Profile of extracellular matrix metalloproteinase in healthy and infected Toggenburg goats with small ruminant lentivirus in Southeast Brazil / Perfil das metaloproteínas de matriz extracelular em cabras Toggenburg sadias e infectadas por lentivírus de pequenos ruminantes no Sudeste brasileiro

Small ruminant lentiviruses (SRLV) are difficult to diagnose due to their escape mechanisms. Therefore, proteomics is an alternative in the search for biomarkers through extracellular matrix metalloproteinases (MMPs), enzymes related to the immune response. In this sense, this study aimed to analyze the profile of MMPs in healthy and infected Toggenburg goats with chronic SRLV infection in Southeast Brazil. Five positive and five negative goats for SRLV were selected using the agar gel immunodiffusion (AGID) microtechnique, western blot (WB), and nested polymerase chain reaction (nPCR). All animals were submitted to blood collection by puncture of the jugular vein, followed by centrifugation to obtain blood plasma, protein quantification by the Bradford method, one-dimensional electrophoretic separation (1D), and identification of protease activity by zymography and confirmation via reverse zymography in the presence of MMP-2 through the action of tissue inhibitors (TIMP-2). The analysis of protein bands was performed using descriptive statistics and densitometry values for zymography were subjected to the Shapiro-Wilk test to determine normality. Little difference was observed in the occurrence of protein bands between groups. Regarding MMPs, no differences were observed in the expression of proMMP-9, MMP-9, and MMP-2 in animals affected by SRLV. TIMP-2 inhibited proMMP-2 and MMP-2 in all animals. Thus, the profile of protein bands does not change in healthy goats with chronic SRLV infection. The TIMP-2 expression allowed proving the existence of MMP-2 in animals chronically infected by SRLV via reverse zymography

Lentivírus de pequenos ruminantes (LVPR) demonstram diagnóstico complexo devido seus mecanismos de escape. Desse modo, a proteômica apresenta-se como alternativa na busca por biomarcadores através das metaloproteinases da matriz extracelular (MMPs), enzimas ligadas a resposta imunológica. Assim, objetivou-se analisar o perfil das MMPs em cabras Toggenburg sadias e com infecção crônica por LVPR no Sudeste brasileiro. Selecionou-se cinco cabras positivas e cinco negativas para LVPR utilizando: microtécnica de imunodifusão em gel de agarose (MIDGA), Western Blot (WB) e reação em cadeia da polimerase nested (nPCR). Todas foram submetidas à coleta de sangue por punção da veia jugular, seguido de centrifugação para obtenção do plasma sanguíneo, quantificação proteica pelo método Bradford, separação via eletroforese unidimensional (1D), e identificação da atividade das proteases por zimografia e confirmação via zimografia reversa na presença da MMP-2 por meio da ação de inibidores teciduais (TIMP-2). A análise das bandas proteicas ocorreu através de estatística descritiva e para a zimografia os valores de densitometria foram submetidos ao teste de Shapiro-Wilk para determinar a normalidade. Observou-se pouca distinção na ocorrência das bandas proteicas entre os grupos. Em relação as MMPs, não houve diferenças na expressão da proMMP-9, MMP-9 e MMP-2 nos acometidos por LVPR. Observou-se que a TIMP-2 inibiu a proMMP-2 e MMP-2 em todos os animais. Dessa forma, o perfil de bandas proteicas não se altera em cabras sadias e com infecção crônica por LVPR. Através da expressão da TIMP-2 foi possível comprovar a existência da MMP-2 em animais cronicamente infectados por LVPR via zimografia reversa

Study of metalloproteinases in the blood of goats experimentally infected with caprine encephalitis arthritis virus / Estudo de metaloproteinases no sangue de caprinos experimentalmente infectados pelo vírus da artrite encefalite caprina

Caprine arthritis encephalitis is a lentiviral disease that leads to considerable losses in goat farming. In the acute phase of viral infection, though antiviral antibodies are produced by the host's immune system, they are not sufficient to be detected by serological tests. Acute infections begin with an incubation period, during which the viral genome replicates and host innate responses are initiated. Matrix metalloproteinases (MMPs) are enzymes that play an important role in the physiological and pathological processes of tissue remodeling. The present study aimed to evaluate the expression of MMPs and their activity in the blood serum of male goats experimentally infected with caprine arthritis encephalitis virus (CAEV). Five dairy male goats, aged 3-4 years, were intravenously inoculated with CAEV Cork strain (titer: 105-6 TCID50/mL) after being tested negative for CAEV thrice at consecutive intervals of 30 days using western blot analysis and nested-PCR. The study included three stages: S1 or pre-infection stage; S2 or seroconversion stage, corresponding to the occurrence of first seroconversion; and S3 or post-seroconversion stage, corresponding to 23 weeks after seroconversion. Zymography was performed for the samples using gelatin zymography gels (12.5%), which were subjected to electrophoresis at 170V, 1A, and 300W for 50-70 min. The density of MMP-2 was found to be lower at S1 (1456.20 pixels) than that at S2 and S3 (1943.80 and 2104.40 pixels, respectively) (P < 0.05); and the density of MMP-9 was found to be lower at S3 (133.60 pixels) than that at S1 and S2 (359.60 and 370.60 pixels, respectively). The density of proMMP-2 was low at S1 and S3 (130.45 and 145.20 pixels, respectively). On the other hand, the density of proMMP-9 was statistically different between S1 and S3 (89.22 vs. 415.60 pixels). Both proMMP-2 and proMMP-9 were absent at S2. Thus, MMP-2 and MMP9 exhibited opposite behaviors depending on the stage [...].(AU)

A encefalite por artrite caprina é uma doença lentiviral que leva a perdas consideráveis na criação de caprinos. Na fase aguda da infecção viral, embora os anticorpos antivirais sejam produzidos pelo sistema imunológico do hospedeiro, eles não são suficientes para serem detectados por testes sorológicos. As infecções agudas começam com um período de incubação, durante o qual o genoma viral se replica e as respostas inatas do hospedeiro são iniciadas. As metaloproteinases da matriz (MMPs) são enzimas que desempenham um papel importante nos processos fisiológicos e patológicos da remodelação tecidual. O presente estudo teve como objetivo avaliar a expressão de MMPs e sua atividade no soro sanguíneo de reprodutores caprinos infectados experimentalmente pelo vírus da encefalite por artrite caprina(CAEV). Cinco machos caprinos, com idades entre 3-4 anos, foram inoculadas por via intravenosa com a cepa de CAEV Cork (título: 105-6 TCID50/mL) após serem testados negativamente para CAEV três vezes em intervalos consecutivos de 30 dias usando a análise de western blot e nested-PCR. O estudo incluiu três etapas: estágio S1 ou pré-infecção; S2 ou estágio de soroconversão, correspondente à ocorrência de primeira soroconversão; e S3 ou pós-soroconversão, correspondendo a 23 semanas após a soroconversão. A zimografia foi realizada para as amostras utilizando gel de gelatina (12,5%), que foram submetidos à eletroforese em 170V, 1A e 300W por 50-70 min. Verificou-se que a densidade de MMP-2era menor em S1 (1456,20 pixels) do que em S2 e S3 (1943,80 e 2104,40 pixels, respectivamente) (P <0,05); e a densidade de MMP-9 foi menor em S3 (133,60 pixels) do que em S1 e S2 (359,60 e 370,60 pixels, respectivamente). A densidade do proMMP-2 era baixa em S1 e S3 (130,45 e 145,20 pixels, respectivamente). Por outro lado, a densidade do proMMP-9 foi estatisticamente diferente entre S1 e S3(89,22 vs. 415,60 pixels). ProMMP-2 e proMMP-9 estavam ausentes no S2. Assim, MMP-2 e [...].(AU)

Study of metalloproteinases in the blood of goats experimentally infected with caprine encephalitis arthritis virus / Estudo de metaloproteinases no sangue de caprinos experimentalmente infectados pelo vírus da artrite encefalite caprina

Caprine arthritis encephalitis is a lentiviral disease that leads to considerable losses in goat farming. In the acute phase of viral infection, though antiviral antibodies are produced by the host's immune system, they are not sufficient to be detected by serological tests. Acute infections begin with an incubation period, during which the viral genome replicates and host innate responses are initiated. Matrix metalloproteinases (MMPs) are enzymes that play an important role in the physiological and pathological processes of tissue remodeling. The present study aimed to evaluate the expression of MMPs and their activity in the blood serum of male goats experimentally infected with caprine arthritis encephalitis virus (CAEV). Five dairy male goats, aged 3-4 years, were intravenously inoculated with CAEV Cork strain (titer: 105-6 TCID50/mL) after being tested negative for CAEV thrice at consecutive intervals of 30 days using western blot analysis and nested-PCR. The study included three stages: S1 or pre-infection stage; S2 or seroconversion stage, corresponding to the occurrence of first seroconversion; and S3 or post-seroconversion stage, corresponding to 23 weeks after seroconversion. Zymography was performed for the samples using gelatin zymography gels (12.5%), which were subjected to electrophoresis at 170V, 1A, and 300W for 50-70 min. The density of MMP-2 was found to be lower at S1 (1456.20 pixels) than that at S2 and S3 (1943.80 and 2104.40 pixels, respectively) (P < 0.05); and the density of MMP-9 was found to be lower at S3 (133.60 pixels) than that at S1 and S2 (359.60 and 370.60 pixels, respectively). The density of proMMP-2 was low at S1 and S3 (130.45 and 145.20 pixels, respectively). On the other hand, the density of proMMP-9 was statistically different between S1 and S3 (89.22 vs. 415.60 pixels). Both proMMP-2 and proMMP-9 were absent at S2. Thus, MMP-2 and MMP9 exhibited opposite behaviors depending on the stage [...].

A encefalite por artrite caprina é uma doença lentiviral que leva a perdas consideráveis na criação de caprinos. Na fase aguda da infecção viral, embora os anticorpos antivirais sejam produzidos pelo sistema imunológico do hospedeiro, eles não são suficientes para serem detectados por testes sorológicos. As infecções agudas começam com um período de incubação, durante o qual o genoma viral se replica e as respostas inatas do hospedeiro são iniciadas. As metaloproteinases da matriz (MMPs) são enzimas que desempenham um papel importante nos processos fisiológicos e patológicos da remodelação tecidual. O presente estudo teve como objetivo avaliar a expressão de MMPs e sua atividade no soro sanguíneo de reprodutores caprinos infectados experimentalmente pelo vírus da encefalite por artrite caprina(CAEV). Cinco machos caprinos, com idades entre 3-4 anos, foram inoculadas por via intravenosa com a cepa de CAEV Cork (título: 105-6 TCID50/mL) após serem testados negativamente para CAEV três vezes em intervalos consecutivos de 30 dias usando a análise de western blot e nested-PCR. O estudo incluiu três etapas: estágio S1 ou pré-infecção; S2 ou estágio de soroconversão, correspondente à ocorrência de primeira soroconversão; e S3 ou pós-soroconversão, correspondendo a 23 semanas após a soroconversão. A zimografia foi realizada para as amostras utilizando gel de gelatina (12,5%), que foram submetidos à eletroforese em 170V, 1A e 300W por 50-70 min. Verificou-se que a densidade de MMP-2era menor em S1 (1456,20 pixels) do que em S2 e S3 (1943,80 e 2104,40 pixels, respectivamente) (P <0,05); e a densidade de MMP-9 foi menor em S3 (133,60 pixels) do que em S1 e S2 (359,60 e 370,60 pixels, respectivamente). A densidade do proMMP-2 era baixa em S1 e S3 (130,45 e 145,20 pixels, respectivamente). Por outro lado, a densidade do proMMP-9 foi estatisticamente diferente entre S1 e S3(89,22 vs. 415,60 pixels). ProMMP-2 e proMMP-9 estavam ausentes no S2. Assim, MMP-2 e [...].

Matrix metalloproteinases 2 and 9 in rabbits with doxorubicin-induced cardiomyopathy / Metaloproteinases de matriz 2 e 9 em coelhos com cardiomiopatia induzida pela doxorrubicina

Some studies have shown the role played by matrix metalloproteinases and their inhibitors in doxorubicin cardiotoxicity. In this study, we sought to investigate how plasma and myocardial MMP 2 and 9 perform in rabbits with doxorubicin-induced cardiomyopathy, searching for a correlation between the activity of these collagenases and cardiac remodeling. Cardiomyopathy was induced by doxorubicin given intravenously twice a week for six consecutive weeks. Plasma MMP activity and the echocardiogram were assessed at baseline, and at 15 and 45 days after first injection of doxorubicin. The myocardial activity of these enzymes was solely evaluated in nine rabbits at 45 days, and results were compared with nine healthy controls. We only identified the full-length forms of both MMP 2 and 9 throughout the study. The plasma pro-MMP 2 reduced along the deterioration of cardiac function, while the pro-MMP 9 increased significantly at T45 as compared to baseline and T15. A negative significant correlation was found to exist between the plasma activity of pro-MMP 2 and mitral E-to-mitral septal annular early diastolic velocity ratio, which is an estimate of mean left atrial pressure and congestion. Only pro-MMP 2 was found in myocardial samples, and mean activity of such enzyme was statistically lower than that recorded for healthy controls. Although no active form was documented for either collagenase, the duration of the treatment with doxorubicin played a role in the alteration of plasma pro-forms activity. However, these changes could not be associated with most echocardiographic parameters that are supportive of cardiac remodeling.(AU)

Alguns estudos já demonstraram o papel exercido pelas metaloproteinases de matriz e seus inibidores na cardiotoxicidade promovida pela doxorrubicina. Assim, este estudo teve como objetivo investigar o comportamento das MMPs 2 e 9 plasmáticas e miocárdicas em coelhos com cardiomiopatia induzida pela doxorrubicina, buscando determinar se há correlação entre a atividade dessas colagenases e o remodelamento cardíaco. A cardiomiopatia foi induzida pela doxorrubicina aplicada por via intravenosa duas vezes por semana ao longo de seis semanas consecutivas. A atividade plasmática das MMPs e o ecocardiograma foram avaliados no momento basal e aos 15 e 45 dias após a primeira aplicação da doxorrubicina. A atividade miocárdica dessas enzimas foi quantificada em apenas nove coelhos aos 45 dias e os resultados comparados com outros nove controles saudáveis. Foram identificadas apenas as formas inativas das MMPs 2 e 9 durante todo o estudo. A pro-MMP 2 plasmática reduziu à medida que a função cardiaca se deteriorou, enquanto a pro-MMP 9 aumentou significativamente em T45 quando comparada aos momentos basal e T15. Houve correlação negativa significativa entre a atividade plasmática da pro-MMP 2 e a relação entre E mitral e a velocidade anular mitral no início da diástole, um parâmetro que permite estimar a pressão atrial esquerda média e a congestão. Apenas a pro-MMP 2 foi documentada nas amostras miocárdicas dos coelhos com cardiomiopatia e atividade media dessa enzima foi estatisticamente menor que aquela observada nos controles saudáveis. Embora a forma ativa de ambas as colagenases não tenha sido identificada, o tempo de tratamento com doxorrubicina interferiu na atividade das formas inativas plasmáticas. Contudo, essas alterações não se associaram com a maioria dos parâmetros ecocardiográficos que indicam remodelamento cardíaco.(AU)

Matrix metalloproteinases 2 and 9 in rabbits with doxorubicin-induced cardiomyopathy / Metaloproteinases de matriz 2 e 9 em coelhos com cardiomiopatia induzida pela doxorrubicina

Some studies have shown the role played by matrix metalloproteinases and their inhibitors in doxorubicin cardiotoxicity. In this study, we sought to investigate how plasma and myocardial MMP 2 and 9 perform in rabbits with doxorubicin-induced cardiomyopathy, searching for a correlation between the activity of these collagenases and cardiac remodeling. Cardiomyopathy was induced by doxorubicin given intravenously twice a week for six consecutive weeks. Plasma MMP activity and the echocardiogram were assessed at baseline, and at 15 and 45 days after first injection of doxorubicin. The myocardial activity of these enzymes was solely evaluated in nine rabbits at 45 days, and results were compared with nine healthy controls. We only identified the full-length forms of both MMP 2 and 9 throughout the study. The plasma pro-MMP 2 reduced along the deterioration of cardiac function, while the pro-MMP 9 increased significantly at T45 as compared to baseline and T15. A negative significant correlation was found to exist between the plasma activity of pro-MMP 2 and mitral E-to-mitral septal annular early diastolic velocity ratio, which is an estimate of mean left atrial pressure and congestion. Only pro-MMP 2 was found in myocardial samples, and mean activity of such enzyme was statistically lower than that recorded for healthy controls. Although no active form was documented for either collagenase, the duration of the treatment with doxorubicin played a role in the alteration of plasma pro-forms activity. However, these changes could not be associated with most echocardiographic parameters that are supportive of cardiac remodeling.(AU)

Alguns estudos já demonstraram o papel exercido pelas metaloproteinases de matriz e seus inibidores na cardiotoxicidade promovida pela doxorrubicina. Assim, este estudo teve como objetivo investigar o comportamento das MMPs 2 e 9 plasmáticas e miocárdicas em coelhos com cardiomiopatia induzida pela doxorrubicina, buscando determinar se há correlação entre a atividade dessas colagenases e o remodelamento cardíaco. A cardiomiopatia foi induzida pela doxorrubicina aplicada por via intravenosa duas vezes por semana ao longo de seis semanas consecutivas. A atividade plasmática das MMPs e o ecocardiograma foram avaliados no momento basal e aos 15 e 45 dias após a primeira aplicação da doxorrubicina. A atividade miocárdica dessas enzimas foi quantificada em apenas nove coelhos aos 45 dias e os resultados comparados com outros nove controles saudáveis. Foram identificadas apenas as formas inativas das MMPs 2 e 9 durante todo o estudo. A pro-MMP 2 plasmática reduziu à medida que a função cardiaca se deteriorou, enquanto a pro-MMP 9 aumentou significativamente em T45 quando comparada aos momentos basal e T15. Houve correlação negativa significativa entre a atividade plasmática da pro-MMP 2 e a relação entre E mitral e a velocidade anular mitral no início da diástole, um parâmetro que permite estimar a pressão atrial esquerda média e a congestão. Apenas a pro-MMP 2 foi documentada nas amostras miocárdicas dos coelhos com cardiomiopatia e atividade media dessa enzima foi estatisticamente menor que aquela observada nos controles saudáveis. Embora a forma ativa de ambas as colagenases não tenha sido identificada, o tempo de tratamento com doxorrubicina interferiu na atividade das formas inativas plasmáticas. Contudo, essas alterações não se associaram com a maioria dos parâmetros ecocardiográficos que indicam remodelamento cardíaco.(AU)

Matrix metalloproteinases 2 and 9 in rabbits with doxorubicin-induced cardiomyopathy

ABSTRACT: Some studies have shown the role played by matrix metalloproteinases and their inhibitors in doxorubicin cardiotoxicity. In this study, we sought to investigate how plasma and myocardial MMP 2 and 9 perform in rabbits with doxorubicin-induced cardiomyopathy, searching for a correlation between the activity of these collagenases and cardiac remodeling. Cardiomyopathy was induced by doxorubicin given intravenously twice a week for six consecutive weeks. Plasma MMP activity and the echocardiogram were assessed at baseline, and at 15 and 45 days after first injection of doxorubicin. The myocardial activity of these enzymes was solely evaluated in nine rabbits at 45 days, and results were compared with nine healthy controls. We only identified the full-length forms of both MMP 2 and 9 throughout the study. The plasma pro-MMP 2 reduced along the deterioration of cardiac function, while the pro-MMP 9 increased significantly at T45 as compared to baseline and T15. A negative significant correlation was found to exist between the plasma activity of pro-MMP 2 and mitral E-to-mitral septal annular early diastolic velocity ratio, which is an estimate of mean left atrial pressure and congestion. Only pro-MMP 2 was found in myocardial samples, and mean activity of such enzyme was statistically lower than that recorded for healthy controls. Although no active form was documented for either collagenase, the duration of the treatment with doxorubicin played a role in the alteration of plasma pro-forms activity. However, these changes could not be associated with most echocardiographic parameters that are supportive of cardiac remodeling.

RESUMO: Alguns estudos já demonstraram o papel exercido pelas metaloproteinases de matriz e seus inibidores na cardiotoxicidade promovida pela doxorrubicina. Assim, este estudo teve como objetivo investigar o comportamento das MMPs 2 e 9 plasmáticas e miocárdicas em coelhos com cardiomiopatia induzida pela doxorrubicina, buscando determinar se há correlação entre a atividade dessas colagenases e o remodelamento cardíaco. A cardiomiopatia foi induzida pela doxorrubicina aplicada por via intravenosa duas vezes por semana ao longo de seis semanas consecutivas. A atividade plasmática das MMPs e o ecocardiograma foram avaliados no momento basal e aos 15 e 45 dias após a primeira aplicação da doxorrubicina. A atividade miocárdica dessas enzimas foi quantificada em apenas nove coelhos aos 45 dias e os resultados comparados com outros nove controles saudáveis. Foram identificadas apenas as formas inativas das MMPs 2 e 9 durante todo o estudo. A pro-MMP 2 plasmática reduziu à medida que a função cardiaca se deteriorou, enquanto a pro-MMP 9 aumentou significativamente em T45 quando comparada aos momentos basal e T15. Houve correlação negativa significativa entre a atividade plasmática da pro-MMP 2 e a relação entre E mitral e a velocidade anular mitral no início da diástole, um parâmetro que permite estimar a pressão atrial esquerda média e a congestão. Apenas a pro-MMP 2 foi documentada nas amostras miocárdicas dos coelhos com cardiomiopatia e atividade media dessa enzima foi estatisticamente menor que aquela observada nos controles saudáveis. Embora a forma ativa de ambas as colagenases não tenha sido identificada, o tempo de tratamento com doxorrubicina interferiu na atividade das formas inativas plasmáticas. Contudo, essas alterações não se associaram com a maioria dos parâmetros ecocardiográficos que indicam remodelamento cardíaco.

Diagnóstico clínico en saliva. Revisión

El odontólogo está llamado a convertirse en un elemento importante en el desarrollo de estudios de fenotipado de poblaciones para impulsar la aplicación de la medicina personalizada y la razón se debe a la saliva, que además de resultar un fluido imprescindible para la conservación de las funciones orales, se perfila con cualidades importantes para el diagnóstico clínico con base genética. Con el desarrollo de potentes técnicas de laboratorio, como el inmunoensayo, electroforesis, fluorescencia, cromatografía (HPLC y CG), espectrometría de masa (EM), la reacción en cadena de la polimerasa (PCR) y pruebas genéticas de fenotipado, se ha podido correlacionar la presencia de ciertos biomarcadores de daño tisular en la saliva con los niveles de esas especies en sangre. Estos biomarcadores constituyen señales del daño tisular o de respuestas del organismo a esas injurias, cuando aun no se pueden observar las evidencias clínicas del mismo. Esta aplicación de la saliva le confiere una importancia especial en lo referente al diagnóstico clínico. Al estudio de los biomarcadores orales se ha unido, de forma más reciente, el desarrollo de protocolos para la obtención de ADN genómico de la saliva que permite, por la viabilidad en la colección de muestras, su conservación y facilidades en su traslado, realizar estudios poblacionales para conocer la funcionalidad de ciertos genes que se relacionan con la biotransformación de los medicamentos, una causa importante de las variaciones interindividuales a los tratamientos farmacológicos. El conocimiento de polimorfismos en genes que expresan las enzimas que metabolizan fármacos específicos, permite realizar cambios en los principios activos o ajustes en las dosis de tratamientos individuales, que es el objetivo de la medicina personalizada o farmacogenética. Estos estudios también permiten conocer la predisposición genética de poblaciones al desarrollo de ciertas enfermedades, tanto orales como sistémicas, lo que propicia el establecimiento de nuevas políticas en la profilaxis y medicina preventiva. El uso de la saliva con estos fines abre nuevas perspectivas en la atención odontológica, y requiere de esfuerzos interdisciplinarios y cooperación en el equipo de salud.

The dentist is called to become an important element in the development of studies phenotyping of populations to advance the implementation of personalized medicine and the reason is because saliva, which besides being a prerequisite for the preservation of oral functions fluid , is emerging with important qualities for clinical diagnosis with genetic basis. With the development of powerful laboratory techniques, such as immunoassay, electrophoresis, fluorescence, chromatography (HPLC and GC), mass spectrometry (MS), polymerase chain reaction (PCR) and genetic testing phenotyping, it has been correlate the presence of certain biomarkers of tissue damage in saliva levels of these species in blood. These biomarkers are signs of tissue damage or agency responses to these injuries, when not even be seen clinical evidence of it. This application saliva attaches special importance with regard to clinical diagnosis. The study of oral biomarkers has joined, more recently, the development of protocols for obtaining genomic DNA from saliva that allows for viability in sample collection, preservation of the same and facilities during transport, conduct population studies to determine the function of certain genes that are related to biotransformation of drugs, a major cause of differences among individuals to drug treatments. Knowledge of polymorphisms in genes that express enzymes that metabolize specific drugs can make changes or adjustments active principles in doses of individual treatments, which is the goal of personalized medicine or pharmacogenetics. These studies also provide insight into the genetic predisposition of populations to the development of certain diseases, both oral and systemic, which favors the establishment of new policies in the prophylaxis and preventive medicine. The use of saliva for this purpose opens new perspectives in care dental and requires interdisciplinary efforts and cooperation in the health team.

Análise comparativa da expressão e atividade das metaloproteinases 2 e 9 e de seus inibidores teciduais nas lesões cutâneas das variantes poiquilodérmica e clássica da micose fungoide / A comparative analysis of the expression and activity of metalloproteinases 2 and 9 and their tissue inhibitors in cutaneous lesions of poikilodermatous and classic variants of mycosis fungoides

Micose fungoide poiquilodérmica (MFp) é uma variante clínica de micose fungoide (MF). É mais indolente e caracterizada pela presença da poiquilodermia. As metaloproteinases (MMP) e seus inibidores específicos TIMP (Tissue Inhibitors of Metaloproteinases) estão envolvidos na oncogênese. Especificamente as MMP2 e MMP9 e seus inibidores, TIMP-2 e TIMP-1, respectivamente, foram relacionados ao prognóstico em tumores. Poucos trabalhos estudaram MMP e nenhum estudou a ação dos TIMP na MF. Objetivos: avaliar a relação entre MMP2 e MMP9 e seus inibidores TIMP2 e TIMP1 e a agressividade da MF e descrever a casuística de micose fungoide poiquilodérmica no ambulatório de linfomas cutâneos da Divisão de Clínica Dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Métodos: análise retrospectiva de 54 casos de MFp, sendo 25 de MFp localizada 14 de MFp generalizada e 15 de MFp mista. Para análise das MMP e TIMP, os grupos de MFp foram comparados com 7 amostras de pele normal (PN), 10 casos de MF clássica inicial (MFi), 9 casos de MF tumoral não-transformada (MFT nt) e 10 de MF tumoral transformada (MFT t). Resultados: A proporção de mulheres: homens foi 2,44. MFp apresentou maior tempo entre os primeiros sintomas e o diagnóstico. MFpG apresentou maior prevalência de lesões do tipo pitiríase liquenoide crônica (PLC) (79%). Houve alta prevalência de MF hipocromiante (62%) no grupo MFp mista. A histologia da MFp apresentou características típicas de MF e, adicionalmente, atrofia, telangectasias e derrame pigmentar, específicos da forma poiquilodérmica. Na imuno-histoquímica predominou o fenótipo CD3+, CD4+, CD7-, CD8- em todos os grupos, e MFp apresentou significantemente menor predomínio do fenótipo CD8+ que o grupo MFi. O grupo MFpG apresentou baixa positividade para pesquisa de clonalidade T da pele (12,5%). A MMP2 esteve mais presente na epiderme em MFi e MFp relativamente a MFT. Na derme superficial, os grupos MFi e MFp...

Poikilodermatous mycosis fungoides (pMF) is a clinical variant of mycosis fungoides (MF). It is more indolent than classic MF and is characterized by the presence of poikiloderma. The matrix metalloproteinases (MMPs) and their specific inhibitors TIMP (Tissue Inhibitors of Metalloproteinases) are involved in oncogenesis. Specifically, MMP2 and MMP9 and their inhibitors, TIMP-2 and TIMP-1, respectively, have been related to prognosis in tumors. There are few studies on MMP and none on the role of TIMPs in MF. Objectives: To evaluate if there is a relationship between the presence and activity of MMP2 and MMP9 and their inhibitors TIMP2 and TIMP1, and the aggressiveness of MF. To describe a casuistic of poikilodermatous mycosis fungoides in an outpatient clinic in the Dermatological Division of Hospital das Clinicas of University of Sao Paulo Medical School. Methods: Retrospective analysis of 54 cases of pMF, this included 25 localized pMF (LpMF), 14 generalized pMF (GpMF) and 15 mixed pMF. For the analysis of MMPs and TIMPs, the pMF groups were compared with 7 normal skin samples (NS), 10 cases of initial classical MF (cMF), 9 cases of non-transformed tumor MF (nt MFT) and 10 transformed tumor MF (t MFT). Results: The proportion of women : men was 2.44. The pMFs groups showed a longer period of time from the first symptoms to the diagnosis than the cMF group. The GpMF group had a higher incidence of pityriasis lichenoides chronica-like lesions (PLC) (79%) than the other groups. There was a high incidence of hypopigmented MF (62%) in the mixed pMF group. Histology showed typical characteristics of MF and, additionally, atrophy, telangiectasia and pigmentary alterations compatible with pMF. At immunohistochemistry the cases were predominantly CD3+, CD4+, CD7-, CD8- phenotype in all groups, and the pMF groups had a significantly lower prevalence of CD8+ phenotype than the cMF group. The GPMF group showed low positivity for clonality of the T-cell...

Evaluation of matrix metalloproteinases in blood from buck naturally infected with Caprine Arthritis Encephalitis in Semiarid region of Brazil / Avaliação das metaloproteinases de matriz no sangue de reprodutores caprinos naturalmente infectados com Artrite Encefalite Caprina na Região Semiárida do Brasil

Background: Studies have attempted to understand the importance of metalloproteinase (MMPs) in the pathogenesis ofdiseases caused by lentiviruses, being the human immunodeficiency virus (HIV) the most investigated. Despite advancesin studies with MMPs in others diseases, research correlating the presence and activity of gelatinases in animals affectedby caprine arthritis encephalitis virus (CAEV), a lentiviruses, are incipient and there is a need for research aiming to understand the dynamic of these enzymes in animals infected and its relation to pathological condition. The aim of this studywas to evaluate the presence and activity of the MMPs in blood serum of chronically infected bucks by CAEV.Materials, Methods & Results: The experiment was constituted by two groups (n = 5 each group). The first one wascomposed of five naturally infected bucks (4-5 years) and second group constituted of five seronegative bucks (3-4 years)for CAE. Serology was performed using the Western Blotting (WB) and confirmed by Polymerase Chain Reaction (PCR).These bucks belong to the experimental flock at Embrapa Goats and Sheep and the seropositive bucks were confirmed forCAE in the first two years. Blood samples were collected by puncturing the jugular vein from animals and evaluated byzymography (SDS-PAGE) using gelatin as substratum. Clinical examination was performed by evaluating the temperature (T), cardiac frequency (FC), respiratory frequency (FR) and clinical articular index (IAC). The IAC was calculatedby measuring the circumference of carpal joint and metacarpal bone height (difference between greater extent carpal andmetacarpal lesser extent). In infected and control groups were found molecular mass bands of 66 kDa (MMP-2), 72 kDa(pro-MMP-2), 86 kDa (MMP-9) and 92 kDa (pro-MMP-9). The correlation...(AU)

Evaluation of matrix metalloproteinases in blood from buck naturally infected with Caprine Arthritis Encephalitis in Semiarid region of Brazil / Avaliação das metaloproteinases de matriz no sangue de reprodutores caprinos naturalmente infectados com Artrite Encefalite Caprina na Região Semiárida do Brasil

Background: Studies have attempted to understand the importance of metalloproteinase (MMPs) in the pathogenesis ofdiseases caused by lentiviruses, being the human immunodeficiency virus (HIV) the most investigated. Despite advancesin studies with MMPs in others diseases, research correlating the presence and activity of gelatinases in animals affectedby caprine arthritis encephalitis virus (CAEV), a lentiviruses, are incipient and there is a need for research aiming to understand the dynamic of these enzymes in animals infected and its relation to pathological condition. The aim of this studywas to evaluate the presence and activity of the MMPs in blood serum of chronically infected bucks by CAEV.Materials, Methods & Results: The experiment was constituted by two groups (n = 5 each group). The first one wascomposed of five naturally infected bucks (4-5 years) and second group constituted of five seronegative bucks (3-4 years)for CAE. Serology was performed using the Western Blotting (WB) and confirmed by Polymerase Chain Reaction (PCR).These bucks belong to the experimental flock at Embrapa Goats and Sheep and the seropositive bucks were confirmed forCAE in the first two years. Blood samples were collected by puncturing the jugular vein from animals and evaluated byzymography (SDS-PAGE) using gelatin as substratum. Clinical examination was performed by evaluating the temperature (T), cardiac frequency (FC), respiratory frequency (FR) and clinical articular index (IAC). The IAC was calculatedby measuring the circumference of carpal joint and metacarpal bone height (difference between greater extent carpal andmetacarpal lesser extent). In infected and control groups were found molecular mass bands of 66 kDa (MMP-2), 72 kDa(pro-MMP-2), 86 kDa (MMP-9) and 92 kDa (pro-MMP-9). The correlation...

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment / Expressão de metaloproteinases 2 e 9 no tecido laminar do casco de equinos após obstrução intestinal e tratamento com hidrocortisona

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg), grupo instrumentado, Ig (sem obstrução intestinal), grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona) e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg), grupo instrumentado, Ig (sem obstrução intestinal), grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona) e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg), grupo instrumentado, Ig (sem obstrução intestinal), grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona) e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

Concomitant chemopreventive and antibacterial effects of some Iranian plants from the genus Cousinia (Asteraceae)

During the past several years, various species of Cousinia (Asteraceae) have been authenticated in Iran. However, data concerning their biological activities remain limited. The main purpose of this research was to assess potential cytotoxicity and matrix metalloproteinases (MMP) inhibitory effects of seven ethanol extracts of Cousinia using a cell line model (Fibrosarcoma-WEHI 164). We further investigated the antibacterial activity of these Cousinia ethanol extracts, using disk diffusion method. Among the ethanol extracts, the total extract of C. sulabadensis elicited significant inhibition of MMP activity in a dose-response fashion (49.2 ± 0.51, p < 0.05). However, this extract exhibited the lowest cytotoxicity effect at all tested concentrations. The concentration necessary to produce a 50 percent cell death rate (IC50) with C. shulabadensis was 304.5 ± 0.61 μg/mL. The calculated IC50 for cytotoxicity of the other Cousinia species extracts ranged between 18.4 ± 0.59 to 87.9 ± 0.58 μg/mL. The highest antibacterial activity was observed for the total extract of Cousinia phyllocephala. In conclusion, this study supports that Cousinia species display a remarkable inhibition of matrix metalloproteinases activity. The concomitant MMP-inhibitory and low cytotoxicity effects observed in C. sulabadensis might coin this extract for future potential anti-invasive herbal medicine studies.

Durante os últimos anos, várias espécies de Cousinia (Asteraceae) têm sido identificadas no Irã. No entanto, dados acerca de suas atividades biológicas permanecem limitados. O principal propósito desta pesquisa foi avaliar a potencial citotoxidade e os efeitos inibitórios de metaloproteinases da matriz (MMP) de sete extratos etanólicos de Cousinia utilizando um modelo de linhagem celular (Fibrosarcoma-WEHI 164). Além disso, investigamos a atividade antibacteriana destes extratos etanólicos de Cousinia, utilizando o método de difusão em disco. Dentre os extratos etanólicos, o extrato total de C. sulabadensis promoveu inibição significativa da atividade de MMP de maneira dose-resposta (49,2 ± 0,51, p < 0,05). Todavia, este extrato exibiu o menor efeito de citotoxicidade em todas as concentrações testadas. A concentração necessária para produzir uma taxa de 50 por cento de morte celular (IC50) com C. shulabadensis foi 304,5 ± 0,61 μg/mL. A IC50 calculada para a citotoxicidade dos outros extratos de espécies de Cousinia situou-se entre 18,4 ± 0,59 e 87,9 ± 0,58 μg/mL. A maior atividade antibacteriana foi observada para o extrato total de Cousinia phyllocephala. Em conclusão, este estudo corrobora que espécies de Cousinia mostram uma notável inibição da atividade de metaloproteinases da matriz. Os concomitantes efeitos inibitórios de MMP e baixa citotoxicidade observados em C. sulabadensis devem direcionar este extrato para futuros estudos de potenciais fitoterápicos anti-invasivos.