrogenic agents markedly affected various metabolic events. The incorporation into cartilage slices of sulfate-S35, glucose-U-C14, and glycine-1-C14 was significantly depressed, as was the production of organic acids, including lactic acid. In general, these effects were more severe under anaerobic conditions. Glutamine restored the activities of the slices treated with lathyrogenic agents to control values obtained in the absence of either lathyrogen or glutamine

A BIOCHEMICAL AND MORPHOLOGIC STUDY OF MYELINATION AND DEMYELINATION : II. LIPOGENESIS IN VITRO BY RAT NERVES FOLLOWING TRANSECTION

Bilateral transection was performed on rat sciatics. At varying intervals after the operation, samples of nerve were taken both distal and proximal to the level of transection, as well as from the tissue which bridged the gap between the stumps. These samples were incubated in Warburg flasks, with glucose and a labelled lipide precursor (acetate or phosphate). The total lipides were then extracted and their radioactivity was measured. Normal rat sciatics served as controls, and the biochemical and histological findings were correlated. In the distal portion undergoing Wallerian degeneration, the lipide content began to fall before any removal of myelin could be detected histologically. It is suggested that there is a period of "non-cellular removal" prior to the physical breakdown of the myelin. Changes in respiration and in lipogenesis from acetate followed a triphasic course, and agreed with the histological findings in that after a period of predominantly passive changes (approximately 1 to 3 days) there follows a period of cellular reaction (4 to 50 days) and a period of atrophy (from 50 days onward). The incorporation of phosphate into the lipides was increased at all stages examined, even as early as 22 hours after section. This increased P32 incorporation could not be reproduced in nerves allowed to degenerate in vitro. It is suggested that the hypertrophying Schwann cells synthesize some lipide moieties at a considerably faster rate than others. Proximal to the level of transection, lipogenesis from acetate was depressed, for as long as 32 days postoperatively. It appears, therefore, that the maintenance of the myelin sheath is impaired also above the level of transection. In the "union tissue" which developed between the stumps, prior to the appearance of histologically visible myelin, lipogenesis was low; later it rose above levels for normal nerve. This pattern of lipogenesis in regenerating nerve is similar to that found in growing nerves

A BIOCHEMICAL AND MORPHOLOGIC STUDY OF MYELINATION AND DEMYELINATION : I. LIPIDE BIOSYNTHESIS IN VITRO BY NORMAL NERVOUS TISSUE

Samples of normal grey matter, white matter, and peripheral nerves obtained from rats were incubated in Warburg vessels with glucose and a labelled lipide precursor (acetate, phosphate, choline, glycerol, glucose). The total lipides were then extracted and their radioactivity measured. The preparations were compared with respect to dry weight, lipide content, O2 uptake, and ability to incorporate the various substrates into the lipides. Grey matter was found to be the least damaged by incubation, white matter the most. Damage to the tissue depressed lipogenesis to a greater extent than respiration. Five substrates were compared with respect to their degree of incorporation into the lipides of the various preparations. White matter, which had a greater oxygen uptake than peripheral nerves, showed the lowest degree of incorporation for most of the substrates studied. The results suggest that there are considerable quantitative differences in the metabolism of central and peripheral myelin. In the sciatic preparations, oxygen uptake and lipogenesis from acetate were found to decrease from the proximal to the distal end of the nerve. This finding may be relevant to the pathogenesis of peripheral neuropathies. The growth and metabolic activity of peripheral nerves were studied in rats aged 1 to 500 days, and the biochemical and histological findings were correlated. The results indicated that the lipogenetic activity of the Schwann cell was lowest in the newborn animal, and reached its peak at about 20 days. Comparative data were also obtained from the cerebral cortex. The growth pattern of peripheral nerves was distinctly different from that of the brain. With respect to changes in tissue weight, respiration, and lipogenesis, growing peripheral nerve correlated with body weight, while the brain matured much more rapidly

THE DEPRESSION OF PHAGOCYTOSIS BY EXOGENOUS CYCLIC NUCLEOTIDES, PROSTAGLANDINS, AND THEOPHYLLINE

The effects of agents that elevate intracellular cyclic adenosine 3',5'-monophosphate (cAMP) have been studied with respect to phagocytosis by guinea pig polymorphonuclear leukocytes. The investigation depends upon the use of a precise method for following ingestion. Theophylline, dibutyryl cAMP, and prostaglandins inhibited the phagocytosis of starch particles. The inhibitions caused by prostaglandins E1, E2, and F2α (PGE1, PGE2, and PGF2α) were synergistic with that due to theophylline. Inhibition by PGA1 and PGA2 was not. At equal concentrations the order of increasing inhibition of phagocytosis (assayed at 10 min) by the prostaglandins was PGE1 < PGF2α < PGE2 < PGA1 = PGA2. Our results are consistent with the hypothesis that increased intracellular levels of cAMP impair the phagocyte's ability to ingest particles. The mechanism of the inhibition has not been defined. The increment in oxidation of [1-14C]glucose to 14CO2 that normally accompanies phagocytosis was found to be depressed in the presence of PGE1 or theophylline, together or individually as expected from the inhibition of phagocytosis. Paradoxically, oxygen consumption although depressed by theophylline or PGE1 plus theophylline, was stimulated by PGE1 alone

STUDIES ON THE INTERACTION BETWEEN PHAGOCYTES AND TUBERCLE BACILLI : II. THE ACTION OF PHAGOCYTES UPON C14-LABELLED TUBERCLE BACILLI

Tubercle bacilli labelled with C14 were prepared by growth on radioactive substrates such as glycerol, CO2, and acetate. These organisms were exposed in vitro to leucocytes (mostly polymorphonuclear leucocytes) from peritoneal exudates of guinea pigs. The respiration of the leucocytes and of the bacilli, alone and together, was followed by determining oxygen uptake and C14O2 production. When heat-killed labelled tubercle bacilli were exposed to leucocytes there was little or no degradation of bacillary material to C14O2 by leucocytic enzymes. On the other hand, conversion of components of sonically disrupted bacilli to C14O2 by leucocytes was significant. It was possible to determine the oxygen uptake and C14O2 production of phagocytized living tubercle bacilli, and it was found that after phagocytosis the bacilli maintained their rates of oxygen consumption and C14O2 production. This finding was in contrast to observations made with Mycobacterium phlei, a saprophytic acid-fast organism, and with Bacillus subtilis. In these cases oxygen consumption and C14O2 production declined after phagocytosis, and bacterial components were converted to carbon dioxide to a significant degree by leucocytic enzymes

STUDIES ON THE INTERACTION BETWEEN PHAGOCYTES AND TUBERCLE BACILLI : I. OBSERVATIONS ON THE METABOLISM OF GUINEA PIG LEUCOCYTES AND THE INFLUENCE OF PHAGOCYTOSIS

As a basis for studies on the interaction of phagocytes and tubercle bacilli experiments were carried out to obtain information on some biochemical characteristics of exudate leucocytes from guinea pigs. It was found that total cellular phosphorus was the most suitable measure of protoplasm. Cell counts were less reliable because of unavoidable clumping in the suspension, and dry weight measurements were less specific when contamination with erythrocytes occurred. The utility of phosphorus measurements in this connection depends upon the fact that an erythrocyte contains only 4 per cent of the phosphorus present in a leucocyte. From measurements on mixed suspensions consisting of varying known proportions of polymorphonuclear and mononuclear leucocytes as determined by differential counting, it was possible to compute true values for these two cell types with respect to oxygen consumption and lactic acid production. Thus it was found that monocytes consumed considerably more oxygen and produced more lactate than polymorphonuclear leucocytes. From the data obtained it is suggested that differences in metabolic activity found when comparing cell suspensions obtained by the use of different irritants are due to different proportions of the two cell types. In particular, the effects of oxygen tension and pH on the activities of the cells were studied. It was found that decreasing the proportion of oxygen in the atmosphere from that of air to 1 per cent reduced oxygen consumption by about 80 per cent, whereas lactic acid production was increased by about 45 per cent. It was also found that decreasing the pH of the medium below pH 7.5 caused a considerable reduction in the respiration, lactate production, and viability of polymorphonuclear leucocytes. The monocytes proved less sensitive to similar changes in pH, especially with regard to lactic acid production and viability. Observations were made of oxygen consumption and lactate production during phagocytosis. During the hour following the addition of heat-killed tubercle bacilli to the phagocytes, the oxygen consumption of suspensions rich in polymorphonuclear leucocytes rose by 60 per cent, and that of suspensions rich in monocytes rose by nearly 100 per cent. Lactic acid production was unchanged during phagocytosis

METABOLIC AND MORPHOLOGICAL OBSERVATIONS ON THE EFFECT OF SURFACE-ACTIVE AGENTS ON LEUKOCYTES

Morphological and metabolic observations have been made on the effects of endotoxin, deoxycholate, and digitonin (at less than 50 µg/ml) on polymorphonuclear leukocytes and mononuclear cells. The agents stimulate the respiration and glucose oxidation of these cells in a manner similar to that seen during phagocytosis. Electron microscopy revealed no morphological changes with the first two agents, but dramatic membrane changes were seen in the case of digitonin. Here tubular projections of characteristic size and shape formed on and split off the membrane. All the agents stimulated uptake of inulin, but efforts to demonstrate increased pinocytosis by electron microscopy have not so far succeeded, probably due to limitations in present experimental techniques

ALTERATIONS OF MACROPHAGE FUNCTIONS BY MEDIATORS FROM LYMPHOCYTES

Sensitized lymphocytes were incubated in vitro with the specific antigen Supernatants from these cultures were chromatographed on Sephadex G-100 columns. Supernatant fractions containing MIF, chemotactic factor, and lymphotoxin, but free of antigen and antibody, were incubated with normal peritoneal exudate macrophages. Macrophage adherence, phagocytosis, spreading, motility, and direct hexose monophosphate oxidation were enhanced, while protein synthesis was unaffected. Thus, antigen-stimulated lymphocytes secrete a factor or factors which enhance certain macrophage functions. Implications for models of cellular immunity and cellular hypersensitivity are discussed

STUDIES ON THE INTERACTION BETWEEN PHAGOCYTES AND TUBERCLE BACILLI : III. SOME METABOLIC EFFECTS IN GUINEA PIGS ASSOCIATED WITH INFECTION WITH TUBERCLE BACILLI

In continuing studies concerning the interactions between phagocytes and tubercle bacilli the effect of tuberculous infection on respiration and glucose utilization was investigated in guinea pigs. Peritoneal exudates rich in polymorphonuclear leucocytes, derived from guinea pigs infected with tubercle bacilli, had a significantly higher rate of respiration than the same cells from normal animals. The difference between cells from normal and infected animals was greater when the animals were infected with a virulent strain (Vallée) than when infected with an attenuated one (R1Rv or BCG). By the use of glucose labelled with C14 at position 1 or 6, or uniformly labelled glucose, it was established that this difference in oxygen uptake between normal and infected cells was probably not caused by a difference in the pathway of glucose utilization. Similarly, the respiration of liver and kidney slices from normal and infected guinea pigs was compared and it was found that liver slices showed differences similar to those shown by leucocytes, but that the kidney slices did not. The possibility has not been ruled out that the difference in rate of respiration of liver slices due to infection might be caused by tuberculous lesions in the livers of infected animals. The mononuclear cells which invade the liver have a higher rate of oxygen uptake than liver cells. The rate of glucose utilization and the total amount of CO2 produced was also determined in intact guinea pigs. Both functions were found not to differ significantly in normal and infected animals. The rate of production of CO2 from C1 and C6 of glucose was the same in both groups of animals. The ratio of the rate of production of C14O2 from C1 and C6 of glucose by the whole animal was found to be about 1.35. It was found to be much higher with polymorphonuclear leucocytes (C1/C6 = 8 in the absence of serum). During the process of phagocytosis this ratio increased from about 25 to about 130 (in the presence of 2 per cent serum) indicating an increase in the direct oxidative pathway of glucose utilization during stimulated cellular activity