Differential biodistribution of native and 2 kGy 60Co irradiated crotoxin in tissues of CBA/J mice.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Irradiation of Crotalus durissus terrificus crotoxin with 60Co gamma-rays induces its uptake by macrophages through scavenger receptors.

PURPOSE: To investigate the action of 2 kGy 60Co gamma-rays on crotoxin and its favoured uptake through scavenger receptor (ScvR) mouse peritoneal macrophages. MATERIALS AND METHODS: Native or irradiated crotoxin (iCTX) (50 microg/ml) dosed with 2 kGy 60Co gamma-rays (dose-rate 540 Gy/h) were offered to mouse peritoneal macrophages; their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. Receptors recognizing irradiated crotoxin were evaluated with specific ScvR blockers (Probucol and dextran sulphate) or with non-specific blocking using foetal calf serum (FCS). RESULTS: Immunohistochemical assays revealed more deeply staining intracytoplasmic vacuoles in macrophages incubated with iCTX. Using in situ ELISA with ScvR specific blockers, it was shown that the increased uptake of iCTX was blocked by Probucol or dextran sulphate, but not by FCS. On the other hand, the uptake of native crotoxin was decreased by FCS, but not affected by ScvR blockers. The morphology and viability of macrophages were preserved during the experiments. CONCLUSIONS: It is concluded that 60Co gamma-rays probably induced oxidative changes in crotoxin, driving this toxin towards ScvR mouse peritoneal macrophages. This suggests a different in vivo route of iCTX away from toxic neural sites by a preferential and rapid internalization and processing by macrophages, leading to the induction of a better immune response.

Immunogenic properties of the M. leprae recombinant 18-kDa antigen purified from Saccharomyces cerevisiae; enhancement of delayed-type hypersensitivity after gamma-irradiation.

In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described [Biotech. Lett. 16 (1994) 1241-1246]. The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100). The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice. Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained. Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response. The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M. leprae infection.

Immunochemical detection of purified crotoxin from Crotalus durissus terrificus venom in the motor end plate of striated muscle in CBA/J mice.

Electrophysiological studies of crotoxin, a potent neurotoxic fraction from Crotalus durissus terrificus venom, have shown a pre- and post-synaptic action. In order to determine the specific site of this activity, we performed an immunohistochemical analysis on striated muscle from CBA/J mice, injected with crotoxin (5 LD50), iv, using a single-step immunoperoxidase assay with peroxidase-conjugated antibodies to whole venom. Control muscle and muscle obtained from treated animals 15 min after injection showed no staining. However, 30 min after injection, the neuromuscular motor end plate of mice who received crotoxin was clearly stained, including thin terminations, without any morphological alteration. Sixty min after administration, the motor end plate was no longer intact, but only roughly formed stained areas without clearly identifiable structures were present. These data show specific crotoxin binding to neural end plates in striated muscle, with a subsequent toxin-induced degeneration of this structure.

Immunochemical detection of purified crotoxin from crotalus durissus terrificus venom in the motor end plate of striaqted muscle in CBA/J mice

Electrophysiological studies of crotoxin, a potent neurotoxic fraction from Crotalus durissus terrificus venom, have shown a pre- and post-synaptic action. In order to determine the specific site of this activity, we performed an immunohistochemicaql analysis on striated muscle from CBA/J mice, injected with crotoxin (5LD50), iv, using a single-step immunoperoxidase assay with peroxidase-conjugated antibodies to whole venom. Control muscle and muscle obtained from treated animals 15 min after injection showed no staining. However, 30 min after injection, the neuromuscular motor end plate of mice who received crotoxin was clearly stained, including thin terminations, without any morphological alteration. Sixty min after administration, the motor end plate was no longer intact, but only roughly formed stained areas without clearly identifiable structures were present. These data show specific crotoxin binding to neural end plates in striated muscle, with a subsequent toxin-induced degeneration of this structure