BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding. (C) 2007 Elsevier Ltd. All rights reserved.

Uso da cola de fibrina na anastomose nervosa : estudo comparativo experimental, em ratos

A procura de um método que permita um melhor resultado na anastomose de um nervo completamente seccionado é antiga. Há muitos anos a técnica, dita convencional, para aproximação dos cotos afastados do nervo lesado, é realizada com pontos de fios microcirúrgicos. Mais recentemente, a utilização da cola de fibrina tem permitido a adesão de tecidos, como pele, fáscia e outras estruturas anatômicas. O uso da cola de fibrina, na aproximação das extremidades nervosas, tem sido propagada pela indústria como um fato incontestável. No entanto, somente com a realização de estudos comparativos clínicos e laboratoriais tornou-se possível comparar a eficácia da cola de fibrina como agente de reconstituição em lesões de nervos periféricos. Com o objetivo de comprovar essa afirmação foi realizado um trabalho através de uma mensuração nas bainhas de mielina de cotos regenerados de nervos entre diferentes tipos de anastomose nervosas em nervo isquiático de ratos da raça Wistar. Com esse mesmo escopo, foi mensurado o número de axônios em regeneração após o uso dessa cola. Foi utilizada uma amostra de 35 ratos divididos em 03 grupos (A, B e C). No grupo A, 25 ratos foram submetidos à cirurgia com o uso da cola de fibrina para a anastomose nervosa. No grupo B, 05 ratos foram submetidos ao mesmo procedimento; entretanto, ao reparo nervoso acrescentou-se a utilização de dois pontos opostos de mononylon 9-0, usando-se cola de fibrina no seu interior. No terceiro grupo (C), 05 ratos foram submetidos a neurorrafia com 6 a 8 pontos de mononylon 9-0 sem auxílio da cola de fibrina (anastomose nervosa convencional). Os animais foram submetidos a um novo procedimento após 90 dias, quando o nervo isquiático tratado foi retirado e encaminhado para o estudo. Após esse procedimento, os animais foram sacrificados. Cabe esclarecer que os espécimens foram submetidas a uma preparação histológica, sendo avaliados. Para tanto, foi realizada uma mensuração da espessura média da bainha de mielina das fibras regeneradas. Também foi realizada uma medição do número de axônios regenerados em um milímetro quadrado. Com base nesses achados, foram comparados os resultados.

Efetividade da terapia fisica descongestiva na cicatrização de úlceras venosas

A venous ulcer is the most serious clinical manifestation of chronic venous failure, and the most frequent in lower limbs, accounting for 70% of all ulcers. Patients may suffer from this infirmity for several years without healing of the lesion if treatment is inadequate. The aim of this investigation was to verify the effectiveness of decongestive physical therapy in the healing of venous ulcers. This is a quasi-experimental, interventionist study, with paired, non-probabilistic sampling, composed of 50 patients divided into two groups: control and intervention, each composed of 25 patients. Both groups were identically treated for six months with daily dressings and the latter also underwent complex physical therapy consisting of a combination of the following techniques: manual lymphatic drainage, compression bandaging, lower limb elevation, myolymphokinetic exercises and skin care. The study was approved (Protocol no. 59/2007) by the Ethics Committee of the State University of Southeast Bahia. The Mann-Whitney and Chi-square tests were applied for data analysis. After statistical analysis the patients who underwent therapy showed a statistically significant difference with respect to wound contraction starting in the second month of treatment. Compared to the control, the intervention group showed a greater reduction in both pain and edema starting in the third and fourth month of therapy, respectively. To reinforce these findings, the mean percentage of tissue present at the base of the ulcer (granulation/fibrin ratio) was calculated. The intervention group showed greater granulation at the base of the ulcer compared to the control, significant from the second month of treatment on. It was observed in this study that lymphotherapy, when compared between the intervention and control groups, accelerated the healing process, and reduced pain and edema in the affected limbs. It is expected, therefore, that these results widen scientific knowledge and we suggest that this therapy be used 78 not only to reduce lymphedema, but also as a treatment option for venous ulcers, given its easy application and low cost. The data, therefore, demonstrated the importance of basic care on the part of a multiprofessional and transdisciplinary health team involved in the healing process of these infirmities, thereby contributing to better quality of life in these individuals

Purification, characterization, crystallization and theoretical molecular modeling of gyroxin fraction from Crotalus durissus terrificus venom (Laurenti, 1768)

The venom of Crotalus durissus terrificus snakes presents various substances, including a serine protease with thrombin-like activity, called gyroxin, that clots plasmatic fibrinogen and promote the fibrin formation. The aim of this study was to purify and structurally characterize the gyroxin enzyme from Crotalus durissus terrificus venom. For isolation and purification, the following methods were employed: gel filtration on Sephadex G75 column and affinity chromatography on benzamidine Sepharose 6B; 12% SDS-PAGE under reducing conditions; N-terminal sequence analysis; cDNA cloning and expression through RT-PCR and crystallization tests. Theoretical molecular modeling was performed using bioinformatics tools based on comparative analysis of other serine proteases deposited in the NCBI (National Center for Biotechnology Information) database. Protein N-terminal sequencing produced a single chain with a molecular mass of similar to 30 kDa while its full-length cDNA had 714 bp which encoded a mature protein containing 238 amino acids. Crystals were obtained from the solutions 2 and 5 of the Crystal Screen Kit (R), two and one respectively, that reveal the protein constitution of the sample. For multiple sequence alignments of gyroxin-like B2.1 with six other serine proteases obtained from snake venoms (SVSPs), the preservation of cysteine residues and their main structural elements (alpha-helices, beta-barrel and loops) was indicated. The localization of the catalytic triad in His57, Asp102 and Ser198 as well as S1 and S2 specific activity sites in Thr193 and Gli215 amino acids was pointed. The area of recognition and cleavage of fibrinogen in SVSPs for modeling gyroxin B2.1 sequence was located at Arg60, Arg72, Gln75, Arg81, Arg82, Lis85, Glu86 and Lis87 residues. Theoretical modeling of gyroxin fraction generated a classical structure consisting of two alpha-helices, two beta-barrel structures, five disulfide bridges and loops in positions 37, 60, 70, 99, 148, 174 and 218. These results provided information about the functional structure of gyroxin allowing its application in the design of new drugs.

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory

Qualidade de vida de pessoas com úlcera venosa: associação dos aspectos sociodemográficos, de saúde, assistência e clínicos da lesão

Venous ulcer (VU) is a lower limbs injury resulting from inadequate return of venous blood in feet or legs. Although it is not a deadly disease, it causes chronic wounds, which seriously undermine patients´ quality of life (QOL) and sometimes leads to drastic family, social, economic and psychological changes. In this sense, there are several aspects that may influence the venous ulcers patients´ QOL. The study´s objective aimed on the association of socio-demographic and health, health care and clinical injury on UV patients‟ QOL. Analytical studies, which consider the complexity of factors involved in changes in UV patients‟ QOL has a cross-sectional and quantitative approach. The HUOL Ethics Committee approved this project (n.279/09). The collection of data lasted a period of 3 months in 2010 and it took place at the clinic of Angiology at Hospital Universitário Onofre Lopes (HUOL). The data sample consisted of 60 patients treated by UV angiologists in the HUOL Surgical Clinic. The results were analyzed with SPSS 15.0 by descriptive and inferential statistics. The study was based on UV patients that were predominantly female, average age of 61.4 years, that had low education level and low family income, with occupations requiring long periods of standing or sitting, but mostly retired, unemployed or laid off due to the disease and/or due to chronic diseases associated with the UV. The study took also into consideration patients that used inappropriate products, that were improperly treated by a professional caregiver, that lacked of adequate guidance and compression therapy, that performed no lifting of the lower limbs and regular exercise, that the time of injury were greater than or equal to six months, that were missing specific laboratory tests. The study‟s reference were on recurrent lesions, medium to large lesions area, bed of the lesion (injuries) with fibrin and/or necrosis, with amount of exudate with medium to large, odorless and no signs of infection, with tissue loss between 1st and 2nd degree, without collecting swab or biopsy and with pain. In general, QOL of researched individuals were considered low, the maximum score was 69 points, which the areas that were mostly influenced were the total scores of QOL functional capacity (0.021), emotional (0.000) and social functioning (0.080). Of the 60 individuals, 53.3% had scores between 40 and 69 points in SF-36, and they had the best scores in sociodemographic and health variables (ρ = 0.049). In respect to the assistance and injury characteristics, patients who scored between 40 and 69 points in SF-36 had better scores on these characteristics. By combining the socio-demographic variables, health, and handling characteristics of the injury, we observed a significant difference (ρ = 0.032) when linking them with the QOL total scores. When analyzing separately the domains of the SF-36 scores on the quality of life, we find that the areas that showed statistical significance were functional ability (ρ = 0.035), appearance (ρ = 0.019), emotional (ρ = 0.000), and mental health (ρ = 0.050). Among the socio-demographic characteristics studied, gender and marital status contributed more to the reduction of QOL and among the variables of assistance and the injury, orientation, reference and area of UV contributed the most. By analyzing these five variables all together in accordance with the overall score obtained in the quality of life, we found a significant correlation (ρ = 0.002); with 6.23 times more chances of patients have better QOL in the presence of these five positive factors. By conducting the Mann Whitney U test between all the five demographic variables, health, and clinical care, we found that this combination also proved to be significant (ρ = 0.006). Therefore, patients with these five variables positive tend to have a better QOL. Based on these results, we reject the null hypothesis (H0) and accept the alternative hypothesis (H1) proposed in this study because we noted that the QOL of patients with UV is associated with sociodemographic and health, health care and clinical aspects of the injury

Ação local do alendronato sódico na reparação óssea de ratos espontaneamente hipertensos (SHR)

FUNDAMENTO: A hipertensão arterial é uma desordem caracterizada por alterações relevantes no tecido ósseo. O alendronato sódico tem indicação no tratamento de doenças ósseas, por causa de sua afinidade pela hidroxiapatita, inibindo as reabsorções ósseas. OBJETIVO: Analisar a ação local do alendronato sódico na reparação óssea de ratos espontaneamente hipertensos (SHR). MÉTODOS: Um defeito ósseo foi criado no fêmur esquerdo de 80 ratos. de acordo com o material utilizado no local, criaram-se quatro grupos: controle (C), amido (Am), alendronato 1 mol (A1) e alendronato 2 mol (A2). Após 7 e 21 dias, os animais foram sacrificados. Foram realizadas análises histológicas e histomorfométricas e os dados foram submetidos a análise de variância (ANOVA) e teste de Tukey (5%). RESULTADOS: Aos 7 dias, observou-se, na área do defeito, tecido conjuntivo com hemorragia e inflamação em todos os grupos. Alguns apresentavam matriz osteóide. Os grupos A1 e A2 apresentaram, ainda, uma rede de fibrina. Aos 21 dias, as trabéculas ósseas fechavam praticamente a extensão do defeito nos grupos C e Am. No grupo A1 de animais machos, observaram-se trabéculas que se irradiavam do canal medular até a área do defeito. Nos grupos A1 e A2, constatou-se apenas a presença de tecido conjuntivo com mínima deposição de osteóide. Um achado histológico marcante foi a formação de tecido ósseo extracortical subperiosteal nos animais dos grupos A1 e A2. CONCLUSÃO: Concluiu-se que a administração do alendronato sódico não contribuiu para o reparo ósseo nos ratos SHR, mas possivelmente tenha sido responsável pelas formações ósseas extracorticais observadas.

Tratamento de linfocele inguinal pós-operatória com injeção de cola de fibrina: relato de caso

O paciente desenvolveu linforragia na região inguinal direita, depois de ponte aorto-bifemoral com enxerto de dácron®. Não respondeu ao tratamento conservador com cuidados locais e compressão. Foi realizado exame de ultra-sonografia Doppler, que evidenciou grande linfocele (6,4 x 3,36 x 6,1 cm), tratada pelo esvaziamento da loja por aspiração e injeção de cola de fibrina (1,6 mL) em seu interior. O paciente se recuperou sem intercorrências e sem recidiva, após 3 meses de seguimento.

Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Treatment of venous ulcers with surgical adhesives derived from snake venom

A Brazilian dictionary states that "ulcer", from the Latin word ulcuserise, is a superficial lesion on skin or mucous membrane characterized by an inflammatory process and loss of tissue. Ulcers are a relatively frequent problem that may be worsen by infection or long-time duration. In these cases, they can represent a serious public health issue, since the increased case number contributes to the burden on the public health service. In recent decades, wound treatment has advanced in technological and scientific aspects regarding both development of new products and techniques. Ongoing research in this area is providing new products for wound treatment. Among newly tested substances that have presented successful results is fibrin glue, a snake venom derivative that promotes reduction of infection and edema as well as bleeding control and pain decrease. Thus, the present study aimed to evaluate fibrin glue effects on ulcers of venous origin in 24 patients that were divided into two groups. The first one (G1) consisted of 11 patients treated without the adhesive while the second group (G2) was composed of 13 persons who were under therapy with the adhesive. The results of this study revealed that patients treated with fibrin glue evolved to a satisfactory healing when compared to the other group. Therefore, fibrin glue is an appropriate alternative for treating leg ulcers with several advantages: easy application, less pain, early hospital discharge, not transmiting diseases, lower cost than the adhesive currently available in Brazil and, finally, it is a Brazilian technology.

Tratamento de feridas cutâneas extensas usando tecido dérmico acelular porcino com e sem cobertura impermeável

FUNDAMENTOS: O tecido dérmico acelular porcino é alternativa para o tratamento de feridas cutâneas. OBJETIVOS: Avaliar a resposta clínica e inflamatória do implante de tecido dérmico acelular porcino, com e sem cobertura impermeável. MÉTODOS: Estudo pareado, longitudinal, criando-se duas feridas cutâneas no dorso de 16 ratos (quatro animais/grupo), em que foi implantado tecido dérmico acelular coberto ou não por impermeável. Os animais foram avaliados e sacrificados sete, 15, 30 e 60 dias após a cirurgia, sendo removidos os tecidos acelulares e adjacentes para avaliação histológica e morfométrica. RESULTADOS: A cobertura impermeável permaneceu sobre o tecido acelular porcino até cerca de 15 dias. O grupo sem impermeável apresentou maior desidratação, com crosta fibrinoleucocitária, edema e reação inflamatória na derme. Sessenta dias após a cirurgia, animais do grupo sem impermeável ainda apresentavam ulcerações, afinamento do epitélio e ausência de queratina, enquanto nos do grupo com impermeável a pele já se encontrava normal. CONCLUSÃO: O tecido dérmico acelular porcino com cobertura impermeável apresentou resultados clínicos e histológicos melhores do que os do tecido dérmico acelular porcino sem impermeável para tratamento de feridas cutâneas extensas.

Fixação de enxerto cutâneo em malha de espessura total com sutura ou cola de fibrina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tissue Reaction to Silver Nanoparticles Dispersion as an Alternative Irrigating Solution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)