Abstract Background Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 (Lys49-PLA2) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation. Methods Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2 myotoxicity. In addition, K115A and K116A mutants – which contribute to decrease cytotoxicity – and the K122A mutant – which decreases both myotoxicity and cytotoxicity – were also used. The H48Q mutant – which does not interfere with membrane damage or myotoxic activity – was used to evaluate if the PLA2 catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls. Results Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response. Conclusions The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain.

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 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.

RESUMO: A crotalfina é um novo peptídeo analgésico que atua em receptores opioides kappa e delta, causando analgesia potente em ratos submetidos a modelos de dorinflamatória, neuropática e oncológica. Este estudo avaliou os efeitos clínicos, comportamental e antinociceptivo da crotalfina em equinos, empregou 18 cavalos da raça Puro Sangue Árabe e foi dividido em três fases. Na Fase I, "efeitos clínicos e comportamentais", a crotalfina não alterou a latência para urinar e defecar; não interferiu nos valores de frequência cardíaca ou respiratória, motilidade intestinal etemperatura retal; e não causou ataxia, ptose labial, palpebral ou de cabeça significativas. Na Fase II, "efeito antinociceptivo na pele íntegra da região escapular ou isquiática", a crotalfina não gerou analgesia significativa. Na Fase III, "efeito antinociceptivo na pele incisada da região escapular ou isquiática", a crotalfina promoveu efeito antinociceptivo efetivo por seis horas e inibição da hiperalgesia por três dias na região isquiática de equinos submetidos ao modelo incisional de dor inflamatória, mas não houve analgesia relevante na região escapular. Conclui-se que uma única injeção de crotalfina (3,8ngkg-1) não causa alterações clínicas ou comportamentais importantes e promove efeito antinociceptivo na região isquiática incisionada durante sete dias em equinos. Além disso, a crotalfina não evoca efeito antinociceptivo relevante na região escapular e nem na pele intacta de cavalos.

ABSTRACT: Crotalphine is a novel analgesic peptide that acts on kappa opioid and delta receptors, causing powerful analgesia in rats submitted to inflammatory, neuropathic or oncologic models of pain. This study evaluated clinical, behavioral and antinociceptive effects caused by crotalphine in horses, employing 18 Arabian horses and it was divided in three phases. In Phase I, "clinical and behavioral effects", crotalphine did not change the latency to urinate and defecate; did not modify the values of cardiac or respiratory rates, intestinal motility and rectal temperature; and did not cause significant ataxia, head, eye and lip ptosis. In Phase II, "antinociceptive effect on intact skin at scapular or ischial region", crotalphine did not cause significant analgesia. In Phase III, "antinociceptive effect on incised skin at scapular or ischial region", crotalphine promoted effective antinociceptive effects for six hours and inhibited hyperalgesia state for three days in the ischial region of horses submitted to incisional model of inflammatory pain, but crotalphine did not evoke relevant analgesic effect on the scapular region. Concluding, intravenous injection of a single dose of crotalphine (3.8ngkg-1) did not cause important clinical or behavioral changes and promotes antinociceptive effect on incised ischial region for seven days in horses. Moreover, crotalphine did not evoke relevant anti nociceptive effect on the scapular region or in intact skin of horses.

Crotalfina é um novo peptídeo analgésico que atua em receptores opioides kappa e delta promovendo potente analgesia em ratos submetidos a modelos de dor inflamatória, neuropática ou oncológica. Talvez a crotalfina possa ser utilizada para tratar a dor em outras espécies. Assim, o objetivo deste estudo foi avaliar a resposta nociceptiva na região escapular e isquiática de cavalos tratados com crotalfina, morfina, U50-488H ou fenilbutazona e submetidos à estimulação térmica na pele íntegra. Dezoito cavalos da raça Puro Sangue Árabe foram alocados em cinco grupos experimentais: GC (5mL NaCl 0,9%), GCRO (3,8mg.kg-1 crotalfina), GK (160 µg.kg-1 U50-488H), GM (0,1mg.kg-1 morfina) e GF (4,4mg.kg-1 fenilbutazona). Os animais foram submetidos ao modelo de dor inflamatória por meio de estimulação térmica (140°C) e durante 24h avaliou-se a latência para o reflexo do frêmito cutâneo na região escapular (LRFCesc) e isquiática (LRFCisq). O U50-488H apresentou efeito antinociceptivo na região isquiática por duas horas, porém, nos demais momentos do grupo GK, bem como nos grupos GC, GCRO, GM e GF, não foi observado efeito antinociceptivo, visto que a LRFCesc e a LRFCisq na pele íntegra de cavalos não aumentaram em 24 horas de avaliação. Portanto, a crotalfina, a morfina, o U50-488H e a fenilbutazona não produziram efeito antinociceptivo relevante em equinos submetidos à estimulação térmica em pele íntegra.

Crotalphine is a novel analgesic peptide that acts on kappa and delta opioid receptors providing powerful analgesia in rats submitted to inflammatory, neuropathic or oncologic pain model. Maybe crotalphine can be used to treat pain in other species. So, the aim of this study was to evaluate nociceptive response at scapular and isquiatic region of horses treated with crotalphine, morphine, U50-488H or phenylbutazone and submitted to thermal stimulation in complete skin. Eighteen Arabian horses were allocated in five experimental groups: GC (5mL NaCl 0.9%), GCRO (3.8ng.kg-1 crotalphine), GK (160 µg.kg-1 U50-488H), GM (0.1mg.kg-1 morphine) and GP (4.4mg.kg-1 phenylbutazone). Animals were submitted to inflammatory pain model by thermal stimulation (140°C) and during 24h latency to skin twitch at scapular and isquiatic region were evaluated. The U50-488H produced antinociceptive effect at isquiatic region along two hours, but, in other moments of GK and in the other groups there was not antinociceptive effect, because LRFCesc and LRFCisq in complete skin of horses did not increase during 24h evaluation. Thus, crotalphine, morphine, U50-488H and phenylbutazone did not cause relevant antinociceptive effect in horses submitted to thermal stimulation in complete skin.

OBJETIVO: Analisar variáveis maternas e infantis associadas à ocorrência de anemia em crianças atendidas por serviços de atenção básica à saúde do município de São Paulo. MÉTODOS: Participaram do estudo 357 crianças, de quatro a 24 meses, de três serviços de atenção básica à saúde da região sul da cidade. As variáveis foram separadas em categorias relacionadas à mãe e à criança. A dosagem de hemoglobina foi realizada por punção digital, com ponto de corte de 11g/dL para o diagnóstico de anemia. O teste t de Student foi aplicado para comparar as médias de hemoglobina de dois grupos independentes; a ANOVA, para três ou mais grupos; e o teste de Mann-Whitney para comparar a velocidade de crescimento e o grupo etário. RESULTADOS: A prevalência de hemoglobina abaixo de 11g/dL foi de 60%. Entre as variáveis maternas, não se constatou associação com a ocorrência de anemia. Houve associação entre anemia, gênero da criança e velocidade de ganho de peso. Quanto à alimentação, não foi encontrada associação entre tempo de aleitamento materno e anemia, mas houve associação com a ingestão quantitativa e qualitativamente pobre em ferro. CONCLUSÕES: A alta prevalência de anemia relacionou-se ao gênero masculino, no qual a velocidade de crescimento em menores de seis meses e naqueles com 18 a 24 meses foi maior, além de se associar à baixa ingestão de ferro na dieta.

OBJECTIVE: To analyze mother and child factors associated with the occurrence of anemia in children assisted at primary health care centers in São Paulo city, Brazil. METHODS: The study enrolled 357 children from four to 24 months assisted at three health care centers in the southern region of the city. Possible factors associated to anemia and related to mother and children characteristics were categorized. Hemoglobin was determined by digital puncture and the cut value for anemia diagnosis was 11g/dL. Student t test was used to compare the hemoglobin means of two independent groups; ANOVA for three or more independent groups, and Mann-Whitney was used to test the association of anemia and age group or speed of growing. RESULTS: The prevalence of anemia was 60%. Anemia was not associated to maternal variables. Weight gain and male gender were associated to a higher prevalence of anemia. Anemia was also associated with poor qualitative or quantitative ingestion of iron. The occurrence of anemia was not avoided by breastfeeding practice. CONCLUSIONS: The high prevalence of anemia was related to masculine gender, which had a faster speed of weight gain in children less than six months old and from 18 to 24 months old. Anemia was also related to low ingestion of iron in the diet.

OBJETIVO: comparar os resultados de ciclos de reprodução assistida em mulheres doadoras de oócitos no ciclo de tratamento com o de mulheres não doadoras. MÉTODOS: foram avaliadas, retrospectivamente, as taxas de gravidez, implantação e abortamento de 50 pacientes que doaram oócitos durante o ciclo de reprodução assistida (grupo de doadoras) e de 50 pacientes que não doaram oócitos (grupo de não-doadoras), em clínica privada de reprodução assistida em São Paulo, entre os anos de 2001 e 2003. Os critérios de inclusão no estudo foram os seguintes: idade menor que 35 anos; ciclos menstruais regulares; dosagem basal de FSH<10 mUI/mL; pacientes submetidas à primeira tentativa de fertilização assistida; recuperação de mais de seis oócitos maduros após a aspiração folicular. Os resultados foram analisados estatisticamente por meio do teste do chi2. RESULTADOS: ambos os grupos eram comparáveis em termos de idade, indicação e duração da infertilidade. A média de idade no grupo de doadoras foi de 30,6 anos e no grupo de não doadoras, 31,1 anos. Todas as pacientes tiveram mais do que seis oócitos aspirados e identificados. No grupo de doadoras foram recuperados 590 oócitos e 215 oócitos foram doados para receptoras (36,5%), levando à transferência de 152 embriões. Foram obtidas 15 gestações (30% por transferência), com 2 abortamentos (13,3%), sendo que a taxa de implantação foi de 11,2%. No grupo de não-doadoras, recuperaram-se 545 oócitos e foram transferidos 153 embriões. Foram obtidas 17 gravidezes (34% por transferência) e houve três abortamentos (17,6%). A taxa de implantação foi de 14,3%. Não houve diferenças estatisticamente significantes entre os dois grupos no que se refere às taxas de gravidez, implantação e abortamento (p>0,05). CONCLUSÃO: em pacientes que recuperam mais de seis oócitos, a doação de oócitos no ciclo de tratamento não prejudica os resultados dos ciclos de reprodução assistida e não eleva as taxas de abortamento.

PURPOSE: to compare the outcome of treatment in patients undergoing assisted reproductive technology (ART) cycles who donated eggs during their own ART treatment with the outcome of patients undergoing ART without egg donation. METHODS: we studied retrospectively the pregnancy and implantation rates of 50 patients who donated eggs during the course of their ART treatment (donor group), and the pregnancy and implantation rates of 50 patients who underwent ART cycles and kept all their eggs (non-donor group). between the years 2001-2003. The inclusion criteria used were as follows: age <35 years old, normal menstrual cycles, FSH<10 mIU/mL, first attempt of ART treatment and more than six mature oocytes retrieved. The results were analyzed statistically using the chi2 test. RESULTS: both groups were comparable in terms of age, indication, and duration of infertility. The mean age was 30.6 years for the donor group and 31.1 years for the non-donor group. All the patients of the donor group produced more than 6 eggs. From the donor group we collected 590 oocytes; 215 eggs were donated to recipients (36.5%) and 152 embryos were transferred. A total of 15 pregnancies were achieved (pregnancy rate per transfer: 30%); the implantation rate was 11.2% and there were 2 miscarriages (miscarriage rate: 13.3%). From the non-donor group, 545 oocytes were collected and 153 embryos were transferred. A total 17 pregnancies were achieved (pregnancy rate per transfer: 34%); the implantation rate was 14.3% and there were 3 miscarriages (miscarriage rate: 17.6%). The pregnancy and implantation rates were similar in both groups, and there were no significant statistical differences regarding the miscarriage rate (p>0.05). CONCLUSION: this study suggests that in patients who produce more than 6 oocytes, egg donation in the treatment cycle does not influence adversely the outcome of ART cycles and does not increase the miscarriage rate.

Foram estudados 59 adolescentes, sendo 37 do sexo feminino e 22 do sexo masculino, que procuraram atendimento no Centro de Saúde Escola da Universidade de Santo Amaro. O objetivo foi verificar se o programa de orientação desenvolvido com adolescentes promoveu conhecimento sobre as questões abordadas durante a realização do referido programa (sexualidade, doenças sexualmente transmissíveis, Aids, drogas). Antes da realização do programa, os adolescentes responderam a um questionário avaliativo contendo questões com temas específicos dessa fase da vida. No final do processo, o questionário foi novamente respondido. O confronto entre as respostas pré e pós-programa serviu como aferição do conhecimento adquirido pelos adolescentes. Os resultados mostraram aumento significativo de conhecimento após o programa desenvolvido por uma equipe multidisciplinar e que os sexos não diferiram significantemente em relação aos conhecimentos adquiridos.

Fifty-nine adolescent were analyzed in this study consisted of 37 girls and 22 boys who looked for help at the school-health center at Santo Amaro University. This study aimed verifying in the orientation program developed with the adolescents promoted knowledge about the questions developed during the execution of the referred program (sexuality, sexually transmitted diseases-STD, AIDS, drugs). The adolescents completed an evaluation questionnaire, which included the questions that subsequently would be asked before beginning the program. At the end of the process, they completed the evaluation questionnaire again. The comparison between the previous and post-program answers served as an assessment of the knowledge acquired by the adolescents. The results showed a significant increase of knowledge about the themes developed by a multidisciplinary staff and the gender did not differ significanthy in relation to the acquired knowledge.

A IRC (insuficiência renal crônica) terminal é acompanhada por alterações sistêmicas, inclusive do eixo hipotálamo-hipofisário-gonadal. Após o aparecimento da EPO (eritropoetina humana recombinante), surgiram relatos de melhora da disfunção sexual em homens, havendo relativa escassez de dados sobre dosagens hormonais e função sexual das mulheres. No Brasil, há poucos dados sobre o perfil hormonal de mulheres em programa crônico de diálise. OBJETIVOS: Avaliação e comparação do perfil hormonal sexual, através das dosagens de FSH (hormônio folículo estimulante), LH (hormônio luteinizante), PRL (prolactina) e E2 (estradiol) de mulheres em programa crônico de diálise, sob terapêutica com EPO. MÉTODOS: Foram avaliadas 47 mulheres (uma em diálise peritoneal intermitente, quatro em diálise peritoneal ambulatorial contínua e 42 em hemodiálise), subdivididas em três grupos: (1) Grupo I, de mulheres com menos de 48 anos de idade e menstruando regularmente; (2) Grupo II, de mulheres com menos de 48 anos de idade e amenorreicas; (3) Grupo III, de mulheres com mais de 48 anos de idade e amenorreicas. Os grupos foram comparados entre si através do teste de Mann-Whitney para duas amostras independentes. RESULTADOS: Todos os grupos mostraram valores normais das dosagens séricas de FSH, LH e E2, e não houve diferença estatisticamente significante nos níveis desses hormônios entre as mulheres que menstruam e as amenorreicas com idade inferior a 48 anos. Os níveis de PRL encontravam-se acima da normalidade em todos os grupos, independentemente da idade e do padrão menstrual, não havendo diferenças estatísticas significantes entre os grupos. As pacientes do grupo III apresentaram perfis hormonais compatíveis com a menopausa, ou seja, níveis séricos de FSH e LH elevados e de E2 diminuídos.

OBJECTIVE: The end stage renal disease has accompaniments in body systems, including hormonal changes. The present study was designed to evaluate the pattern of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and estradiol (E2) in 47 women with end stage renal disease (ESRD), on regular dialysis and receiving recombinant human erythropoietin (r-HuEPO). METHODS: One patient was on intermitent peritoneal dialysis, 4 on continuous ambulatory peritoneal dialysis and 42 on maintenance haemodialysis. They were divided into: Group I consisted of 10 women with regular menses, aged lesser than 48 years; Group II consisted of 15 women with amenorrhea, aged lesser than 48 years and; Group III, consisted of 22 women with amenorrhea, aged 48 years or more. Levels of FSH, LH, PRL e E2 were obtained by ELISA. RESULTS: All groups showed FSH, LH and E2 levels in the normal range for normal women without renal failure, and there was no statistical significance in the levels of these hormones between women with regular menses or women with amenorrhea aged lesser than 48 years. The serum concentrations of PRL were above the normal range in all groups. The patients aged 48 or more showed hormonal profile consistent with post-menopausal (i.e. high FSH, high LH and low estradiol). CONCLUSIONS: We conclude that ovarian function is intact on ESRD and we have to search for other causes for amenorrhea in women that do not have na abnormal hormonal sex profile.

Foram estudados 59 adolescentes, sendo 37 do sexo feminino e 22 do sexo masculino, que procuraram atendimento no Centro de Saúde Escola da Universidade de Santo Amaro. O objetivo foi verificar se o programa de orientação desenvolvido com adolescentes promoveu conhecimento sobre as questões abordadas durante a realização do referido programa (sexualidade, doenças sexualmente transmissíveis, Aids, drogas). Antes da realização do programa, os adolescentes responderam a um questionário avaliativo contendo questões com temas específicos dessa fase da vida. No final do processo, o questionário foi novamente respondido. O confronto entre as respostas pré e pós-programa serviu como aferição do conhecimento adquirido pelos adolescentes. Os resultados mostraram aumento significativo de conhecimento após o programa desenvolvido por uma equipe multidisciplinar e que os sexos não diferiram significantemente em relação aos conhecimentos adquiridos.

Fifty-nine adolescent were analyzed in this study consisted of 37 girls and 22 boys who looked for help at the school-health center at Santo Amaro University. This study aimed verifying in the orientation program developed with the adolescents promoted knowledge about the questions developed during the execution of the referred program (sexuality, sexually transmitted diseases-STD, AIDS, drugs). The adolescents completed an evaluation questionnaire, which included the questions that subsequently would be asked before beginning the program. At the end of the process, they completed the evaluation questionnaire again. The comparison between the previous and post-program answers served as an assessment of the knowledge acquired by the adolescents. The results showed a significant increase of knowledge about the themes developed by a multidisciplinary staff and the gender did not differ significanthy in relation to the acquired knowledge.

A mionecrose é um dos efeitos causados pelo veneno de Bothrops jararacussu. Uma miotoxina com homologia estrutural à fosfolipase A2 (PLA2), mas sem atividade enzimática, foi isolada desse veneno. O veneno de Crotalus durissus terrificus apresenta também atividade miotóxica, que vem sendo atribuída à crotoxina e à PLA2 (crotoxina B), o componente básico do complexo crotoxina. O veneno de Bothrops jararacussu apresenta três proteínas, que têm identidade imunológica com a PLA2 da crotoxina. O presente trabalho comparou a eficiência dos antivenenos polivalentes comerciais produzidos pelo Instituto Butantan - o antiveneno botrópico (AB) e o antiveneno botrópico-crotálico (AB/C) - na neutralização das atividades letal, hemorrágica, coagulante e miotóxica do veneno de B. jararacussu. Os dois antivenenos neutralizaram de maneira semelhante a atividade hemorrágica, mas o AB/C foi três vezes mais potente que o AB em neutralizar a ação miotóxica e duas vezes mais potente na neutralização da letalidade e na ação coagulante do veneno de B. jararacussu. Os dados sugerem que a utilização do AB/C pode ser vantajosa no tratamento de pacientes picados por serpentes dessa espécie

Myonecrosis is one of the effects of Bothrops jararacussu venom, from which a myotoxin was isolated showing structural homology to phospholipase A2 (PLA2), but without enzimatic activity. Such myotoxic activity is also present in the Crotalus durissus terrificus venom, and is atributed to crotoxin and to PLA2 (crotoxin B), the basic component of the crotoxin complex. The Bothrops jararacussu venom showed three proteins with immunologic identity to PLA2 from crotoxin. The bothropic (AB) and the bothropic/crotalic (AB/C) antivenoms, two commercial polyvalent antivenoms produced at Instituto Butantan, were compared in order to assess their capacity for neutralization of the lethal, hemorrhagic, coagulant and myotoxic activities of Bothrops jararacussu venom. Both antivenoms showed the same level of hemorrhagic activity neutralization. However, AB/C was about three times more efficient than AB in neutralizing the myotoxic activity, and two times more potent for neutralization of lethality and coagulant activity of Bothrops jararacussu venom. These data suggest that the use of AB/C could be of value in the treatment of patients bitten by snakes of this species