Molecular investigations of the effect of thermal manipulation during embryogenesis on muscle heat shock protein 70 and thermotolerance in broiler chickens

The objective of this study was to elucidate the optimum protocol timing of thermal manipulation (TM) during embryogenesis, which underline genetic improvement of muscle thermotolerance acquisition. For the present study, 1,440 fertile eggs were divided randomly and equally into control (37.8 °C with 56% relative humidity) and four thermally manipulated groups (TM1, TM2, TM3, and TM4) subjected to 39 °C for 18 h with 65% relative humidity daily during different embryonic periods. Then, at day 35 post-hatch, all groups were subjected to thermal challenge at 43 °C for 6 h to identify the level of thermotolerance acquisition differences between them. Hsp70 mRNA expression was evaluated by using a relative quantitatively RT-qPCR. Single nucleotide polymorphisms sequence of the Hsp70 gene was evaluated by Sanger's sequencing method. Pectoral and thigh muscles samples were subjected to immunohistochemistry to detect Hsp70. Among TM conditions that were investigated, TM1 (39 °C for 18 h during embryonic days (ED) 7­11) induced a significant improvement in thermotolerance parameters (body temperature and T3 levels) during thermal challenge combined with an increase in the levels of Hsp70 mRNA and its protein with a high stability of nucleotide sequences in both pectoral and thigh muscles. The partial DNA sequence of Hsp70 gene in TM1 was reported, and nucleotide sequences were deposited in NCBI GenBank database with the accession numbers (MK852579) and (MK852580). Thigh muscle thermotolerance acquisition was higher than pectoral muscle during thermal challenge at 43 °C for 6 h. Thus, TM during ED7­11 may improve thermotolerance acquisition without adversely affecting performance.(AU)

Dietary vegetable oils inclusion on the performance, hormonal levels and hsp 70 gene expression in broilers under heat stress

The aim of the present study was to compare the effects of unsaturated and saturated oils on the performance, hormonal levels and hsp gene expression in broiler chickens exposed to heat stress. 300 one-day male broiler chicks were assigned to 4 treatments (Diets containing palm, corn, linseed or olive oils) with 5 replicates. At day 28 of age, 2 chickens were removed from each replicate, then blood samples and liver tissue samples were collected for analyses. Feeding linseed and olive oil reduced feed conversion ratio compared to corn and palm oils. The lowest level of insulin was for chickens fed linseed oil and corn oil. The highest level of corticosterone was found in chickens fed palm oil and the lowest level was for those received linseed oil. Chickens received linseed and corn oils had the highest levels of T3 and T4 and those fed palm and olive oils had the lowest levels. The highest HSP 70 gene expression was for chickens fed diet containing olive and linseed oils and the lowest one was for those fed corn and palm oils. It was concluded that olive oil and linseed oil could improve performance and heat tolerance of chickens under heat stress.(AU)

Dietary vegetable oils inclusion on the performance, hormonal levels and hsp 70 gene expression in broilers under heat stress

The aim of the present study was to compare the effects of unsaturated and saturated oils on the performance, hormonal levels and hsp gene expression in broiler chickens exposed to heat stress. 300 one-day male broiler chicks were assigned to 4 treatments (Diets containing palm, corn, linseed or olive oils) with 5 replicates. At day 28 of age, 2 chickens were removed from each replicate, then blood samples and liver tissue samples were collected for analyses. Feeding linseed and olive oil reduced feed conversion ratio compared to corn and palm oils. The lowest level of insulin was for chickens fed linseed oil and corn oil. The highest level of corticosterone was found in chickens fed palm oil and the lowest level was for those received linseed oil. Chickens received linseed and corn oils had the highest levels of T3 and T4 and those fed palm and olive oils had the lowest levels. The highest HSP 70 gene expression was for chickens fed diet containing olive and linseed oils and the lowest one was for those fed corn and palm oils. It was concluded that olive oil and linseed oil could improve performance and heat tolerance of chickens under heat stress.

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

ABSTRACT The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.(AU)

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.

HSP70 genotypes and heat tolerance of commercial and native chickens reared in hot and humid conditions

Heat tolerance in poultry production was obtained attention due to the need for genetic lines that can withstand climate changes. This study aimed at investigating heat tolerance in commercial and native broiler genetics, as well as the physiological and growth performance responses of HSP70 genotypes submitted to heat stress. In Experiment I, heterophil:lymphocyte (H:L) ratio, as an indicator of heat tolerance, was compared between commercial broilers (n = 100) and Thai native chickens (n = 100). Growing chickens (with similar initial weight) of each genetic strain were randomly divided into two groups: 1) thermoneutral environment (26 oC ± 2 oC) and 2) heat stress (36 oC ± 2 oC). The results showed that native chickens originating from a tropical environment presented lower H:L ratio and mortality rate compared with commercial broilers. In Experiment II, HSP70 genotypes were compared. PCR-RFLP was applied to identify the genotypes (C1C1, n = 38; C1C2, n = 38; and C2C2, n = 28). Ten-week-old chickens of each genotype were evaluated in the same environments described in Experiment I. Heat-stress indicators - respiratory rate (RR), cloacal temperature (CT), packed cell volume (PCV), and average daily gain (ADG) - were measured for three weeks. The significant difference in PCV indicated that C2C2 chickens were less tolerant to heat stress compared to other genotypes. The RR, CT, and ADG were not significantly different among all genotypes. Since the C2C2 genotype was shown to be sensitive to heat stress, C1C1 and C1C2 could be used as markers for heat-tolerant genetic strains of Thai indigenous chickens and hybrid commercial lines.(AU)

HSP70 genotypes and heat tolerance of commercial and native chickens reared in hot and humid conditions

Heat tolerance in poultry production was obtained attention due to the need for genetic lines that can withstand climate changes. This study aimed at investigating heat tolerance in commercial and native broiler genetics, as well as the physiological and growth performance responses of HSP70 genotypes submitted to heat stress. In Experiment I, heterophil:lymphocyte (H:L) ratio, as an indicator of heat tolerance, was compared between commercial broilers (n = 100) and Thai native chickens (n = 100). Growing chickens (with similar initial weight) of each genetic strain were randomly divided into two groups: 1) thermoneutral environment (26 oC ± 2 oC) and 2) heat stress (36 oC ± 2 oC). The results showed that native chickens originating from a tropical environment presented lower H:L ratio and mortality rate compared with commercial broilers. In Experiment II, HSP70 genotypes were compared. PCR-RFLP was applied to identify the genotypes (C1C1, n = 38; C1C2, n = 38; and C2C2, n = 28). Ten-week-old chickens of each genotype were evaluated in the same environments described in Experiment I. Heat-stress indicators - respiratory rate (RR), cloacal temperature (CT), packed cell volume (PCV), and average daily gain (ADG) - were measured for three weeks. The significant difference in PCV indicated that C2C2 chickens were less tolerant to heat stress compared to other genotypes. The RR, CT, and ADG were not significantly different among all genotypes. Since the C2C2 genotype was shown to be sensitive to heat stress, C1C1 and C1C2 could be used as markers for heat-tolerant genetic strains of Thai indigenous chickens and hybrid commercial lines.

CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular infiltrate of canine mammary carcinoma in mixed tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes).[...]

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and

CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular infiltrate of canine mammary carcinoma in mixed tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes).[...](AU)

ASPECTOS MOLECULARES DE OÓCITOS DE FÊMEAS BOS INDICUS E BOS TAURUS SUBMETIDOS AO ESTRESSE TÉRMICO

Com o objetivo de avaliar a influência do estresse térmico sobre a competência oocitária, bem como sobre parâmetros fisiológicos de fêmeas bovinas de diferentes raças, foram realizados três experimentos. No experimento I, foram avaliados complexo cumulus-oócitos (COCs) de vacas da raça Simental maturados in vitro em diferentes temperaturas (37°C, 38,5°C ou 40°C). Antes da maturação, os oócitos foram corados com Brilhante Cresilblue (BCB) e, classificados em BCB+ e BCB-. Os COCs marcados (BCB+) eram considerados de melhor qualidade. Após a maturação, foram produzidos embriões in vitro e os oócitos foram avaliados através de PCR em tempo real e imunofluorescência, para avaliação das sirtuínas, BCL11A e p53. A alteração da temperatura (±1,5°C) de maturação do oócito afetou negativamente a taxa de blastocisto após a fertilização in vitro, além de influenciar a expressão de gene importantes para a proteção celular. A expressão dos genes SIRT1, SIRT2, SIRT3, SIRT5, BCL11a e p53 foi alterada pela temperatura de maturação, em oócitos e células da granulosa. No experimento II foram avaliados complexo cumulus-oócitos (COCs) de fêmeas da raça Nelore (Bos Taurus indicus) maturados in vitro em diferentes temperaturas (37°C, 38,5°C e 40°C). Após esse período parte dos COCs foram submetidos a fecundação e cultivo dos embriões e a outra parte, após determinação da taxa de maturação, foram fixados em paraformoldeído para análise de HSP70 e HSP90 por western blott. O estresse térmico não influenciou a taxa de maturação nuclear dos oócitos, porém influenciou a taxa de clivagem e de blastocisto. A intensidade do sinal da HSP70 foi aumentado nos oócitos submetidos a estresse, em relação ao grupo controle, além disso as células cumulus mostraram um aumento significativo da intensidade de sinal dessa proteína à 40°C. A HSP90 mostrou maior quantidade em oócitos maturados à 37°C, porém se mantendo estável nas demais temperaturas. A menor taxa de blastocistos e a ação paralela das HSP´s demonstram que a produção in vitro de embriões é afetada quando a maturação in vitro ocorre em situações de estresse crônico (24 hs de MIV; 37°C e 40°C). E no experimento III foram avaliados complexos oócitos-cumulus (COCs) recuperados através da aspiração folicular guiada por ultrassonografia (OPU), de vacas Bos taurus x Bos indicus (Girolando) e Bos taurus (Pantaneira). Parte dos COCs viáveis foram submetidos a análise de imunofluorescência sob microscopia confocal para identificação das proteínas HSP70 e HSP90. Antes da realização de cada OPU, foi aferida a temperatura retal (TR) e frequência respiratória (FR) de cada animal. O estresse térmico foi estimado pelo Indice de Temperatura do Globo e Umidade (ITGU) que foi calculado de acordo com a fórmula descrita por Buffington et al. (1981) e classificados de acordo com o National Weather Service, 90 dias antes de cada OPU. A rusticidade das raças Girolando e Pantaneira pode ser confirmada pela manutenção da TR e FR em nívies fisiológicos em ITGUs de até 94. A HSP nos oócitos foi influenciada pelo ITGUs para as duas raças, porem com comportamentos contrários. No entanto, a raça Pantaneira sofreu efeitos negativos em ITGUs <78 demonstrando estresse em temperaturas mais amenas. Por outro lado, observa-se que ITGUs >78 apresentam efeito deletério na qualidade do oócitos de vacas Girolando. Aparentemente a raça Pantaneira reage negativamente em temperaturas mais baixas, já que apresentou menor viabilidade oocitária com ITGUs <78. Por outro lado observa-se que ITGUs de >79 aos 90 dias antes da OPU apresentam um efeito deletério na qualidade do oócitos de vacas Girolando.

Aiming to evaluate the effects of in vivo and in vitro thermal stress on oocyte competence and early embryonic development as well as on physiological parameters of cows of different breeds, they were conducted three experiments. In the experiment I, the cumulus-oocyte complex (COCs) of Simmental cows matured in vitro at different temperatures (37°C, 38.5°C and 40°C) were evaluated. Before maturation the oocytes were stained with Brilliant Cresilblue (BCB) and then categorized according to staining in BCB + and BCB-. The labeled COCs (BCB +) were considered to be of better quality. After maturation, embryos were produced in vitro and oocytes were evaluated by realtime PCR and immunofluorescence, for the evaluation of sirtuins, BCL11A and p53. The change in temperature (±1,5°C) of oocyte maturation adversely affects the blastocyst rate after in vitro fertilization, in addition to influencing gene expression important for cell protection. Expression of the SIRT1, SIRT2, SIRT3, SIRT5, BCL11a and p53 genes were influenced by the maturation temperature in oocytes and granulosa cells. In the experiment II, cumulus-oocyte complexes (COCs) of Nellore (Bos Taurus indicus) females were evaluated in vitro at different temperatures (37°C, 38,5°C and 40°C). After this period part of the COCs were submitted to embryo fertilization and culture and the other part, after determining the maturation rate, were fixed in paraformoldeído for analysis of HSP70 and HSP90 by western blot. Thermal stress did not influence the maturation rate of oocytes, but influenced the rate of cleavage and blastocyst. The signal intensity of HSP70 was increased in stressed oocytes compared to the control group, and cumulus cells showed a significant increase in the signal intensity of this protein at 40°C. HSP90 showed higher numbers in oocytes matured at 37 ° C, but remained stable in the other temperatures. The lower rate of blastocysts and the parallel action of HSPs demonstrate that the in vitro production of embryos is affected when in vitro maturation occurs in situations of chronic stress (24 hours of IVM) by cold and heat. In the experiment III, cumulative oocytes (COCs) recovered through ultrasound-guided follicular aspiration (OPU), Bos taurus x Bos indicus (Girolando) and Bos taurus (Pantaneira) cows were evaluated. Part of the viable COCs were submitted to immunofluorescence analysis under confocal microscopy for identification of HSP70 and HSP90 proteins. Before each OPU, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. Thermal stress was estimated by the Black Globe Humidity Index (BGHI) which was calculated according to the formula described by Buffington et al. (1981) classified according to the National Weather Service, 90 days before each OPU. The rusticity of the Girolando and Pantaneira breeds can be confirmed by the maintenance of the RT and RF in physiological levels in BGHIs of up to 94. The HSP in the oocytes was influenced by the BGHIs for both breeds, but with opposite behaviors. However, the Pantaneira breed had negative effects on BGHIs <78 demonstrating stress at warmer temperatures. On the other hand, it is observed that BGHIs >78 have a deleterious effect on the quality of Girolando cow oocytes. It appears that the Pantaneira breed reacts negatively at lower temperatures, since it presented lower oocyte viability with BGHI <78. On the other hand it is observed that ITGUs of> 79 at 90 days before OPU have a deleterious effect on the quality of Girolando cow oocytes.

Avaliação da atividade imunoterapêutica do probiótico LLHsp65 na asma experimental.

A asma alérgica é uma doença pulmonar de inflamação crônica caracterizada por uma resposta imune do tipo Th2 e uma das principais abordagens terapêuticas para o seu tratamento no futuro, poderia ser a imunoterapia baseada na modulação da resposta imune Th2 para um perfil Th1 e anti-inflamatório. Nosso grupo já demonstrou a eficácia de uma imunoterapia, baseada em um plasmídeo de DNA contendo o gene hsp65 de M. Leprae (DNAHsp65) em modelo murino de asma alérgica. No entanto, apesar dos excelentes resultados, o grupo está procurando outras alternativas imunoterapêuticas, usando a Hsp65 micobacteriana, para futura utilização clinica na área de saúde humana e veterinária. Efeitos benéficos na prevenção e tratamento de doenças alérgicas vêm sendo obtidos com o uso de probióticos. Nossa hipótese é de que uma nova formulação terapêutica, como o probiótico Lactococcus lactis expressando Hsp65 micobacteriana (LLHsp65), apresentaria vantagens significativas no desenvolvimento de pesquisas translacionais. Diante disso, este estudo teve como objetivo avaliar se o probiótico LLHsp65 apresenta atividades imunoterapêuticas no modelo de asma experimental induzida por ovalbumina (OVA). Em diferentes grupos experimentais, 5x109 CFU de LLHsp65 ou de L. lactis selvagem (LLSELV), foram administrados por via oral durante 10 dias consecutivos aos camundongos BALB/c previamente sensibilizados e desafiados com OVA. Em seguida, investigamos os efeitos do tratamento na inflamação alérgica, modulação do padrão de citocinas, produção de anticorpos específicos, hiper-responsividade das vias aéreas e inflamação pulmonar. Nossos resultados demonstraram que o tratamento oral com LLhsp65 modula a resposta imune alérgica de padrão Th2 para o perfil Th1 com aumento dos níveis de IFN-, IL-12, TNF-, IL-10, IL-6 e IL-17, e com a redução das citocinas IL-4, IL-5 e IL-13. Os níveis de IgE e IgG1 anti-OVA no soro também foram significativamente diminuídos. Como consequência desses resultados, também observamos diminuição significativa do infiltrado de eosinófilos no lavado bronco-alveolar, na hiper-responsividade nas vias aéreas e a atenuação da inflamação e produção de muco no tecido pulmonar, quando comparados com o grupo controle (OVA/SAL). Por conseguinte, nossos resultados demonstraram que a administração oral de LLHsp65 proporciona uma melhora significativa do processo inflamatório alérgico induzido por OVA, sendo portanto, uma estratégia terapêutica promissora para o desenvolvimento de pesquisa translacional no tratamento de asma alérgica.

Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response and one of the main therapeutic approaches to its treatment in the future could be immunotherapy based on the modulation of the Th2 immune response to a Th1 and anti-inflammatory profile. Our group has already demonstrated the efficacy of an immunotherapy based on a DNA plasmid carrying the mycobacterial hsp65 gene (DNAHsp65) in murine model of allergic asthma. However, despite the excellent results, our group is looking for other immunotherapeutic alternatives, using mycobacterial Hsp65, for future clinical use in the area of human and veterinary health. Beneficial effects in the prevention and treatment of allergic diseases have been obtained with the use of probiotics. Our hypothesis is that a new therapeutic formulation, such as the probiotic Lactococcus lactis expressing mycobacterial Hsp65 (LLHsp65), would present significant advantages in the development of translational research. The objective of this study was to evaluate whether the probiotic LLHsp65 has immunotherapeutic activities in the ovalbumin-induced asthma model. In different experimental groups, 5x109 CFU of LLHsp65 or control L. lactis (ctLL) were administered orally for 10 consecutive days to BALB/c mice previously sensitized and challenged with OVA. We then investigated the effects of treatment on allergic inflammation, modulation of the cytokine pattern, production allergen-specific antibodies, airway hyper-responsiveness and pulmonary inflammation. Our results demonstrated that oral treatment with LLhsp65 modulates the allergic Th2 immune response to Th1 profile with increased levels of IFN-, IL-12, TNF-, IL-10, IL-6 and IL-17 and with the reduction of IL-4, IL-5 and IL-13 cytokines. Serum anti-OVA IgE and IgG1 levels were also significantly decreased. Correspondingly with these results, we also observed a significant decrease in eosinophil infiltrate in bronchoalveolar lavage, airway hyper responsiveness and attenuation of inflammation and mucus production in lung tissue when compared to the control group (OVA/SAL). Therefore, our results demonstrated that oral administration of LLHsp65 provides a significant improvement of the OVA-induced allergic inflammatory process and is therefore a promising therapeutic strategy for the development of translational research in the treatment of allergic asthma.

CLONAGEM DO GENE DA PROTEÍNA DE CHOQUE TÉRMICO DE 70 kDa (HSP70) DE Trypanosoma evansi

BOAVENTURA, Bruna Larissa. CLONAGEM DO GENE DA PROTEÍNA DE CHOQUE TÉRMICO DE 70 kDa (HSP70) de Trypanosoma evansi.66f. Tripanossomoses são doenças causadas por protozoários pertencentes ao gênero Trypanosoma que podem infectar uma ampla gama de animais, inclusiveo homem. OTrypanosoma evansi é um hemoparasito que acomete várias espécies de animais, principalmente equinos, sendo responsável pela enfermidade conhecida como tripanossomose equina, tendo relato também de sua ocorrência em humanos,sendo, por tanto uma zoonose. Possui ampla distribuição geográfica, e é importante causa de prejuízos econômicos em criações de equinos. O controle do agente é feito por meio da utilização de tripanocidas, porém, ao longo dos anos têm sido descritas cepas resistentes às drogas atualmente disponíveis. As proteínas de choque térmico da família HSP 70 são componentes centrais da rede celular, auxiliando em diversas funções como dobramento de proteínas e se destacando como importantes alvos para terapias gênicas e biomarcadores por serem proteínas altamente conservadas em diversos parasitos. A realização deste trabalho teve como objetivo clonar o gene codificante para a proteína HSP70 de T. evansi (TEHSP70) e realizar estudos in silico com a proteína TEHSP70. A clonagem do gene TEHSP70 em pGEM- T easy®, foi bem sucedida e a partir do seu sequenciamento obteve-se a sequencia de nucleotídeos que, traduzida, forneceu a sequencia primária da proteína alvo TEHSP70. A sequência de TEHSP70 foi comparada com de ortólogos de genes de Trypanosomas disponíveis em banco de dados públicos e os resultados mostraram que TEHSP70 possui alta similaridade filogenética com as espécies de Trypanosomas de maior ocorrência. A análise de bioinformática realizada na HSP70 e sua co-chaperona HSP40 demonstrou que a região C-terminal da HSP70 pode atuar como um potente alvo para interrupção do ciclo biológico do parasito.O estudo de proteínas funcionais, envolvidas no metabolismo do parasito, pode servir de base para o desenvolvimento de novas drogas.

BOAVENTURA, Bruna Larissa. CLONING OF HEAT SHOCK PROTEIN HSP70 GENE IN Trypanosoma evansi. 2016. 66p. Trypanosomes are diseases caused by protozoa belonging to the genus Trypanosoma that can infect a wide range of animals, including man.Trypanosoma evansi is a hemoparasite that affects several species of animals, mainly equines, being responsible for the disease known as equine trypanosomiasis, and also reports of its occurrence in humans, being, therefore, a zoonosis.It has a wide geographical distribution, and is an important cause of economic losses in equine breeding. Control of the agent is done through the use oftrypanocides, however, over the years, resistant strains of drugs currently available have been described.The heat shock proteins of the HSP 70 family are central components of the cellular network, assisting in several functions such as protein folding and stand out as important targets for gene therapies and biomarkers because they are proteins highly conserved in several parasites.The objective of this work was to clone the gene coding for the HSP70 protein of T. evansi (TEHSP70) and to perform in silico studies with the TEHSP70 protein.Cloning of the TEHSP70 gene in pGEM-T easy® was successful and from its sequencing the nucleotide sequence was obtained which translated gave the primary sequence of the target protein TEHSP70. The sequence of TEHSP70 was compared with that of orthologs of Trypanosome genes available in public database and the results showed that TEHSP70 has high phylogenetic similarity with the most frequent species of Trypanosomes.The bioinformatics analysis performed on HSP70 and its HSP40 co-chaperone demonstrated that the C-terminal region of HSP70 may act as a potent target for disruption of the parasite's life cycle.The study of functional proteins, involved in the metabolism of the parasite, can serve as the basis for the development of new drugs.

Brain and hepatic Hsp70 protein levels in heat-acclimated broiler chickens during heat stress

In the present study we have investigated the effects of heat acclimation on brain and hepatic Hsp70 protein levels and body temperature of broiler chickens in response to gradual heat stress. Two groups of broilers were raised up to 47 days of age under distinct temperature conditions: thermoneutral (TN, according to bird age) or hot environmental (HS, 31-33°C). At 46 days of age, the birds reared at high ambient temperature were transferred to thermoneutrality conditions. After 18 h, these birds and the birds reared at thermoneutral temperature were submitted to gradual heat stress in a climatic chamber so that environment temperature was increased from 28 to 40ºC at a rate of 2ºC/h. Colonic temperature was measured using a thermometer sensor probe at each two hours, and hepatic and brain tissues were collected immediately after slaughter in order to assess Hsp70 protein level by Western blotting analysis. The colonic temperatures of birds reared at high temperature increased steeply during the first 2 h of heat stress (1.06ºC/h) and more slowly thereafter (0.59ºC/h). Broilers reared at thermoneutral temperature showed a small increase in the first 4 h of heat stress (0.18ºC/h) and then colonic temperature increased sharply (0.72ºC/h). Nevertheless, both groups presented similar final colonic temperature by the end of the stress period. Hsp70 levels (ng Hsp70 µg total protein-1) did not change in the liver or brain of the birds reared at high temperature. On the other hand, both liver and brain Hsp70 levels increased significantly during heat stress in the animals reared at thermoneutrality, with a higher expression of this peptide in brain tissue.

Brain and hepatic Hsp70 protein levels in heat-acclimated broiler chickens during heat stress

In the present study we have investigated the effects of heat acclimation on brain and hepatic Hsp70 protein levels and body temperature of broiler chickens in response to gradual heat stress. Two groups of broilers were raised up to 47 days of age under distinct temperature conditions: thermoneutral (TN, according to bird age) or hot environmental (HS, 31-33°C). At 46 days of age, the birds reared at high ambient temperature were transferred to thermoneutrality conditions. After 18 h, these birds and the birds reared at thermoneutral temperature were submitted to gradual heat stress in a climatic chamber so that environment temperature was increased from 28 to 40ºC at a rate of 2ºC/h. Colonic temperature was measured using a thermometer sensor probe at each two hours, and hepatic and brain tissues were collected immediately after slaughter in order to assess Hsp70 protein level by Western blotting analysis. The colonic temperatures of birds reared at high temperature increased steeply during the first 2 h of heat stress (1.06ºC/h) and more slowly thereafter (0.59ºC/h). Broilers reared at thermoneutral temperature showed a small increase in the first 4 h of heat stress (0.18ºC/h) and then colonic temperature increased sharply (0.72ºC/h). Nevertheless, both groups presented similar final colonic temperature by the end of the stress period. Hsp70 levels (ng Hsp70 µg total protein-1) did not change in the liver or brain of the birds reared at high temperature. On the other hand, both liver and brain Hsp70 levels increased significantly during heat stress in the animals reared at thermoneutrality, with a higher expression of this peptide in brain tissue.

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and