Efeito da exposição à hidroquinona na resposta imune adaptativa induzida pela vacina contra a influenza / Effects of hydroquinone exposure on the adaptive immune response induced by the influenza vaccine

A gripe é causada pelo vírus Influenza e é um problema de saúde pública mundial, que pode levar a problemas sérios em idosos e crianças. O Brasil implantou a vacinação anual contra influenza a partir de 1999, como ação preventiva contra a doença. A vacina é produzida pelo Instituto Butantan e contém três cepas diferentes do vírus Influenza fragmentado para induzir resposta imune adaptativa, com produção de anticorpos específicos e neutralizantes. A literatura tem mostrado que a exposição à xenobióticos com potencial imunossupressor pode comprometer a eficácia de imunizações ativas, como a imunização contra a gripe. Nosso grupo de pesquisa tem mostrado que a exposição à hidroquinona (HQ), um composto tóxico presente em altas concentrações na fumaça do cigarro, prejudica a resposta imune inata e adquirida. Assim, este trabalho avaliou o efeito da exposição à HQ sobre a resposta imune à vacinação contra influenza. Camundongos machos da linhagem C57BL/6 foram diariamente expostos à HQ (2500 ppm) ou PBS, por 1 hora, por nebulização, por um período de 8 semanas. Durante este período, foram imunizados nas semanas 6 e 8 do início das exposições, pela injeção i.m. de 100µL da vacina. Os parâmetros tóxicos e imunológicos foram avaliados 7, 35 e 70 dias após a segunda dose da vacina. A exposição à HQ não alterou o peso corpóreo dos animais e nem causou alterações morfológicas no pulmão, fígado e rins (histologia por H&E); reduziu a frequência de hemácias (11%), hematócrito (14%), hemoglobina (14%) e volume celular (4%); causou estresse oxidativo no baço (citometria de fluxo); aumentou a área dos folículos de células B no baço e linfonodomegalia (histologia por H&E). Em conjunto, os dados aqui obtidos mostram que a exposição à HQ afetou mecanismos envolvidos na gênese da imunidade ativa contra influenza. Assim, os dados deste trabalho mostram mecanismos tóxicos ainda não descritos para a HQ, e ressalta a HQ como um poluente ambiental que deve ser considerado nas avaliações de risco

The flu is a health problem worldwide which is caused by the Influenza virus and may result in severe illness in infants and the elderly. The annually vaccination against influenza was implemented in Brazil in 1999 as a preventive measure. The vaccine is produced by Butantan Institute and contains three different strains of the inactivated Influenza virus which induce the adaptive immune response along with production of specific and neutralizing antibodies. The literature has shown that exposure to immunosuppressive xenobiotics may compromise the efficacy of active immunizations, such as influenza. Our research group has shown that exposure to hydroquinone (HQ), a toxic constituent of cigarette smoke, impairs both innate and adaptive immune response. Thus, the aim of this work was to evaluate the effects of HQ on the immune response induced by the influenza vaccine. Male C57BL/6 mice were daily exposed to HQ (2500 ppm) or PBS by nebulization, for 1 hour, for 8 weeks. During the exposure period, the animals were vaccinated on weeks 6 and 8 with 100µL of the vaccine. Toxicologic and immunological parameters were assessed 7, 35 and 70 days after boost administration. HQ exposure did not alter body weight and did not cause morphological alterations in the lungs, liver and kidneys (H&E staining); reduced the frequency of erythrocytes (11%), hematocrit (14%), hemoglobin (14%) and cellular volume (4%) and caused oxidative stress on the spleen (Flow Cytometry); increased the area of B cell follicles in the spleen and increased the size of draining lymph nodes (H&E staining). Altogether, these data show that HQ exposure affected mechanisms involved in the genesis of the adaptive immune response. Thus, the data presented in this work show toxic mechanisms of HQ that have not yet been described, and it also points out HQ as an environmental pollutant which should be considered on risk assessments

Interferência do TSPO sobre as vias de ativação de adipócitos da linhagem 3T3-L1 / Interference of TSPO upon the activation pathways of 3T3-L1 adipocytes

A obesidade está associada a um processo inflamatório crônico de baixa intensidade e representa um dos fatores de risco para o desenvolvimento de uma série de comorbidades. A proteína TSPO está envolvida em inúmeras funções celulares, incluindo biossíntese e transporte de esteróides, transporte de porfirinas, apoptose, biossíntese do heme, processos oxidativos e imunomodulação. Ademais, a presença e a função da proteína TSPO no tecido adiposo e na inflamação ainda não estão bem estabelecidas. Deste modo, o objetivo do presente estudo foi validar a expressão e função do TSPO durante a diferenciação de células 3T3-L1, e investigar se o tratamento de adipócitos 3T3-L1 com diazepam, um benzodiazepínico de ação central (GABAA) e periférica (TSPO), é capaz de modular os efeitos inflamatórios induzidos pela incubação das células 3T3-L1 com TNF-α. Nossos resultados evidenciaram que, em nosso estudo, o tratamento de pré-adipócitos com diazepam não modulou a adipogênese. Entretanto, apesar de o diazepam per se não modular o acúmulo de triacilglicerol e a expressão gênica e protéica de PPAR-γ; em células estimuladas pelo TNF-α, o tratamento com diazepam foi capaz de reverter a diminuição da expressão gênica e protéica de PPAR-γ induzida pelo TNF-α. Ademais, o tratamento dos adipócitos com diazepam foi capaz de modular positivamente a expressão protéica de TSPO, efeito este que não observamos em células tratadas pelo clonazepam, um benzodiazepínico de ação exclusivamente central. Em resumo, os dados obtidos neste estudo, pela primeira vez, demonstram a possível relação entre as vias que controlam a sinalização de TSPO, TNF-α e PPAR-γ. Assim, nos é possível inferir que a ativação de TSPO pelo seu ligante diazepam foi capaz de modular a ativação de NF-kB induzida pelo TNF-α, promovendo, com a diminuição da lipólise e aumento da expressão gênica de TSPO e gênica e protéica de PPAR-γ, o reestabelecimento da homeostase celular, o que aumentaria a sobrevida das células, a atividade mitocondrial, e a atividade adipogênica dos adipócitos

Obesity is associated with a chronic low-grade inflammation and these represents one of the risk factors to development of other non-communicable diseases. TSPO 18 kDa is involved in several cellular functions, including biosynthesis and steroids transport, porphyrin transport, apoptosis, heme biosynthesis, oxidative metabolism and immunomodulation. Furthermore, the TSPO expression and function on adipose tissue and in the chronic low-grade inflammation have not been established. Thus, the aim of present study was to validate the TSPO expression and function on the 3T3-L1 differentiation process and to investigate whether diazepam treatment is able to modulate the TNF-α induced inflammatory effects on 3T3-L1 cells. Our results showed that diazepam treatment of preadipocytes was not able to modulate the adipogenesis. However, although diazepam treatment per se does not modulate the triacylglycerol accumulation and gene and protein expression of PPAR-γ; in TNF-α stimulated adipocytes, the treatment with diazepam was able to modulate the decreased of PPAR-γ gene and protein expression induced by TNF-α. In addition, the diazepam treatment of adipocytes was able to positively modulate the TSPO protein expression, an effect that we did not observe in cells treated with clonazepam, a central benzodiazepine ligand. In summary, the data obtained in this study, for the first time, demonstrate the possible relationship between the pathways that control the TSPO, TNF-α and PPAR-γ signaling. Thus, it is possible that the activation of TSPO by diazepam was able to modulate TNF-α-induced activation of NF-kB, promoting the reduction of lipolysis and increased of TSPO gene expression and PPAR-γ gene and protein expression, reestablishment of cellular homeostasis, which would increase cell survival, mitochondrial activity, and adipogenic activity of adipocytes

Immunotoxicological Effects of Aripiprazole: In vivo and In vitro Studies

Aripiprazole (ARI) is a commonly prescribed medication used to treat schizophrenia and bipolar disorder. To date, there have been no studies regarding the molecular pathological and immunotoxicological profiling of aripiprazole. Thus, in the present study, we prepared two different formulas of aripiprazole [Free base crystal of aripiprazole (ARPGCB) and cocrystal of aripiprazole (GCB3004)], and explored their effects on the patterns of survival and apoptosis-regulatory proteins under acute toxicity and cytotoxicity test conditions. Furthermore, we also evaluated the modulatory activity of the different formulations on the immunological responses in macrophages primed by various stimulators such as lipopolysaccharide (LPS), pam3CSK, and poly(I:C) via toll-like receptor 4 (TLR4), TLR2, and TLR3 pathways, respectively. In liver, both ARPGCB and GCB3004 produced similar toxicity profiles. In particular, these two formulas exhibited similar phospho-protein profiling of p65/nuclear factor (NF)-kappaB, c-Jun/activator protein (AP)-1, ERK, JNK, p38, caspase 3, and bcl-2 in brain. In contrast, the patterns of these phospho-proteins were variable in other tissues. Moreover, these two formulas did not exhibit any cytotoxicity in C6 glioma cells. Finally, the two formulations at available in vivo concentrations did not block nitric oxide (NO) production from activated macrophage-like RAW264.7 cells stimulated with LPS, pam3CSK, or poly(I:C), nor did they alter the morphological changes of the activated macrophages. Taken together, our present work, as a comparative study of two different formulas of aripiprazole, suggests that these two formulas can be used to achieve similar functional activation of brain proteins related to cell survival and apoptosis and immunotoxicological activities of macrophages.

Evaluation of the immunotoxicological effects of Dimorphandra mollis Benth., Fabaceae, in rats / Avaliação dos efetos imunotoxicológicos de Dimorphandra mollis Benth., Fabaceae, em ratos

Dimorphandra mollis Benth., Fabaceae, also known as "faveira" or "fava-d'anta", is a plant common to the central woodsy meadow region of Brazil. It is well known for its antioxidant, antiplatelet and, principally, vasoprotective properties. Its principal component is rutin. The objective of this study is the evaluation of the safety of the use of the dried D. mollis extract in rodents. The rutin content of the standardized extract was 76.0±3 percent. With respect to the biochemical and hematological parameters evaluated, no alterations in the groups of rats that received 1000 and 2000 mg/kg doses of D. mollis were observed, but an increase in eosinophiles occurred. Hyperactivity of the white splenic pulp was detected in the group that received the 2000 mg/kg dose of D. mollis. In the evaluation of the lymphproliferative response with 1000 and 2000 mg/kg, no alterations were observed, and a decrease in IgG was only observed in the studies with a 2000 mg/kg dose. The results obtained with rodents suggest that no toxicity exists with the administration of dried D. mollis extract in a 1000 mg/kg dose.

A Dimorphandra mollis Benth., Fabaceae, conhecida como faveira ou fava-d'anta, é uma planta comum do cerrado central do Brasil, muito utilizada por suas propriedades antioxidante, antiplaquetária e, principalmente, como vasoprotetora. Seu principal marcador é a rutina. Este estudo teve como objetivo avaliar a segurança da utilização do extrato seco de D. mollis em roedores. O extrato foi extraído, padronizado e quantificado apresentando teor de 76,0±3 por cento de rutina. Nos parâmetros bioquímicos e hematológicos avaliados, não se observou alterações nos grupos de machos e fêmeas que receberam a dose de 1000 e 2000 mg/kg de D. mollis, mas observou-se um aumento de eosinófilos. Nos estudos histopatológicos detectou-se hiperreatividade da polpa branca esplênica, no grupo que recebeu a dose de 2000 mg/kg de D. mollis. Na avaliação da resposta linfoproliferativa, com 1000 e 2000 mg/kg não foram observadas alterações, e somente nos estudos com a dose de 2000 mg/kg se observou diminuição de IgG. Os resultados obtidos, utilizando roedores, sugerem que nenhuma toxicidade existe na administração de extrato seco de D. mollis na dose de 1000 mg/kg.

Humoral immune alterations caused by lead: studies on an adult toad model

There is evidence that environmentalmetal levels affect the immune function. In the particular case of the impact of heavy metals, information available suggests that the immune system is a target for low-dose Pb exposure. Among vertebrates it was shown that amphibians are capable of forming antibodies against a variety of antigens, causing several responses such as anaphylactic response and rejectinggrafts. In this study, the production of antibodies was assessed against sheep red blood cells (SRBC) in the anuran Bufo arenarum after six weekly injections of sublethal doses of lead (50 mg.kg-1, as lead acetate). Natural antibodies (natural heteroagglutinins)were also quantified against SRBC. Both assessments were carried out employing an ELISA method developedto this end, measuring absorbance (A). For natural anti-SRBC antibodies in both control (C) and Pb treated (T) toads, there was a non significant tendency to increase the initial absorbances (C initial: 0.69+0.39 A; T initial: 0.54+0.30 A), relative to those registered at the end of the experiments (C final: 0.89+0.49 A; T final: 0.76+0.31A); the T/C ratios also did not show changes. The only significant difference was found between initial and final samples from lead-treated toads (p<0.014). The immune anti-SRBC antibody levels of toads immunized with SRBC showed a significant lower increase (p<0.05) in lead-treated animals (T final: 0.66+0.36 A), as compared to control toads (C final: 0.91+0.50 A) at the end of the experiment. It was thus concluded thatthe changes due to the assayed doses of Pb in the levels of antibodies cannot be explained on the basis of only one singleaction mechanism of the metal, but as the result of a conjunction of effects over different immunocompetent cell subpopulations. These different responses suggest that factors affecting animals exposed to a foreign stimulus are different from those influencing the response of wild animals.

Existe evidencia de que los niveles de metal ambientales afectan la función inmune. En el caso particular del impacto de metales pesados, la información disponible sugiere que el sistema inmune es un blanco para la exposición a bajas dosis de Pb. Entre los vertebrados, se ha mostrado que los anfibios son capaces de formar anticuerpos contra una variedad de antígenos, que causan diversasrespuestas, tales como respuesta anafiláctica y rechazo de injertos. En este estudio, la producción de anticuerpos fue evaluada contra eritrocitos de oveja (EO) en el anuro Bufo arenarum, luego de seis inyecciones semanales de dosis subletales de plomo (50 mg.kg-1, como acetato de Pb). Los anticuerpos naturales (heteroaglutininas naturales) fueron también cuantificados contra EO. Ambas evaluaciones fueron llevadas a cabo empleando un método de ELISA desarrollado a este fin, midiendo laabsorbancia (A). Para los anticuerpos anti-EO naturales, tanto en sapos controles (C) como en sapos tratados con Pb (T), hubo una tendencia significativa a incrementar las absorbancias iniciales (C inicial: 0,69+0,39 A; T inicial: 0,54+0,30 A); la relación T/C tampoco mostró cambios. La única diferencia significativa se encontró entre las muestras inicial y final de los sapos tratados con plomo (p<0,014). Los niveles de anticuerpos anti-EO inmune de sapos inmunizados con EO mostraron un bajo incrementosignificativo (p<0,05) en los animales tratados con plomo (T final: 0,66+0,36 A), al compararse con sapos control (C final: 0,91+0,50 A) al final del experimento. De este modo, se concluye que los cambios debidos a las dosis analizadas de Pb en los niveles de anticuerpos no pueden explicarse sólo sobre la base de un único mecanismo de acción del metal, sino como resultadode una conjunción de efectos sobre diferentes subpoblaciones de células inmunocompetentes. Estas diferentes respuestas sugieren que los factores que afectan los animales expuestos a un estimulo externo son diferentes...