Light emitting diode (LED) therapy reduces local pathological changes induced by Bothrops asper snake venom.

The therapeutic effect of the Light Emitting Diode (LED) treatment in two wavelengths (635 or 945 nm) was evaluated in the local pathological alterations induced by Bothrops asper snake venom. Mice received irradiation of infrared LED (120 mW, 945 nm) or red LED (110 mW, 635 nm) applied immediately, 1 and 2 h after venom injection. LED treatment reduced edema formation in the plantar region and gastrocnemius muscle and significantly reduced neutrophil migration and hyperalgesia after the venom injection. Also, both infrared LED and red LED treatment significantly reduced myonecrosis, as revealed by muscle CK and plasma CK levels. Histological analysis corroborated the reduction in the extent of venom-induced myonecrosis. In conclusion, our data demonstrates that PBM with LED light in both red and infrared wavelengths, when applied after envenomation in mice, reduces the extent of myotoxicity, edema, inflammatory infiltrate and hyperalgesia, suggesting that photobiomodulation is a potential therapeutic approach that should be further investigated for the treatment of local effects of Bothrops snakebite.

New nanostructured silica adjuvant (SBA-15) employed to produce antivenom in young sheep using Crotalus durissus terrificus and Apis mellifera venoms detoxified by cobalt-60.

Equine antivenom is considered the only treatment for animal-generated envenomations, but it is costly. The study aimed to produce Apis mellifera (Africanized honeybee) and Crotalus durissus terrificus (C.d.t.) antivenoms using nanostructured silica (SBA-15) as adjuvant and cobalt-60 ((60)Co)-detoxified venoms utilizing young sheep. Natural and (60)Co-irradiated venoms were employed in four different hyperimmunization protocols. Thus, 8 groups of 60- to 90-d-old sheep were hyperimmunized, enzyme-linked immunosorbent assay (ELISA) serum titers collected every 14 d were assessed clinically daily, and individual weight were measured, until d 84. Incomplete Freund's (IFA) and nanostructured silica (SBA15) adjuvants were compared. The lethal dose (LD(50)) for both venoms was determined following intraperitoneal (ip) administration to mice. High-performance liquid chromatography on reversed phase (HPLC-RP) was used also to measure the (60)Co irradiation effects on Apis venom. At the end of the study, sheep were killed in a slaughterhouse. Kidneys were histologically analyzed. LD(50) was 5.97 mg/kg Apis and 0.07 mg/kg C.d.t. for native compared to 13.44 mg/kg Apis and 0.35 mg/kg C.d.t. for irradiated venoms. HPLC revealed significant differences in chromatographic profiles between native and irradiated Apis venoms. Native venom plus IFA compared with SBA-15 showed significantly higher antibody titers for both venoms. Apis-irradiated venom plus IFA or SBA-15 displayed similar antibody titers but were significantly lower when compared with native venom plus IFA. Weight gain did not differ significantly among all groups. (60)Co irradiation decreased toxicity and maintained venom immunogenic capacity, while IFA produced higher antibody titers. SBA-15 was able to act as an adjuvant without producing adverse effects. Hyperimmunization did not affect sheep weight gain, which would considerably reduce the cost of antiserum production, as these sheep were still approved for human consumption even after being subjected to hyperimmunization.

Study of irradiated bothropstoxin-1 with60Co gamma rays: immune system behavior

Ionizing radiation has been successfully employed to modify the immunological properties of biomolecules. Very promising results were obtained when crude animal venoms, as well as isolated toxins, were treated with 60Co gamma rays, yielding toxoids with good immunogenicity. The achievement of modified antigens with lower toxicity and preserved or improved immunogenicity can be very useful. Ionizing radiation has already been proven to be a powerful tool to attenuate snake venom toxicity without affecting, and even increasing, their immunogenic properties. However, little is known about the modifications that irradiated molecules undergo and even less about the immunological response that such antigens elicit. In the present work, we investigated the immunological behavior of bothropstoxin-1, a K49 phospholipase, before and after irradiation. Structural modifications of the toxin were analyzed by SDS-PAGE. Isogenic mice were immunized with either the native or the irradiated toxin. The circulating antibodies were isotyped and titrated by ELISA. According to our data, irradiation promoted structural modifications in the toxin characterized by higher molecular weight forms of proteins (aggregates and oligomers). The results also indicated that irradiated toxins were immunogenic and antibodies elicited by them were able to recognize the native toxin in ELISA. These findings suggest that irradiation of toxic proteins can promote significant modifications in their structures; however they still retain many of the original antigenic and immunological properties of native proteins. Also, our data indicate that irradiated proteins induce higher titers of IgG2a and IgG2b, suggesting that Th1 cells are predominantly involved in the immune response.

60Co gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction.

The ability of gamma radiation from 60Co (2000 Gy) to attenuate the toxic effects of Bothrops jararacussu venom was investigated on mouse neuromuscular preparations in vitro. A comparative study between the effects of native and irradiated venoms was performed on both phrenic--diaphragm (PD) and extensor digitorum longus (EDL) preparations by means of myographic, biochemical and morphological techniques. Native venom (10 and 20 micro g/ml) induced a concentration--dependent paralysis of both directly and indirectly evoked contractions on PD preparations. At 20 micro g /ml, it also caused a pronounced myotoxic effect on the EDL muscle preparation that was characterized by an increase of creatine kinase release and by several morphological changes of this preparation. By contrast, irradiated venom, even at concentrations as high as 40 micro g/ml, induced neither paralyzing nor myotoxic effects. It was concluded that 60Co gamma radiation is able to abolish both the paralyzing and the myotoxic effects of B. jararacussu venom on the mouse neuromuscular junction. These findings support the hypothesis that gamma radiation could be an important tool to improve antisera production by reducing toxicity while preserving immunogenicity.

Effects of (60)Co gamma radiation on crotamine.

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Influence of ionizing radiation on crotoxin: biochemical and immunological aspects.

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Quantitative assessment of radiation damage in a thin protein crystal.

Radiation damage is a limiting factor for high resolution structural determination of protein crystals. We have used the median and quartile values of Ne exposures and the structural disordering factor (delta B) to describe the crystalline disordering due to radiation damage in thin crystals of crotoxin complex embedded in glucose at room and low temperatures and in ice embedded crystals. By approximating the radiation damage to follow first order kinetics, we applied a correction to the diffraction intensities from patterns with high accumulated exposures. We used the structural similarity factor (R) to show that, for some data, the effectiveness of the correction can be as good as those in protein X-ray crystallography.