W. Dias da Silva

The activation of the complement system by Paracoccidioides brasiliensis in vitro: its opsonic effect and possible significance for an in vivo model of infection.

Abstract The yeast phase of Paracoccidioides brasiliensis is poorly phagocytosed by “stimulated” mouse peritoneal macrophages in culture. However, either heterologous or homologous antibodies greatly enhanced the phagocytosis of P. brasiliensis by macrophages. The same effect was observed when the fungus was pretreated with fresh normal sera. The following treatments abolished the opsonic activity of fresh normal serum: heating at 56°C for 30 min, addition of 10 mM EDTA. CoF treatment, and properdin or factor-B depletion. Conversely, EGTA had no effect. These data indicate that the alternative pathway of complement activation is involved in the expression of this phenomenon. When yeast cells of P. brasiliensis were incubated with fresh normal serum, the third component of complement changed its electrophoretic mobility of β1C to β1A. In an attempt to correlate these data with the in vivo conditions, fungi were inoculated into the subcutaneous tissues of normal mice. The resultant lesions reached their maximal size 48 hr after inoculation. Histological studies of these lesions showed a massive influx of polymorphonucleocytes. The histological picture was not modified conclusively when the mice were pretreated with CoF or when the fungi were inoculated in C5-deficient mice. The hypothesis that the fungus might secrete a chemotactic factor was tested in an in vitro system. It was verified that the activation of the complement system by P. brasiliensis elicited the formation of chemotactic substances for mouse polymorphonucleocytes. However, when the supernatant of cultures of the fungus in medium 199 was used as a chemotactic source, no effect could be detected. Thus, neither complement activation nor a fungus-secreted factor seems to explain the initial events of the lesion formation induced by P. brasiliensis.

Purification of F(ab′)2 anti-snake venom by caprylic acid: A fast method for obtaining IgG fragments with high neutralization activity, purity and yield

Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab)2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab)2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab fragments were obtained by reduction and alkylation of F(ab)2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab)2B], 4800 [F(ab)2M] and 3200 (FabB). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab)2M was free of IgG and of other plasma proteins, whereas that containing F(ab)2B was not. One milligram of either F(ab)2B, F(ab)2M or FabB was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.

Cell-mediated cytotoxicity to Trypanosoma cruzi: I. Antibody-dependent cell mediated cytotoxicity to trypomastigote bloodstream forms

Abstract The antibody-dependent, cell-mediated cytotoxicity (ADCC) to trypomastigote blood-stream forms of Trypanosoma cruzi was examined, utilizing trypanosomes isolated from mice infected with “Y” and “CL” stocks, normal mouse spleen (NMSC), or peritoneal (NMPC) cells and homologous immune serum (IMS). The incubations were performed at 35°C for 18 hr and the cytotoxicity was expressed as the percentage reduction in the number of motile trypanosomes. At an effector-target ratio of 40:1, NMSC induced a significant reduction in the number of motile trypanosomes of both “Y” and “CL” stocks. With trypanosomes obtained from nonirradiated mice this effect was frequently observed even in absence of IMS in the incubation mixture. In contrast, with trypanosomes obtained from irradiated mice, the presence of IMS was a prerequisite for the reaction. The loss in the motility of trypanosomes correlates well with their capacity to infect C3H/He mice, a strain that is highly susceptible to T. cruzi infection. ADCC to trypanosomes was also detected when NMPC were used, and the cytotoxic cells were found only in the nonadherent cell population. A striking difference was observed when immune mouse spleen cells (IMSC) were substituted for normal spleen cells: IMSC were only able to mediate cytotoxicity against trypanosomes raised in irradiated mice, an effect blocked by the simultaneous presence of IMS in the incubation mixture. It appears, therefore, that in infected mice the cells endowed with the capacity to mediate ADCC are blocked and that the immune cytotoxic cells are ineffective when the parasites are covered with specific antibodies.

Phospholipase A2 injection in mice induces immunity against the lethal effects of Crotalus durissus terrificus venom

Phospholipase A2, purified from crotoxin obtained from C. d. terrificus venom, alone or incorporated in Freunds complete adjuvant (FCA) or in Al(OH)3 was used as an antigen to immunize mice against the lethal effects of C. d. terrificus venom. The animals were intracutaneously (i.c.) or subcutaneously (s.c.) injected with 60 micrograms of phospholipase A2, divided into three equal doses and injected every 7 days. Samples of blood were collected just before each injection and the sera used to determine the antibodies against whole venom by the ELISA method. The animals were s.c. challenged with 8 LD50 or with 16 LD50 28 or 95 days after immunization. The animals that received two s.c. doses of antigen followed by a third i.c. dose were partially resistant to 8 LD50 (58% protection). This resistance increased when the first two injections consisted of phospholipase A2, the third of whole venom, all i.c., all in Al(OH)3 (67% of protection). The maximal protection (90%) was attained when the animals were i.c. injected with phospholipase A2 in Al(OH)3 in all three immunizing doses. Antibodies against whole venom were detected 15 days after immunization, reaching a plateau on the twenty-eighth day and remaining unchanged at least until the ninety-fifth day after immunization.

Study of the mechanism of inhibition produced by hexoses on histamine release activity of dextran

Die Hemmung der Histaminfreisetzung durch Hexosen wurde untersucht. Es gelang, nachzuweisen bzw. graphisch darzustellen, dass ein Zusammenhang zwischen dem Prozentgehalt der Histaminfreisetzung und der Dextrankonzentration besteht, wenn nachLineweaver undBurk analysiert wurde.

Trypanosoma cruzi: Antibody-dependent killing of bloodstream trypomastigotes by mouse bone marrow-derived mast cells and by mastocytoma cells

Two different populations of mast cells, that is, mastocytoma cells (P815) that were maintained either in vitro or in vivo, and mast cells obtained by differentiation of bone marrow precursor cells (MMC) in conditioned medium, were used as effector cells in antibody-dependent cytotoxic reactions (ADCC) against bloodstream trypomastigotes (BT) of Trypanosoma cruzi. The assay consisted of incubating effector cells with parasites that had been previously sensitized with immune mouse sera, immune IgG isotypes, or with medium. After the incubation period, the number of live BT was assessed. It was found that (a) cytotoxicity is antibody dependent; (b) the main isotypes involved are IgG1, IgG2a, and IgG2b; (c) both types of mast cells (mastocytoma and MMC cells) are equally efficient in killing BT; (d) mastocytoma cells degranulated by pretreatment with compound 48/80 are still able to effect ADCC; (e) on optical microscope examination, large numbers of parasites were often seen attached to the cells, but only when anti-T. cruzi antibodies were present; and (f) on electron microscope examination, no integral or ruptured parasites were seen inside the cells. We conclude that both T dependent and T independent mast cells are capable of mediating ADCC by a mechanism that is probably not dependent on granule extrusion.