Diazepam, em dose única, inibe a migração celular, a estimulação macrofágica e a atividade de TNF-α na reação inflamatória aguda induzida por LPS em camundongos

Benzodiazepines are one of the most frequently prescribed drugs due to their anxiolytic properties. The aim of this study was to evaluate the effects of diazepam on lipopolysaccharide-induced peritoneal acute inflammatory responses. Swiss mice were treated with diazepam in a single dose of 1 or 10 mg/kg- subcutaneously 1 h before an intraperitoneal injection of lipopolysaccharide or sterile saline solution. The mice were killed 16 h after and the cells were washed from the peritoneal cavity to determine the total number of cells and the mononuclear and polimorfonuclear subpopulations, as well as the TNF-alpha activity and percentage of spread macrophages. Our results showed that the diazepam treatment (1 and 10 mg/kg) induced a significant reduction in the LPS-induced macrophage stimulation and TNF-α activity. Diazepam (10 mg/kg) also reduced the inflammatory cellular migration when compared to the control. It can be concluded that the diazepam treatment in a single dose is able to influence the inflammatory cellular influx, macrophage stimulation and TNF-α activity in the acute inflammatory response in mice, having possible implications on the anti-infectious response efficiency.

Migração e invasão celular in vitro de células humanas expressando variantes da oncoproteína LMP1 do vírus de Epstein-Barr (EBV)

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

Adição de ácido linolênico e L-carnitina na maturação oocitária: efeitos sobre o metabolismo celular, potencial de desenvolvimento e criotolerância de embriões bovinos produzidos in vitro

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

Viabilidade celular, fagocitose e espraiamento de fagócitos mononucleares, e liberação de peróxido de hidrogênio por leucócitos de glândulas mamárias bovinas sadias e infectadas

O presente estudo objetivou avaliar a viabilidade celular, a capacidade de fagocitose e espraiamento pelos fagócitos mononucleares, e a liberação de peróxido de hidrogênio (H2O2) por leucócitos oriundos de glândulas mamárias bovinas sadias e infectadas. Deste modo, 94 amostras foram divididas de acordo com os resultados da cultura bacteriológica e da contagem de células somáticas (CCS). O presente estudo não encontrou diferenças na viabilidade celular, e nos índices de fagocitose e espraiamento entre os diferentes grupos. No entanto, a liberação de H2O2 oriundos dos quartos mamários infectados, infectados por Streptococcus spp. ou Corynebacterium spp. foi menor do que nas amostras de leite provenientes dos quartos mamários sadios. Ao estimar a concentração de H2O2 mL-1 leite observou-se que as amostras de quartos mamários positivos no exame bacteriológico, infectados por Staphylococcus spp. e negativos no exame bacteriológico com alta celularidade foram maiores que aquelas provenientes de quartos mamários sadios. Observou-se também correlação positiva entre a CCS e a viabilidade celular e os índices de fagocitose e espraiamento; e correlação negativa entre a liberação de H2O2 e a CCS e a viabilidade celular. Conclui-se que a CCS, assim como a sua viabilidade e função, são conceitos intimamente relacionados com a saúde da glândula mamária.

Effects of MK-801 and amphetamine treatments on allergic lung inflammatory response in mice

Glutamate acts as a neurotransmitter within the Central Nervous System (CNS) and modifies immune cell activity. In lymphocytes, NMDA glutamate receptors regulate intracellular calcium, the production of reactive oxygen species and cytokine synthesis. MK-801, a NMDA receptor open-channel blocker, inhibits calcium entry into mast cells, thereby preventing mast cell degranulation. Several lines of evidence have shown the involvement of NMDA glutamate receptors in amphetamine (AMPH)-induced effects. AMPH treatment has been reported to modify allergic lung inflammation. This study evaluated the effects of MK-801 (0.25mg/kg) and AMPH (2.0mg/kg), given alone or in combination, on allergic lung inflammation in mice and the possible involvement of NMDA receptors in this process. In OVA-sensitized and challenged mice, AMPH and MK-801 given alone decreased cellular migration into the lung, reduced IL-13 and IL10 levels in BAL supernatant, reduced ICAM-1 and L-selectin expression in granulocytes in the BAL and decreased mast cell degranulation. AMPH treatment also decreased IL-5 levels. When both drugs were administered, treatment with MK-801 reversed the decrease in the number of eosinophils and neutrophils induced by AMPH in the BAL of OVA-sensitized and challenged mice as well as the effects on the expression of L-selectin and ICAM-1 in granulocytes, the IL-10, IL-5 and IL-13 levels in BAL supernatants and increased mast cell degranulation. At the same time, treatment with MK-801, AMPH or with MK-801+AMPH increased corticosterone serum levels in allergic mice. These results are discussed in light of possible indirect effects of AMPH and MK-801 via endocrine outflow from the CNS (i.e., HPA-axis activity) to the periphery and/or as a consequence of the direct action of these drugs on immune cell activity, with emphasis given to mast cell participation in the allergic lung response of mice.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Efeito da desnutrição proteica sobre aspectos da mobilização, migração e sinalização celular. Papel da glutamina na modulação desses processos

A desnutrição é uma condição nutricional que pode afetar muitos aspectos da resposta imunológica, como alterações na migração celular, na fagocitose, na resposta bactericida, mudanças na produção de radicais livres e espécies de nitrogênio e na produção de citocinas pró-inflamatórias. Logo, indivíduos desnutridos apresentam maior susceptibilidade a infecções. Visto que a glutamina é um aminoácido de extrema importância para a funcionalidade de diversas células do sistema imune e que as mesmas apresentam aumento da utilização desse aminoácido durante processos infecciosos, investigou-se, neste trabalho, quais os efeitos da glutamina sobre alguns aspectos da mobilização, migração e sinalização celular em um modelo experimental de desnutrição proteica. Para tanto, utilizou-se camundongos da linhagem BALB/c machos, os quais foram divididos em dois grupos, Controle e Desnutrido, que passaram a receber dietas isocalóricas contendo 12% (normoproteica) e 2% de caseína (hipoproteica), respectivamente, durante 5 semanas. Para as avaliações in vivo, animais de ambos os grupos receberam por via endovenosa 100µL de solução contendo 1,25µg de LPS e após 1 hora 0,75mg/Kg de L-glutamina (GLUT). Após o período de desnutrição ou de indução ao processo inflamatório, os animais foram eutanasiados e as amostras biológicas coletas. Foram avaliados nos animais estimulados in vivo hemograma, mielograma, as citocinas IL-10 e TNF-α circulantes e a expressão de CD11b/CD18 nos granulócitos do sangue periférico. Foi avaliado, in vitro, a capacidade migratória, a expressão de CD11b/CD18 de polimorfonucleados da medula óssea e do sangue periférico, bem como a síntese de citocinas IL-1α, IL-6, IL-10, IL-12 e TNF-α e a expressão de NF-κB e IκBα em células cultivadas em meio com 0; 0,6; 2 e 10 mM de GLUT. Os animais desnutridos apresentaram anemia, leucopenia, hipoplasia medular e diminuição na concentração sérica de proteínas, albumina e pré-albumina. A GLUT, in vitro, apresentou capacidade de reduzir a produção de IL-1α e IL-6, bem como a ativação da via do NF-κB. No modelo in vivo a GLUT, em animais estimulados com LPS, alterou a cinética de migração neutrofílica e reduziu a expressão de CD18, bem como diminuiu os níveis de TNFα circulantes.

Avaliação do efeito da inibição do reparo de sítios abásicos na resposta inflamatória celular

Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor κB (NF-κB) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-κB and tumor necrosis factor (TNF-α) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

Altered regulatory response of Rab7a and Rab9 in MM1 and VV2 subtype of Creutzfeldt-Jakob disease

The present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. We performed a target search for lysosomal network protein, Rab7a and Rab9, in frontal cortex and cerebellum of human brain from patients with sCJD-MM1 and sCJD-VV2. The intracellular level of Rab7a was increased significantly, when compared with healthy age-matched control. Interactions of PrPC and Rab7a/Rab9 were further investigated by using confocal laser scanning microscopy. Immunofluorescence results suggested potential interactions of Rab7a and PrPC. siRNA against the Rab7a gene was used to knockdown the expression of Rab7a protein in primary cell culture of cortical neurons from wild type mice. This depleted Rab7a resulted an impairment of PrPC trafficking leading to an accumulation of PrPC in the endocytosis pathway. Furthermore, interactions of Tau and Rab7a were investigated by using western blot analysis and confocal laser scanning microscopy. Cell cultures of cortex of wildtype mice were treated with siRNA-Tau, siRNA-Rab7 and control siRNA followed by immunofluorescence. The results of immunofluorescence suggested potential interaction of Tau and Rab7a. Cells lines treated with siRNA-Tau, the intracellular levels of Rab7a and Rab9 significantly increases and their localization is also modified. When we transfected this cells lines with siRNA-rab7a the accumulation of Tau decreases in cytosolic region and their localization was also modified when compared with control cells. In conclusion, this study may help to understand and characterize the subtype specific disease progression in CJD cases. Furthermore, it could be a step ahead to development of new treatment strategies for diseases subtype specific manner.

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal