Thalita Rocha

Production of the First Effective Hyperimmune Equine Serum Antivenom against Africanized Bees

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)´2-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

The greater black krait (Bungarus niger), a newly recognized cause of neuro-myotoxic snake bite envenoming in Bangladesh

Prospective studies of snake bite patients in Chittagong, Bangladesh, included five cases of bites by greater black kraits (Bungarus niger), proven by examination of the snakes that had been responsible. This species was previously known only from India, Nepal, Bhutan and Burma. The index case presented with descending flaccid paralysis typical of neurotoxic envenoming by all Bungarus species, but later developed generalized rhabdomyolysis (peak serum creatine kinase concentration 29,960 units/l) with myoglobinuria and acute renal failure from which he succumbed. Among the other four patients, one died of respiratory paralysis in a peripheral hospital and three recovered after developing paralysis, requiring mechanical ventilation in one patient. One patient suffered severe generalized myalgia and odynophagia associated with a modest increase in serum creatine kinase concentration. These are the first cases of Bungarus niger envenoming to be reported from any country. Generalized rhabdomyolysis has not been previously recognized as a feature of envenoming by any terrestrial Asian elapid snake, but a review of the literature suggests that venoms of some populations of Bungarus candidus and Bungarus multicinctus in Thailand and Vietnam may also have this effect in human victims. To investigate this unexpected property of Bungarus niger venom, venom from the snake responsible for one of the human cases of neuro-myotoxic envenoming was injected into one hind limb of rats and saline into the other under buprenorphine analgesia. All animals developed paralysis of the venom-injected limb within two hours. Twenty-four hours later, the soleus muscles were compared histopathologically and cytochemically. Results indicated a predominantly pre-synaptic action (β-bungarotoxins) of Bungarus niger venom at neuromuscular junctions, causing loss of synaptophysin and the degeneration of the terminal components of the motor innervation of rat skeletal muscle. There was oedema and necrosis of extrafusal muscle fibres in envenomed rat soleus muscles confirming the myotoxic effect of Bungarus niger venom, attributable to phospholipases A₂. This study has demonstrated that Bungarus niger is widely distributed in Bangladesh and confirms the risk of fatal neuro-myotoxic envenoming, especially as no specific antivenom is currently manufactured. The unexpected finding of rhabdomyolysis should prompt further investigation of the venom components responsible. The practical implications of having to treat patients with rhabdomyolysis and consequent acute renal failure, in addition to the more familiar respiratory failure associated with krait bite envenoming, should not be underestimated in a country that is poorly equipped to deal with such emergencies.

Inflammation and apoptosis induced by mastoparan Polybia-MPII on skeletal muscle

Mastoparan firstly described as an inducer of mast cell granules exocytosis has been also related to many essential mechanisms of cell function. In skeletal muscle tissue the best characterization of mastoparan effect was induction of myonecrosis. We examined the ability of mastoparan Polybia-MPII from Polybia paulista wasp venom to induce apoptosis and inflammation in mouse tibial anterior muscle. The activation of caspase 3 and 9, the expression of TNF-alpha, IFN-gamma, CD68 and CD163 proteins, specific of resident and migrant macrophages, respectively, were examined (3h to 21d). TUNEL-positive nuclei were found both in damaged and normal-looking muscle fibres, whereas the caspases, cytokines and macrophages proteins were only in damaged fibres. The caspase 3 and 9 expression and the immunolabelled areas of TNF-alpha and IFN-gamma were significantly higher compared to control. TUNEL-positive nuclei and TNF-alpha expression were also present in regenerating fibres. CD68 and CD163 signalize necrotic debris removal, release of chemo-attractants and cytokines which have been considered a pre-requisite for muscle regeneration. High levels of cytokines coincided with the intense muscle proteolysis by mastoparan (3-24h) and the climax of regeneration (3 d) whereas cytokines decline corresponded to periods of tissue remodeling and intense fibre protein synthesis (7-21 d). We conclude that the mastoparan Polybia-MPII causes myonecrosis and apoptosis, the latter probably involving caspases signalling, corroborated by mitochondrial damage, and cytokines activation.

Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom

Bmaj-9, a basic PLA2 (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on ¼-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA2. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA2 from other snake venoms. It also exhibited a pronounced phospholipase A2 activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 µg/mL); 40 ± 6 and 90 ± 2 (5 µg/mL); 30 ± 3 and 70 ± 5 (10 µg/mL); 42 ± 1 and 60 ± 2 (20 µg/mL), with no effect on the contractures elicited by either exogenous ACh (110 µM) or KCl (20 mM). Bmaj-9 (10 µg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 µg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA2 family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade.

Ultramorphological characteristics of Chrysomya megacephala (Diptera, Calliphoridae) eggs and its eclosion

Chrysomya blowflies are originally from Africa and Australasia and were introduced in the American Continent, probably as a sheep parasite, in the 1970s. These flies are extremely important for medical-sanitary purposes and are useful to forensic entomology being used as an indicative of decomposition time in human corpses. The morphology of the larval and pupal stages of some species has been already studied by different authors demonstrating that it is possible to identify them in premature stages. In this study, Chrysomya megacephala egg ultramorphology was analyzed to describe its structure, generating data for further comparison between different species and genera. The blowflies were collected in Rio Claro city, SP, Brazil. Flies were attracted by fish carcasses and maintained in net cages; small portions of minced meat were placed in plastic pots for egg laying. Eggs were collected, fixed in alcoholic Bouin, prepared according to SEM routine and observed under Philips scanning electron microscope. C. megacephala eggs are oval with one flat face and another convex, measuring approximately 0.52 (+/-0.03) mm of length and 0.12 (+/-0.02) mm of width. The micropyle is circular in shape, with two perforations enclosed by countless small projections and is located in one of the extremities in the interface between the flat and convex faces. On the convex surface hexagonal imprints were observed, this surface is impermeable to water and gases. The flat surface has numerous round projections with different sizes, creating a permeable surface with a thinner chorion from which the larvae hatches. The larvae presents itself all wrinkled and with numerous cuticular projections with a thorn shape facing the posterior region. The cuticle projections with a thorn shape from C. megacephala larvae are probably a vestige of the thorns found in larvae of parasite Diptera.

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.

Ultramorphology and histology of the Polistes versicolor (Oliver) (Vespidae) thorax salivary gland compared with other Hymenoptera

The salivary system of the Hymenoptera consists of the mandible, hypopharynx and thoracic salivary glands. It is very important because it is related to various aspects of the life of the insects, such as pheromone production, feeding the young, food digestion and nest building. Adult Polistes versicolor (Olivier) individuals were dissected, the thoracic salivary glands removed and processed for scanning electronic microscopy and histological examination. The P. versicolor thoracic salivary gland presents alveolar secretory units, consists of pseudoacines and does not have a reservoir. Four types of cells are present in the gland. The T1 and T2 cells make up the pseudoacines and differ mainly by the many secretory vessels in T2. There is a cluster of T3 cells at the base of the gland duct collectors, also with secretory characteristics. The secretion produced in the pseudoacines is conducted by canals and ducts to the outside, and the latter are made of T4 cells. The comparison of these characteristics with those of different Hymenoptera species, already studied, showed that the thoracic salivary gland cannot be used as a single comparison factor in evolutionary studies.

Revealing the functional structure of a new PLA2 K49 from Bothriopsis taeniata snake venom employing automatic "de novo" sequencing using CID/HCD/ETD MS/MS analyses

Snake venoms are composed of approximately 90% of proteins with several pharmacological activities having high potential in research as biological tools. One of the most abundant compounds is phospholipases A2 (PLA2), which are the most studied venom protein due to their wide pharmacological activity. Using a combination of chromatographic steps, a new PLA2 K49 was isolated and purified from the whole venom of the Bothriopsis taeniata and submitted to analyses mass spectrometry. An automatic “de novo” sequencing of this new PLA2 K49 denominated Btt-TX was performed using Peaks Studio 6 for analysis of the spectra. Additionally, a triplex approach CID/HCD/ETD has been performed, to generate higher coverage of the sequence of the protein. Structural studies correlating biological activities were made associating specific Btt-TX regions and myotoxic activity. Lysine acetylation was performed to better understand the mechanism of membrane interaction, identifying the extreme importance of the highly hydrophobic amino acids L, P and F for disruption of the membrane. Our myotoxical studies show a possible membrane disruption mechanism by Creatine Kinase release without a noticeable muscle damage, that probably occurred without phospholipid hydrolyses, but with a probable penetration of the hydrophobic amino acids present in the C-terminal region of the protein.

Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.