RESUMO

Environmental contaminants have been suggested as a possible cause of fibropapillomatosis (FP) in green sea turtles. In turn, a reduced concentration of serum cholesterol has been indicated as a reliable biomarker of malignancy in vertebrates, including marine turtles. In the present study, metal (Ag, Cd, Cu, Fe, Ni, Pb and Zn) concentrations, oxidative stress parameters [antioxidant capacity against peroxyl radicals (ACAP), protein carbonyls (PC), lipid peroxidation (LPO), frequency of micronucleated cells (FMC)], water content, cholesterol concentration and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity were analyzed in the blood/serum of juvenile (29.3-59.5cm) female green sea turtles (Chelonia mydas) with FP (n=14) and without FP (n=13) sampled at Ubatuba coast (São Paulo State, southeastern Brazil). Green sea turtles were grouped and analyzed according to the severity of tumors. Individuals heavily afflicted with FP showed significantly higher blood Cu, Pb and Fe concentrations, blood LPO levels, as well as significantly lower serum cholesterol concentrations and HMGR activity than turtles without FP. Significant and positive correlations were observed between HMGR activity and cholesterol concentrations, as well as LPO levels and Fe and Pb concentrations. In turn, Cu and Pb concentrations were significantly and negatively correlated with HMGR activity and cholesterol concentration. Furthermore, Cu, Fe and Pb were positively correlated with each other. Therefore, the reduced concentration of serum cholesterol observed in green sea turtles heavily afflicted with FP is related to a Cu- and Pb-induced inhibition of HMGR activity paralleled by a higher LPO rate induced by increased Fe and Pb concentrations. As oxidative stress is implicated in the pathogenesis of viral infections, our findings support the idea that metal contamination, especially by Cu, Fe and Pb, may be implicated in the etiology of FP in green sea turtles through oxidative stress generation.

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Os papilomas são pequenas excrescências verrugosas de etiologia viral que ocorrem mais frequentemente nas narinas, nos lábios e condutos auriculares de todas as espécies animais. São relativamente frequentes em animais de menor faixa etária, durante o período de aleitamento e desmame. Sua disseminação não é bem determinada, porém há fatores imunológicos envolvidos, acreditando-se que o contato direto entre os animais, o uso de materiais contaminados e insetos hematófagos são formas de transmissão. Normalmente o tratamento é cirúrgico, seguido de vacinação autógena. Alguns autores indicam a aplicação de sangue autólogo muscular e da hemo-acupuntura como alternativas terapêuticas. O objetivo do trabalho foi determinar a eficácia hemoterapia em pontos de acupuntura no tratamento de uma borrega com papilomas na face, narinas e ventre. Uma fêmea ovina, mestiça, com cerca de 70 dias de idade, foi encaminhada para Laboratório de Inovação em Cirurgia, Hemoterapia e Terapias Celulares Veterinárias da UFAL (LABINOVET- UFAL) com muitas excrescências verrugosas em narinas, face e ventre, sendo direcionada para o tratamento com acupuntura. As coletas de sangue foram na jugular externa esquerda, com aplicação imediata do sangue no mesmo lado, sendo utilizada a aplicação de 3,5 mL de sangue autólogo em: 1,5 mL ponto tireóide ou imunidade (localizado caudal;-/]mente ao músculo tríceps braquial), 0,5 mL Rim 3 ( localizado medialmente entre o calcâneo e epicôndilo tibial), 0,5 ,L em B12 (na Lina média entre a 5º vértebra torácica e escápula) e 0,5 mL 3m Ho Hai ou VG1( entre ânus e cauda). O animal foi avaliado no momento da primeira aplicação (M1), sendo que as aplicações foram a cada 4 dias, num total de 4 aplicações. As avaliações da melhora do quadro foram realizadas em M2 (4 dias), M3 (8 dias) e M4 (12 dias). Em M2 as lesões haviam diminuído exsudação e ainda apresentavam-se presentes. Em M3, as excrescências haviam diminuído em 50% e com aspecto de cicatrização. Em M4 restavam apenas uma lesão em regressão no lábio. Com a melhora, o paciente não retornou mais para novas sessões de acupuntura. Conclui-se que a hemoterapia em acupontos foi eficaz no tratamento de papilomatose em borrega, necessitando de estudos em maiores populações, para indicação da técnica pouco onerosa na rotina de rebanhos infectados.

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Bovine papillomavirus (BPV) is an oncogenic virus with mucous and epithelial tropism. Possible productive virus infection in other tissues, such as blood, has been hypothesized. In order to investigate this possibility, three samples of skin papillomas and blood were collected from bovines with BPV infection and five samples of peripheral blood and one sample of normal tissue were collected from a calf without BPV infection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and examined by reverse transcription-polymerase chain reaction, immunofluorescence, in situ hybridization, and electron microscopy. The tissue samples were examined for histopathological and immunohistochemical features. The skin papillomas showed the presence of DNA sequences of BPV-2, BPV-11, and a putative virus type. The blood samples showed DNA sequences of BPV-1, 2, and 4 simultaneously. Immunohistochemistry showed BPV L1 protein in both epithelium and stroma and BPV E2 protein in koilocytes. In situ hybridization confirmed the presence of BPV DNA in PBMCs and immunofluorescence showed nuclear labeling of E2 and L1 BPV proteins in PBMCs. The transcription analysis revealed transcripts of BPV-1 L1, BPV-2 L2, and BPV-4 E7 in blood and papilloma samples of BPV-infected cattle. The comet assay revealed high levels of host cell DNA damage upon BPV infection. Electron microscopy analysis of PBMCs identified the presence of particles in the cytoplasm that are consistent with papillomavirus in size and shape. The productive infection of PBMCs with BPV has been previously discussed and this study provides evidence indicating that PBMCs are a target of BPV.

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Packed cell volume (PCV), plasma biochemistry, visual body condition (BC), and calculated body condition index (BCI) were evaluated in 170 wild juvenile green sea turtles Chelonia mydas from an aggregation in the effluent canal of a steel mill in Brazil. Occurrence of cutaneous fibropapillomatosis (FP) was observed in 44.1% of the animals examined. BCI alone did not differ significantly between healthy animals and those afflicted with FP. However, all turtles with low BCI were severely afflicted and were uremic, hypoglycemic, and anemic in relation to healthy animals. Severe FP was not always reflected by a poor health condition of the individual. Clinical evaluation and plasma biochemistry indicated that most animals afflicted with FP were in good health condition. Differences in FP manifestations and associated health conditions in different geographic regions must be assessed by long-term health monitoring programs to help define priorities for conservation efforts.

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La infección persistente por ciertos tipos de alto riesgo oncogénico de virus papiloma humano (VPHAR) es el principal factor de riesgo para el desarrollo de cáncer de cuello uterino y sus lesiones precursoras. Los VPHAR inducen alteraciones moleculares durante todo el proceso de carcinogénesis cervical, que provocan la acumulación de errores genéticos, con la consecuente inestabilidad genética y transformación maligna. Estas alteraciones son producidas por la acción directa de las oncoproteínas virales E6 y E7 sobre las principales proteínas celulares supresoras de tumor, p53 y pRb, respectivamente, y pueden ser monitoreadas durante el surgimiento de la lesión neoplásica, mediante el uso de biomarcadores. En este artículo se revisan las últimas tendencias sobre el uso del estudio inmunocitoquímico, como una prueba complementaria a la citología y a la detección y tipificación de VPHAR en la evaluación de la expresión de biomarcadores como la proteína inhibidora de la proliferación celular p16INK4a, marcador único o combinada con otros biomarcadores, que puedan contribuir eficazmente en la detección de las pacientes con mayor riesgo a desarrollar neoplasia del cuello uterino asociada a la infección por VPHAR, durante la pesquisa de cáncer de cuello uterino de rutina y en el manejo clínico adecuado y oportuno.

Persistent infection with certain types of high oncogenic risk human papillomavirus (HR-HPV) is the main risk factor for developing cervical cancer and its precursor lesions. HR-HPV induces molecular changes during cervical carcinogenesis, causing the accumulation of genetic anomalies, with subsequent genetic instability and malignant transformation. These alterations are produced by the direct action of the E6 and E7 viral oncoproteins on principal tumor cell suppressor proteins, p53 and pRb, respectively, and can be monitored during growth of the neoplastic lesion using biomarkers. In this paper we review the latest trends on the use of immunocytochemistry as a complementary test to cytology and HR-HPV detection and typing in evaluating expression of biomarkers such as the p16INK4a cell proliferation inhibitor protein, as a single marker or combined with other biomarkers, which can contribute effectively to the detection of patients with increased risk of developing cervical neoplasia associated with HR-HPV infection during routine screening for cervical cancer and in appropriate clinical management.

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Ten types of bovine papillomavirus (BPV) have been described and there are reports of viral transmission via blood. The presence of viral DNA in lymphocytes was described to be associated with chromosome instability in these cells. This study presents an evaluation of chromosome instability in short-term peripheral lymphocyte cultures from cows presenting skin papillomatosis, compared with asymptomatic infected animals and non-infected healthy bovines. In a total of 2203 cells, 918 (42%) showed at least one chromosome aberration: 42.7 (± 7.8) in animals with papillomatosis (BPV + W), 40.2 (± 11) in asymptomatic animals (BPV-W) and 4 (± 2) in control animals. Significant differences were found between the infected group (with or without symptoms) and the control group (P < 0.0001). The increased frequencies of chromosome aberrations suggest an interaction between the virus and host cell chromatin.

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The green turtle Chelonia mydas feeds and nests in the Brazilian coastal area and is considered an endangered species by the World Conservation Union (IUCN 2009) and threatened by the Red List of Brazilian Fauna (Ministério do Meio Ambiente 2009). Fibropapillomatosis is a disease characterized by benign skin tumors (fibropapillomas), and it is one of the main threats to the survival of this species. Studies suggest the involvement of viruses as infectious agents associated with environmental and genetic factors. Blood samples were collected from 45 turtles captured in the coastal area of the state of Sao Paulo, Brazil. From these, 27 were affected by fibropapillomas and 18 were tumor free. Biometrical data on the turtles, size, location and quantity of tumors were recorded. The area occupied by fibropapillomas per animal was calculated and four groups were determined according to severity of the disease or its absence. The objective of the study was to compare hemogram results of the sea turtles classified in these four groups. The lowest hematocrit value was observed in severely affected animals. In the hemoglobin assay, the highest value was observed in the group of tumor free turtles and the lowest, in animals severely affected. Lymphocyte counts and curved carapace length were on the verge of statistical significance.

Chelonia mydas, denominada tartaruga verde, é uma tartaruga marinha que frequenta o litoral brasileiro para alimentação e nidificação e é considerada em perigo de extinção pela IUCN (World Conservation Union, 2009) e ameaçada pela Lista Vermelha da Fauna Brasileira (Ministério do Meio Ambiente, 2009). A fibropapilomatose, doença caracterizada por tumores cutâneos benignos (fibropapilomas), é uma das mais importantes ameaças à sobrevivência dessa espécie. Pesquisas sugerem o envolvimento de agentes infecciosos virais em associação com fatores ambientais e genéticos. Foram colhidas amostras sanguíneas de 45 tartarugas provenientes do litoral do estado de São Paulo, Brasil, sendo 18 sem fibropapilomas e 27 acometidas. Dados de biometria das tartarugas, quantidade, localização e tamanho dos tumores foram anotados. Foi realizado o cálculo da área de fibropapilomas por animal e foram estipulados 4 grupos de acordo com o grau de acometimento e sua ausência. O objetivo foi realizar uma comparação entre os hemogramas das tartarugas marinhas classificadas nos 4 grupos. Animais de grau grave apresentaram o menor valor para hematócrito. Para dosagem de hemoglobina, observou-se que o maior valor foi para o grupo de tartarugas sem fibropapilomas e o menor para o de grau grave. Os valores de linfócitos e comprimento curvilíneo da carapaça beiraram a significância estatística.

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RESUMO

The green turtle Chelonia mydas feeds and nests in the Brazilian coastal area and is considered an endangered species by the World Conservation Union (IUCN 2009) and threatened by the Red List of Brazilian Fauna (Ministério do Meio Ambiente 2009). Fibropapillomatosis is a disease characterized by benign skin tumors (fibropapillomas), and it is one of the main threats to the survival of this species. Studies suggest the involvement of viruses as infectious agents associated with environmental and genetic factors. Blood samples were collected from 45 turtles captured in the coastal area of the state of Sao Paulo, Brazil. From these, 27 were affected by fibropapillomas and 18 were tumor free. Biometrical data on the turtles, size, location and quantity of tumors were recorded. The area occupied by fibropapillomas per animal was calculated and four groups were determined according to severity of the disease or its absence. The objective of the study was to compare hemogram results of the sea turtles classified in these four groups. The lowest hematocrit value was observed in severely affected animals. In the hemoglobin assay, the highest value was observed in the group of tumor free turtles and the lowest, in animals severely affected. Lymphocyte counts and curved carapace length were on the verge of statistical significance.(AU)

Chelonia mydas, denominada tartaruga verde, é uma tartaruga marinha que frequenta o litoral brasileiro para alimentação e nidificação e é considerada em perigo de extinção pela IUCN (World Conservation Union, 2009) e ameaçada pela Lista Vermelha da Fauna Brasileira (Ministério do Meio Ambiente, 2009). A fibropapilomatose, doença caracterizada por tumores cutâneos benignos (fibropapilomas), é uma das mais importantes ameaças à sobrevivência dessa espécie. Pesquisas sugerem o envolvimento de agentes infecciosos virais em associação com fatores ambientais e genéticos. Foram colhidas amostras sanguíneas de 45 tartarugas provenientes do litoral do estado de São Paulo, Brasil, sendo 18 sem fibropapilomas e 27 acometidas. Dados de biometria das tartarugas, quantidade, localização e tamanho dos tumores foram anotados. Foi realizado o cálculo da área de fibropapilomas por animal e foram estipulados 4 grupos de acordo com o grau de acometimento e sua ausência. O objetivo foi realizar uma comparação entre os hemogramas das tartarugas marinhas classificadas nos 4 grupos. Animais de grau grave apresentaram o menor valor para hematócrito. Para dosagem de hemoglobina, observou-se que o maior valor foi para o grupo de tartarugas sem fibropapilomas e o menor para o de grau grave. Os valores de linfócitos e comprimento curvilíneo da carapaça beiraram a significância estatística.(AU)

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Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles, Alexa Fluor 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

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RESUMO

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Chelonia mydas é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e uma doença denominada fibropapilomatose é uma das mais importantes ameaças à sobrevivência dessa espécie. Desta forma, a definição de uma metodologia que permita analisar a função dos leucócitos sangüíneos torna-se extremamente necessária. Foram utilizadas amostras sangüíneas de C. mydas com e sem fibropapilomas capturadas no litoral norte do estado de São Paulo. As amostras sangüíneas foram colocadas em tubos contendo heparina sódica e transportadas em meio de cultura celular RPMI 1640 estéril e sob refrigeração. Os leucócitos foram obtidos por gradiente de densidade usando Ficoll-PaqueTM Plus, Amershan Biociences®. Os estímulos aplicados foram Miristato Acetato de Phorbol (PMA) para avaliação de burst oxidativo e Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate para avaliação de fagocitose. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo.

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