Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom

Abstract

The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A₂, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts.     

Cell death induced by Bothrops asper snake venom metalloproteinase on endothelial and other cell lines

Two adherent cell lines, BAEC and HeLa, and non-adherent Jurkat, were treated with snake venom metalloproteinase BaP1 to determine whether cytotoxicity, previously reported for this toxin, could be mediated by the process of anoikis. It was observed that there was no correlation between the ability of this toxin to induce loss of adherence, and the cytotoxic effect, since concentrations that do not induce loss of adherence (3–6 μg/mL), were able to trigger 50% of cytotoxicity in BAEC. In the case of HeLa, where toxicity was very low (less than 20% at maximun concentrations and times of exposure), significant detachment and no toxicity was observed at concentrations of 1.5 μg/mL, showing also no correlation between both events.We also observed differences between BAEC toxicity measured by XTT reduction and DNA fragmentation determined by flow cytometry (as an indicator of apoptosis), since concentrations that induce 100% of cytotoxicity barely showed any DNA fragmentation (12% at 24 h), suggesting that if apoptosis was involved, DNA damage is still not present, although chromatin condensation, another indicator of apoptosis, is observed in 40% of the cells. Inhibition of BAEC cytotoxicity by caspase inhibitors indicate that apoptosis is playing a role in this process, but other mechanisms of cell death could be participating also.Another way to determine whether the mechanism of cell death was related to anoikis was using a non-adherent cell line, which should show substrate independence. We determined by TUNEL that at 50 μg/ml BaP1 triggered 50% of apoptosis at 96 h, an effect that was seen earlier, suggesting also that if this toxin was inducing apoptosis in a non-adherent cell line, the mechanism could not be related to loss of attachment. Cell cycle arrest in S phase was also observed in Jurkat cells, an effect that could be leading to apoptosis.In conclusion, since there was no correlation between cell detachment and cytotoxicity (and apoptosis) in adherent cell lines and due to the ability of BaP1 to induce apoptosis in a non-adherent cell line, we suggest that this enzyme is toxic by a mechanism not related to anoikis, and that in the case of Jurkat cells, it is likely to be related to its ability to induce cell cycle arrest. Processes other than apoptosis could be also involved in the cell death mechanism mediated by BaP1 on BAEC.     

Role of nitric oxide in the local and systemic pathophysiological effects induced by Bothrops asper snake venom in mice

Objective To assess the role of nitric oxide in the most relevant local and systemic manifestations in mice injected with the venom of the snake Bothrops asper. Mice were pretreated with nitric oxide synthase inhibitors, and the modifications of the pathological effects induced by the venom were tested. Results Inhibition of NO synthesis did not affect acute local myonecrosis and hemorrhage in muscle tissue upon intramuscular injection of venom. Local footpad edema was reduced in mice pretreated with the NO synthase inhibitor L-NAME, and a reduction in the extent of inflammatory infiltrate in muscle tissue was observed after envenomation in mice pretreated with L-NAME and aminoguanidine. The most pronounced effect of NOS inhibition by L-NAME was an increment in the lethal activity of the venom, when injected by the intraperitoneal route. Conclusion Nitric oxide does not seem to play a significant role in the local acute pathological alterations (hemorrhage and myonecrosis) induced by B. asper venom in mice, although it contributes to edema and inflammatory infiltrate. Nitric oxide exerts a protective role in the systemic pathophysiological manifestations leading to lethality. Received 7 November 2005; returned for revision 19 December 2005; returned for final revision 3 February 2006; accepted by M. Katori 11 February 2006     

Ultrastructural alterations in mouse capillary blood vessels after experimental injection of venom from the snake Bothrops asper (Terciopelo).

Histological and ultrastructural alterations in capillary blood vessels were studied at various time intervals after im injection of 50 micrograms of Bothrops asper snake venom in mouse gastrocnemius muscle. Hemorrhage was observed as early as 5 min after envenomation, as abundant erythrocytes appeared in the interstitial space. Ultrastructural observations revealed two different patterns of pathological changes: in the majority of damaged capillaries, endothelial cells had blebs and cytoplasmic projections pinching off to the lumen. This phenomenon was observed together with a decrease in the number of pinocytotic vesicles, with endothelial cells becoming very thin. As an apparent consequence of this process, some endothelial cells had evident gaps in their continuity. In addition, basal laminae surrounding these capillaries were altered and discontinuous. Other endothelial cells underwent a morphologically different process of degeneration, characterized by swelling of the endoplasmic reticulum and of the cytosol. These cells had a diffuse appearance and their basal laminae were discontinuous or absent. No major changes in the intercellular junctions were noticed in damaged endothelial cells. Samples obtained 30 and 60 min after venom injection were devoid of normal capillaries in many areas, and only diffuse remnants of their structure were found. Many altered capillaries had platelet aggregates and fibrin, the latter also being observed in the interstitial space. It is concluded that B. asper venom induces rapid and drastic pathological effects on capillaries leading to hemorrhage per rhexis i.e., erythrocytes probably escape through gaps in damaged endothelial cells and not through widened intercellular junctions.     

Immunodiagnosis of snake venom poisoning

Uncertainty as to the species diagnosis remains a serious problem in the management of snake venom poisoning. This is particularly so in areas inhabited by numerous species, the venoms of which elicit similar pharmacological effects and clinical symptoms and against which para-specific cross-neutralizing antivenom is not available. Attempts have been made to eliminate some of this ambiguity through the development of various immunodiagnostic tests. Tests based on ELISA are sensitive, specific and even quantitative and adaptable to field application. In the development of diagnostic tests for use in developing countries, however, practical consideration must be given to speed, cost, simplicity in terms of equipment and expertise, and stability to the climate and storage conditions. This may dictate further modification or selection of more suitable alternative methodologies. Furthermore, the test may have to allow more flexibility in accommodating local species distributions and to address probable complications of heterophile antibodies in test samples from rural people.     

Changes in myofibrillar components after skeletal muscle necrosis induced by a myotoxin isolated from the venom of the snake Bothrops asper

The effects of a myotoxic phospholipase A2 isolated from the venom of the crotaline snake Bothrops asper on skeletal muscle myofibrils were studied by histological, ultrastructural, immunohistochemical, and biochemical parameters. Myotoxin induced a rapid and prominent muscle necrosis after intramuscular injection in mice. In this process, myofibrils were affected and three main changes were observed: (A) Initially, they were hypercontracted, eventually forming "clumped," dense masses which alternated with spaces devoid of myofilaments in the cytoplasm. This initial stage is probably due to hypercontraction resulting from a calcium influx after toxin-induced sarcolemmal damage. (B) A second change occurred between 3 and 6 hr, when the clumped or hypercontracted pattern changed to a "hyaline" pattern in which myofilaments were relaxed and had a more uniform distribution in the cellular space. Although there was not a widespread degradation of myofibrillar components at this stage, desmin started to be lost in samples obtained as early as 15 min after toxin injection, and alpha-actinin was almost absent by 7 hr. Thus, it is proposed that this shift may be due to a selective proteolytic degradation of structurally relevant components, particularly alpha-actinin. As a consequence, the mechanical integration of myofilaments is impaired, precluding hypercontraction. (C) Finally, at later time periods (24, 48, and 72 hr), there was widespread degradation of myofibrillar proteins, probably caused by proteases derived from inflammatory cells such as neutrophils and macrophages, whose numbers in necrotic muscle increased markedly at these time periods.     

Vipera lebetina venom contains all types of snake venom metalloproteases

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.     

Degenerative and regenerative changes in murine skeletal muscle after injection of venom from the snake Bothrops asper: a histochemical and immunocytochemical study.

The degenerative and regenerative changes in murine skeletal muscle after injection of Bothrops asper venom were studied by histological, lectin histochemical and immunocytochemical techniques. According to our observations, the process was divided into four main stages: (a) During the first 3 days prominent degenerative events took place in skeletal muscle fibres, capillaries, arteries, veins and intramuscular nerves. An inflammatory infiltrate was abundant after the first day and removal of necrotic material was well advanced by the third day. (b) Muscle regeneration was evident by the fourth day. From 4 to 6 days there were two populations of regenerating muscle fibres, one of apparently normal fibres located in areas where capillary vessels were abundant, and another population of groups of regenerative fibres showing signs of degeneration. This second type of fibre was predominant in areas where the number of capillaries was greatly reduced. (c) One and 2 weeks after envenomation areas of small regenerative fibres of normal morphology and areas of degenerating regenerative fibres were observed. The latter were abundant in regions of dense fibrotic tissue and scarce capillaries. (d) Finally, at 4 and 8 weeks after envenomation there were both areas of fibrosis and areas where regenerating muscle fibres predominated. However, the diameter of these fibres was abnormally small, an indication that they may have been atrophic fibres. It is suggested that muscle regeneration is partially impaired after myonecrosis induced by Bothrops asper venom, probably due to the damage induced by this venom on muscle microvasculature and nerves.     

Hypersensitivity to airborne spitting cobra snake venom.

BACKGROUND: Although the cytolytic, neurotoxic, and hemolytic actions of snake venoms are well known, the ability of airborne inhaled snake venom of the spitting cobra to induce asthma in snake handlers has not been reported. OBJECTIVE: To report the allergenicity of inhaled snake venom in a snake handler who developed increasing hypersensitivity to airborne venom, produced by spitting cobras during public demonstrations. METHODS: Serum samples were obtained from 2 handlers (our study patient and another snake handler who reported developing wheezing when handling spitting cobras), and desiccated venom was obtained from 9 species to which the handlers were exposed. Serum from an asymptomatic and nonatopic snake handler exposed to the same snake species was used as a control. Phosphate-buffered saline extracts were prepared from the desiccated venom, proteins in the venom extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting was performed. Inhibition enzyme-linked immunosorbent assays (ELISAs) were performed to demonstrate cross-reactivity. RESULTS: The study patient had never been previously bitten by a cobra. Wheezing occurred rapidly on inhalational exposure and was reversed by inhalation of salbutamol. The patient had developed IgE antibodies to 9 different snake venoms on Western immunoblots, with major IgE binding proteins of 59 to 63 kDa and 8 to 15 kDa. The cross-reactive nature of the IgE epitopes in the venoms in the different species was also confirmed by 50% inhibition of IgE binding in an ELISA by preincubation with unrelated species. Life-threatening sensitivity of the patient was sustained after a long period of avoidance. CONCLUSIONS: We propose that aerosolized snake venom be considered a new potential source of allergens that may result in anaphylaxis on subsequent exposure. Further studies of the development of specific IgE sensitization following snakebites and the risks of such sensitization should be conducted on snake handlers, particularly those who demonstrate the spitting species.     

Inflammatory pathogenesis of snake venom metalloproteinase-induced skin necrosis

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were induced experimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL-1betaR, IL-6 and iNOS. Jararhagin-induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL-6(-/-) mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase-induced effects are independent of these pro-inflammatory mediators. The contribution of both TNF receptors and IL-6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.     

Pathogenesis of myonecrosis induced by crude venom and a myotoxin of Bothrops asper

The pathogenesis of skeletal muscle necrosis induced by crude Bothrops asper venom and isolated myotoxic phospholipase was studied using light and electron microscopy. White mice were injected intramuscularly with a dose of 2.5 micrograms/g and tissue samples were taken at 30 min and 1, 3, 6, 12, 24, and 48 hr. Toxin-injected muscle showed localized wedge-shaped lesions ("delta lesions") by 30 min, which included disrupted plasma membranes. At 1 and 3 hr the predominant type of necrotic cell contained clumped myofibrils in which individual myofilaments were indistinguishable. At later time periods there was a relaxation and redistribution of myofilaments resulting in a more homogeneous and hyaline appearance of necrotic cells. Some mitochondria were swollen and had flocculent densities, and most of them were disrupted, having only one membrane and vesiculated cristae. The basal lamina was intact at all time intervals. Phagocytosis of muscle cell debris started at 3 hr and was prominent by 24-48 hr. In crude venom-injected muscle many cells showed pathologic features identical to those observed after myotoxin injection. Crude venom also induced hemorrhage which was evident 30 min after injection, reaching its highest level by 12 hr. At 3, 6, and 12 hr some cells were undergoing different pathologic changes which appeared to be due to ischemia. Although these cells were irreversibly damaged, as indicated by ruptured plasma membrane, their myofibrillar structure was better preserved than that of toxin-affected cells. The Z line was absent, but A, I, H, and M bands were intact. As a result of Z line loss, sarcomeres were disoriented. It is proposed that the myotoxin induces myonecrosis by first altering the integrity of the plasma membrane, thereby increasing the permeability to calcium, other ions, and molecules which leads to death of the cell. Crude venom affects muscle cells in two ways: by direct action of myotoxin (s) and by ischemia due to hemorrhage.     

蛇毒的抗肿瘤活性成分研究进展

Many snake venoms contain complex mixtures of pharmacologically important molecules,some of which show potential therapeutic value in the treatment of cancer and other human disorders. In this review,we mainly reports the effects of snake venom active components, such as disintegrins and lectins in paralyzing cancer cells, blocking on cell migration, interaction with integrins, inhibition of tumor dissemination and angjogenesis. The advanced researches on the snake venom‘s apoptosis- inducing components on tumors are also introduced.     

Crotalus durissus terrificus snake venom regulates macrophage metabolism and function

In the present study, we examined the effect of Crotalus durissus terrificus venom on rat macrophage metabolism and function. Two hours after subcutaneous injection of the venom, peritoneal resident (unstimulated), elicited (thioglycollate-stimulated), and activated Mycobacterium bovis strain bacille Calmette Guérin (BCG) macrophages were collected, and their functional and metabolic parameters were analyzed. The venom inhibited spreading and phagocytosis of macrophages. On the other hand, this treatment increased H(2)O(2) and NO production, candidacidal activity, and the activities of key enzymes of glycolysis and glutaminolysis. We also investigated whether the venom could affect macrophage activation by thioglycollate or BCG. The administration of venom 2 h before injection of thioglycollate and BCG or 2 or 3 days after injection of the thioglycollate or BCG, respectively, did not modify the previous observations. These findings suggest that crotalic venom leads the macrophage to an activated state, with high production of oxygen- and nitrogen-reactive species. This cell activation state does not include inflammatory properties of spreading and phagocytosis.     

Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.     

Development of specific IgE antibodies after repeated exposure to snake venom

A patient who had been working with snakes for many years developed urticarial lesions on contact with the venom of the poisonous rinkals (Haemachates haemachatus). More recently, the patient complained of generalized allergic reactions occurring within minutes of exposure to the venom. The patient's serum, but not control sera, contained IgE antibodies that reacted with the specific snake venom in an ELISA and was demonstrated to associate with a 66 kd component of the venom with Western blotting. With an ELISA, the patient's serum was also demonstrated to contain IgG antibodies to the specific snake venom and to venom from three other snakes with which the patient had previously been in contact. The possibility of an acute allergic reaction should be considered in individuals continuously working with snakes or in individuals who have previously been bitten by snakes.