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PubMed1982

Selective modification of rat peritoneal macrophage lysosomal hydrolases by inflammatory stimuli.

Lysosomal acid hydrolases were surveyed in elicited and non-elicited rat peritoneal macrophages to determine the types of enzymes present and optimal assay conditions. Adherent peritoneal cells (primarily macrophages) were cultured 24 hours prior to use. Intracellular distribution of enzymes was determined by differential centrifugation of whole cell homogenates into nuclear, cytoplasmic, and lysosomal fractions. The acid glycosidase, acid phosphatase, acid protease, and lysozyme were largely sedimentable in the lysosomal fraction. Much enzyme activity was latent, being activated by addition of Triton X-100. Chymotrypsin-like protease activity in cell fractions was apparently due to low level mast cell contamination. Elicited macrophages had elevated total cell protein as compared to non-elicited cells, but changes in intracellular enzyme levels were selective depending on the enzyme and the stimulus used to elicit macrophages. Thioglycollate-elicited cells showed elevations of most acid hydrolases compared to non-elicited cells, whereas enzyme levels in zymosan-elicited cells were similar to those in non-elicited cells. All elicited cells showed marked decreases in total cellular alpha-D-mannosidase and alpha-L-fucosidase compared to non-elicited cells. Intracellular lysozyme levels also varied between different rat strains. Cultured macrophages exhibited increasing intracellular levels and extracellular secretion of acid hydrolases, especially extracellular lysozyme (10-25 mug/10(6) cells/day), over 72 hours. No significant intra- or extracellular elastinolytic activity was detected.

Animals↗
PubMed1983

Role of reduced sulfur compounds in nutrition of Propionibacterium acnes.

In a study of the nutritional requirements of Propionibacterium acnes, acid hydrolyzed casein was shown to be the preferred nitrogen source in a glycerol-salts medium. Of 22 individual amino acids tested, only cysteine supported growth in the glycerol-salts medium. This indicated a requirement not only for amino nitrogen but also for reduced sulfur. In the presence of an auxiliary nitrogen source, thioglycolate or thiosulfate could replace sulfur-containing amino acids. The apparent requirement of P. acnes for reduced sulfur possibly could be satisfied in vivo by the constant availability of this substance in the form of sulfhydryl groups in the sebaceous follicle during keratinization. The relationship between P. acnes and its nutritional substrate may give this organism a selective advantage to exist in this ecological niche.

Amino Acids↗
PubMed1979

Development of a chemically defined liquid medium for growth of Legionella pneumophila.

A chemically defined liquid medium has been developed for the study of the physiology and antigen production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, alpha-ketoglutaric acid, pyruvate, 0.05% l-cysteine, 0.05% glutathione, and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium in shake culture at 37 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum cell surface antigen as distinguished by specific fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2, pyruvate, and alpha-ketoglutarate strongly stimulated growth; that cysteine and methionine were required for growth; and that serine, threonine, histidine, tyrosine, and tryptophane were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not use glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate, and ethylenediaminetetraacetic acid totally inhibited growth. Beta-mercaptoethanol, thioglycolate, dithiothreitol, and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and antigen production.

Aerobiosis↗
PubMed1996

Isolation of presumptive Streptobacillus moniliformis from abscesses associated with the female genital tract.

We report three cases in which Streptobacillus moniliformis was isolated from abscesses. Abscess material in each case contained small, pleomorphic, gram-negative to gram-variable bacilli. Anaerobic blood agar cultures yielded pinpoint colonies adjacent to small gray-white colonies. The pinpoint colonies did not gram stain, and the gray-white colonies varied from gram-variable coccobacilli to long, curly, gram-variable rods. The pinpoint colonies microscopically resembled L-forms on Dienes-stained agar preparation. Subculture to serum-supplemented thioglycolate broth demonstrated "puff ball" colonies. Fatty acid profiles obtained with use of gas chromatography coupled with mass spectrometry showed major peaks for C16:0, C18:2, C18:1, and C18:0 fatty acids, a profile characteristic of S. moniliformis. Results of biochemical testing of each isolate were equivocal. S. moniliformis, bacterial L-forms, and common isolates from genital tract abscesses are discussed.

Abscess↗
PubMed1993

Water soluble complex of palmitic acid in media for cultivation of leprosy-derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues.

Palmitic acid and palmitates were transformed into water soluble complexes with crystalline heptakis-2,6-di-0-methyl-beta-cyclodextrin. This formulation was incorporated into liquid and solid chemically well-defined media. The fatty acid served as C and energy source, ammonium thioglycolate as the sole source of N with the SH group as further source of energy. Minute amount of dimethyl-sulfoxide added was used for its known effect on cell membrane permeability. The media were inoculated with host grown Mycobacterium leprae cells isolated from human, armadillo and Nu mice foot pad lepromata. No growth occurred in the liquid medium at 22 or 32 degrees C, but cultures and subcultures of acid fast rods were grown at 10 degrees C. Bacilli in the cultures were solid, strongly acid fast rods, growing in clumps like globi. Growth on the semisolid media was visible as smooth round colonies, of white to ivory in colour, slowly expanding flatly at the periphery of the colony on the agar surface. Colonies developed within 2-3 weeks and reached maximum size at 50-80 days depending on the size of inoculum. Subcultures grow faster and more abundantly with adaptation to the media. No growth was seen without the water soluble complexes of palmitic acid or palmitates in the media. The free fatty acid or its salts had an equal growth supporting effect. Identical psychrophilic cultures were obtained from 7 out of 9 armadillo, 12 out of 12 Nu mice and 1 out of 2 human lepromata. None of the cultures grow on Loewenstein, Dubos or 7H9 media at 10 degrees C, 20 degrees C or 32 degrees C, respectively. The tested 4th to 7th subcultures of the strains were strongly positive for phenolic glycolipid-1. Heat killed suspensions of up to 7th subcultures gave negative late skin reaction in all of 16 LL cases. In 19 I, B and T cases the late skin reactions were all similar to that obtained with authentic human lepromin.

Animals↗
PubMed1966

The enzymic oxidation of chlorogenic acid and some reactions of the quinone produced.

1. Partially purified preparations of tobacco-leaf o-diphenol oxidase (o-quinol-oxygen oxidoreductase; EC 1.10.3.1) oxidize chlorogenic acid to brown products, absorbing, on average, 1.6atoms of oxygen/mol. oxidized, and evolving a little carbon dioxide. 2. The effect of benzenesulphinic acid on the oxidation suggests that the first stage is the formation of a quinone; the solution does not go brown, oxygen uptake is restricted to 1 atom/mol. oxidized, and a compound is produced whose composition corresponds to that of a sulphone of the quinone derived from chlorogenic acid. 3. Several other compounds that react with quinones affect the oxidation of chlorogenic acid. The colour of the products formed and the oxygen absorbed in their formation suggest that the quinone formed in the oxidation reacts with these compounds in the same way as do simpler quinones. 4. Some compounds that are often used to prevent the oxidation of polyphenols were tested to see if they act by inhibiting o-diphenol oxidase, by reacting with quinone intermediates, or both. 5. Ascorbate inhibits the enzyme and also reduces the quinone. 6. Potassium ethyl xanthate, diethyldithiocarbamate and cysteine inhibit the enzyme to different extents, and also react with the quinone. The nature of the reaction depends on the relative concentrations of inhibitor and chlorogenic acid. Excess of inhibitor prevents the solution from turning brown and restricts oxygen uptake to 1 atom/mol. of chlorogenic acid oxidized; smaller amounts do not prevent browning and slightly increase oxygen uptake. 7. 2-Mercaptobenzothiazole inhibits the enzyme, and also probably reacts with the quinone; inhibited enzyme is reactivated as if the inhibitor is removed as traces of quinone are produced. 8. Thioglycollate and polyvinylpyrrolidone inhibit the enzyme. Thioglycollate probably reduces the quinone to a small extent.

Ascorbic Acid↗
PubMed1978

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.

Animals↗
PubMed1997

Effect of dietary flaxseed on fatty acid composition, superoxide, nitric oxide generation and antilisterial activity of peritoneal macrophages from female Sprague-Dawley rats.

The impact of ground flaxseed (FS) or flaxseed meal (FSM) diets on the fatty acid composition and functions of rat peritoneal exudate cells (PEC) was determined. Female weanling Sprague-Dawley rats (10/group) were fed isocaloric AIN-76 diets supplemented with 0.0, 10.0% (w/w) FS or 6.2% (w/w) FSM. At the end of 56-days, rat serum and thioglycollate-elicited PEC were analyzed for total lipid fatty acids. Production of nitric oxide (NO) and superoxide (O2-), Listeria monocytogenes (LM) phagocytic index and antilisterial activity of resident PEC were also assessed. A significant increase in alpha-linolenic (C18:3), eicosapentanoic (C20:5) and docosahexanoic (C22:6) acids, as well as a significant reduction in arachidonic acid (C20:4) was observed in the serum of rats fed 10% FS. Dietary FS caused a significant reduction in palmitic acid (C16:0) and an increase in stearic acid (C18:0) of PEC. Defatted FSM produced a significant increase in long chain fatty acids, which included eicosadienoic acid (C20:2) in PEC and C22:6 in serum. PEC from rats fed 10.0% FS produced significantly less (about 50%) O2- in response to phorbol myristate acetate (PMA), than did PEC from control animals; dietary treatment had no effect on O2- in response to LM. FSM had no impact on the O2- production by PEC in response to PMA or LM. Antilisterial activity of PEC was determined by comparing bacterial uptake after 1 hr with recovery 24 hrs later. Despite comparably equivalent bacterial uptake, few viable intracellular LM were recovered at T = 24 for all test samples, indicating that, regardless of the dietary treatment, PEC were able to handle the in vitro LM infection. This bacterial clearance was accompanied by equivalent NO generation by PEC from each dietary group in response to LM. Summarily, dietary FS produced significant changes in fatty acid composition of serum and PEC, inhibited O2- generation by PEC, and was ineffectual to both NO production by and antilisterial activity of PEC.

Animals↗
PubMed1996

Fucoidin, a potent inhibitor of leukocyte rolling, prevents neutrophil influx into phorbol-ester-induced inflammatory sites in rabbit lungs.

The polysaccharide fucoidin, a homopolymer of sulfated L-fucose, is known, by interfering with the function of L-selectin, to inhibit leukocyte rolling, which is an early and essential step in the process of leukocyte extravasation into inflamed sites. We tested the inhibitory effect of fucoidin on neutrophil accumulation in lung inflammation induced by phorbol myristate acetate and peritoneal inflammation induced by thioglycollate in rabbits. Pretreatment (10 mg/kg) followed by continuous administration (10 mg/kg/h) of fucoidin dramatically reduced the number of neutrophils recruited into the lungs (determined by neutrophil counts in bronchoalveolar lavage fluid; 97.7 +/- 0.52% reduction) and peritoneal cavity (determined by neutrophil counts in peritoneal lavage fluid; 98.4 +/- 1.29% reduction). Fucoidin treatment also increased the systemic neutrophil count. These results suggest that the inhibition of leukocyte rolling, which may be a primary function of fucoidin, leads to a reduction of neutrophil accumulation at inflammatory sites, which may be beneficial for attenuating neutrophil-mediated tissue injury.

Animals↗
PubMed1972

Use of shake cultures in a semisolid thioglycolate medium for differentiating staphylococci from micrococci.

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.

Aerobiosis↗
PubMed1977

Further data on the characters of Listeria strains.

The composition of a minimal medium suitable for the demands of modern genetical work is described. The nutritional requirements of some 15 strains of L. monocytogenes were studied on this medium. DL-6,8-thioctic acid was not essential. The organisms also grew without thiamine but for a convenient colony size this vitamin had to be used. The presence of Fe++, Mg++, and Ca++ ions as well as a low redox potential supplied by thioglycollate was necessary. There was some difference in the amino acid requirements of the strains. The most common demand was for leucine, iso-leucine, valine, and cysteine. The serotype, agglutinability, hemolytic effect, virulence, and nutritional requirements of the Listeria were compared. It seems that these properties are not consistently linked. Two 'EMS' mutants were also studied. The arginine, glutamine, and methionine metabolism of the auxotrophic mutant 025/4/4 was damaged and its virulence was lost. Data connected with the virulence markers of Listeria are discussed.

Culture Media↗
PubMed1991

Poly I:C-induced anti-herpes simplex virus type 1 activity in inflammatory macrophages is mediated by induction of interferon-beta.

We examined the mechanism by which Polyriboinosinic:Polyribocytidylic acid (Poly I:C) augments resistance of thioglycolate-elicited inflammatory macrophages to infection with herpes simplex virus type 1 (HSV-1). We show that Poly I:C-induced antiviral activity is completely abrogated by antibodies to interferon-beta (IFN-beta) whereas antibodies to other interferons or to other cytokines have no effect. Furthermore, treatment of inflammatory macrophages with exogenous IFN renders them resistant to HSV-1, whereas treatment with other cytokines does not. In addition, we demonstrate that supernatants from macrophages treated with Poly I:C contain IFN-beta but not IFN-alpha. Taken together these data indicate that the antiviral effects of Poly I:C in inflammatory macrophages are mediated solely by IFN-beta, which acts in an autocrine manner to induce resistance to HSV-1.

Animals↗
PubMed2000

Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages.

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised low-density lipoprotein (LDL) via scavenger receptors. Here we investigate whether some of the anti-atherosclerotic effects ascribed to dietary fish oil are mediated through effects on macrophage intercellular adhesion molecule 1 (ICAM-1) and scavenger receptor expression. Mice were fed on a low fat diet (containing 25 g/kg corn oil) or on high fat diets containing 200 g/kg coconut oil, safflower oil or fish oil. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by gas chromatography. Macrophage scavenger receptor A (MSR-A) type I+type II and ICAM-1 expression were measured by flow cytometry and the levels of mRNA coding for MSR-A type I, MSR-A type II and ICAM-1 were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant changes in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. Macrophages from the fish oil fed mice had the lowest expression of ICAM-1 and MSR-A at the level of both mRNA and cell surface expression. The reduced expression of ICAM-1 and MSR-A on macrophages from mice fed on a fish oil-rich diet supports our hypothesis that part of the protective effect of fish oil against atherosclerosis might be due to an altered macrophage phenotype and function ameliorating macrophage-induced plaque formation.

Analysis of Variance↗
PubMed2001

An olive oil-rich diet reduces scavenger receptor mRNA in murine macrophages.

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised LDL via scavenger receptors. Here we investigated whether some of the anti-atherosclerotic effects ascribed to an olive oil rich-diet are mediated through effects on macrophage scavenger receptors (MSR). Male C57 Bl6 mice aged 6 weeks were fed for 12 weeks on a low-fat diet (containing 25 g corn oil/kg) or on high-fat diets containing 200 g coconut oil, olive oil or safflower oil/kg. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by GC and the levels of mRNA coding for three MSR (MSRA type I, MSRA type II and CD36) were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant differences in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. These differences were accompanied by a lower level of mRNA for MSRA type I, MSRA type II and CD36 in macrophages from mice fed an olive-oil-enriched diet compared with the mice fed on the low-fat diet. These data suggest that part of the protective effect of olive oil against atherosclerosis might be via reducing macrophage uptake of oxidised LDL. Whether this effect is due to the downregulation of gene transcription directly by unsaturated fatty acids or is the result of the effect of monounsaturated fatty acids or other components of olive oil on LDL composition and oxidation remains to be ascertained.

Analysis of Variance↗
PubMed2006

VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.

T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.

Amino Acid Sequence↗
PubMed1988

Cytoplasmic pH regulation in macrophages by an ATP-dependent and N,N'-dicyclohexylcarbodiimide-sensitive mechanism. Possible involvement of a plasma membrane proton pump.

Cytoplasmic pH (pHi) regulation was studied in thioglycolate-elicited murine macrophages using fluorescent probes. Acid-loaded macrophages regained normal pHi by extrusion of H+ equivalents across the plasma membrane. A fraction of this recovery was due to Na+/H+ exchange, as evidenced by its partial Na+ dependence and amiloride sensitivity. The residual, Na+-independent pHi recovery (approximately 50% of the total) persisted in the nominal absence of HCO3- and was insensitive to disulfonic stilbenes, ruling out mediation by anion exchange. In contrast, intracellular alkalinization and H+ extrusion from the cells were inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide or by prior depletion of intracellular ATP. These observations are consistent with the existence of a H+-pumping ATPase in the plasma membrane of macrophages. The mechanism of activation of the ATP-dependent H+ extrusion process was also investigated. In other systems, Ca2+ mobilization has been suggested to signal an exocytic insertion of H+ pumps into the plasma membrane. Acid loading of macrophages was accompanied by an elevation of the cytosolic Ca2+ concentration ([Ca2+]i), measured using indo-1. These results are consistent with a role for Ca2+ mobilization in the activation of H+ extrusion.

Adenosine Triphosphate↗