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Effect of thioglycollate on macrophage lysosomal enzymes.

Mice peritoneal macrophages obtained after i.p. injection of a medium containing thioglycollate (Institut Pasteur, Paris) have increased levels of beta glucuronidase and acid phosphatase, when compared with macrophages obtained without previous thioglycollate injection. The maximum increase is observed between 7 and 10 days after injection and reaches up to 3 to 4 times the normal activity. When using separately the various components of the medium, the same phenomenon may be observed but never to the same degree. The injection of thioglycollate also produces: a) an increase in total number of peritoneal cells, and b) an increase in the percentage of macrophages among these cells. Kinetic studies suggest that thioglycollate injection first induces an increase in the number of peritoneal macrophages and subsequently macrophage modifications resulting in lysosomal enzyme increase.

Acid Phosphatase

Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages.

We have examined the Fc- and complement-receptor function of resident and thioglycollate-elicited mouse peritoneal macrophages plated on surfaces coated with rabbit antibody-antigen complexes and with complement. We derive four major conclusions from these studies. (a) The trypsin-resistant Fc receptors of resident and thioglycollate-elicited macrophages are completely modulated when these cells are plated on rabbit antibody-antigen complexes. Residual Fc receptor activity is a result of the incomplete modulation of trypsin-sensitive IgG2a receptors. (b) The complement receptors of thioglycollate-elicited macrophages, but not of resident macrophages, are modulated when these cells are plated on complement-coated surfaces. The capacity of the two cell types to modulate their complement receptors is correlated with their ability to ingest complement-coated erythrocytes. (c) The complement and Fc receptors of both types of macrophages move independently of one another. (d) Complement masks the Fc segments of IgG in immune complexes thereby rendering them ineffective as ligands for macrophage Fc receptors.

Animals

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated macrophages.

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated mouse peritoneal macrophages was studied. Thioglycolate-activated macrophages exhibited a lower percent phagocytosis than did normal macrophages. Differences in phagocytosis could not be attributed to differences in macrophage viability, minor variations in the concentration of adherent macrophages, or a general depression in activated macrophage phagocytosis. Thioglycolate-activated macrophages required heat-labile opsonins for optimal phagocytosis of non-encapsulated cryptococci, whereas nonactivated macrophages did not require heat-labile opsonins for phagocytosis of the yeast. Both types of macrophages exhibited similar sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The results show that depletion of heat-labile opsonins from serum or inactivation of yeast-bound, heat-labile opsonins by polysaccharide cannot account for the phagocytosis-inhibiting properties of cryptococcal polysaccharide.

Animals

Conversion of agroclavine to setoclavine and isosetoclavine in cell-free extracts from Claviceps sp. SD 58 and in a thioglycolate-iron (II) system.

Agroclavine was converted to setoclavine and isosetoclavine in crude extracts from Claviceps sp. SD 58. The ratio of setoclavine to isosetoclavine was 0.95. The conversion with boiled crude extract was 68% of the conversion with unboiled extract. In a thioglycolate-iron (II) system at 40 degrees for 5 hr, 45.5% of agroclavine was converted to setoclavine and isosetoclavine. At 70 degrees for 4 hr in the thioglycolate-iron (II) system, 4% of 4-dimethylallyltryptophan was converted to clavicipitic acid.

Alkaloids

Aerobically incubated thioglycolate broth disk method for antibiotic susceptibility testing of anaerobes.

The anaerobic broth disk (AnBD) method of Wilkins and Thiel and a new modification, designated the thioglycolate broth disk method, were compared with an agar dilution technique. The thioglycolate broth disk method was incubated aerobically (AeTBD) or anaerobically (AnTBD). One hundred anaerobic bacteria representing 15 species were tested with clindamycin, chloramphenicol, erythromycin, penicillin, and tetracycline. Agreement of results by the two methods with minimal inhibitory concentration determinations were: AnBD, 95.2%; AnTBD, 91.5%; AeTBD, 94.5%. With clindamycin, chloramphenicol, and penicillin, the agreement of the AeTBD and agar dilution results was 100%, 100%, and 95%, respectively. Using the AeTBD method, only 1.1% of all tests gave false susceptible readings, whereas 4.4% gave false resistant readings. All susceptibility testing errors occurred with tetracycline, erythromycin, and, to a lesser extent, penicillin. For each method, the changes in designation of bacteria as being susceptible or resistant to an antibiotic between trials primarily involved strains with minimal inhibitory concentrations which were +/- one dilution of the respective breakpoint value. The same situation was true for most bacteria that yielded false resistant readings within each trial. False resistant readings with tetracycline were determined to be unrelated to excess cation content of test media. These results reaffirm the reliability of the AnBD method and indicate that the AeTBD modification is equally reliable. The greater convenience and lower cost of the AeTBD method should make possible more widespread performance of susceptibility testing for anaerobic bacteria in hospital laboratories.

Anaerobiosis

[Effect of thioglycolic acid on Allium sativum L. root meristems].

Thioglycolic acid is, in the Allium test, somewhat less cytotoxic than mercaptoethanol but the characteristics of cytotoxicity are the same in both cases : the mitoclasic effect cannot be separated from a strong mitodepressive effect and is quickly followed by a lethal effect.

Mercaptoethanol

Release of cartilage proteoglycan degrading enzyme activity by thioglycollate stimulated mouse peritoneal macrophages in culture.

Media from cultured mouse peritoneal macrophages were tested for cartilage proteoglycan degrading activity using S35-labelled rabbit ear cartilage. Media samples collected at 2-day intervals contained increasing amounts of activity between days two and six. This activity was activated by trypsin and antagonized by chelating agents. The macrophage products induced release of the proteoglycan component of cartilage as determined by biochemical and histological methods without affecting the collagen component. Media from cells incubated with hydrocortisone were devoid of proteoglycan degrading activity.

Animals

Cell surface changes in mouse peritoneal macrophages after induction with proteose peptone or thioglycollate.

Some five to six per cent of mouse PEC spontaneously form rosettes with SRBC. This weak intercellular interaction is most likely mediated by a "receptor" for SRBC located on the surface of peritoneal macrophages. Manifestation of the receptor is influenced by the genetic background and the H-2 haplotype of PEC donors; a high proportion of RFC is associated with the H-2s haplotype. PEC derived from intact mice markedly differ in morphological and functional characteristics from those of PP- or TG-pretreated donors. Formation of rosettes by induced macrophages depends on the time interval between stimulation and PEC harvesting and on the type of the inducing agent; it is also radiosensitive and more responsive to the action of colchicine. TG-induced macrophages have a significantly reduced capacity to bind syngeneic lymphoid cells. The difference in adhesivity between intact and stimulated PEC can be abolished by glutaraldehyde prefixation. In vivo induction results in modified morphological and functional properties of macrophages, including transformation of their cell surface.

Animals

The effect of reducing and other agents on the motility of Treponema pallidum in an acellular culture medium.

The maintenance of Treponema pallidum motility was investigated in an acellular medium based on T. pallidum immobilization test medium. The acellular medium contained cysteine, glutathione, thioglycollate and dithiothreitol as reducing agents and had a redox potential of -275 +/- 25 mV at pH 7.3. In an atomosphere containing 3% O2, motile treponemes survived four times longer when calf serum and bovine serum albumin were added to the medium. The selective omission of glutathione and, particularly, thioglycollate prolonged the survival of motile treponemes almost fivefold. In addition, stored medium, in which thioglycollate had become inactive, sustained motile treponemes for longer than did freshly prepared medium. Thus, thioglycollate is toxic for the organisms. It may be omitted from the medium because low redox potentials can be achieved without it.

Culture Media

Use of a macrophage cytotoxicity system to show macrophage activation by Listeria monocytogenes cell wall fraction.

Experiments were conducted to determine whether a partially purified listeria cell wall fraction could stimulate macrophages to high levels of activation. To detect activation of macrophages, a macrophage-mediated cytotoxicity system were established. The data demonstrate that listeria cell wall components are capable of activating thioglycollate-induced adherent peritoneal exudate cells to be cytotoxic for 51Cr-labelled target tumour cells, and that the listeria fraction is as effective as bacterial lipopolysaccharide in inducing cytotoxicity. The listeria fraction can also induce peritoneal exudate cells from congenitally thymusless nude mice to become cytotoxic, suggesting that mature T cells are not required. Furthermore, thioglycollate-induced adherent peritoneal exudate cells from mice hyperimmunized to live Listeria organisms are already stimulated to be cytotoxic for tumour cells, and do not need to be activated in vitro. Additional data are presented which characterize the system. These data demonstrate that a critical concentration of adherent peritoneal cells is required for in vitro activation. Moreover, only peritoneal cells induced with aged batches of thioglycollate, and not uniduced peritoneal cells or those induced with fresh thioglycollate or with protease peptone can be activated in vitro to kill tumour cells. Evidence is presented which suggests that the cytotoxic cell is a macrophage.

Animals

Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages.

Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.

Adenosine Triphosphate

Effect of in vivo stimulation of mice on the secretion of factor B of the alternate complement pathway by peritoneal macrophages.

After in vivo treatment of mice with thioglycollate medium, the amount of native factor B which could be detected in vitro in culture supernatants of peritoneal macrophages was much lower than that found in supernatants of macrophages taken from untreated mice. However, when the macrophages from thioglycollate medium-treated mice were cultured on a plastic surface covered with glutardialdehyde-linked bovine serum albumin, the culture supernatants contained larger quantities of native factor B than culture supernatants of macrophages from untreated mice under the same conditions. Thus, the effect of in vivo thioglycollate medium treatment on the in vitro secretion of factor B by peritoneal macrophages could be modulated by the culture conditions. Factor B in culture supernatants of macrophages obtained from both untreated and thioglycollate medium-treated mice was stable. It remained functionally active, and therfore uncleaved over a long incubation period at 37 degrees C. In addition, factor D activity was never detected in any culture supernatant.

Animals