Irradiated Venom Research Articles

Scavenger receptors mediate increased uptake of irradiated T.gondii extracts by J774 macrophages.

ABSTRACT PurposeProtein extracts developed increased immunogenicity without the aid of adjuvants after gamma irradiation. Gamma irradiation of snake venom increased antivenin production by detoxification and enhanced immunity, probably due preferential uptake of irradiated venoms by macrophage scavenger receptors. We studied this uptake of irradiated soluble Toxoplasma gondii extract (STag) by the J774 macrophage cell line similar to antigen presenting cells. Material and Methods: We labeled STag by biosynthesis in living tachyzoites with radioactive amino acids before purification and irradiation or by adding labels as biotin or fluorescein in stored STag, for quantitative studies or subcellular distribution visualization. Results: There was enhanced binding and uptake of irradiated STag into the cells compared to non-irradiated STag. Using fluorescein labelled antigens and morphological assays, we confirmed that cells avidly ingested both native and irradiated proteins but native STag were digested after ingestion while irradiated proteins remained in the cell, suggesting diverse intracytoplasmic pathways. Native or irradiated STag present the same in vitro sensitivity to three types of peptidases. Inhibitors of scavenger receptors (SRs) such as Dextran sulfate (SR-A1 blocker) or Probucol (SR-B blocker) affect the specific uptake of irradiated antigens, suggesting its association with enhanced immunity. Conclusions: Our data suggests that cell SRs recognize irradiated proteins, mainly SRs for oxidized proteins, leading to antigen uptake by an intracytoplasmic pathway with fewer peptidases that prolongs presentation to nascent major histocompatibility complex I or II and enhances immunity by better antigen presentation.

Improvement of Naja haje snake antivenom production using gamma radiation and a biotechnological technique

This study aimed to improve antivenom production using gamma radiation and calcium phosphate nanoparticles (CPN) as adjuvant. Toxicological and immunological properties of the Naja haje venom before and after exposure to radiation were estimated. The immune response of immunized rabbits was evaluated. Toxicity of irradiated venom was reduced six times as compared to native venom. Moreover, no change in antigenic reactivity between native and irradiated venoms. Serum titer produced with irradiated venom loaded on CPN showed highest titer and neutralization capacity as compared to other sera. Thus, irradiation and nanoparticles adjuvant could be a new approach for snake antivenom production.

THE EFFECT OF DIFFERENT DOSES OF GAMMA IRRADIATION ON THE ECHIS COLORATUS VENOM TOXICITY IN MICE

Snake venoms are complex mixtures of proteins and non proteins components. This study evaluated the toxicity effect of LD50 of native Echis coloratus venom and the LD50 of dry venom irradiated with different doses of gamma radiation (2, 5 & 10 KGy) on Swiss albino mice. Measurement of some biochemical parameters and histological alterations in liver and kidney was investigated. The biochemical parameters showed highly significant increase (P< 0.001) in levels of ALT, AST, urea and CK in all groups compared to control group. Levels of serum creatinine showed highly significant increase (P< 0.001) in groups treated with LD50 of crude venom and 5 times LD50 of irradiated venom at 2 and 10 KGy, But, showed significant (P< 0.05) with groups treated with 5 times LD50 of irradiated venom at 5KGy & LD50 of irradiated venomat 10 KGy showed non-significant in group treated with LD50 of 5KGy & 2KGy compared to control group. Histological examination showed alterations in liver, kidney in mice injected with crude venom compared to control group. Also, the group of mice which injected with different doses of irradiated venom showed no histopathological alteration in liver and kidney tissues, except the mice group injected with 5 times LD50 of irradiated venom at 2 KGy and the group which injected with 5 times LD50 of irradiated venom at 10 KGy which showed alterations in liver and kidney tissues.

Vaccine approach against scorpion envenoming: Comparative study between the efficiency of MF59 adjuvant and alginate nanoparticles associated to irradiated venom

Vaccine approach against scorpion envenoming: Comparative study between the efficiency of MF59 adjuvant and alginate nanoparticles associated to irradiated venom

The impact of low doses of gamma radiation on Echis coloratus venom and its fractions

Echis coloratus viper (painted saw-scaled viper) is considered as one of the most venomous snakes in Egypt, its bite may cause severe health complications as coagulopathy, nephropathy, cardiopathy, necrosis and death. Ionizing radiation is one of the methods used to reduce the venom toxicity. The present study aims to study the effect of low doses of gamma radiation on Echis coloratus venom and its fractions. Two proteases (FNI, FNII and FRI, FRII) were partially purified on sephadex G-75 gel filtration chromatography of native and 3 kGy gamma irradiated venoms, respectively. The specific activities of proteases of FRI and FRII fractions were decreased from 20 ± 0.46 to 8 ± 0.64 U/mg and from 10 ± 0.29 to 5 ± 0.23U/mg, respectively, similarly, the PLA2 specific activities of FRII and FRIII fractions were decreased from 10 ± 0.23 to 3.5 ± 0.29U/mg and from 12 ± 0.35 to 5 ± 0.52U/mg, respectively, compared with the corresponding fractions of native one. In addition, the edema activities induced by 3 kGy irradiated whole venom and its isolated fractions were reduced compared to native venom and its corresponding isolated fractions. Further, the coagulant activity of venom and the isolated fractions were significantly reduced after exposure of venom to 3 kGy gamma radiation showing anticoagulant activities except FNI and FRI fractions. However, similar antigenic determinants of 3 kGy irradiated venom and its isolated fractions were detected by double immunodiffusion using horse polyvalent antivenin. Therefore, these findings indicate that low doses of gamma irradiation may offer an effective method for preparing an effective antivenin using either the whole or purified components of venom with minimal toxic effects caused to the hyper-immunized animals.

New and safe formulation for scorpion immunotherapy: Comparative study between saponin and FCA adjuvants associated to attenuated venom

Envenoming by scorpion is a major health problem in Maghreb regions as well as in several regions of the world. Immunotherapy is the only effective treatment for scorpion stings. The immune sera are obtained from hyper-immunized animals with a formulation of venom associated to Freund’s Complete Adjuvant (FCA). This formulation seems to protect against several alterations in immunized animals leading to worsening of their health due to added toxicity of native venom and FCA adjuvant. This study aims to provide a more efficient and non-toxic alternative to this formulation. Two formulations of saponin or FCA associated to irradiated venom of Androctonus australis hector (Aah) were used to compare their safety and their efficiency to better enhance the antibody titers against toxic antigens.Both of these formulations were used in immunization schedule of three months. Blood samples were collected every week, cell count, myeloperoxydase (MPO) and eosinophil peroxidase (EPO) activities and specific antibody titers were evaluated. Four months after the last immunization, rabbits were challenged with increased doses of native Aah venom.Results showed that immunization with saponin formulation induced lower inflammatory cell activation as well as reduced MPO and EPO activities compared to that using FCA. The formulation of irradiated venom with saponin seems also to be more efficient in the activation of lymphocytes resulting in higher titers of specific IgG. The immunoprotective effect evaluation showed that the formulation using saponin seems to protected animals until 3 LD50 of native venom compared to that using FCA which protected only until 2 LD50. These results indicate that saponin formulation with irradiated antigen could be more efficient and safe immunizing preparation for the production of sera against scorpion envenomation.

Assessment of the neutralizing potency of antisera raised against native and γ-irradiated Naja nigricollis (black-necked spitting cobra) venom in rabbits, concerning its cardiotoxic effect.

The present study was designed to prepare a specific safe antiserum for Naja nigricollis using γ-irradiated (1.5KGy and3KGy) venoms. Rabbits were used for active immunization using irradiated venoms (1.5 and 3 kGy) as a toxoid, mice were used for determination of LD50 post immunization and the rats were used for neutralization of the cardiotoxic effect of venom. Results of the immunodiffusion test indicated that the sera of rabbits raised against non-irradiated, 1.5- and 3-kGy γ-irradiated venom, had the same results of precipitin bands. A significant inhibition of phospholipase A2 activities was obtained when neutralized with native, γ-irradiated (1.5KGy and3KGy) venoms. On the other hand, preincubation of the venom ½ LD50 (0.154 mg/kg i.p.) with each antiserum (non-irradiated or irradiated venom) at 37°C for 1 h in a ratio (1:4) produced a significant reduction in the values of creatine kinase and creatine kinase isoenzyme-MB. However, significant elevation in aspartate aminotransferase level and no change in lactate dehydrogenase level were observed. So the results of this study indicated that the irradiated venom treatment reduces the cardiotoxic effect of venom in immunized immunization animals for preparing vaccines.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

BackgroundSnakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.MethodologyC2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.ResultsIn non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.ConclusionLLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

Pathophysiological effects of Cerastes cerastes and Vipera lebetina venoms: Immunoneutralization using anti-native and anti-60Co irradiated venoms

Cerastes cerastes and Vipera lebetina are the most medically important vipers in Algeria. Their bite induces several pathological effects on victims of accidental envenomation. In this study we analyzed the pathogenesis induced after an experimental envenomation. Indeed, we determined, in vitro, venom enzymatic activities and we analyzed, in vivo, pathological effects induced on liver, heart, lung and skin. In addition we investigated the neutralizing potency of four experimental antivenoms elicited against native and irradiated venoms. Results revealed that V. lebetina and Cerastes cerastes venoms presented strong hemorrhagic, oedematic and necrotic activities. Histopathological study showed that both venoms induced deep damage in tissue structures leading to organ dysfunction. They also increased cellular peroxidases activities, indicating an inflammatory process that is known to amplify tissue damage. Western-blot analysis evidenced that anti-irradiated venoms recognized most components of native venoms. Antivenoms were effective in neutralizing all tested activities, with an increased protective effect obtained with anti-irradiated venoms. Anti-irradiated venoms reduced cellular peroxidases activities indicating a reduction of the inflammatory response. These results may improve our understanding of Algerian Viperidae bite pathogenesis and would encourage further studies planning to provide more proofs on the effectiveness of anti-irradiated venoms administration in the treatment of envenomation.

Enhanced immune sera and vaccine: Safe approach to treat scorpion envenoming

Irradiation of Androctonus australis hector venom using a dose of 2 kGy has successfully abolished toxicity without reducing its antigenic or immunogenic properties. Toxicity of irradiated antigen was abolished until 20 times of LD(50) of native venom. Analysis of physiopathological effects induced by native and irradiated venoms was assessed by the analysis of tissue damage, immunohistochemistry and metabolical analysis in the organs (heart, lungs and liver). Immunological response of Aah venom using native or irradiated venom showed high titers of IgG1 in the plasma of immunized animals with native venom suggesting that Th2 cells were predominantly involved in the immune response. In the other hand, irradiated venom induced high titers of IgG2, indicating a predominantly Th1 type response. A protective effect of immunized mice with irradiated venom was evaluated. Immunized mice were protected from the toxic effects of native venom doses at one, three and six months after immunization. Mice were protected against a challenge of 4 LD(50) doses of native venom, one month after immunization. This protective effect was improved and effective at 3 and 6 months, all immunized mice were protected respectively against 6 and 10 LD(50) of native venom. At the one-month time point, the protective effect of mice was associated with high levels of antibodies in the plasma of immunized mice. However, despite the persistence of higher protection levels, the antibody titers decreased in a time-dependent manner. These results suggest that additional factors other than circulating antibodies provided the long-term protective activity produced by immunization with irradiated venom.

Modulation of the Pharmacological and Biochemical Actions of Leiurus quinquestriatus (L.q) Scorpion Venom by Exposure to Gamma Radiation

Back ground This study was undertaken to evaluate the effect of gamma radiation (1.5 KGy & 3 KGy) on L.q scorpion venom. This was carried out by studying the toxicological, biochemical & immunological properties of the venom before and after exposure to gamma radiation. Material and methods Animals, venom, antivenin, gamma radiation, 125I. Results Data revealed that the toxicity of irradiated venom (1.5 KGy & 3 KGy) decreased as compared to that of the native one. LD50 of irradiated venom were 3.5 mg/kg & 7.5 mg/kg respectively while, that of the native venom was (0.39 mg/kg). Moreover, the distribution of 125I-labeled L.q venom was studied in male Swiss mice tissue using chloramine-T method by being injected intravenously. At various time intervals, urine and blood were collected and the animals were sacrificed. Brain, lungs, heart, liver, kidneys, spleen, intestine, bone and muscle were isolated in order to determine the radioactivity content. The highest contents of 125I-labeled L.q venom were found in the liver and kidney that were quickly excreted into the urinary tract. Trial to label irradiated (1.5 & 3 KGy) L.q venom was unsuccessful due to its decomposition. For that reason the utilization of the labeled irradiated L.q venom is of no meaning. In immunodiffusion technique, both irradiated and native venoms exhibited prominent precipitin bands indicating high concentration of specific antibodies against polyvalent antivenin with venom. Furthermore, the effect of half the LD50 of native or irradiated (1.5 KGy) L.q venom was studied on the activities of certain enzymes: ASAT, ALAT, LDH, CPK, CPK-MB, and the levels of total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and the oxidative stress biomarkers (plasma MDA & blood GSH). Biochemical measurements showed that scorpion venom envenomation caused significant (p < 0.05) elevation in serum ASAT, ALAT, LDH, CPK as well as CPK-MB activities, blood GSH level while, caused significant (p < 0.05) reduction in triglyceride, HDL-cholesterol. In contrast, the 1.5 KGy gamma irradiated L.q venom showed no alterations except in HDL-cholesterol that significantly decreased compared to that of the non-envenomated normal rats. Conclusion These results indicated that 1.5 KGy gamma irradiation of venom offer an effective method for reducing the toxic effect of venom.

Modulation of the Pharmacological and Biochemical Actions of Leiurus quinquestriatus (L.q) Scorpion Venom by Exposure to Gamma Radiation

Back ground This study was undertaken to evaluate the effect of gamma radiation (1.5 KGy & 3 KGy) on L.q scorpion venom. This was carried out by studying the toxicological, biochemical & immunological properties of the venom before and after exposure to gamma radiation. Material and methods Animals, venom, antivenin, gamma radiation, 125I. Results Data revealed that the toxicity of irradiated venom (1.5 KGy & 3 KGy) decreased as compared to that of the native one. LD50 of irradiated venom were 3.5 mg/kg & 7.5 mg/kg respectively while, that of the native venom was (0.39 mg/kg). Moreover, the distribution of 125I-labeled L.q venom was studied in male Swiss mice tissue using chloramine-T method by being injected intravenously. At various time intervals, urine and blood were collected and the animals were sacrificed. Brain, lungs, heart, liver, kidneys, spleen, intestine, bone and muscle were isolated in order to determine the radioactivity content. The highest contents of 125I-labeled L.q venom were found in the liver and kidney that were quickly excreted into the urinary tract. Trial to label irradiated (1.5 & 3 KGy) L.q venom was unsuccessful due to its decomposition. For that reason the utilization of the labeled irradiated L.q venom is of no meaning. In immunodiffusion technique, both irradiated and native venoms exhibited prominent precipitin bands indicating high concentration of specific antibodies against polyvalent antivenin with venom. Furthermore, the effect of half the LD50 of native or irradiated (1.5 KGy) L.q venom was studied on the activities of certain enzymes: ASAT, ALAT, LDH, CPK, CPK-MB, and the levels of total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and the oxidative stress biomarkers (plasma MDA & blood GSH). Biochemical measurements showed that scorpion venom envenomation caused significant (p < 0.05) elevation in serum ASAT, ALAT, LDH, CPK as well as CPK-MB activities, blood GSH level while, caused significant (p < 0.05) reduction in triglyceride, HDL-cholesterol. In contrast, the 1.5 KGy gamma irradiated L.q venom showed no alterations except in HDL-cholesterol that significantly decreased compared to that of the non-envenomated normal rats. Conclusion These results indicated that 1.5 KGy gamma irradiation of venom offer an effective method for reducing the toxic effect of venom.

Influence of Ionizing Radiation on Echis pyramidium Snake Venom: Biochemical and Immunological Aspects

The effect of a single LD50 dose of native Echis pyramidum venom (27.69μg/mouse) on the activities of certain serum enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), creatine kinase isoenzyme (CK-MB) were studied. Samples from the serum were collected 4hr following LD50 venom dose intraperitonealy injected in male Swiss albino mice. The activities of these enzymes showed significant elevation compared to the non-envenomated group. In contrast, an equivalent dose of 1.5 kGy γ irradiated Echis pyramidum venom (27.69g/mouse) did not cause any significant increase compared to non-envenomated group. The effect of a dose that is equivalent to 1⁄2 LD50 (13.8 μg/50 μl) of native Echis pyramidum venom on plasma creatine phosphokinase (CPK) induced a significant increase of creatine phosphokinase (CPK) level compared to normal control (P<0.01). In contrast, an equivalent dose of 1.5 kGy γ irradiated Echis pyramidum venom showed non significant difference in creatine phosphokinase activity when compared to the normal control. Light microscopic examinations of gastrocenemius muscles of mice injected with native Echis pyramidum venom (1⁄2 LD50; 13.8μg/50μl) showed fragmentation, disorganization, loss of myofibrils in some of the muscle fibers, hemorrhage in-between the muscle fibers and mononuclear cellular infiltration. While light microscopic examinations of gastrocenemius muscles of mice injected with 1.5 kGy γ irradiated Echis pyramidum venom (13.8μg/50μl; a dose identical to that used from native venom) showed that most muscle fibers were of normal appearance except for small area of fragmentation and disorganized myofibrils and oedema of the intercellular connective tissue. Double immunodiffusion test revealed a similar reactivity for native, 1 kGy, 1.5 kGy and 3 kGy γ irradiated Echis pyramidum venoms against a commercial polyvalent Egyptian antivenin. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners, indicating that there was no change in their antigenic reactivity. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3 kGy. Both antivenins raised against native or 1.5 kGy γ irradiated venoms recognized Echis pyramidum venom when submitted to protein blotting, but the anti 1.5 kGy γ irradiated venom show a higher intensity bands than the antivenin raised against native Echis pyramidum venom, in spite of having less neutralizing activity (native neutralize 50 LD50, 1.5 kGy γ irradiated neutralize 40 LD50), this indicates that antibodies were formed not only for toxic fraction but also for non toxic fraction. Irradiation of the whole Echis Pyramidium Venom with 1.5KGy reduced its lethality 12.5 times though keeping its immunogenicity. The 1.5KGy dose was shown to be the best radiation dose to promote detoxification without significantly affecting its immunogenicity. Thus results of this study confirm the conclusion that γ radiation is a suitable way to detoxify Echis Pyramidium Venom without affecting its immunogenicity provided that proper dose is used.

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 (60Co)-detoxified venoms utilizing young sheep. Natural and 60Co-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 (LD50) 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 60Co irradiation effects on Apis venom. At the end of the study, sheep were killed in a slaughterhouse. Kidneys were histologically analyzed. LD50 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. 60Co 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.

Resposta imune e capacidade de neutralização de anticorpos produzidos em ovinos jovens imunizados com veneno de Crotalus durissus terrificus nativo e irradiado com Cobalto 60

A técnica de Elisa foi utilizada para avaliar e comparar a resposta imune humoral de ovinos jovens para a produção de soro anticrotálico. Durante o processo de soroprodução, foi realizada a avaliação clínica dos animais. A capacidade de neutralização do soro produzido a partir de veneno de serpente Crotalus durissus terrificus, nativo (VN) e irradiado (VIr) com Cobalto-60 foi verificada por meio de desafios in vitro. Um grupo de seis animais recebeu veneno nativo, o segundo grupo recebeu veneno irradiado e o terceiro grupo foi o controle. Os animais receberam seis imunizações durante 84 dias com intervalo de 14 dias. Houve diferença significativa (p&lt;5%) no teste de ELISA do perfil de anticorpos produzidos pelos grupos experimentais (VN &lt; VIr). O grupo imunizado com veneno irradiado apresentou perfil de anticorpos maior que o grupo imunizado com veneno nativo. A capacidade de neutralização do soro produzido a partir do VIr foi cinco vezes maior quando comparado ao soro produzido com VN. Estes resultados justificam o uso da radiação gama na destoxicação do veneno de Crotalus durissus terrificus como alternativa na produção de antiveneno.

The Ability of Low Level Laser Therapy to Prevent Muscle Tissue Damage Induced by Snake Venom

Antivenom therapy has been ineffective in neutralizing the severe local fast developing tissue damage following snakebite envenoming. Herein, some effects of in situ helium neon (HeNe) laser irradiation on rat nerve-muscle preparation injected with Bothrops jararacussu venom are described. The tibialis anterior muscle was injected with venom diluted in 0.9% saline solution (60 microg/0.02 mL) or saline solution alone. Sixty minutes after venom injection, laser (HeNe) treatment was administered at three incident energy densities: dose 1, a single exposure of 3.5 J cm(-2); dose 2, three exposures of 3.5 J cm(-2); dose 3, a single exposure of 10.5 J cm(-2). Muscle function was assessed through twitch tension recordings whereas muscle damage was evaluated through histopathologic analysis, morphometry of area of tissue affected and creatine kinase (CK) serum levels, and compared to unirradiated muscles. Laser application at the dose of 3.5 J cm(-2) reduced the area of injury by 64% (15.9 +/- 1.5%vs 44.2 +/- 5.7%), decreased the neuromuscular blockade (NMB) by 62% (11.5 +/- 2.5%vs 30.4 +/- 5.2%) and reduced CK levels by 58% (from 455 +/- 4.5% to 190.3 +/- 23.4%) when compared with unirradiated controls. Dose 2 showed a poorer benefit than dose 1, and dose 3 was ineffective in preventing the venom effects. Measurements of the absorbance of unirradiated and irradiated venom solution showed no difference in absorption spectra. In addition, no difference in the intensity of partial NMB in nerve-muscle preparation was shown by unirradiated and irradiated venom. The results indicate that the laser light did not alter venom toxicity. We conclude that HeNe laser irradiation at a dosage of 3.5 J cm(-2) effectively reduces myonecrosis and the neuromuscular transmission blocking effect caused by B. jararacussu snake venom. Thus, low level laser therapy may be a promising tool to minimize the severity of some of the local effects of snake envenoming.

Irradiated Cerastes cerastes Venom as a Novel Tool for Immunotherapy

Immunotherapy is the most effective treatment for the snake bites. The antivenoms are commonly obtained by hyperimmunization of animals that suffer from venom toxicity. The present report describes gamma irradiation effects on Cerastes cerastes venom. Doses of 1 kGy and 2 kGy gamma radiations were used for venom detoxification. These treated venoms did not have any residual lethal effects until 10 LD50. Immunological analysis of sera raised against native and irradiated venoms, showed that elicited antibodies to irradiated venoms were able to recognize native venom. Anti-2 kGy irradiated venom had more protective ability than anti-native venom, as tested in mice.

Biochemical and hematological study of goats envenomed with natural and 60Co-irradiated bothropic venom

Venoms from snakes of the Bothrops genus are proteolytic, coagulant, hemorrhagic and nephrotoxic, causing edema, necrosis, hemorrhage and intense pain at the bite site, besides systemic alterations. Many adjuvants have been added to the venom used in the sensitization of antiserum-producer animals to increase antigenic induction and reduce the envenomation pathological effects. Gamma radiation from 60Co has been used as an attenuating agent of the venoms toxic properties. The main objective was to study, comparatively, clinical and laboratory aspects of goats inoculated with bothropic (Bothrops jararaca) venom, natural and irradiated from a 60Co source. Twelve goats were divided into two groups of six animals: GINV, inoculated with 0.5mg/kg of natural venom; and GIIV, inoculated with 0.5mg/kg of irradiated venom. Blood samples were collected immediately before and one, two, seven, and thirty days after venom injection. Local lesions were daily evaluated. The following exams were carried out: blood tests; biochemical tests of urea, creatinine, creatine kinase (CK), aspartate amino-transferase (AST) and alanine amino-transferase (ALT); clotting time; platelets count; and total serum immunoglobulin measurement. In the conditions of the present experiment, irradiated venom was less aggressive and more immunogenic than natural venom.

Immunological assessment of mice hyperimmunized with native and Cobalt-60-irradiated Bothrops venoms

ELISA was used to evaluate, accompany, and compare the humoral immune response of Swiss mice during hyperimmunization with native and Cobalt-60-irradiated (60Co) venoms of Bothrops jararaca, Bothrops jararacussu and Bothrops moojeni. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by in vivo challenge, and the side effects were assessed. The animals immunization with one LD50 of each venom occurred on days 1, 15, 21, 30, and 45, when blood samples were collected; challenges happened on the 60th day. Results showed that ELISA was efficient in evaluating, accompanying and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co-irradiation. The sera produced with native venom showed neutralizing potency and capacity similar to those of the sera produced with irradiated venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom, however, necrosis and death occurred in animals inoculated with native venom.

Immunization with native and cobalt 60-irradiated Crotalus durissus terrificus venom in swiss mice: assessment of the neutralizing potency of antisera

ELISA was used to evaluate, follow, and compare the humoral immune response of Swiss mice during hyperimmunization with natural and Cobalt 60-irradiated (60Co) Crotalus durissus terrificus venom. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by "in vivo" challenge and the side effects were assessed. The animals immunization with one LD50 of the venom was on days one, 15, 21, 30, and 45, when blood samples were collected; the challenges occurred on the 60th day. Results showed that ELISA was efficient in evaluating, following, and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co irradiation. Serum produced from Crotalus durissus terrificus irradiated venom showed higher potency and neutralization capacity than that from natural venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom.