A biocomplex to repair experimental critical size defects associated with photobiomodulation therapy

Abstract Background: The association of scaffolds to repair extensive bone defects can contribute to their evolution and morphophysiological recomposition. The incorporation of particulate biomaterials into three-dimensional fibrin bioproducts together with photobiomodulation therapy (PBM) has potential and can improve regenerative medicine procedures. The objective of this experiment was to evaluate the effects of PBM therapy on critical size defects filled with xenogenic bone substitute associated with fibrin biopolymer. Methods: A critical defect of 8 mm was performed in 36 Wistar male adult rats that were divided into four groups. Groups BC and BC-PBM were defined as controls with defects filled by a clot (without or with PBM, respectively) and groups XS and XS-PBM that comprised those filled with biocomplex Bio-OssTM in association with fibrin biopolymer. PBM was applied immediately after the surgery and three times a week every other day, with the parameters: wavelength of 830 nm, energy density 6.2 J/cm2, output power 30 mW, beam area of 0.116 cm2, irradiance 0.258,62 W/cm2, energy/point 0.72 J, total energy 2.88 J. Fourteen and 42 days after the surgery, animals were euthanatized and subjected to microtomography, qualitative and quantitative histological analysis. Results: The BC-PBM and XS-PBM groups had a similar evolution in the tissue repair process, with a higher density of the volume of new formed bone in relation to the groups without PBM (p = 0.04086; p = 0.07093, respectively). Intense vascular proliferation and bone deposition around the biomaterial particles were observed in the animals of the groups in which biocomplex was applied (XS and XS-PBM). Conclusion: PBM therapy allowed an improvement in the formation of new bone, with a more organized deposition of collagen fibers in the defect area. Biocomplex favored the insertion and permanence of the particulate material in bone defects, creating a favorable microenvironment for accelerate repair process.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Abstract Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

A biocomplex to repair experimental critical size defects associated with photobiomodulation therapy

The association of scaffolds to repair extensive bone defects can contribute to their evolution and morphophysiological recomposition. The incorporation of particulate biomaterials into three-dimensional fibrin bioproducts together with photobiomodulation therapy (PBM) has potential and can improve regenerative medicine procedures. The objective of this experiment was to evaluate the effects of PBM therapy on critical size defects filled with xenogenic bone substitute associated with fibrin biopolymer. Methods: A critical defect of 8 mm was performed in 36 Wistar male adult rats that were divided into four groups. Groups BC and BC-PBM were defined as controls with defects filled by a clot (without or with PBM, respectively) and groups XS and XS-PBM that comprised those filled with biocomplex Bio-OssTM in association with fibrin biopolymer. PBM was applied immediately after the surgery and three times a week every other day, with the parameters: wavelength of 830 nm, energy density 6.2 J/cm2, output power 30 mW, beam area of 0.116 cm2, irradiance 0.258,62 W/cm2, energy/point 0.72 J, total energy 2.88 J. Fourteen and 42 days after the surgery, animals were euthanatized and subjected to microtomography, qualitative and quantitative histological analysis. Results: The BC-PBM and XS-PBM groups had a similar evolution in the tissue repair process, with a higher density of the volume of new formed bone in relation to the groups without PBM (p = 0.04086; p = 0.07093, respectively). Intense vascular proliferation and bone deposition around the biomaterial particles were observed in the animals of the groups in which biocomplex was applied (XS and XS-PBM). Conclusion: PBM therapy allowed an improvement in the formation of new bone, with a more organized deposition of collagen fibers in the defect area. Biocomplex favored the insertion and permanence of the particulate material in bone defects, creating a favorable microenvironment for accelerate repair process.(AU)

Launching a CDMO in Brazil aiming to develop biopharmaceuticals for clinical trials

The innovation timeline is expensive, risky, competitive, time-consuming, and labor-intensive. In order to overcome such challenges and optimize financial resources, pharmaceutical companies nowadays hire contract development and manufacturing organizations (CDMO) to help them. Based on the experience acquired first from the development of two biopharmaceuticals, the Heterologous Fibrin Sealant and the Apilic Antivenom, and more recently, during their respective clinical trials; the Center for the Study of Venoms and Venomous Animals (CEVAP) proposed to the Ministry of Health the creation of the first Brazilian CDMO. This groundbreaking venture will assist in converting a candidate molecule - from its discovery, proof of concept, product development, up to pilot batch production - into a product. The CDMO impact and legacy will be immense, offering service provision to the public and private sector by producing validated samples for clinical trials and academic training on translational research for those seeking a position in pharmaceutical industries and manufacturing platforms.(AU)

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)

Traceability of animal protein byproducts in ruminants by multivariate analysis of isotope ratio mass spectrometry to prevent transmission of prion diseases

Ruminant feed containing animal byproduct proteins (ABPs) is prohibited in many countries due to its risk of transmitting prion diseases (PD). In most cases the entire herd is sacrificed, which causes great harm to the producer countries by preventing their exportation of ruminant derived-products. Methods: We used stable isotope ratio mass spectrometry (IRMS) of carbon (13C/12C) and nitrogen (15N/14N) to trace the animal protein in the blood of 15 buffaloes (Bubalus bubalis) divided into three experimental groups: 1 - received only vegetable protein (VP) during 117 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets containing 13.7% bovine meat and bone meal (MBM) added to a vegetable diet (from days 21-117 in the AVP group and until day 47 in the AVPR group, when MBM was removed). Results: On the 36th day, differences were detectable in the feeding profile (p <0.01) among the three experimental groups, which remained for a further 49 days (85th day). The AVPR group showed isotopic rate reversibility on the 110th day by presenting values similar to those in the control group (VP) (p> 0.05), indicating that it took 63 days to eliminate MBM in this group. Total atoms exchange (> 95%) of 13C and 15N was observed through incorporation of the diet into the AVP and AVPR groups. Conclusions: IRMS is an accurate and sensitive technique for tracing the feeding profile of ruminants through blood analysis, thus enabling investigation of ABP use. enabling investigation of ABP use.(AU)

Honey bee envenoming in Santa Catarina, Brazil, 2007 through 2017: an observational, retrospective cohort study

Abstract INTRODUCTION: We evaluated the epidemiological and clinical profile of reported cases of bee sting incidents in Santa Catarina, Brazil. METHODS: This retrospective cohort study included all reported cases of bee sting incidents among the population of Santa Catarina from 2007 to 2017. RESULTS: In total, 8,912 cases were reported, corresponding to an overall rate of 12.3/100,000 population. The mean age was 29,8 years with 60.2% men. The lethality rate was 0.2%. CONCLUSIONS: Santa Catarina has a high incidence rate of bee stings, which is higher than the national average. The data presented in this study may be underestimated.

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.(AU)

In vitro activity of phospholipase A 2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction (<3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 μg/mL for PLA2 and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 μg/mL and 16.98 μg/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.(AU)

Africanized honeybee stings: how to treat them / Picadas de abelhas africanizadas: como tratá-las?

INTRODUCTION: In 1956, Africanized honeybees (AHB) migrated from Brazil to other regions of the Western Hemisphere, including South, Central, and North America, except for Canada. Despite being productive, they are highly aggressive and cause fatal accidents. This study aimed to evaluate patients at the Clinical Hospital of Botucatu Medical School (HC-FMB) and to propose treatment guidelines. METHODS: From 2005 to 2006, the clinical and laboratorial aspects of 11 patients (7 male and 4 female) and the anatomopathological aspects of one patient who had died in 2003 were analyzed. RESULTS: The age of the surviving patients varied from 5 to 87 years, with a mean of 42.5 years. The majority of accidents occurred in the afternoon, and the number of stings ranged from 20 to 500. The principal signs and symptoms were pain and local inflammatory signs, nausea, tachycardia, and vomiting. Biochemical findings presented increased levels of creatine phosphokinase, lactate dehydrogenase, and aspartate/alanine aminotransferase. An 11-year-old male patient died upon entering the attic of a two-storey building where he was attacked by a swarm, receiving more than 1,000 stings. He was sent to HC-FMB where he was treated, but he died 24h later. Observed at the autopsy were erythematous-purpuric skin lesions besides necrosis at the sting locations, rhabdomyolysis, focal myocardial necrosis, tubular hydropic degeneration and focal tubular acute necrosis of the kidneys, myoglobinuria, and centrolobular necrosis in the liver. CONCLUSIONS: Accidents caused by multiple AHB stings always constitute a medical emergency. As there is no specific antivenom, we have developed guidelines, including first aid, drugs, and the proper removal of stingers.

INTRODUÇÃO: As abelhas africanizadas (AHBs) migraram do Brasil em 1956 para todo o continente Americano. Apesar de produtivas, são agressivas causando acidentes fatais. O objetivo foi avaliar pacientes atendidos no Hospital das Clínicas da Faculdade de Medicina de Botucatu (HC-FMB) e propor um roteiro de tratamento. MÉTODOS: Entre 2005 e 2006, foram analisados os aspectos clínicos e laboratoriais de 11 pacientes e anatomopatológicos de um que foi a óbito em 2003. RESULTADOS: A idade dos pacientes variou entre 5 e 87 com média de 42,5 anos. Sete eram do sexo masculino e quatro do feminino. O número de picadas variou entre 20 e 500. Nove deles receberam mais de 50 picadas. Os principais sinais e sintomas foram dor local, náuseas, taquicardia e vômitos. Os exames hematológicos mostraram leucocitose, neutrofilia, anemia e desvio à esquerda escalonado. Os exames bioquímicos revelaram níveis elevados de creatinofosfoquinase, desidrogenase lática e aspartato/alanina aminotransferase. O paciente que foi a óbito 24h após o atendimento tinha 11 anos, era do sexo masculino e foi atacado ao adentrar um edifício de dois andares recebendo mais de 1.000 picadas. O exame anatomopatológico mostrou lesões eritemato-purpúricas, além de necrose nos locais das picadas. Apresentou também rabdomiólise, necroses focais do miocárdio, degeneração hidrópica acompanhada de necrose tubular renal aguda, mioglobinúria e necrose centrolobular no fígado. CONCLUSÕES: Os pacientes acometidos por múltiplas picadas necessitam de tratamento imediato e por não dispormos de um soro específico desenvolvemos um roteiro que inclui os primeiros socorros, as drogas a serem empregadas e a retirada dos ferrões corretamente.

Colaboración docente on-line en educación universitaria / Online teaching collaboration in university education / Colaboração docente online na educação universitária

La colaboración docente on-line permite la creación de comunidades virtuales. Las herramientas virtuales de un equipo en red permiten crear clases virtuales en diferentes localizaciones geográficas. Estos ambientes permiten, en la Educación, diseñar transferencia de conocimientos entre alumnos y docentes incluyendo actividades sincrónicas y asincrónicas. En el presente trabajo describimos nuestra experiencia en colaboración docente on-line usando herramientas virtuales en educación universitaria. Los programas de conexión utilizados fueron 1- Skype: software de aplicación para llamadas en Internet (VoIP). 2- LAN. Aula virtual digital usando la URL: http://www.cevap.unesp.br/abertura. htm. Con los diferentes medios de comunicación disertó el docente colaborador a distancia en diferentes días y sitios de emisión y recepción. Los temas fueron: 1-"Cómo preparar una videoconferencia", 2-"Importancia de las TIC en docencia universitaria", 3-"Plataformas virtuales como bases de datos automatizadas en la búsqueda bibliográfica". 4- "Uso del laboratorio virtual en la docencia de Biología Celular, Histología y Embriología". Las herramientas virtuales permiten la colaboración on-line de docentes situados en diferentes lugares geográficos y la formación de recursos humanos que usarán los entornos virtuales en el desempeño de la docencia universitaria.

Online teaching collaboration allows the creation of virtual communities. Virtual tools in a networked team allow creating virtual classes in different geographic locations. These environments allow knowledge transfer between students and faculty, including synchronous and asynchronous activities. In this article, we describe our experience with online teaching collaboration using virtual tools in university education. The software packages were Skype, an application for voice over Internet (VoIP) and LAN, a virtual digital class available at http://www.cevap.unesp.br/abertura.htm. These media were used on different days and at different locations for webcasting and receiving. The themes were: 1) "How to prepare a videoconference", 2) "The importance of ICTs in university teaching", 3) "Virtual platforms such as automated databases in literature searches", and 4) "Using the virtual laboratory in teaching Cell Biology, Histology, and Embryology". Virtual tools allow online collaboration among teachers at different geographic locations, in addition to training human resources to use such tools in order to improve performance in university education.

A colaboração docente on-line permite a criação de comunidades virtuais. As ferramentas virtuais de uma equipe em rede permitem criar aulas virtuais em diferentes localizações geográficas. Estes ambientes permitem na educação a transferência do conhecimento entre alunos e docentes, incluindo atividades síncronas e assíncronas. Neste trabalho descrevemos nossa experiência em colaboração docente on-line, utilizando ferramentas virtuais na educação universitária. Os softwares utilizados foram 1-Skype: Software de aplicação para chamadas na Internet (VoIP). 2- LAN: Aula virtual digital disponibilizado em http://www.cevap.unesp.br/abertura.htm. Estas mídias foram utilizadas em dias e locais de transmissão e recepção diferentes. Os temas foram: 1-"Como preparar uma videoconferência", 2-"Importância das TICs na docência Universitária", 3-"Plataformas virtuais com bases de dados automatizadas na busca bibliográfica", 4-"Uso do laboratório virtual no ensino de Biologia Celular, Histologia e Embriologia". As ferramentas virtuais permitem a colaboração on-line de docentes localizados em diferentes localizações geográficas, além de formar recursos humanos que as usarão para melhorar o desempenho da educação universitária.

Comparação da resposta imune humoral, da capacidade de neutralização dos soros anticrotálicos produzidos em ovinos jovens, avaliação clínica e ponderal entre animais inoculados com veneno de serpente Crotalus durissus terrificus nativo ou irradiado com Cobalto-60 / Comparison of the humoral immune response, the capacity of neutralization of sera anticrotálicos produced in young sheep, clinical and weight evaluation between animals inoculated with snake venom of Crotalus durissus terrificus native or irradiated

A técnica de Elisa foi utilizada para avaliar e comparar a resposta imune humoral de ovinos jovens para a produção de soro anticrotálico. Durante o processo de soroprodução, foi realizada a avaliação clínica e ponderal dos animais. Os parâmetros utilizados foram hemograma completo, dosagem de uréia, creatinina, aspartato aminotransferase, proteínas totais, albumina, globulina. O peso dos animais foi aferido quinzenalmente durante o experimento. A capacidade de neutralização do soro produzido a partir de veneno de serpente Crotalus durissus terrificus, nativo (VN) e irradiado (Vlr) com Cobalto-60 foi verificada por meio de desafios in vitro. Um grupo de seis animais recebeu veneno nativo, o segundo grupo recebeu veneno irradiado e o terceiro grupo foi o controle. Os animais receberam seis imunizações durante 84 dias com intervalo de 14 dias. Houve diferença significativa (p menor que 5%) no teste de ELISA do perfil de anticorpos produzidos pelos grupos experimentais (VN menor que Vlr). Não houve diferença significativa (p menor que 5%) para os testes bioquímicos, hemograma e peso dos animais entre os três grupos testados. O grupo imunizado com veneno irradiado apresentou perfil de anticorpos maior que o grupo imunizado com veneno nativo. A capacidade de neutralização do soro produzido a partir do Vlr foi cinco vezes maior quando comparado ao soro produzido com VN. A avaliação clínica e ponderal, mostrou que ovinos em fase pós-desmame, não tiveram alterado seus perfis fisiológicos e apresentaram um excelente ganho de peso durante o período experimental. Estes resultados demonstram uma nova perspectiva para a utilização de ovinos visando a produção comercial de soro anticrotálico, que poderá ser aplicado no tratamento do envenenamento de seres humanos e animais. O custo para sua produção poderá ser reduzido pela posterior utilização dos ovinos hiperimunizados na alimentação humana.