Effect Of Angiotensin Ii And Losartan On The Phagocytic Activity Of Peritoneal Macrophages From Balb/c Mice.

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.99167-7

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10-14 to 10-7 M) and/or losartan, 10-16 to 10-6 M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cytotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10-13 M and 10-12 M AII concentrations. The addition of losartan (up to10-14 M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.16717

Analise dos proteoglicanos e proteinas não colagenicas presentes nas cartilagens do tibiotarso e tarsometatarso de frango

Orientador: Laurecir GomesDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Já foram descritos os componentes da matriz extracelular de diferentes cartilagens de mamíferos, mas sobre aves são encontradas poucas informações. Nosso trabalho, teve como objetivo extrair e identificar componentes da matriz extracelular de duas cartilagens articulares de frango (tibiotarso e tarsometatarso). As cartilagens articulares foram homogeneizadas e os componentes extraídos com Gu-HCI4M. Após obtenção do extrato total foi realizada uma ultracentrifugação em gradiente de c1oreto de césio, onde foram obtidas as frações DI, D2, D3 e D4. O conteúdo protéico foi maior na fração D4, onde em tarsometatarso a extração foi mais eficiente (2,64 mg/rnl) do que em tibiotarso (1,6 mglrnl). Com relação à dosagem de ácido urônico, a concentração maior foi encontrada na fração Dl-tarsometatarso (1,52 mglrnl) em relação à Dl-tibiotarso (0,80 mg/rnl). A fração D4 foi aplicada em DEAE-Sephacel em tampão Tris-HCI 20mM pH 7,2 com uréia 7M. O material que se ligou ao DEAE foi eluído com um gradiente de O a 1,5M de NaCI no mesmo tampão. A análise destas frações em SDS-PAGE mostraram que somente em tibiotarso há um componente com 250 kDa, que na presença de 2-Me migra com 59 kDa. Após o teste de "immunoblotting" e CEC/azul de alcian foi possível mostrar que se trata do pequeno proteoglicano fibromodulim, em condições redutoras e não redutoras. Em tarsometatarso a proteina com 59 kDa se apresentou proeminente em condições redutoras. Uma proteína, apresentou uma banda polidispersa em tomo de 70 kDa e foi encontrada em ambas as regiões. Provavelmente, se trata do pequeno proteoglicano decorim. Proteínas não colagênicas com Mr de 46, 36 e 30 kDa foram observadas em ambas regiões. A fração DI foi analisada em Sepharose CL-6B. O perfil cromatográfico de tibiotarso e tarsometatarso foi muito semelhante. Ambas regiões apresentaram um único pico que eluiu logo após o volume morto, com Kav de 0,08. Pela análise em agarosepoliacrilamida nas duas regiões analisadas, pode ser visto uma única população de grandes proteoglicanos. O tipo de glicosaminoglicano presente em cada região foi analisado através de gel de agarose-propileno. O glicosaminoglicano predominante para as duas regiões foi condroitim-sulfato, embora tenha apresentado características diferentes para cada cartilagem. Este resultado foi confirmado após incubação com condroitinase ABCAbstract: The extracellular matriz (ECM) components of different cartilages of mammals is already kwown, but little information is found for ECM of avian cartilage. The purpose of this work was to identi:fy the components of the ECM of tibiotarsal and tarsometatarsal cartilage of chicken. The cartilage was homogeneized in PBS and the fragments extracted with 4M Gu-HCI. The extract was submitted to ultracentrifugation in CsCI gradient, resulting in DI, D2, D3 e D4 fractions. The protein contents was greater in the D4 fraction and in tarsometatarsal extract the concentration of proteins (2,64 mglml) was larger than in tibiotarsal extract (1,6 mglml). With respect to uronic acid, we found more in Dltarsometatarsal (1,51 mg/ml) than in Dl-tibiotarsal (0,80 mg/ml). D4 fraction was dialysed, applied on DEAE-SepOOcel and eluted with 7M urea in 20mM Tris-HCI pH 7,2. Bound material was eluted with a gradient ranging from O to 1,5M NaCI in the same buffer. SDS-P AGE of DEAE fractions of tibiotarsal cartilage, showed a component with 250 kDa, which in presence of 2-Me appears to migrate as a 59 kDa protein. After immunoblotting and CEC/alcian blue, it was demonstrated to be the small proteoglycan fibromodulin in reducing and non-reducing conditions. In tarsometatarsal fractions this protein, migrating as 59 kDa, was detected only in reducing conditions. Another protein migrating as a polidisperse band around 70 kDa was detected in both cartilages. Probably it is the small proteoglycan decorin. Non-collagenous proteins with 46,36 e 30 kDa were detected in both cartilages. DI fration was analysed in Sepharose CL-6B. The cromatography profiles were similar for tibiotarsal and tarsometatarsal cartilages. Both regions showed only one peak tOOt eluted with Kav=0,08. Analysis of fractions in SDS-P AGE and agarose-polyacrilamide gel electrophoresis showed the presence oflarge proteoglycans in tibiotarsal and tarsometatarsal material. The glicosaminoglycans were analysed in agarose gel electrophoresis for each region. The predominant glicosaminoglycan was condroitin sulfate in both cartilages, but its migration in agarose gel was different for tibiotarsal and tarsometatarsal samples. This result was confirmed using chase ABC digestionMestradoBiologia CelularMestre em Ciências Biológica

Ação de angiotensina II sobre macrofagos peritoneais de camundongos normais e diabeticos

Orientador: Jose Francisco FigueiredoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Sistema Renina Angiotensina (SRA) é um mecanismo importante para a regulação da homeostase cardiovascular via balanço de sódio em indivíduos normais e hipertensos. Recentemente, diversos trabalhos têm comprovado a existência de um SRA local em diferentes tecidos, inclusive em células do Sistema Imunológico. Nosso estudo teve como objetivo analisar em cultura a ação de Angiotensina II (AlI) sobre algumas funções de macrófagos provenientes do peritôneo de camundongos (BALB/c e NOD - Diabéticos Não Obesos). Primeiramente, determinamos a curva dose-resposta de Angiotensina II (AII) com relação à atividade fagocitária nos períodos de 1 e 3 horas de tratamento. Verificamos que a dose de AII que estimulou a fagocitose foi 10-12 M. Para verificarmos a presença de receptores de AII, determinamos também uma curva dose-resposta do antagonista do receptor ATl, DuP 753, conhecido comercialmente como Losartan®. O protocolo com DuP 753 foi realizado na presença e ausência de AII. Nossos resultados mostraram que apesar de inibirmos a atividade fagocitária em diferentes concentrações de DuP, a maior inibição, tanto em animais normais quanto em animais diabéticos, foi com a dose de 1 O-6 M. Em seguida resolvemos avaliar o efeito citotóxico de DuP 753 para comprovarmos que seu efeito inibitório não foi causado por uma alteração da viabilidade celular. Realizamos 3 testes de citotoxicidade: MTT (Brometo de [3-( 4,5-dimetiltiazol-2-il]-2,5 difeniltetrazolio), análise do conteúdo de ácidos nucléicos (CAN) e a liberação de lactato desidrogenase (LDH). Os 3 testes comprovaram que DuP 753 nas concentrações utilizadas não exerceu efeito tóxico para macrófagos em cultura. Além da atividade fagocitária, analisamos também a produção do radical superóxido através da redução do ferricitocromo c. Nossos resultados demonstraram que a dose estimuladora de AII (10-12 M) e a dose inibitória de DuP (10-6 M) com relação à atividade fagocitária das células, não alteraram a produção do ânion superóxido. Isto mostra que a função fagocitária e a produção do radical superóxido são ativadas provavelmente por concentrações diferentes de AII. Finalmente demonstramos por imunofluorescência os tipos de receptores de AII presentes em macrófagos BALB/c e NOD. Verificamos a existência de receptores do tipo ATI e AT2 e que o tratamento das células com AII e DuP parece alterar o número destes receptores. Isto comprova a influência do SRA sobre a função fagocitária de uma das células do Sistema ImunológicoAbstract: The Renin Angiotensin System (RAS) is an important regulatory mechanism of cardiovascular homeostasis in normal and hypertensive subjects. Recently, several reports have been showed the existence of a local RAS in different cells and tissues, inclusive immunological cells. The aim of this study was evaluated in culture the effect of Angiotensin II (AII) on peritoneal mice macrophages (BALB/c and Non Obese Diabetic - NOD). The dose response curve of AII on phagocytic activity was determined during 1 and 3 hours of treatment and the stimulant dose of the phagocytosis was AII 10-12 M. To verify if this stimulus was obtained through AT1 receptor, the dose response curve of DuP 753 was determined. The protocol with DuP 753 was realized in the presence and absence of AII. The results showed that the inhibition of the phagocytic activity occurred in different concentrations of DuP, but the higher inhibition, in normal and diabetic animals, it was with 10-6 M. In order to evaluate the cytotoxic effect of DuP 753 and to show that the inhibition effect was not caused by an alteration of the cellular viability, were realized 3 cytotoxic assays: MTT reduction, analysis of nucleic acid content (NAC) and lactate dehydrogenase release (LDH). All assays indicated that cell viability was maintained afier treatment with DuP 753 in concentrations used. In addition of the phagocytic activity, the effect of AII and DuP 753 in production of superoxide anions was also investigate through the ferricytochrome c reduction. The results demonstrated that the phagocytic stimulant dose of AII (10-12 M) and the inhibition phagocytic dose of DuP (10-6 M) did not influence the superoxide production of macrophages. These results showed that the phagocytic function and superoxide production are probably activated by different concentrations of AII. Finally, an immunofluorescence study was performed in order to identify the type of AII receptors are present in BALB/c and NOD macrophages. The treatment of the cells with AII and DuP changed the immunoreactive of AT1 and AT2 receptors. This shows the influence of RAS on the function and metabolism of lmmunological Cell SystemDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10-14 to 10-7 M) and/or losartan, 10-16 to 10-6 M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cytotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10-13 M and 10-12 M AII concentrations. The addition of losartan (up to10-14 M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection

Análise citogenética em material de abortamento espontâneo Cytogenetic analysis of material from spontaneous abortion

OBJETIVO: Descrever as anormalidades cromossômicas em material de abortamento espontâneo. MÉTODOS: Realizou-se compilação retrospectiva da análise de cariótipo em lâmina corada com Banda G por microscopia óptica e em material de 428 produtos de abortamento encaminhados para estudo. RESULTADOS: Foram observados 145 resultados normais (33,9%) e 237 resultados anormais (55,4%). Em 46 amostras não houve crescimento celular (10,7%). As anormalidades numéricas foram as mais frequentes, destacando-se a trissomia do 16 (41 casos), a triplodia (27 casos), a monossomia do X (26 casos), a tetraploidia (13 casos) e a trissomia do 15 (13 casos). CONCLUSÃO: As alterações citogenéticas representam importante causa de perdas gestacionais e sua detecção auxilia o aconselhamento genético do casal. A trissomia do cromossomo 16 é a alteração mais frequentemente encontrada.<br>OBJECTIVE: To describe chromosomal abnormalities in spontaneous abortion material. METHODS: A retrospective compilation of karyotype analysis of slides stained with Band G was carried out by optical microscopy with materials of 428 abortion products referred for study. RESULTS: There were 145 normal results (33.9%) and 237 abnormal results (55.4%). In 46 samples there was no cell growth (10.7%). Numerical abnormalities were the most frequent, especially trisomy 16 (41 cases), triplodia (27 cases), monosomy X (26 cases), tetraploidy (13 cases) and trisomy 15 (13 cases). CONCLUSION: Cytogenetic alterations are an important cause of pregnancy loss and their detection helps the genetic counseling to the couple. Trisomy 16 is the most often found change