Mini-dissertation submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy in the Faculty of Health Sciences at the Durban University of Technology, 2008. / The aim of this study was to elicit and document the effects, in the form of signs and symptoms produced in relatively healthy volunteers, of the venom of Hemachatus haemachatus, prepared in accordance with the methods set out in the homoeopathic pharmacopoeia. These signs and symptoms form the indications for the prescription of the remedy, according to the homoeopathic Law of Similars. A further aim of this study was a comparative analysis of symptoms produced by Hemachatus haemachatus 30ch with existing remedies derived from snake venom used in homeopathy, with the aim of highlighting the similarities and differences between them. The homoeopathic drug proving of Hemachatus haemachatus 30ch took the form of a double-blind, placebo controlled trial. The proving population consisted of 30 healthy subjects who met with the necessary inclusion criteria (Appendix B). Eighty percent (24 subjects) served as the experimental group, receiving the active verum in a randomised manner, while twenty percent (6 subjects) formed the placebo group, receiving non-medicated placebo powders in a randomised manner. Provers were unaware of either the nature or potency of the substance. Verum and placebo were indistinguishable from each other, and neither researcher nor volunteers knew who received verum and who received placebo. Intra-individual control was achieved through a pre-proving observation period of a week’s duration, during which provers recorded the signs and symptoms of their normal state. This symptom picture served as a baseline for comparison with symptoms noted after administration of the remedy. Verum and placebo were dispensed in the form of six powders to be taken sublingually three times a day for a period of two days, or until the onset of symptoms. Data was primarily collected in the form of a diary or journal kept by each prover in which they recorded symptoms on a daily basis. Provers were closely monitored by the researchers during this period. Data collected by the researchers during daily telephonic contacts, as well as during the pre-proving consultation, was also considered. Information obtained from the journals was then assessed by the researchers for suitability for inclusion in the materia medica of Hemachatus haemachatus. The data did not require statistical analysis. In a concurrent study of similar methodology, Cahill (2008) conducted a comparison of the symptom complex produced in the proving of Hemachatus haemachatus, with other homeopathic remedies which scored highest on repertorisation. Symptoms from both studies were collated and included in the materia medica and repertory of Hemachatus haemachatus. The investigation supported the hypothesis that Hemachatus haemachatus would produce clear and observable signs and symptoms in healthy proving volunteers. During the course of this study provers experienced a wide range of mental, emotional and physical symptoms. The highest number of symptoms was produced on the mental and emotional level. Provers experienced elation, joy, increased confidence, enthusiasm and energy. On the other hand, there was lack of confidence, vulnerability, anxiety, decreased motivation, decreased concentration, lethargy, depression and indifference. There were sudden changes in mood and provers experienced marked irritability. On the physical level, many provers experienced headaches, irritation of the eyes, symptoms resembling allergic rhinitis, sore throats, nausea, heartburn, abdominal pain and flatulence, menstrual disturbances, lumbar pain, rheumatic joint pains, and skin eruptions. There were disturbances in normal sleep patterns, subjective perceptions of increased body temperature with hot flushes, and generalised tiredness and lethargy. Symptoms obtained from the proving of Hemachatus haemachatus were analysed as part of a comparative study with other remedies derived from snake venom: Lachesis muta, Naja tripudians, Elaps corallinus, Naja mossambica and Bitis arietans arietans. This comparison highlighted both the similarities and differences between these remedies and Hemachatus haemachatus.

Thesis (M. Phil.)--University of Hong Kong, 1998. / Includes bibliographical references (leaf 92-113).

Mini-dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Homoeopathy at the Durban Institute of Technology, 2002. / Dream provings are considered to be a new era in Homoeopathy and as such are met with a lot of scepticism. They involve getting in touch with the dynamic influence of the remedy and observing this influence on the vital force in the form of symptoms (Dam, 1998: 128). Dreams are a main focus of the proving as they are considered to be the 'royal way to the psycho-dynamic depth of the state of the remedy being proved' (Dam, 1998: 128). The motivations for their acceptance or rejection are both reasonable. To determine if they are provings that should appear in the Materia Medica and Repertory it needed to be seen if they revealed the same features of a remedy that a classical proving provides. / M

Pseudoscorpions are a diverse lineage of arachnids with a rich history of taxonomic study. However, they remain one of the lesser-known groups of arachnids and many questions about these enigmatic arthropods remain. The present work revises the taxonomy and systematics of the family Neobisiidae in the Southwest, documenting the existence of several new species and a hitherto unknown clade from the Sky Island region of southeastern Arizona. It also describes the venom of a pseudoscorpion for the first time, through comparative transcriptomic studies. Seven new species are described and assigned to the genus Globocreagris, extending the known range of this genus from California into Arizona, Oregon, and Washington. The monophyly of the subfamily Neobisiinae was tested using two genes (COI and 28S). Molecular phylogenetic analysis of both genes and the pattern of trichobothrial placement on the chelae supports removing Parobisium from the subfamily Neobisiinae, and placing it within the subfamily Microcreagrinae, a reassignment here made. Although it has long been known that most pseudoscorpions possess venom glands in their pincers which they use to subdue their arthropod prey, the components of the venom have never been identified. Using comparative transcriptomics from the pedipalps of Globocreagris the first putative venom proteins in pseudoscorpions were identified. Putative venom proteins include astacin-like metalloproteases, chitinases, cysteine-rich secretory proteins, Kunitz-type serine protease inhibitors, phospholipase A2, and scorpion La1-like peptides.

Stress is a physiological state induced by disturbance or adverse environmental conditions and is modulated by the glucocorticoid hormone corticosterone (CORT) in reptiles. Stressors can have various impacts on vertebrate trait expression and may affect survival or reproduction. Little is known about the effects of chronically elevated CORT in free-ranging reptiles, or the effect of disturbance stress on venom composition in captive snakes. In chapter 1, we investigated the effects of researcher induced disturbance on CORT levels and venom composition in a group of captive Northern Pacific rattlesnakes (Crotalus oreganus). Venom protein concentration and plasma CORT levels were compared before and after two weeks of unpredictable bouts of cage vibration, and to a non-vibrated control group. CORT levels were also assessed one week into vibration treatment. We found no effect of vibration treatment on CORT levels or on venom composition, and within-snake relative protein abundance was highly repeatable, although some variation was observed. We found a strong correlation between changes in relative abundance of several proteins and CORT. These results led us to believe that while differential forms of researcher-induced disturbance may not affect venom composition, significant changes in baseline CORT, or chronic stress, may affect the venom phenotype. In the next study, we investigated the effects of chronically elevated CORT in a wild population of radio-telemetered Southern Pacific rattlesnakes (C. helleri). Snakes were implanted intra-coelomically with either crystalline CORT or sham implants. Prior to implant and for two week periods thereafter, we sampled blood, venom, defensive behavior, and body temperature (Tb). Thermal data logger implants recorded snake Tb each hour. Snakes were tracked daily for one month, and detectability, defensive behavior, movement, home range size and thermal parameters were calculated for each group during the periods between samples. Stress reactivity was assessed as change in CORT from baseline after one hour of acute confinement stress. CORT implants led to elevated baseline CORT for at least two weeks in treatment snakes, showing that our treatment was successful. Chapter 2 describes the effects of CORT treatment on venom parameters. Increased baseline CORT was associated with increased activity of venom protein phospholipase A2, indicating that CORT may have direct effects on regulating venom protein activity. Overall, venom activity was repeatable within individual snakes. Chapter 3 describes the effect of CORT on behavioral, ecological, and physiological variables. Implant treatment led to decreased average Tb in weeks two and three. We detected a trend for lower baseline CORT to predict a greater magnitude of acute stress response. Snakes with higher testosterone levels exhibited higher defensive behavior scores. Overall, there were no other effects of implant treatment. Our results suggest that rattlesnake thermoregulation is impacted by chronic stress, which could affect other aspects of their metabolism and ecology. Results of both studies suggest baseline CORT may direct both the activity and relative abundance of venom proteins in different manners, a hypothesis which deserves further investigation using proteomic tools. When responding to an acute stressor, rattlesnakes may secrete CORT until a threshold response is reached, regardless of baseline levels. Overall, rattlesnakes appear resilient to the effects of researcher-induced disturbance in the laboratory and to two weeks of chronically elevated CORT in the field, as no change was detected in many of the parameters investigated.

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Durban University of Technology. 2013. / This study was a homoeopathic drug proving of Bitis atropos 30CH (derived from Berg adder venom) with a subsequent comparison of the proving symptoms to known venom toxicology and existing remedies from the materia medica, that on repertorisation, yielded the greatest similarities in the Mental, General, Physical and unique symptomatology of Bitis atropos. Methodology : The proving was carried out in the form of a double-blinded, placebo controlled trial on healthy subjects who were administered the proving substance or placebo. The resultant influence of this substance on the health of provers (i.e. symptoms produced) was recorded in journal format and formed the materia medica and ultimately the clinical indications thereof according to the Law of Similars. Twenty eight healthy consenting provers who meet the inclusion criteria (Appendix B), were randomly split into two groups, one being the experimental group comprising 22 provers, and the other a placebo control group comprising 6 provers. This was further split between the researcher and co-researcher, each responsible for 11 provers receiving verum and 3 receiving placebo. The researchers and the individual provers were unaware of their respective group allocation and the provers were unaware of the identity of the proving substance. The fresh venom sourced from a wild, Berg adder, was processed according to the German Homoeopathic Pharmacopoeia (Appendix G) to produce the 30CH Homoeopathic potency thereof. Six lactose powders were dispensed to each prover (either placebo or verum) and taken sublingually three times a day or until the onset of symptoms. Symptoms were recorded by the provers in journals over 4 weeks and were closely supervised by the researcher. When the symptoms subsided, the combined journals were collected, collated, analysed, interpreted and validated. Accepted symptoms were converted to materia medica and Repertory format. Results : The proving yielded a total of 903 rubrics, of which 18 were newly created. The systems mostly affected were Dreams, Mind, Head and Eye. Comparison of proving symptoms to that of venom toxicology, as seen in case studies of envenomation by Bitis atropos, yielded similar results, as the sensations experienced in provers closely matched that of known venom toxicology. On repertorisation of the proving symptoms, the existing remedies that were closely related were Sepia officinalis, Lachesis mutus and Argentum nitricum. Further repertorisation of toxicological symptoms indicated a further relation to Belladonna, Natrum muriaticum and Hyoscyamus niger. Conclusion : Clearly observable signs and symptoms were produced by healthy provers in response to administration of Bitis atropos 30CH, in addition there was a significant degree of similarity between proving symptoms and that of known toxicology of the crude substance. The researcher identified Sepia officinalis, Lachesis mutis and Argentum nitricum as the three most similar existing homoeopathic remedies and a detailed comparison thereof was conducted. A further repertorisation of the toxicological symptoms of envenomation by the snake, yielded the remedies Belladonna, Natrum muriaticum and Hyoscyamus niger which were also compared to Bitis atropos.

Acidentes ofídicos constituem um problema de saúde publica. Estudos prévios indicam que constituintes do veneno de Crotalus durissus terrificus injetados sistemicamente promovem, agudamente, aumento dos níveis de ansiedade em ratos; se injetados topicamente na formação hipocampal, região intimamente ligada a processos de memória espacial e ansiedade, induzem alterações citoestruturais. O objetivo deste trabalho foi avaliar, em ratos, efeitos comportamentais decorrentes da injeção sistêmica de veneno bruto de Crotalus durissus terrificus e a eficácia do soro anticrotálico em prevenir esses prejuízos quando administrado variáveis intervalos de tempo depois do envenenamento. Ratos submetidos a uma única injeção sistêmica de veneno bruto 7 dias antes do inicio dos testes foram avaliados nas tarefas de memória de referencia e de memória operacional no labirinto aquático de Morris, e também a uma tarefa envolvendo uma plataforma visível no mesmo aparelho. A seguir, os animais foram submetidos ao paradigma do teste-reteste no labirinto em cruz elevado. Os resultados mostraram que houve prejuízos de memória de referencia e de memória operacional; este último efeito ocorreu quando o intervalo entre as tentativas foi de 10 minutos, mas não quando foi de zero minutos. Potenciais efeitos sensoriais e motores foram excluídos. Alem disso, houve substancial aumento nos níveis de ansiedade. A administração de soro anti-crotálico preveniu os principais prejuízos de memória desde que realizada em ate 10 horas apos a injeção do veneno (foram testados intervalos de 0, 0,5, 2, 10 e 24 horas, em grupos independentes de animais)..O grupo tratado com soro anti-crotálico 24 horas depois do envenenamento apesar de prejudicado em relação aos grupos controle (um injetado com salina e o outro apenas com soro), exibiu desempenho melhor do que o grupo tratado apenas com veneno. Assim, o presente conjunto de resultados representam a primeira demonstração de que (1) uma única dose sistêmica do veneno crotálico produz prejuízos de memória espacial em ratos e aumenta os níveis de ansiedade avaliados 4 semanas apos a injeção, e (2) os prejuízos de memória podem ser prevenidos pela administração de soro anticrotálico desde que essa administração ocorra em ate 10 horas apos o envenenamento. / Snakebites constitute a serious public health problem in Brazil. Prior studies have shown that systemic injections of venom fractions of Crotalus durissus terrificus produce acute increase in anxiety levels in rats; when injected topically within the hippocampal formation, a brain region underlying processes of spatial memory and anxiety, induce damage. The aims of this study included to investigate, in rats, behavioral effects of a single systemic injection of crude venom on performance of spatial memory tasks and on anxiety, and the efficacy and time course of the antivenom administration to prevent memory disruption. Rats subjected to a single systemic injection of venom 7 days before the beginning of behavioral testing were evaluated in modified versions of the reference and working memory tasks in the water maze, and also to a version of the task in which the platform is visible. Then, the subjects were submitted to the test-retest paradigm in the elevated plus maze. Rats injected with the venom exhibited disruption of performance both in reference and working memory versions of the water maze task; in this latter task, however, disruption occurred when the intertrial interval was 10 minutes but not when the it was zero minutes. Anti-crotalic serum injection prevented memory disruptions when its administration occurred up to 10 hours after injection of the venom (time intervals evaluated included 0, 0.5, 2, 10 and 24 hours, in independent groups of rats). Subjects that received anti-crotalic serum 24 hours after venom injection exhibited disruption of memory relative to control groups (one of them treated with saline and the other with anti-crotalic serum only); however, performance of those animals was better when compared to subjects receiving only venom administration. These results show, to our knowledge for the first time, that (1) a single systemic injection of crotalic venom induces disruption of spatial memory and increases anxiety evaluated 4 weeks after injection, and (2) major spatial memory disruptions may be prevented by administration of the anti-crotalic serum up to 10 hours after the venom injection.

Background: Myrmecia pilosula (the jack jumper ant, JJA) is the principal cause of ant venom anaphylaxis in Australia. Whereas honeybee and wasp venom allergy can be treated by venom immunotherapy (VIT), no such treatment is available for ant sting allergy. In addition, information on the natural history of JJA sting allergy is required to identify those most likely to benefit from immunotherapy. The main objectives of this research were to establish: (i) the prevalence, natural history and determinants of reaction severity for JJA allergy, and; (ii) the efficacy and tolerability of JJA VIT. Methods: A search of the Royal Hobart Hospital (RHH) forensic register, a random telephone survey, and a review of emergency department (ED) presentations were performed. Three hundred eighty-eight JJA allergic volunteers were assessed, including serum venom-specific IgE RAST, and then followed up for accidental stings over a 4-year period. Finally, a randomised double-blind, placebo-controlled, crossover trial of JJA VIT was performed. Laboratory parameters measured during the trial were; leukocyte stimulation index (SI), IL-4 production, IgE RAST, histamine release test (HRT), leukotriene release test (LRT) and basophil activation test (BAT). Intradermal venom skin testing (VST) was also performed at trial entry. Findings: The prevalence of JJA sting allergy was 2.7% in the Tasmanian population, compared to 1.4% for honeybee. People aged 35 or older had a greater risk of both sting allergy and hypotensive reactions. Four deaths were identified, all in adults with significant comorbidities. During follow-up, 79 (70%) of 113 accidental jack jumper stings caused systemic reactions. Only prior worst reaction severity predicted the severity of follow-up reactions, with the majority of people experiencing similar or less severe reactions when stung again. Sixty-eight otherwise healthy JJA allergic adult volunteers were enrolled in the clinical trial. Systemic reactions to therapy were recorded in 34% during VIT. Objectively defined systemic reactions to sting challenges arose in 1/35 after VIT (mild self-limiting urticaria only) versus 21/29 in the placebo group. Treatment with oxygen, intravenous adrenaline infusion and volume resuscitation was effective and well tolerated. Hypotension was always accompanied by a relative bradycardia, which was severe and treated with atropine in two patients. In the placebo group, only VST and HRT were predictive of sting challenge results. Although IgE RAST, leukocyte SI and IL-4 production, LRT and BAT all correlated well with VST, they did not predict sting challenge outcome. After successful VIT, venom-induced leukocyte IL-4 production tended to fall, whereas IgE RAST increased and a natural decline in HRT reactivity was reversed. Interpretation: VIT is highly effective in prevention of JJA sting anaphylaxis and is likely to be of most benefit to people with a history of severe systemic reactions, which usually occur in people aged over 35. Neurocardiogenic mechanisms &/or direct cardiac effects may be important factors in some anaphylaxis deaths. Systemic reactions to immunotherapy are common and require immediate access to resuscitation facilities. The HRT warrants further investigation as a test for selecting those most likely to benefit from VIT. None of the tests evaluated appear to be reliable markers of successful VIT.

The aim of my thesis is the introduction of the systemic mastocytosis and discussion about results of allergen immunotherapy in patients with systemic mastocytosis and its preventive effects against recurrent anaphylactic reactions. Patients with systemic mastocytosis are more prone to severe systemic anaphylactic reactions after Hymenoptera stings than in the general insect venom allergic population patients without elevated basal tryptase. This severe reaction can result in the death of the patient, it is important to prevent it prophylactically. The medication of choice in insect venom allergic patients is hyposensibilization therapy - allergen immunotherapy, which uses venom allergens of causal Hymenoptera (honey bee, yellow jacket). The thesis aims to summarize the results obtained so far about the appropriateness of this treatment in patients with systemic mastocytosis, side-effects during VIT, optimal dosing schedule and duration of treatment in these patients.

Orientador: Stephen Hyslo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T15:32:56Z (GMT). No. of bitstreams: 1 RochaeSilva_ThomazAugustoAlvesda_D.pdf: 9683821 bytes, checksum: 9eaf69e00f5ad25d739e7f014effdaed (MD5) Previous issue date: 2008 / Resumo: A aranha Vitalius dubius pertence à família Theraphosidae, que compreende todas as caranguejeiras. Sua distribuição é restrita à região Sudeste do Brasil, principalmente em São Paulo e Minas Gerais. Trata-se de uma espécie urbana, pouco agressiva e que não apresenta registros de acidentes com relevância clínica. O presente trabalho teve como objetivo descrever o aparato venenífero e caracterizar bioquímica e farmacologicamente a peçonha desta aranha. A estrutura do aparato venenífero consiste em duas robustas quelíceras anteriores, com grandes ferrões para imobilização de presas e inoculação de peçonha. Cada quelícera é preenchida por musculatura estriada, responsável pela mobilidade dos ferrões, além de abrigar a glândula de peçonha que, por sua vez, encontra-se imersa nesta musculatura, sem estar ligada à mesma. A glândula é envolta por duas túnicas musculares helicoidais entrelaçadas, responsáveis pela contração da glândula para expulsão da peçonha através de um duto interno ao ferrão. Interna à musculatura, uma camada basal rica em elastina sustenta uma complexa rede epitelial produtora de peçonha. Este epitélio extratificado possui células anastomosadas, com núcleos periféricos e prolongamentos citoplasmáticos voltados para a luz da glândula, organizado principalmente por filamentos de actina. Estes prolongamentos formam reservatórios envolvidos na biossíntese de peçonha. A análise ultra-estrutural deste epitélio revelou a presença de organelas envolvidas na síntese protéica, como núcleo com abundante eucromatina, nucléolo, retículo endoplasmático rugoso, polissomos, além de abundantes: retículo liso, aparato de Golgi, mitocôndrias e vacúolos autofágicos. Durante a fase de produção de peçonha, as principais alterações ultra-estruturais observadas foram compactação e remodelamento do epitélio, abundância de organelas e deformidade de núcleos, bem como a presença de hemócitos nos últimos estágios. A extração de peçonha revelou que as fêmeas possuem rendimento maior, em quantidade absoluta [23,1 ± 2,3 (n=11) e 12,5 ± 0,7 (n=16) mg de peçonha/aranha/extração, para fêmeas e machos, respectivamente]. No entanto, quando a quantidade de peçonha é relacionada ao o peso do animal, os machos possuem maior índice. Além disso, as fêmeas adultas com maior peso apresentaram rendimento de peçonha menor que as mais jovens. A caracterização bioquímicofarmacológica da peçonha revelou através de eletroforese que a peçonha de V. dubius possui poucas proteínas de elevada massa molecular, mas é rica em peptídeos e pequenas moléculas. A peçonha contém bastante hialuronidase, mas é destituída de atividade proteolítica, assim como não foi observada ação hemolítica. No imunoensaio de ELISA, a peçonha apresentou reatividade cruzada com soro antiaracnídico do Instituto Butantan, preparado contra as peçonhas de Loxosceles gaucho, Phoneutria nigriventer e Tityus serrulatus. No entanto, o immunoblotting demonstrou que apenas componentes da peçonha maiores que 30 kDa foram responsáveis por esta reação. A peçonha de V. dubius (10 - 300 µg/mL) foi citotóxica para células leucêmicas em cultura e exerceu forte ação edematogênica (10 µg/sítio causou o extravasamento de 90 ± 20 µL de plasma, n = 4) em pele de ratos, mediada em parte por receptores serotoninérgicos e a formação do óxido nítrico. A peçonha não apresentou significativa toxicidade quando testada em coração semi-isolado de barata. Em músculo liso (íleo de cobaia) a peçonha não causou contratura, mas produziu uma pequena potencialização da ação da bradicinina, e o bloqueio parcial de contrações eletroestimuladas em músculo anococígeo de ratos mediado por uma ação pré-sináptica da peçonha. Em preparação de nervo frênico-diafragma de camundongo, a peçonha (80 µg/mL) bloqueou as contrações por estímulos indiretos (95 ± 6% de bloqueio em 56 ± 28 minutos, n = 3). Uma maior potência de bloqueio foi observada em preparação de biventer cervicis de pintainho, onde 25 µg/mL causou 85 ± 4% de bloqueio de contrações indiretamente estimuladas em 52 ± 6 minutos (n = 4); a peçonha também produziu contratura estável. Além disso, as respostas contráteis desta preparação à Ach e KCl foram diminuídas de forma concentração-dependente pela peçonha. O fracionamento da peçonha resultou em dois grupos de proteínas ativas e um inativo tanto no edema quanto na atividade neuromuscular. Estes dados sugerem que ao menos duas toxinas são responsáveis pelo aumento da permeabilidade vascular local. Além disso, o fracionamento da peçonha indicou a presença de, no mínimo, duas toxinas neuromusculares com ações possivelmente de antagonismo nicotínico reversível e promoção de danos à estrutura funcional muscular, respectivamente. / Abstract: Vitalius dubius is a medium-sized, non-aggressive tarantula found in southeastern Brazil. In this work, we examined the histological organization of the venom apparatus of V. dubius and investigated some biochemical and pharmacological properties of this spider's venom. The venom apparatus consisted of two chelicerae fitted with large fangs for prey immobilization and venom injection. Each chelicera contained bundles of striated muscle involved in fang movement and also housed one venom gland that was surrounded by, but unattached to, these muscle bundles. The gland was closely associated with a helicoidally arranged muscle layer responsible for gland compression and venom extrusion through an inner fang duct that opened on the anterior face of the gland. Within the gland, an elastic basal membrane attached to muscles supported the secretory epithelium. This epithelium consisted of anastomosed cells, with peripheral nuclei and cytoplasmic elongations extending towards the gland lumen in an arrangement organized mainly by F-actin filaments. These elongations supported a complex network of vesicles and cisternae involved in venom biosynthesis. Ultrastructural analysis showed an abundance of organelles involved in protein synthesis, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes. Nuclei with evident nucleoli were seen. During venom production, the main ultrastructural changes were epithelial compactation, greater organelle abundance and alterations in nuclear shape. Venom was milked regularly by electrical stimulation. In absolute terms, females yielded more venom than males [23.1+2.3 mg (n=11) versus 12.5+0.7 mg (n=16) of venom/spider/milking], although when expressed in terms of body weight, males had slightly but significantly greater yields. Venom yield decreased with successive milkings and with spider age. SDS-PAGE showed that the venom contained few high molecular mass proteins, but a variety of small molecules (peptides). The venom was devoid of proteases but contained considerable hyaluronidase activity (estimated molecular mass: 45 kDa). ELISA showed that the venom reacted with immunoglobulins from commercial anti-arachnid antivenom raised against the venoms of Loxosceles gaucho (spider), Phoneutria nigriventer (spider) and Tityus serrulatus (scorpion). Immunoblotting showed that only venom components >30 kDa were responsible for this immunoreactivity. Vitalius dubius venom had no hemolytic activity but was cytotoxic to cultured leukemic cells (up to 300 µg/mL). The venom caused potent, dose-dependent (0.1-100 µg/site) dorsal skin edema in rats that was mediated by serotonin and nitric oxide but not by bradykinin or histamine. The edema-forming activity was not neutralized by commercial anti-arachnid antivenom. The venom (100 µg) was weakly cardiotoxic in the cockroach isolated heart and did not contract non-vascular smooth muscle (isolated guinea pig ileum and rat anoccocygeus), nor did it significantly alter the contractile responses to a variety of agonists in these preparations. However, electrically-induced muscle contractures in the anoccocygeus were attenuated by co-incubation with the venom. Reversible neuromuscular blockade was seen in indirectly stimulated chick biventer cervicis preparations (10-50 µg/mL), with a less potent action in mouse phrenic nerve-diaphragm preparations (at 80 µg/mL). Attenuation of the responses to exogenous acetylcholine (110 µM) and KCl (20 mM) in avian preparations suggested that the venom affected postsynaptic nicotinic receptors and had a direct action on muscle. Marked muscle contracture was also seen with the venom. Fractionation of the venom by reverse-phase chromatography yielded three major groups of proteins, two of which produced both edema and neuromuscular blockade while the third was inactive in these assays. These findings indicate that V. dubius venom contains at least two toxins that cause edema and/or produce neuromuscular blockade. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural