Early pregnancy affects expression of Toll-like receptor signaling members in ovine spleen

Abstract Toll-like receptors (TLRs) are involved to the maternal immune tolerance. The spleen is essential for adaptive immune reactions. However, it is unclear that early pregnancy regulates TLR-mediated signalings in the maternal spleen. The purpose of this study was to investigate the effects of early pregnancy on expression of TLR signaling members in the ovine spleen. Ovine spleens were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of pregnancy (n = 6 for each group). Real-time quantitative PCR, western blot and immunohistochemistry analysis were used to detect TLR signaling members, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). The results showed that expression levels of TLR2, TLR4 and IRAK1 were downregulated, but expression levels of TLR3, TLR5, TLR7, TLR9, TRAF6 and MyD88 were increased during early pregnancy. In addition, MyD88 protein was located in the capsule, trabeculae and splenic cords of the maternal spleen. This paper reports for the first time that early pregnancy has effects on TLR signaling pathways in the ovine spleen, which is beneficial for understanding the maternal immune tolerance during early pregnancy.

Early pregnancy affects expression of Toll-like receptor signaling members in ovine spleen

Toll-like receptors (TLRs) are involved to the maternal immune tolerance. The spleen is essential for adaptive immune reactions. However, it is unclear that early pregnancy regulates TLR-mediated signalings in the maternal spleen. The purpose of this study was to investigate the effects of early pregnancy on expression of TLR signaling members in the ovine spleen. Ovine spleens were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of pregnancy (n = 6 for each group). Real-time quantitative PCR, western blot and immunohistochemistry analysis were used to detect TLR signaling members, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). The results showed that expression levels of TLR2, TLR4 and IRAK1 were downregulated, but expression levels of TLR3, TLR5, TLR7, TLR9, TRAF6 and MyD88 were increased during early pregnancy. In addition, MyD88 protein was located in the capsule, trabeculae and splenic cords of the maternal spleen. This paper reports for the first time that early pregnancy has effects on TLR signaling pathways in the ovine spleen, which is beneficial for understanding the maternal immune tolerance during early pregnancy.(AU)

Quantitative analysis of expression patterns of different cell surface Toll-like receptor genes imparting heat stress resilience to indigenous Osmanabadi and Salem Black goat breeds

The study was conducted to establish the differences in the expression pattern of different cell surface toll-likereceptors (TLRs) between indigenous Osmanabadi and Salem Black goats subjected to summer season induced heat stress. The primary objective of the study is to assess the thermotolerance ability of these two breeds based on maintaining the immune status during exposure to heat stress. The results indicated that the expression of TLR1, TLR4, TLR5, TLR6, and TLR10 genes in both the breeds showed different expression pattern. The expression pattern of all these genes did not differed between the control (OC) and heat stress (OHS) group in Osmanabadi breed but significantly higher (P<0.05) expression of these genes were reported in heat stress group (SBHS) as compared to control group (SBC) in Salem Black breed. However, the striking difference was established in the expression pattern of TLR2 between these breeds. The TLR expression was significantly higher in heat stress group of Osmanabadi breed. But TLR2 did not express in Salem Back breed. The higher expression of most of the cell surface TLRs in the SBHS group as compared to OHS group indicates the superior resilient capacity of Salem Black goats to maintain immune status even during exposure to adverse environmental condition. Further, these TLRs could serve as indicators to reflect better thermo-tolerance of Salem Black breed over Osmanabadi breed.(AU)

Quantitative analysis of expression patterns of different cell surface Toll-like receptor genes imparting heat stress resilience to indigenous Osmanabadi and Salem Black goat breeds

The study was conducted to establish the differences in the expression pattern of different cell surface toll-likereceptors (TLRs) between indigenous Osmanabadi and Salem Black goats subjected to summer season induced heat stress. The primary objective of the study is to assess the thermotolerance ability of these two breeds based on maintaining the immune status during exposure to heat stress. The results indicated that the expression of TLR1, TLR4, TLR5, TLR6, and TLR10 genes in both the breeds showed different expression pattern. The expression pattern of all these genes did not differed between the control (OC) and heat stress (OHS) group in Osmanabadi breed but significantly higher (P<0.05) expression of these genes were reported in heat stress group (SBHS) as compared to control group (SBC) in Salem Black breed. However, the striking difference was established in the expression pattern of TLR2 between these breeds. The TLR expression was significantly higher in heat stress group of Osmanabadi breed. But TLR2 did not express in Salem Back breed. The higher expression of most of the cell surface TLRs in the SBHS group as compared to OHS group indicates the superior resilient capacity of Salem Black goats to maintain immune status even during exposure to adverse environmental condition. Further, these TLRs could serve as indicators to reflect better thermo-tolerance of Salem Black breed over Osmanabadi breed.

Endometrial transcription of microbial molecular patterns receptors in Gyr and F1 Holstein x Gyr postpartum cows / Transcrição endometrial de receptores de padrões moleculares microbianos em vacas Gir e F1 Holandês x Gir após o parto

Zebu and Holstein x Zebu crossbred have low incidence of uterine infection when compared to Holstein cows. Resistance to uterine infections may be associated with the ability to recognize invading microorganisms. Endometrial transcription of microbial molecular patterns receptors has been investigated in the postpartum period of Holstein cows, but it is completely unknown in Zebu or Holstein x Zebu cows. In this study, 9 Gyr and 12 F1 Holstein x Gyr cows were submitted to endometrial biopsies at the first and seventh days postpartum, with the objective to measure transcription levels of toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2). There was a significant (P<0.05) decrease in transcription of TLR5 in Gyr, and an increase in transcription of TLR9 in F1 cows, between the first and seventh day postpartum. Both groups had low incidences of uterine infections up to 42 days postpartum. Uterine involution completed at 27.7 ± 10.1 and 25.1 ± 4.7 days postpartum for Gyr and F1 cows, respectively. In Gyr cows, higher transcription levels of TLR1/6 and NOD1 correlated to a longer period required for uterine involution. In F1 cows, lower levels of TLR1/6, TLR2 and NOD2 correlated to a longer period required for uterine involution. In conclusion, some pathogen recognition receptors associated significantly with the time required for uterine involution in Gyr and F1 cows.(AU)

Vacas Zebu e mestiças Holandês x Zebu apresentam baixas incidências de infecções uterinas quando comparadas às Holandesas. A resistência às infecções uterinas pode estar relacionada com a capacidade de reconhecimento dos microrganismos invasores. A transcrição endometrial de receptores de padrões moleculares microbianos tem sido investigada em vacas Holandesas recém-paridas, porém ainda é desconhecida em vacas Zebu e mestiças Holandês x Zebu. No presente estudo, nove vacas Gir e 12 F1 Holandês x Gir foram submetidas a biópsias endometriais no primeiro e no sétimo dia após o parto, com o objetivo de mensurar os níveis de transcrição gênica dos receptores tipo Toll (TLRs) 1/6, 2, 4, 5 e 9; receptores tipo NOD 1 e 2; e dos coreceptores CD14 e MD-2. Houve diminuição significativa (P < 0,05) do nível de transcrição de TLR5 em vacas Gir e aumento da transcrição de TLR9 em vacas F1, entre o primeiro e o sétimo dia após o parto. Os dois grupos apresentaram baixas incidências de infecções uterinas até 42 dias pós-parto. O período de involução uterina foi de 27,7 ± 10,1 e 25,1 ± 4,7 dias pós-parto, para vacas Gir e F1, respectivamente. No grupo de vacas Gir, altos níveis de transcrição de TLR1/6 e NOD1 tiveram correlação significativa com o prolongamento do período de involução uterina. No grupo de vacas F1, baixos níveis de transcrição de TLR1/6, TLR2 e NOD2 foram associados a maiores períodos de involução uterina. Portanto, os níveis de transcrição endometrial de alguns receptores de padrões moleculares microbianos na primeira semana após o parto podem estar relacionados com o tempo requerido para ocorrência da involução uterina em vacas Gir e F1.(AU)

Endometrial transcription of microbial molecular patterns receptors in Gyr and F1 Holstein x Gyr postpartum cows / Transcrição endometrial de receptores de padrões moleculares microbianos em vacas Gir e F1 Holandês x Gir após o parto

Zebu and Holstein x Zebu crossbred have low incidence of uterine infection when compared to Holstein cows. Resistance to uterine infections may be associated with the ability to recognize invading microorganisms. Endometrial transcription of microbial molecular patterns receptors has been investigated in the postpartum period of Holstein cows, but it is completely unknown in Zebu or Holstein x Zebu cows. In this study, 9 Gyr and 12 F1 Holstein x Gyr cows were submitted to endometrial biopsies at the first and seventh days postpartum, with the objective to measure transcription levels of toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2). There was a significant (P<0.05) decrease in transcription of TLR5 in Gyr, and an increase in transcription of TLR9 in F1 cows, between the first and seventh day postpartum. Both groups had low incidences of uterine infections up to 42 days postpartum. Uterine involution completed at 27.7 ± 10.1 and 25.1 ± 4.7 days postpartum for Gyr and F1 cows, respectively. In Gyr cows, higher transcription levels of TLR1/6 and NOD1 correlated to a longer period required for uterine involution. In F1 cows, lower levels of TLR1/6, TLR2 and NOD2 correlated to a longer period required for uterine involution. In conclusion, some pathogen recognition receptors associated significantly with the time required for uterine involution in Gyr and F1 cows.(AU)

Vacas Zebu e mestiças Holandês x Zebu apresentam baixas incidências de infecções uterinas quando comparadas às Holandesas. A resistência às infecções uterinas pode estar relacionada com a capacidade de reconhecimento dos microrganismos invasores. A transcrição endometrial de receptores de padrões moleculares microbianos tem sido investigada em vacas Holandesas recém-paridas, porém ainda é desconhecida em vacas Zebu e mestiças Holandês x Zebu. No presente estudo, nove vacas Gir e 12 F1 Holandês x Gir foram submetidas a biópsias endometriais no primeiro e no sétimo dia após o parto, com o objetivo de mensurar os níveis de transcrição gênica dos receptores tipo Toll (TLRs) 1/6, 2, 4, 5 e 9; receptores tipo NOD 1 e 2; e dos coreceptores CD14 e MD-2. Houve diminuição significativa (P < 0,05) do nível de transcrição de TLR5 em vacas Gir e aumento da transcrição de TLR9 em vacas F1, entre o primeiro e o sétimo dia após o parto. Os dois grupos apresentaram baixas incidências de infecções uterinas até 42 dias pós-parto. O período de involução uterina foi de 27,7 ± 10,1 e 25,1 ± 4,7 dias pós-parto, para vacas Gir e F1, respectivamente. No grupo de vacas Gir, altos níveis de transcrição de TLR1/6 e NOD1 tiveram correlação significativa com o prolongamento do período de involução uterina. No grupo de vacas F1, baixos níveis de transcrição de TLR1/6, TLR2 e NOD2 foram associados a maiores períodos de involução uterina. Portanto, os níveis de transcrição endometrial de alguns receptores de padrões moleculares microbianos na primeira semana após o parto podem estar relacionados com o tempo requerido para ocorrência da involução uterina em vacas Gir e F1.(AU)

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Agaricus brasiliensis é um cogumelo medicinal com atividades imunomoduladoras e antitumorais atribuídas aos ß-glucanos presentes na fração polissacarídica de seu corpo de frutificação. Uma vez que os ß-glucanos aumentam a imunorresponsividade celular, neste estudo objetivamos avaliar o efeito de uma fração rica em polissacarídeos tratados com ácido (ATF) de A. brasiliensis sobre a capacidade de monócitos humanos de aderir / fagocitar células de levedura C. albicans . expressão de receptores de reconhecimento de padrões e sua capacidade de produzir citocinas. Métodos: A adesão / fagocitose de C. albicans marcada com FITC foi avaliada por citometria de fluxo. As células foram incubadas com anticorpos marcados com fluorocromo específicos para TLR2 e 4, ßGR e MR e também avaliadas por citometria de fluxo. Os monócitos foram cultivados com ATF, e os sobrenadantes da cultura foram coletados para análise da produção de citocinas in vitro por ELISA (TNF-α, IL-1ß, IL-12 e IL-10). Resultados: ATF aumentou significativamente a aderência / fagocitose de C. albicans por monócitos e isso foi associado com expressão aumentada de TLR2 e TLR4, enquanto nenhum efeito foi observado em ßGR ou MR. Além disso, a expressão de TLR4 e TLR2 foi associada a níveis mais elevados de produção in vitro de TNF-α e IL-1, respectivamente. A produção de IL-10 também foi aumentada pelo tratamento com ATF, mas não encontramos associação entre sua produção e a expressão de receptores Toll-like. Conclusão: Nossos resultados nos forneceram evidências de que polissacarídeos de A. brasiliensis afetam monócitos humanos provavelmente através da modulação de receptores Toll-like.(AU)

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Agaricus brasiliensis é um cogumelo medicinal com atividades imunomoduladoras e antitumorais atribuídas aos ß-glucanos presentes na fração polissacarídica de seu corpo de frutificação. Uma vez que os ß-glucanos aumentam a imunorresponsividade celular, neste estudo objetivamos avaliar o efeito de uma fração rica em polissacarídeos tratados com ácido (ATF) de A. brasiliensis sobre a capacidade de monócitos humanos de aderir / fagocitar células de levedura C. albicans . expressão de receptores de reconhecimento de padrões e sua capacidade de produzir citocinas. Métodos: A adesão / fagocitose de C. albicans marcada com FITC foi avaliada por citometria de fluxo. As células foram incubadas com anticorpos marcados com fluorocromo específicos para TLR2 e 4, ßGR e MR e também avaliadas por citometria de fluxo. Os monócitos foram cultivados com ATF, e os sobrenadantes da cultura foram coletados para análise da produção de citocinas in vitro por ELISA (TNF-α, IL-1ß, IL-12 e IL-10). Resultados: ATF aumentou significativamente a aderência / fagocitose de C. albicans por monócitos e isso foi associado com expressão aumentada de TLR2 e TLR4, enquanto nenhum efeito foi observado em ßGR ou MR. Além disso, a expressão de TLR4 e TLR2 foi associada a níveis mais elevados de produção in vitro de TNF-α e IL-1, respectivamente. A produção de IL-10 também foi aumentada pelo tratamento com ATF, mas não encontramos associação entre sua produção e a expressão de receptores Toll-like. Conclusão: Nossos resultados nos forneceram evidências de que polissacarídeos de A. brasiliensis afetam monócitos humanos provavelmente através da modulação de receptores Toll-like.(AU)

Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle

The aim of this study was to elucidate the presence of components of the innate immune system inthe bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3–6), mature CL (mCL; n = 5, approx. days 8–12), and regressing CL (rCL; n = 5, approx. days 17–19). An upregulation of TLR2 mRNA was detected only in rCL(P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCLand rCL compared to dCL(P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.

Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle

The aim of this study was to elucidate the presence of components of the innate immune system inthe bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3–6), mature CL (mCL; n = 5, approx. days 8–12), and regressing CL (rCL; n = 5, approx. days 17–19). An upregulation of TLR2 mRNA was detected only in rCL(P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCLand rCL compared to dCL(P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.(AU)

Upregulation of tlr4 mrna expression levels in broiler chickens under acute heat stress

Toll-like receptors (TLRs) are well-characterized in mice and rats, but little is known on their role in broiler chickens during acute heat stress (AHS). The aim of the present study was to estimate the change in TLR4 mRNA expression in the liver, kidney's pleen, heart, and small intestine of broiler chickens under AHS. A total of 240 healthy Arbor Acres (AA) broiler chickens were randomly divided into four groups: control group (22±1°C; 0h), HS2, HS5 and HS10 (38±1°C; 2h, 5h and 10h of heat stress, respectively). As AHS duration increased, TLR4 mRNA expression slightly decreased at HS2 and HS5, but was dramatically elevated in HS10 (p 0.001) compared with the control group in the small intestine, as well as in the spleen at HS2 (p=0.001) and HS10 (p 0.001). The mRNA expression levels of TLR4 significantly increased in the liver, heart, and kidneys (p 0.001) at HS10, and in the kidneys at HS5 (p=0.003). It is found that TLR4 mRNA expression at HS10 in different organs was significantly higher (p 0.001) compared with HS2 and HS5. The results of the present study suggest that AHS may modulate the functional responses of the liver, kidney, spleen, heart, and small intestine of broilers by regulating TLR4 mRNA expression.

Upregulation of tlr4 mrna expression levels in broiler chickens under acute heat stress

Toll-like receptors (TLRs) are well-characterized in mice and rats, but little is known on their role in broiler chickens during acute heat stress (AHS). The aim of the present study was to estimate the change in TLR4 mRNA expression in the liver, kidney's pleen, heart, and small intestine of broiler chickens under AHS. A total of 240 healthy Arbor Acres (AA) broiler chickens were randomly divided into four groups: control group (22±1°C; 0h), HS2, HS5 and HS10 (38±1°C; 2h, 5h and 10h of heat stress, respectively). As AHS duration increased, TLR4 mRNA expression slightly decreased at HS2 and HS5, but was dramatically elevated in HS10 (p 0.001) compared with the control group in the small intestine, as well as in the spleen at HS2 (p=0.001) and HS10 (p 0.001). The mRNA expression levels of TLR4 significantly increased in the liver, heart, and kidneys (p 0.001) at HS10, and in the kidneys at HS5 (p=0.003). It is found that TLR4 mRNA expression at HS10 in different organs was significantly higher (p 0.001) compared with HS2 and HS5. The results of the present study suggest that AHS may modulate the functional responses of the liver, kidney, spleen, heart, and small intestine of broilers by regulating TLR4 mRNA expression.(AU)

AVALIAÇÃO DA EXPRESSÃO DA VIA PD-1/PDL-1 E DAS PROTEÍNAS CTLA-4, RANK-RANK-L E IFN-y EM MASTOCITOMAS CUTÂNEOS DE ALTO GRAU EM CÃES: NOVAS PERSPECTIVAS DE USO DA IMUNOTERAPIA

Os mastócitos caninos são considerados os tumores malignos cutâneos mais frequentes. São potencialmente metastáticos. A escolha do tratamento dependerá da presença ou ausência dos fatores prognósticos negativos, incluindo metástases dos linfonodos regionais, entretanto, até o momento não existe um marcador prognóstico aplicado em linfonodos que pudesse estar associado aos demais fatores prognósticos. A imunoterapia em medicina veterinária está sendo desenvolvida recentemente. Os pontos de controle imunológicos (CTLA-4, PD-1 / PD-L1) desempenham um papel vital na inibição das respostas imunes e na promoção da auto-tolerância através da modulação da atividade das células T, ativando a apoptose de células T específicas do antígeno e inibindo a apoptose de células T regulatórias. Estratégias terapêuticas que modulam o microambiente tumoral e reforçam a migração de células T para o tumor são fundamentais no controle de diversos tipos de câncer. Uma dessas estratégias é o uso de moduladores da imunidade inata, como os agonistas do receptor toll-like (TLRs). Nesse cenário, a nanoimunoterapia Onco- Therad induz a via de sinalização do interferon mediada por TLR4, resultando em potentes efeitos antitumorais. Além disso, a inibição da via de sinalização RANK / RANK-L regulou negativamente a expressão de PD-1 / PD-L1 no microambiente tumoral. O objetivo deste estudo é avaliar a expressão dos checkpoints e anticorpos RANK - RANK-L e IFN- em MCT de alto grau e correlacionados com a expressão de PD-1 / PD-L1 em linfonodos associada a características clínicas.

Canine mast cell tumors (MCTs) being considered the most frequent cutaneous malignant tumor. Are potentially metastatic. The choice of treatment will depend of the presence or absence of negative prognostic factors, including regional lymph nodes metastasis, however, until now does not exist a prognostic marker applied in lymph nodes that could be associated with the others prognostic factors. Immunotherapy in veterinary medicine recently being developed. The immune checkpoints (CTLA-4 , PD-1/PD-L1) plays a vital role in inhibiting immune responses and promoting self-tolerance through modulating the activity of T-cells, activating apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells. Therapeutic strategies that modulate the tumor microenvironment and reinforce the migration of T cells to the tumor are fundamental in the control of several types of cancers. One of these strategies is the use of innate immunity modulators, such as agonists of toll-like receptor (TLRs). In this scenario, OncoTherad nano-immunotherapy induces TLR4-mediated interferon signaling pathway, resulting in potent antitumor effects. Also, inhibition of RANK/ RANK-L signaling pathway downregulated PD-1/PD-L1 expression on tumor microenvironment. The main of this study is evaluate the expression of checkpoints and antibodies RANK - RANK-L and IFN- in MCT high grade and correlated with the expression of PD-1/PD-L1 in lymph nodes associated with clinical characteristics.

Phenotypic and genotypic features of the Salmonella enterica serovar Heidelberg strain UFPR1 and insights about intestinal microbiota in feral and broiler chickens

Salmonelas paratíficas são patógenos relevantes sob uma perspectiva clínica e de saúde pública. A Salmonella enterica sorovar Heidelberg (SH) frequentemente causa doenças transmitidas por alimentos em humanos e é frequentemente encontrada em avicultura em vários países, causando perdas econômicas e preocupações relacionadas à saúde pública. Neste cenário, gerou-se uma riqueza de informações sobre uma cepa emergente brasileira de SH, UFPR1, a fim de compreender sua variação genética, patogenicidade, resistência e medidas alternativas de controle. No Capítulo 1, o primeiro genoma completo da cepa SH UFPR1 é relatado, revelando 11 fragmentos genômicos ausentes em comparação a uma cepa SH resistente a múltiplos fármacos, o que explica a alta suscetibilidade a antibióticos e resistência a ácidos orgânicos de cadeia curta. No Capítulo 2, um probiótico composto por três cepas de Bacillus subtilis melhorou o desempenho animal quando alimentado a 250 g/ton e reduziu a colonização de SH UFPR1 no trato gastrintestinal. O probiótico mobilizou as células imunes e promoveu importantes alterações histológicas, relacionada à ativação da resposta de defesa e absorção intestinal. Além disso, a suplementação de probiótico aumentou a diversidade da microbiota cecal e alterou alguns grupos bacterianos comensais no íleo. Estudos sobre o microbioma intestinal e a imunidade inata em frangos geneticamente diferentes e inseridos em ambientes mais naturais são raros e podem nos fornecer ideias sobre a evolução e a ecologia dos patógenos, comensais e hospedeiro. No Capítulo 3, estudamos duas populações de aves feralizadas em Bermudas e no Havaí, comparando a composição da microbiota intestinal e a resposta imune inata a um grupo de frangos de corte comerciais. O sequenciamento de alto rendimento revelou a presença de uma microbiota central e algumas comunidades exclusivas em aves feralizadas e comerciais. A perda progressiva da diversidade microbiana no grupo de frangos de corte pode estar correlacionada a uma resposta imune inata vulnerável e interessantemente mediada por toll-like receptors (TLRs). Mais estudos serão necessários para distinguir características genotípicas e fenotípicas de diferentes cepas e variantes de Salmonella, a fim de melhorar e identificar medidas específicas de controle. Além disso, mais pesquisas envolvendo a feralização são úteis para esclarecer e explorar possíveis elos relacionados a mudanças evolutivas concomitantes, visando melhorar a produtividade e produzir produtos avícolas mais seguros.

Non-typhoidal Salmonella are relevant pathogens under a clinical and public health perspective. Salmonella enterica serovar Heidelberg (SH) frequently causes food-borne illness in humans and are frequently found in broiler operations in several countries, causing economic losses and health-related concerns. This research effort has generated a wealth of information on an emergent Brazilian SH, UFPR1 strain, in order to understand its genetic variation, pathogenicity, resistance and alternative measures of control. In the Chapter 1, the first complete genome of SH UFPR1 strain is reported, revealing 11 missing genomic fragments in comparison with a multidrug resistant SH strain, which explains the high susceptibility to antibiotics and short-chain organic acids resistance. In the Chapter 2, a probiotic composed by three strains of Bacillus subtilis improved animal performance when fed at 250 g/ton and reduced SH UFPR1 colonization in the gastrointestinal tract. The probiotic mobilized immune cells and promoted important histologic alteration, related to activation of defense response and gut absorption. In addition, the supplementation of probiotic increased the diversity of cecal microbiota and increased some commensal bacterial groups in ileum. Studies about gut microbiome and innate immunity in chickens genetically different and inserted in more natural environments are rare and can provide us insights about evolution and ecology of the pathogens, commensals and the host. In the Chapter 3, we studied two populations of feral chickens in Bermuda and Hawaii, comparing their gut microbiota composition and innate immune response to a group of modern broiler chickens. High throughput sequencing revealed the presence of a core microbiota and some exclusive taxa in feral and broiler chickens. The progressive loss of the microbial diversity in the broilers group may be correlated to a vulnerable and interesting innate immune response mediated by TLRs. Further work will be needed to distinguish genotypic and phenotypic features of different Salmonella serovars and strains in order to enhance and identify specific measures of control. Also, more research involving feralization is useful to clarify and explore possible links related to concomitant evolutionary changes, aiming to improve productivity and produce safer poultry products.

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.(AU)

Expressão gênica de mediadores associados a neuropatogênese causada pelo BVH5

O herpes vírus bovino 5 (HVB-5) é um agente infeccioso pertencente a família Herpesviridae, sub-família Alphaherpesvirinae e gênero Varicellovirus. É um vírus neurotrópico que causa doenças neurológicas principalmente em animais jovens em todo mundo, especialmente nos países Sul-americanos, resultado em significantes perdas econômicas. A infecção pelo herpes vírus bovino 5 em gado jovemestá associado a doença neurológica que é, usualmente, fatal, sendo um ótimo modelo para estudar a patogênese da meningoencefalite induzida por vírus. O HVB5 replica na mucosa nasal, e invadesistema nervoso central (SNC), principalmente por meio do sistema olfatório. A resposta imune inata desencadeada pelo hospedeiro frente à replicação do vírus por meio da via olfativa não é totalmente entendida. Estudos verificaram as variações nos níveis de expressão de receptores Toll-Like (TLRs) em diferentes regiões do sistema nervoso central bovino durante a infecção aguda e reativação de bovinos infectados por HVB-1 e HVB-5-.Uma nova perspectiva para entender a relação do patógeno e o hospedeirorelacionando os microRNAs(miRNAs) que interagem com a resposta imune inatadurante infecções virais neurotrópicas.

Bovine herpes virus 5 (BHV-5) is an infectious agent belonging to the family Herpesviridae, subfamily Alphaherpesvirinae and genus Varicellovirus. It is a neurotropic virus that causes neurological diseases mainly in young animals worldwide, especially in the South American countries, resulting in significant economic losses. Bovine herpesvirus 5 infection in young cattle is associated with neurological disease, which is usually fatal and is a good model for studying the pathogenesis of virus-induced meningoencephalitis. The BHV5 replicates in the nasal mucosa, and invades the central nervous system (CNS), mainly through the olfactory system. The innate immune response triggered by the host against virus replication via the olfactory route is not fully understood. Studies have found variations in the levels of Toll-Like receptor (TLR) expression in different regions of the bovine central nervous system during acute infection and reactivation of BoHV-1 and BoHV-5 infected bovines. A new perspective to understand the relationship of the pathogen and the host relating the microRNAs (miRNAs) that interact with the innate immune response during neurotropic viral infections.

Investigação do perfil de expressão gênica de receptores tipo toll e citocinas inflamatórias no encéfalo e no baço de cães com leishmaniose visceral

A leishmaniose visceral (LV) é uma doença parasitária que apresenta distribuição mundial e que pode afetar homens e animais, sendo que o cão é considerado o principal hospedeiro da doença. Cães infectados pelo parasito Leishmania podem apresentar-se assintomáticos ou com desordens generalizadas, incluindo alterações neurológicas. Existem alguns relatos do acometimento do encéfalo durante a infecção, mas a neuropatogenia da doença não foi completamente elucidada. Há evidências do comprometimento das barreiras encefálicas e da presença do DNA do parasito no encéfalo. Os receptores tipo Toll (TLRs) são sensores do sistema imune inato capazes de detectar padrões moleculares associados aos patógenos (PAMPs), desencadeando uma resposta inflamatórias com produção de diversos mediadores inflamatórios, incluindo citocinas. Desta forma, o objetivo deste estudo foi avaliar o perfil de expressão gênica dos Tolls 1-10, assim como a produção de citocinas pró-inflamatórias TNF-, IFN-, IL-1 e IL-6 no encéfalo e no baço de cães com leishmaniose visceral. No baço houve aumento de expressão gênica de TLR-5 e TLR-9, enquanto no encéfalo houve aumento de TLR-4 em uma pequena população de cães infectados. Em relação às citocinas, todas as citocinas foram detectadas nos dois tecidos avaliados, com excessão de IL-6. Nos cães infectados, TNF- e IL-1 estavam presentes em maiores concentrações no encéfalo e no baço, respectivamente. Este estudo fornece suporte para explicar o envolvimento de TLRs na LV e nossos dados confirmam o envolvimento encefálico durante a doença.

Visceral leishmaniasis (VL) is a parasitic disease that presents world distribution, affecting humans and animals. Dogs are considered the main hosts of the disease. Infected dogs with the Leishmania parasite can be asymptomatic or present generalized disorders, including neurological alterations. There are some reports of brain commitment during infection. Nevertheless, neuropathogenesis of VL is not completely elucidated. There are evidences of brain barriers breakdown and of the presence of Leishmania DNA in the brain. Toll-like receptors (TLRs) are innate immune sensors capable of detecting pathogen-associated molecular patterns (PAMPs), trigger an inflammatory response with production of several inflammatory mediators, including cytokines. Therefore, the aim of this study was to evaluate gene expression profile of TLRs1-10, along with the production of proinflammatory cytokines in both brain and spleen in dogs with VL. In spleen there was an up-regulation of TLR-5 and TLR-9 while in the brain there was up-regulation of TLR-4 in a few number of infected animals. Regarding cytokines, all cytokines were detected in both tissues, except IL-6. In the infected dogs, TNF- and IL-1 were present at higher concentrations in the brain and spleen, respectively. This study provides support to explain the involvement of TLRs in VL and our data confirm the brain as an affected organ in this disease.

Peptidoglycan from Staphylococcus aureus induces the overexpression of TRLs 1-8 mRNA in corneal fibroblasts, but its lipoteichoic acid and muramyl dipeptide only induced the overexpression of TLR5 or TRL9

Lipopolysaccharide induces TLR-1-8 mRNAs over-expression in corneal fibroblast. Analyzing if other TLR-ligands can do the same, we found that peptidoglycan does, but not muramyldipeptide, lipoteichoic acid and polyI:C. This suggests that the recognition of lipopolysaccharide and peptidoglycan is enough to alert these cells against microorganisms through the over-expression of the majority TLRs.

PAPEL DO ADAPTADOR INDUTOR DE INTERFERON- CONTENDO DOMÍNIO TIR (TRIF) NA RESISTÊNCIA DE CAMUNDONGOS A INFECÇÃO POR Neospora caninum

Neospora caninum é um parasito intracelular que tem como hospedeiro definitivo o cão e outros mamíferos, especialmente bovinos, como hospedeiros intermediários. Economicamente, a neosporose é uma doença de grande importância na medicina veterinária por induzir relevantes sinais clínicos, como abortos em bovinos e paralisia neuromuscular em cães. O objetivo deste estudo foi avaliar o papel da molécula adaptadora TRIF da via de sinalização dos TLRs na resistência contra a infecção por N. caninum. Para isso, experimentos in vitro com macrófagos derivados de medula óssea (BMDMs) obtidos de camundongos do tipo selvagem (WT) e TRIF knockout (TRIF-/-) estimulados com taquizoítos e infecções in vivo foram realizadas a fim de se investigar a produção de citocinas e anticorpos, parasitismo celular e tecidual, alterações histológicas durante diferentes fases da infecção e análise de sobrevida. Nós observamos que BMDMs TRIF-/- apresentaram reduções significativas na produção de citocinas inflamatórias em relação a macrófagos WT. Adicionalmente, foi visto que as concentrações de NO, IL-12p40, IFN- e TNF foram diminuídas no lavado peritoneal e nos pulmões de camundongos TRIF-/-, enquanto que IL-2, IFN-, TNF e IL-17 foram reduzidas no soro desses animais em comparação aos WT. Alta carga parasitária foi encontrada nas células peritoneais, pulmões e cérebro durante as fases aguda e crônica da infecção, associada com alterações teciduais inflamatórias significativas nos pulmões. Além disso, camundongos TRIF-/- tiveram uma taxa de sobrevida 2 vezes menor quando comparada aos animais WT. Concluindo, nossos resultados mostram que TRIF é requerido para a resistência contra a infecção induzida por N. caninum, regulando a produção de citocinas chave do perfil Th1 de resposta imune e participando no controle do parasitismo tecidual e das lesões inflamatórias oriundas desta infecção.

Neospora caninum is an intracellular parasite that has the dog as its definitive host and other mammals, especially cattle, as intermediate hosts. Economically, neosporosis is an important disease in Veterinary medicine due to the induction of relevant clinical signs, as abortions in cattle and neuromuscular paralysis in dogs. The aim of this study was to evaluate the role of the TLR adaptor protein TRIF in the resistance against N. caninum infection. For this, in vitro experiments with bone marrow derived macrophages (BMDMs) from C57BL/6 wild-type (WT) and TRIF knockout (TRIF-/-) mice, stimulated by tachyzoites and in vivo infections, were performed in order to investigate the production of cytokines and antibodies, cellular and tissue parasitism, histological changes during different phases of infection and survival analysis. We observed that TRIF-/- BMDMs presented notable defects in inflammatory cytokine production in relation to WT macrophages. Additionally, we found that the concentration of NO, IL-12p40, IFN- and TNF were decreased in peritoneal fluids and lungs of TRIF-/- mice, while IL-2, IFN-, TNF and IL-17 were reduced in sera of these animals compared to WT mice. Higher parasite burden was observed in peritoneal cells, lungs and brain during the acute and chronic phases of infection, which were associated with inflammatory changes in the analyzed tissues, while TRIF-/- mice survival rate decreased 2-fold compared to WT. In conclusion, our results show that TRIF is required for resistance against the infection induced by N. caninum, regulating the production of key Th1 cytokines and participating in the control of the tissue parasitism and inflammatory lesions induced against the parasite.

ATIVAÇÃO DE RECEPTOTRES TOLL-LIKE (TLRS) E DE GENES INDUTORES DE ÁCIDO RETINÓICO I (RIG-I) POR ANÁLOGOS VIRAIS EM CÉLULAS ENDOMETRIAIS BOVINAS

De modo geral, o objetivo deste estudo foi determinar se as células endometriais bovinas responderam a análogos virais de padrões moleculares associados a patógenos (PAMPs) mediante a produção de citocinas pró-inflamatórias após ativadas pelos receptores Toll-Like (TLRs) no endossoma celular e no citoplasma celular pelo genes indutores de ácido retinóico tipo I (RIG-I). No primeiro experimento, amostras uterinas de vacas de corte mestiças pós-púberes foram dissectadas para obtenção de células endometriais epiteliais e estromais. Um controle negativo e quatro PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW foram utilizados. Dois grupos de tratamentos (transfectados e não transfectados) foram analisados durante 24 horas. Em outro experimento, células endometriais foram tratadas apenas com o PAMP Poly (I:C) LMW e um grupo Controle Negativo. Neste, os grupos foram incubados às 0, 2, 6, 12, 24, 36, 48 e 72 horas. Sobrenadantes foram colhidos para desenvolver o teste de ELISA para IL-6 e IL-8. Células epiteliais produziram IL-6 em resposta ao PolyI:C (HMW) quando comparadas com o Controle (Grupo DOTAP positivo; P< 0.05), enquanto que o LPS induziu produção de IL-6 e IL-8 em células estromais (P< 0.05). O uso de um reagente de transfecção entre as células e tratamentos demonstrou efeito (P> 0.05). Ainda, células estromais tratadas por PolyI:C (LMW) demonstraram uma maior produção de IL-6 às 48 e 72 horas (P< 0.05), e para o IL-8 às 6, 12, 24, 36, 48 e72 horas quando comparadas com o grupo Controle (P< 0.05). No segundo experimento, outras amostras uterinas de vacas de corte pós-púberes foram utilizadas. A obtenção de células endometriais estromais e epiteliais foram isoladas pelo mesmo protocolo do primeiro experimento. O PAMP Poly (I:C) LMW e um controle negativo foram utilizados. Proteínas para o RIG-I e p65 foram colhidas após 12, 24, 48 e 72 horas de tratamento. Em resposta a Poly (I:C) LMW, células estromais ativaram o RIG-I às 48 hours (P< 0.05) quando comparadas com o grupo controle. Enquanto que, as células epiteliais não foram suficientemente estimuladas pelo Poly (I:C) LMW na ativação do RIG-I em nenhum momento testado (P> 0.05). A proteína p65 depois de estimulada pela Poly (I:C) LMW foi ativada às 12 horas pelas células estromais (P< 0.05) e às 24 horas pelas células epiteliais (P< 0.05). Conclui-se que, células endometriais bovinas foram essenciais na ativação das vias exercidas tanto pelos TLR como RIG-I com função de iniciar uma defesa imunológica contra infecções virais.

In general, the objective of this study was to determine if bovine endometrial cells replied to virus analogs of pathogen associated molecular pattern (PAMPs) by production of proinflammatory cytokines after Toll-Like Receptor (TLR) activation in the cell endosome and after retinoic acid inducible gene I (RIG-I) stimulation in the cell cytoplasm. In the first experiment, uterine samples from post pubertal cross-breed beef cows were dissected using a protocol to obtain epithelial and stromal cells. A negative control and four different PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW were used. Two treatments (transfected and non-transfected) groups were investigated during 24 hours. In the other experiment, endometrial cells were treated with only Poly (I:C) LMW and a negative Control group. All incubated at 0, 2, 6, 12, 24, 36, 48 and 72 hours. Supernatants were collected to develop Elisa for IL-6 and IL-8. Epithelial cells produced IL-6 in response do Poly I:C (HMW) compared to Control (P< 0.05), otherwise, LPS induced IL-6 and IL-8 in stromal (P< 0.05). The transfection Reagent differ between cells and treatments (P> 0.05). Still, in stromal cells treated by Poly I:C (LMW) the production of IL-6 was higher at 48 and 72 hours (P< 0.05), and for IL-8 at 6, 12, 24, 36, 48 and 72 hours when compared to the Control (P< 0.05). In the second experiment, uterine samples from others post pubertal mixed-breed beef cows were used. To obtain stromal and epithelial cells, uterine samples were dissected with the same protocol as the first experiment. The PAMP Poly (I:C) LMW and a negative control were used. Proteins for RIG-I and p65 were collected after 12, 24, 48 and 72 hours. In response to Poly (I:C) LMW induction, stromal cells activated RIG-I at 48 hours (P< 0.05) were compared to the Control group. On the other hand, epithelial cells were not sufficient stimulated Poly (I:C) LMW to activate RIG-I at any time point evaluated (P> 0.05). The protein p65 after stimulated by Poly (I:C) LMW was activated at 12 hours by stromal (P< 0.05) and at 24 hours by epithelial cells (P< 0.05). In conclusion, bovine endometrial cells were elemental factors in the activation of both TLR and RIG-I pathway in order to start an immune defense against viral infection.