Lipopolysaccharide effects on activation and apoptosis of primordial ovarian follicles in heifers / Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Down-regulation of complement genes in lipopolysaccharide-challenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis

Abstract Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 g/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 g/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 g/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.

Resumo Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 g/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 g/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 g/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).

Down-regulation of complement genes in lipopolysaccharide-challenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis / Regulação negativa dos genes do complemento em larvas de peixe-zebra (Danio rerio) desafiadas com lipopolissacarídeo expostas à própolis indonésia

Abstract Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.

Resumo Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).

Down-regulation of complement genes in lipopolysaccharidechallenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis / Regulação negativa dos genes do complemento em larvas de peixe-zebra (Danio rerio) desafiadas com lipopolissacarídeo expostas à própolis indonésia

Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.

Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).

Down-regulation of complement genes in lipopolysaccharidechallenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis / Regulação negativa dos genes do complemento em larvas de peixe-zebra (Danio rerio) desafiadas com lipopolissacarídeo expostas à própolis indonésia

Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.(AU)

Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).(AU)

Ultrasound assessment of sheep stifle joint undergone lipopolysaccharide-induced synovitis / Ultrassonografia da articulação femorotibiopatelar em ovinos submetidos à indução de sinovite por lipopolissacarídeos

Synovitis can be induced in animals through the application of bacterial wall lipopolysaccharide and has similar signs to naturally-occurring synovitis. Several studies have been using the sheep species as an experimental model to understand osteoarticular diseases of the femorotibiopatellar (FTP) joint in humans. There are echographic studies on the standardization of normality of the femorotibiopatellar joint in sheep. However, there is a gap in the literature for changes such as acute synovitis. The objective was to serially describe the sonographic aspects of the synovitis process induced by intra-articular infiltration of Escherichia coli (E. coli) lipopolysaccharide in the femorotibiopatellar joint of sheep. Twelve healthy crossbred sheep (Santa Inês x Dorper) were used. Induction of synovitis was performed only in the right FTP joints, which were serially evaluated using ultrasound examination at baseline moment (M0) and 12 (M12), 24 (M24), 48 (M48), 72 (M72), and 120 (M120) hours after lipopolysaccharide infiltration for synovitis induction. Intra-articular application of E. coli lipopolysaccharide resulted in one or more echographic signs of synovitis (increased synovial fluid volume, folding of the synovial membrane, and cellularity in the joint cavity), which were identified early, 12 hours after inoculation, and regressed over the evaluated times (p=0.0001) until disappearing after 120 hours of inoculation.

A sinovite pode ser induzida em animais por meio da aplicação de lipopolissacarídeo de parede bacteriana, e apresenta sinais semelhantes à sinovite causada de forma natural. Diversos estudos têm sido realizados utilizando a espécie ovina como modelo experimental na compreensão das enfermidades osteoarticulares da articulação femorotibiopatelar (FTP) em humanos. Existem estudos ecográficos quanto a padronização da normalidade da articulação femorotibiopatelar em ovinos. Porém, para as alterações, como a sinovite aguda há lacuna na literatura. Objetivou-se descrever, de forma seriada, os aspectos ultrassonográficos do processo de sinovite induzida por infiltração intra-articular de lipopolissacarídeo de Escherichia coli (E. coli) na articulação femorotibiopatelar de ovinos. Foram utilizados 12 ovinos mestiços (Santa Inês x Dorper), hígidos. A indução da sinovite foi realizada apenas nas articulações FTP direitas, as quais foram avaliadas, por meio do exame ultrassonográfico de forma seriada, nos momentos basal (M0) e às 12 (M12), 24 (M24), 48 (M48), 72 (M72) e 120 (M120) horas após a infiltração com lipopolissacarídeo para a indução de sinovite. A aplicação intra-articular de lipopolissacarídeo de E. coli resultou em um ou mais sinais ecográficos de sinovite (aumento de volume do fluido sinovial, pregueamento da membrana sinovial e celularidade na cavidade articular), os quais foram identificados precocemente, 12 horas após a inoculação, e regrediram ao longo dos tempos avaliados (p=0,0001), até desaparecerem após 120 horas da inoculação.

Ganoderma Lucidum Extract Regulates Gut Morphology and Microbial Community in Lipopolysaccharide-Challenged Broilers

This study was conducted to investigate the effect of Ganoderma lucidum extract (GLE) on the gut morphology and cecal microbial community of broilers challenged with lipopolysaccharide (LPS). 144 one-day-old unsexed broiler chicks were randomly distributed into four treatments: non-challenged broilers fed a basal diet; LPS-challenged broilers fed a basal diet; LPS challenged broilers fed a basal diet supplemented with 1 mL/L of GLE in the drinking water; and LPS challenged broilers fed a basal diet supplemented with 1.33 mL/L of GLE in the drinking water. Results showed that supplementationwith1.33 mL/L of GLE alleviated intestinal inflammatory gene expression in LPS-challenged broilers (p≤0.05). Supplementation of GLE (1 and 1.33 mL/L) increased the villus height in the jejunum and ileum of LPS-challenged broilers (p≤0.001). Weighted principal coordinate analysis, heat map of species abundance, and microbial function pathway revealed distinct separation between the groups treated with LPS only and LPS in combination with GLE supplementation (1 and 1.33 mL/L). The abundance of the genus Faecalibacterium was increased in the cecal digesta of LPS-challenged broilers receiving GLE(1 and 1.33 mL/L) compared with the LPS challenge-only group (p≤0.001). The growth performance parameter of broilers was positively associated with the abundance of the genus Faecalibacterium in the cecal digesta. In conclusion, GLE supplementation could modulate gut morphology and cecal microbiota composition of broilers under inflammatory challenge.(AU)

Effect of OPG-supplemented diet on immune and stress responses and attenuation of LPS-induced damage in the liver of carp (Cyprinus carpio haematopterus)

Effects of oxidized peptidoglycan (OPG) on immune and stress responses and lipopolysacchari-de (LPS)-induced damage in the liver of carp were investigated in this study. Four hundred carps (Cyprinus carpio haematopterus) were fed with five experimental diets supplemented with 0, 100, 200, 400, and 800 mg kg-1 OPG for 28 days. Each group had four replicates and 20 fish per replication. LPS challenge (injection of 40 mg kg-1 saline or LPS) occurred at day 29. The supplementation with OPG linearly increased (p<0.05) plasma total protein, immunoglobulin M (IgM), complement 4 (C4), cortisol, and lactate on day 14. Dietary supplementation with OPG linearly increased (p<0.05) plasma and complement 3 (C3); quadratically improved (p<0.05) alkaline phosphatase (ALP) and lysozyme (LYS) activities; linearly increased hepatic superoxide dismutase (SOD) and catalase (CAT) activities; increased malondialdehyde (MDA) contents; and improved (p<0.05) hepatic anti-superoxide anion (ASA) and anti-hydroxy radical (AHR) contents on days 14 and 28. Dietary OPG significantly preven-ted the increase of interleukin 1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) by inhibiting the excessive activation of TLR2-Myd88 signaling pathway; downregulating TLR2, Myd88, and NF-κB p65; and upregulating nuclear factor erythroid-2-related factor 2 (Nrf2) and Keap1 mRNA expression (p<0.05). Therefore, this study indicated that dietary OPG improves the plasma immune response, regulates the hepatic antioxidant status, and attenuates LPS-induced negative effects in the carp at the optimal dose of 400 mg kg-1(AU)

Lysozyme activity as an indicator of innate immunity of tilapia (Oreochromis niloticus) when challenged with LPS and Streptococcus agalactiae

The objective of this study was to fully describe the protocol with standardized modifications and evaluate the lysozyme activity, an indicator of innate immunity in tilapia, to compare lipopolysaccharide (LPS) with Streptococcus agalactiae injections. Lysozyme was determined in serum using the turbidimetric method, in which lysozyme activity was evaluated by Micrococcus lysodeikticus lysis, with modifications for microplate assay. The experiment was conducted in a completely randomized design. Juvenile tilapia was divided in the following six treatments: challenged with phosphate buffer PBS (control) and 100, 250, 500, and 600 µg kg−1 LPS and S. agalactiae. All treatments were challenged for 72 h and seven days and then sampled to determine lysozyme activity. After 72 h or seven days, concentrations of LPS promoted changes in lysozyme production, either lesser or equal, depending on concentration when compared with fish injected with S. agalactiae. It was possible to standardize the analysis and determine that the treatment with LPS promotes immunomodulation at a concentration of 250 µg kg−1 LPS, this response being similar to challenge with S. agalactiae.

Effects of increasing lipopolysaccharide concentrations on in vitro developmental competence of ovine oocytes

Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewe’s ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.

Effects of increasing lipopolysaccharide concentrations on in vitro developmental competence of ovine oocytes

Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewes ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.(AU)

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Clinical, hematological and histopathological aspects of experimental induction of laminitis in cattle through lipopolysaccharide infusion / Aspectos clínicos, hematológicos e histopatológicos da indução experimental por lipopolissacarídeos de laminite em bovinos

The purpose of this research was to confirm the changes occurring in the foot system of the heifers challenged with lipopolysaccharides (LPS), at the clinical, serum and histological levels. We studied 16 clinically healthy heifers, 14 months of age, placed in a confinement system. All the animals were provided with an accelerometer collar to establish their activity. They were categorized into two groups: the LPS group (n=8), or those which were administered two intravenous applications of 2 mL containing 0.5 g/kg of body weight of LPS, with a 24-hour interval and the Control group (n=8) which were given two infusions of 2 mL of saline solution in the same time interval. General clinical examination and blood collection were done at 0, 4 and 8 hours post the LPS challenges and analyses of the hemograms and paroxonese-1 were performed. The animals were then slaughtered on day 4 and the laminar tissue was collected for histological analysis. The LPS group revealed a lower total leukocyte count with heart rate and greater activity. None of the animals revealed any abnormal signs symptomatic of foot pathology after histological analysis. Hence, the challenge with LPS failed to induce any clinical and histological changes in the foot tissue compatible with laminitis.(AU)

O objetivo deste trabalho foi verificar alterações do sistema podal á nível clínico, hematológico e histológico de animais desafiados com lipopolissacarídeo (LPS). Foram utilizadas 16 novilhas de corte com 14 meses de idade, clinicamente saudáveis, em sistema de confinamento. Todos os animais continham uma coleira com acelerômetro para verificar atividade, sendo divididos em 2 grupos: LPS (n= 8), que recebeu duas aplicações de 2 mL contendo 0,5 g/kg de peso corporal de LPS via intravenosa, com intervalo de 24 horas e Controle (n= 8) que recebeu duas aplicações de 2 mL de solução salina com o mesmo intervalo de tempo. Foram realizados exame clinico geral e coletas de sangue às 0, 4 e 8 horas após os desafios com LPS para análise de hemogramas e paroxonase-1. Os animais foram abatidos aos 5 dias sendo executada a coleta de tecido laminar para análise histológica. A análise estatística foi executada com a utilização do programa NCSS(2004) através de ANOVA-medidas repetidas, considerando o efeito do tratamento do período e sua interação (tratamento*horas), sendo considerados significativos valores de P 0,05. O grupo LPS apresentou superior contagem total de leucócitos com maior frequência cardíaca e atividade. Nenhum animal apresentou sinal de anormalidade compatível com laminite através da análise histológica. Entretanto, o desafio com LPS foi capaz de gerar alterações a nível clínico, constatado pelas alterações nos parâmetros de frequência cardíaca, respiratória e temperatura corporal, além de mudanças no leucograma.(AU)

TNF-alpha inhibitor adalimumab attenuates endotoxin induced cardiac damage in rats

Purpose To investigate the effects of adalimumab pretreatment on the lipopolysaccharide-mediated myocardial injury. Methods Twenty-eight Wistar rats were randomized into four groups (n=7). Control (C) group animals were injected once a day with intraperitoneal (i.p) 0.9 % saline for two days. In the Adalimumab (Ada) group, adalimumab was injected at a dose of 10 mg/kg/ day (i.p) for two days. Lipopolysaccharide (Lps) group rats were injected with a dose of 5 mg/kg (i.p) lipopolysaccharide. Lipopolysaccharide + Adalimumab (Lps+Ada) group rats received adalimumab before the administration of lipopolysaccharide. The animals were sacrificed 24 h after the last injection and blood samples were obtained for determination of biochemical cardiac injury markers and circulating levels of TNF-α and interleukin-6 (IL-6). Hearts were harvested for histological examination. Results Endotoxin exposure resulted in significant increases in serum cardiac injury markers, serum cytokines and histological myocardial injury scores in the Lps group. The levels of circulating cytokines, cardiac injury markers and histological injury scores for myocardial necrosis, perivascular cell infiltration, and inflammation were significantly reduced in Lps+Ada as compared to Lps group (p<0.05). Conclusions Adalimumab pretreatment reduces endotoxin-induced myocardial damage in rats. This beneficial effect is thought to be related to the reduction of cytokine release.(AU)

Lymphocyte proliferative responses in dairy cows supplemented with an immunomodulatory feed additive and administered polyvalent vaccination / [Proliferação de linfócitos em vacas leiteiras suplementadas com um aditivo alimentar imunomodulador e vacinadas com vacina polivalente]

O presente estudo avaliou o efeito da suplementação com OmniGen-AF® na proliferação de linfócitos e títulos de anticorpos após vacinação em bovinos leiteiros. Amostras de sangue periférico foram coletadas de 32 vacas leiteiras para quantificação dos títulos de anticorpos anti-Leptospira, e amostras de sangue periférico de 16 vacas leiteiras foram também coletadas para avaliação da proliferação de linfócitos. Observou-se que a suplementação com OmniGen-AF® aumentou a proliferação basal de linfócitos (sem estímulos) 21 dias após a vacinação (P=0,03), apesar de reduzir a proliferação de linfócitos B quando estimulada com Leptospira borgpetersenii serovar Hardjo inativada pelo calor (P=0,03). Ademais, nenhum efeito da suplementação sobre a proliferação de linfócitos no momento imediatamente anterior à vacinação e nos títulos de anticorpos anti-Leptospira foi encontrado. Além disso, a proliferação de linfócitos estimulada com lipopolissacarídeos foi maior em vacas multíparas que em primíparas 21 dias após a vacinação (P=0,03). Desse modo, o presente estudo demonstra que a suplementação com OmniGen-AF® não afetou de forma robusta a proliferação de linfócitos e os títulos de anticorpos anti-Leptospira após vacinação em vacas leiteiras sadias.(AU)

Evaluation of cell damage and modulation of cytokines TNF-, IL-6 and IL-10 in macrophages exposed to PpIX-mediated photodynamic therapy / Avaliação do dano celular e da modulação das citocinas TNF-alpha, IL-6 e IL-10 em macrófagos expostos à terapia fotodinâmica mediada pela PpIX

Little is known regarding whether photodynamic therapy (PDT)-induced cell death can substantially compromise macrophages (M), which are important cells in PDT-induced immune responses. Here, parameters of PDT-mediated M cytotoxicity and cytokine production in response to protoporphyrin IX (PpIX) were evaluated. Peritoneal M from BALB/c mice were stimulated in vitro with PDT, light, PpIX, or lipopolysaccharide (LPS). After that, cell viability, lipid peroxidation, Nitric Oxide (NO), DNA damage, TNF-, IL-6 and IL-10 were evaluated. Short PDT exposure reduced cell viability by 1030%. There was a two-fold increase in NO and DNA degradation, despite the non-increase in lipoperoxidation. PDT increased TNF- and IL-10, particularly in the presence of LPS, and decreased the production of IL-6 to 10-fold. PDT causes cellular stress, induces NO radicals and leads to DNA degradation, generating a cytotoxic microenvironment. Furthermore, PDT modulates pro- and anti-inflammatory cytokines in M(AU)

Pouco se sabe se a morte celular induzida pela terapia fotodinâmica (PDT) compromete os macrófagos (M), envolvidos nas respostas imunes induzidas pela PDT. Neste estudo, foram avaliados parâmetros de citotoxicidade dos M mediada pela PDT e a produção de citocinas, frente à protoporfirina IX (PpIX). M peritoneais de camundongos BALB/c foram estimulados in vitro com PDT, luz, PpIX ou lipopolissacarídeo (LPS). Após isto, a viabilidade celular (VC), a lipoperoxidação, os níveis de óxido nítrico (NO), de DNA degradado, de TNF-, IL-6 e IL-10 foram avaliados. A exposição curta à PDT reduziu a VC em 10-30%. Os níveis de NO e de DNA degradado duplicaram, sem aumento da lipoperoxidação. Houve aumento de TNF- e IL-10, sendo maior na presença de LPS. Já a produção de IL-6 reduziu em dez vezes. A PDT induz estresse celular, gera radicais NO e causa dano ao DNA, tornando o microambiente citotóxico. Ainda, modula citocinas pró e anti-inflamatórias em M(AU)

Evaluation of cell damage and modulation of cytokines TNF-, IL-6 and IL-10 in macrophages exposed to PpIX-mediated photodynamic therapy

Abstract Little is known regarding whether photodynamic therapy (PDT)-induced cell death can substantially compromise macrophages (M), which are important cells in PDT-induced immune responses. Here, parameters of PDT-mediated M cytotoxicity and cytokine production in response to protoporphyrin IX (PpIX) were evaluated. Peritoneal M from BALB/c mice were stimulated in vitro with PDT, light, PpIX, or lipopolysaccharide (LPS). After that, cell viability, lipid peroxidation, Nitric Oxide (NO), DNA damage, TNF-, IL-6 and IL-10 were evaluated. Short PDT exposure reduced cell viability by 1030%. There was a two-fold increase in NO and DNA degradation, despite the non-increase in lipoperoxidation. PDT increased TNF- and IL-10, particularly in the presence of LPS, and decreased the production of IL-6 to 10-fold. PDT causes cellular stress, induces NO radicals and leads to DNA degradation, generating a cytotoxic microenvironment. Furthermore, PDT modulates pro- and anti-inflammatory cytokines in M.

Resumo Pouco se sabe se a morte celular induzida pela terapia fotodinâmica (PDT) compromete os macrófagos (M), envolvidos nas respostas imunes induzidas pela PDT. Neste estudo, foram avaliados parâmetros de citotoxicidade dos M mediada pela PDT e a produção de citocinas, frente à protoporfirina IX (PpIX). M peritoneais de camundongos BALB/c foram estimulados in vitro com PDT, luz, PpIX ou lipopolissacarídeo (LPS). Após isto, a viabilidade celular (VC), a lipoperoxidação, os níveis de óxido nítrico (NO), de DNA degradado, de TNF-, IL-6 e IL-10 foram avaliados. A exposição curta à PDT reduziu a VC em 10-30%. Os níveis de NO e de DNA degradado duplicaram, sem aumento da lipoperoxidação. Houve aumento de TNF- e IL-10, sendo maior na presença de LPS. Já a produção de IL-6 reduziu em dez vezes. A PDT induz estresse celular, gera radicais NO e causa dano ao DNA, tornando o microambiente citotóxico. Ainda, modula citocinas pró e anti-inflamatórias em M.

Involvement of TLR4 in the protective effect of intra-articular administration of curcumin on rat experimental osteoarthritis

Purpose: In view of the principal role of Toll-like receptor 4 (TLR4) in mediating sterile inflammatory response contributing to osteoarthritis (OA) pathogenesis, we used lipopolysaccharide (LPS), a known TLR4 activator, to clarify whether modulation of TLR4 contributed to the protective actions of intra-articular administration of curcumin in a classical rat OA model surgically induced by anterior cruciate ligament transection (ACLT). Methods: The rats underwent ACLT and received 50μl of curcumin at the concentration of 1 mg mL-1and 10 μg LPS by intra-articular injection once a week for 8 weeks. Morphological changes of the cartilage and synovial tissues were observed. Apoptotic chondrocytes were detected using TUNEL assay. The concentrations of IL-1β and TNF-ɑ in synovial fluid were determined using ELISA kits. The mRNA and protein expression levels of TLR4 and NF-κB p65 were detected by real-time PCR and Western blotting, respectively. Results: Intra-articular administration of curcumin significantly improved articular cartilage injury, suppressed synovial inflammation and down-regulated the overexpression of TLR4 and its downstream NF-κB caused by LPS-induced TLR4 activation in rat osteoarthritic knees. Conclusion: The data suggested that the inhibition of TLR4 signal might be an important mechanism underlying a protective effect of local curcumin administration on OA.(AU)

Effects of dietary supplementation with tributyrin and coated sodium butyrate on intestinal morphology, disaccharidase activity and intramuscular fat of lipopolysaccharide-challenged broilers

The objectives of this study were to investigate the effects of tributyrin (TB) and coated sodium butyrate (CSB) on intestinal morphology, disaccharidase activity and intramuscular fat of broilers challenged with lipopolysaccharide (LPS). A total of 160 1-day-old healthy Cobb broilers were randomly allocated into four groups: (1) control; (2) LPS, in which broilers were fed a basal diet and intraperitoneally injected with 500 g/kg LPS on days 38, 40 and 42; (3) TB, in which LPS-challenged broilers were fed basal diet supplemented with 500 mg/kg TB; and (4) CSB, in which LPS-challenged broilers were fed basal diet supplemented with 877 mg/kg CSB. Addition of TB and CSB inhibited (p<0.05) the decrease in villus height in the duodenum and ileum of LPS-challenged broilers, respectively. Both TB and CSB increased (p<0.05) activity of maltase in the small intestine, and TB increased (p<0.05) activity of isomaltase in the ileum. Additionally, dietary addition of TB and CSB decreased (p<0.05) the content of intramuscular fat. In conclusion, dietary supplementation of TB was more effective than CSB in improving intestinal morphology and disaccharidase activity of LPS-challenged broilers, and they both reduced intramuscular fat in the breast and legs.(AU)

Effects of dietary supplementation with tributyrin and coated sodium butyrate on intestinal morphology, disaccharidase activity and intramuscular fat of lipopolysaccharide-challenged broilers

The objectives of this study were to investigate the effects of tributyrin (TB) and coated sodium butyrate (CSB) on intestinal morphology, disaccharidase activity and intramuscular fat of broilers challenged with lipopolysaccharide (LPS). A total of 160 1-day-old healthy Cobb broilers were randomly allocated into four groups: (1) control; (2) LPS, in which broilers were fed a basal diet and intraperitoneally injected with 500 g/kg LPS on days 38, 40 and 42; (3) TB, in which LPS-challenged broilers were fed basal diet supplemented with 500 mg/kg TB; and (4) CSB, in which LPS-challenged broilers were fed basal diet supplemented with 877 mg/kg CSB. Addition of TB and CSB inhibited (p<0.05) the decrease in villus height in the duodenum and ileum of LPS-challenged broilers, respectively. Both TB and CSB increased (p<0.05) activity of maltase in the small intestine, and TB increased (p<0.05) activity of isomaltase in the ileum. Additionally, dietary addition of TB and CSB decreased (p<0.05) the content of intramuscular fat. In conclusion, dietary supplementation of TB was more effective than CSB in improving intestinal morphology and disaccharidase activity of LPS-challenged broilers, and they both reduced intramuscular fat in the breast and legs.