Alpha-type phospholipase A2 inhibitors from snake blood

Abstract It is of popular and scientific knowledge that toxins from snake venom (among them the PLA2 and myotoxins) are neutralized by various compounds, such as antibodies and proteins purified from animal blood. Venomous and nonvenomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be self-protection against the enzymes of their own venom, which eventually could reach the circulatory system. However, the presence of PLIs in non-venomous snakes suggests that their physiological role might not be restricted to protection against PLA2 toxins, but could be extended to other functions, as in the innate immune system and local regulation of PLA2s. The present study aimed to review the currently available literature on PLA2 and myotoxin alpha inhibitors present in snake plasma, thus helping to improve the research on these molecules. Furthermore, this review includes current information regarding the mechanism of action of these inhibitors in an attempt to better understand their application, and proposes the use of these molecules as new models in snakebite therapy. These molecules may help in the neutralization of different types of phospholipases A2 and myotoxins, complementing the conventional serum therapy.

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patients lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 g/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 g/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNF, IL-1, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Alpha-type phospholipase A2 inhibitors from snake blood

It is of popular and scientific knowledge that toxins from snake venom (among them the PLA2 and myotoxins) are neutralized by various compounds, such as antibodies and proteins purified from animal blood. Venomous and nonvenomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be self-protection against the enzymes of their own venom, which eventually could reach the circulatory system. However, the presence of PLIs in non-venomous snakes suggests that their physiological role might not be restricted to protection against PLA2 toxins, but could be extended to other functions, as in the innate immune system and local regulation of PLA2s. The present study aimed to review the currently available literature on PLA2 and myotoxin alpha inhibitors present in snake plasma, thus helping to improve the research on these molecules. Furthermore, this review includes current information regarding the mechanism of action of these inhibitors in an attempt to better understand their application, and proposes the use of these molecules as new models in snakebite therapy. These molecules may help in the neutralization of different types of phospholipases A2 and myotoxins, complementing the conventional serum therapy.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.(AU)

Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

A myotoxin phospholipase A2 homologue, BmooMtx, was isolated from the venom of Bothrops moojeni by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. SDS-PAGE showed the enzyme to be a monomer with a molecular weight of 16,500. BmooMtx induced release of creatine kinase and morphological analyses indicated that it provoked an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 hours after injection. Anti-BmooMTx antibodies partially neutralized the myotoxic activity of BmooMtx and crude B. moojeni venom, as judged by determination of plasma creatine kinase levels and histological evaluation of skeletal muscle in mice. Anti-BmooMTx antibodies were effective in reducing the plasma creatine kinase levels of crude B. alternatus and B. leucurus venoms, evidencing immunological cross-reactivity between BmooMTx and other bothropic venoms. Intraplantar (i.pl.) injection of BmooMtx (1 to 15 ìg/animal) caused a dose- and time-dependent hyperalgesia and edematogenic responses. Dexamethasone (0.4 mg/kg), meloxicam (2 mg/kg) and promethazine (5 mg/kg) markedly reduced the hyperalgesia. Our data suggest that these drugs may likely serve as complementary therapies in cases of accidents with Bothrops moojeni, provided that such pharmacological treatments are administered immediately after the incident.

Histologic Analysis of the Myotoxic Change by Bupivacaine and Ricin mAb 35 on Extraocular Muscle in Rabbits

PURPOSE: To analyze the acute histologic change of extraocular muscles (EOM) induced by injection of bupivacaine or Ricin mAb 35. METHODS: The superior rectus and inferior rectus of white rabbits were injected with either bupivacaine (0.4 mg in 0.3ml) or Ricin mAb 35 (0.2 micro gram/kg in 0.3 ml). One, two, and four weeks after injection, the rectus muscles were harvested and the post-injection changes were histologically analyzed. RESULTS: Both the orbital and the global layers of EOM showed myotoxic changes induced by bupivacaine and Ricin mAb 35. However, the inflammation and destruction of myofiber by bupivacaine injection were localized to the injection site, whereas changes induced by Ricin mAb 35 were diffuse. Regenerating myofibers with a central nucleus were found at one week after myotoxin injection. Four weeks after injection, the acute changes induced by these two toxins were much recovered with prominent myofiber regeneration. Bupivacaine-induced myotoxic change was more prominent in the global layer in contrast to the more prominent damage in the orbital layer induced by Ricin mAb 35. CONCLUSIONS: We found that EOM have a superb ability to recover from the acute injury induced by bupivacaine or Ricin mAb 35 and that the two myotoxins cause unique damage including the predilection of muscle layers and the duration for which the damage persisted. Further investigation into the functional change during recovery from the myotoxin-induced injury of EOM is needed.

Myonecrosis induced in rat by a myotoxin isolated from the venom of Bothrops neuwiedifrom Alfenas, MG

Bothrops venoms are composed by several protein fractions. Among these fractions there are myotoxins which induce an important muscle lesion. The purification of this component involves some steps, although providing a pure material, is time consuming. In the present study, we describe a quick method for myotoxin fraction isolation from the venom of Bolhrops nsuwiedi using one-step high performance liquid chromatography (HPLC). The complete procedure took 30 min. The pur6+y of the two myotoxic fractions isolated was assessed by SDS-PAGE. Upon i.m. injection into rat tibialis anterior, both toxins induced early morphological changes, indicating that the plasma membrane was the first cellular structure affected. Afterwards, the lesion was typically myonecrotic and inflammatory infiltrate was present.

Os venenos de Bolhrops são compostos por várias frações protéicas. Dentre elas, as miotoxinas induzem lesões musculares importantes. A purificação deste componente envolve várias etapas e, embora forneça frações puras, é muito trabalhosa. Neste trabalho foi descrito um método rápido para o isolamento da fração miotóxica do veneno de Bothrops neuwiedi (jararaca pintada), utilizando-se uma etapa única de purificação por Cromatografia Liquida de Alta Eficiência (CLAE). O procedimento completo demorou apenas 30 min. Foram isoladas duas frações com atividade miotóxica, cuja pureza foi verificada por eletroforese em gel de poliacrilamida-SDS. A injeção intramuscular no músculo tibial anterior de ratos induziu alterações morfológicas precoces, indicando que a membrana plasmática foi a primeira estrutura celular afetada pela fração miotóxica. A lesão muscular obtida foi tipicamente mionecrótica e foi observado infiltrado inflamatório.