Comparative analysis of the hormone production and gene expression profiles in ovine uterus tissue during oestrus cycle synchronized using medroxyprogesterone acetate plus eCG and prostaglandin analogue / Análise comparativa da produção de hormônios e perfis de expressão gênica em tecido uterino ovino durante o ciclo estral sincronizado usando acetato de medroxiprogesterona mais eCG e análogo de prostaglandina

The combination of medroxyprogesterone acetate (MPA) and gonadotropin chorionic (eCG) has been widely used to synchronize oestrus cycle in sheep, but their effects on the gene expression in uterine tissue are yet to be elucidated. To evaluate the effect of MPA + eCG or prostaglandin analogue (PA) treatments on the rate of oestrus cycle synchronization, as well as further hormone production and gene expression profiles in uterine tissue, 14 Santa Inês ewes were randomly selected. The MPA + eCG group (n=7) received intravaginal insertion of MPA-impregnated sponges for 14 days and was administered 350 IU eCG on the day of sponge withdrawal. The PA group (n=7) was administered two doses of 100 µg of PA separated by 12 days. The ewes were assessed for the rate of oestrus cycle synchronization and the serum concentrations of progesterone (P4) and estradiol (E2). Additionally, the expression of estrogen receptor (ERα), progesterone receptor (P4R), and immunolocalization of interferon receptor (IFNAR1) in the uterine tissue samples collected 15th day post-mating were examined. The rate of oestrus cycle synchronization was 100% (n=7/7) and 57.14% (n=4/7) in the MPA + eCG and PA groups, respectively. Moreover, the MPA + eCG group exhibited higher serum concentration of P4 than the PA group (p < 0.05). However, the E2 serum concentration did not differ between the two groups (p > 0.05). The relative expression of P4R and ERα mRNA analyzed using real-time PCR and immunodetection of IFNAR1 were similar between the two groups tested (p > 0.05). Conclusively, MPA + eCG treatment improved the rate of oestrus cycle synchronization and endogenous P4 production; however, it did not affect the expression of sex steroid receptors and IFNAR1 in uterine ovine tissue.(AU)

A combinação de acetato de medroxiprogesterona (MPA) com gonadotrofina coriônica (eCG) é amplamente utilizada para sincronizar o estro de ovelhas, mas seus possíveis efeitos sobre a expressão gênica em tecido uterino não foram elucidados. Para avaliar o efeito dos protocolos MPA + eCG ou análogo de prostaglandina (PA) sobre a taxa de sincronização do estro, bem como na futura produção hormonal e expressão gênica em tecido uterino, 14 ovelhas Santa Inês foram selecionadas. O grupo MPA + eCG (n=7) recebeu a inserção de esponjas impregnadas de MPA por via intravaginal por 14 dias e 350 UI de eCG no dia da retirada da esponja. O grupo PA recebeu duas doses de 100 µg de PA administradas com 12 dias de intervalo. As ovelhas foram avaliadas quanto à taxa de sincronização do estro, concentração sérica de progesterona (P4) e estradiol (E2). Além disso, foram examinadas a expressão do receptor de estradiol (ERα), receptor de progesterona (P4R) e localização do receptor de interferon (IFNAR1), a partir de amostras de tecido uterino coletadas 15 dias após o acasalamento. A taxa de sincronização do estro foi 100% (n = 7/7) e 57,14% (n = 4/7) nos grupos MPA + eCG e PA, respectivamente. Além disso, o grupo MPA + eCG apresentou maior concentração sérica de P4 em comparação com o grupo PA (P < 0,05). No entanto, a concentração sérica de E2 não diferiu entre os grupos testados (P > 0,05). A expressão relativa de RNAm para P4R e ERα analisado por PCR em tempo real e imunodetecção de IFNAR1 foi semelhante entre os grupos testados (P > 0,05). Em conclusão, o tratamento com MPA + eCG melhora a taxa de sincronização do estro e a produção de progesterona endógena; contudo, não afeta a regulação da expressão de receptores de esteroides sexuais e IFNAR1 no tecido uterino de ovinos.(AU)

Use of Giemsa staining for the immunohistochemical counterstaining in canine melanomas: an "old and forgotten" method

Melanoma is a neoplasm originating from melanocytes and represents 7% of skin tumors and the most common in oral cavity of dogs. Melanoma may present melanocytic pigment in its cytoplasm in varying quantity and its characterization by immunohistochemistry (IHC) is challenging due to the use of chromogen diaminobenzidine (DAB) which itself produces a brown product what makes difficult to distinguish from melanin pigment in this technique. To demonstrate a reliable technique, the use of the Giemsa counterstaining was performed in the IHC of melanomas with different degrees of pigmentation for different cytoplasmic, membrane and nuclear markers. The modification in the IHC technique by the counterstaining of Giemsa allows observable differences under the microscope between melanic pigment(in a blue-green stain), while the DAB chromogen will be observed in brown. With this technique, the prognostic and predictive interpretation of markers in canine melanomas may be more reliable in definitions of clinical behaviors and inexperimental analyzes.(AU)

Use of Giemsa staining for the immunohistochemical counterstaining in canine melanomas: an "old and forgotten" method

Melanoma is a neoplasm originating from melanocytes and represents 7% of skin tumors and the most common in oral cavity of dogs. Melanoma may present melanocytic pigment in its cytoplasm in varying quantity and its characterization by immunohistochemistry (IHC) is challenging due to the use of chromogen diaminobenzidine (DAB) which itself produces a brown product what makes difficult to distinguish from melanin pigment in this technique. To demonstrate a reliable technique, the use of the Giemsa counterstaining was performed in the IHC of melanomas with different degrees of pigmentation for different cytoplasmic, membrane and nuclear markers. The modification in the IHC technique by the counterstaining of Giemsa allows observable differences under the microscope between melanic pigment(in a blue-green stain), while the DAB chromogen will be observed in brown. With this technique, the prognostic and predictive interpretation of markers in canine melanomas may be more reliable in definitions of clinical behaviors and inexperimental analyzes.

Imunodetecção de Toxoplasma gondii em tecido placentário de cabras naturalmente infectadas / Immunodetection of Toxoplasma gondii in placenta tissue of naturally infected goats

A imuno-histoquímica (IHQ) é considerada uma ferramenta rápida e precisa para a identificação de protozoários, como Toxoplasma gondii, em tecidos fetais e placentários. Neste estudo foi avaliada a imunodetecção de Toxoplasma gondii em tecido placentário de cabras naturalmente infectadas. Foram coletadas e analisadas 80 amostras de placentas de cabras procedentes de único rebanho com sorologia positiva para T. gondii na técnica de ELISA. Na histopatologia, 27/80 amostras apresentaram lesões sugestivas de infecção por protozoários. Após a avaliação histopatológica, procedeu-se à realização da técnica de imuno-histoquímica, obtendo-se 85,2% (23/27) de amostras com marcação positiva. A imunodetecção ocorreu no epitélio de revestimento das vilosidades coriônicas e foi classificada de acordo com o grau de intensidade da imunomarcação. Também foi evidenciada imunomarcação no interior dos vasos sanguíneos fetais em 8,69% (2/23) das amostras. Este estudo demonstrou que a técnica de IHQ se comportou como uma ferramenta valiosa no diagnóstico da infeção por T. gondii em tecido placentário de cabras naturalmente infectadas e complementou, de forma decisiva, o diagnóstico, além de agregar maior valor aos resultados obtidos nas análises histopatológica e sorológica.(AU)

Immunohistochemistry (IHC) is considered to be a rapid and accurate tool for the identification of protozoa such as Toxoplasma gondii in fetal and placental tissues. In this study, we evaluated the immunodetection of Toxoplasma gondii in placental tissue from naturally infected goats. A total of 80 samples of goat placentas from a single herd with positive ELISA serology for T. gondii were collected and analyzed. In the histopathology, 27/80 samples presented lesions suggestive of protozoal infection. After the histopathological evaluation, the immunohistochemistry technique was performed, obtaining 85.2% (23/27) of samples with positive marking. Immunodetection occurred in the lining epithelium of the chorionic villi and was classified according to the degree of intensity of the immunostaining. Immunostaining within the fetal blood vessels was also evidenced in 8.69% (2/23) of the samples. This study demonstrated that the IHQ technique behaved as a valuable tool in the diagnosis of T. gondii infection in placental tissue of naturally infected goats completing the diagnosis in a decisive way and adding greater value to the results obtained in the histopathological and serological analysis.(AU)

Imunodetecção de Toxoplasma gondii em tecido placentário de cabras naturalmente infectadas / Immunodetection of Toxoplasma gondii in placenta tissue of naturally infected goats

A imuno-histoquímica (IHQ) é considerada uma ferramenta rápida e precisa para a identificação de protozoários, como Toxoplasma gondii, em tecidos fetais e placentários. Neste estudo foi avaliada a imunodetecção de Toxoplasma gondii em tecido placentário de cabras naturalmente infectadas. Foram coletadas e analisadas 80 amostras de placentas de cabras procedentes de único rebanho com sorologia positiva para T. gondii na técnica de ELISA. Na histopatologia, 27/80 amostras apresentaram lesões sugestivas de infecção por protozoários. Após a avaliação histopatológica, procedeu-se à realização da técnica de imuno-histoquímica, obtendo-se 85,2% (23/27) de amostras com marcação positiva. A imunodetecção ocorreu no epitélio de revestimento das vilosidades coriônicas e foi classificada de acordo com o grau de intensidade da imunomarcação. Também foi evidenciada imunomarcação no interior dos vasos sanguíneos fetais em 8,69% (2/23) das amostras. Este estudo demonstrou que a técnica de IHQ se comportou como uma ferramenta valiosa no diagnóstico da infeção por T. gondii em tecido placentário de cabras naturalmente infectadas e complementou, de forma decisiva, o diagnóstico, além de agregar maior valor aos resultados obtidos nas análises histopatológica e sorológica.(AU)

Immunohistochemistry (IHC) is considered to be a rapid and accurate tool for the identification of protozoa such as Toxoplasma gondii in fetal and placental tissues. In this study, we evaluated the immunodetection of Toxoplasma gondii in placental tissue from naturally infected goats. A total of 80 samples of goat placentas from a single herd with positive ELISA serology for T. gondii were collected and analyzed. In the histopathology, 27/80 samples presented lesions suggestive of protozoal infection. After the histopathological evaluation, the immunohistochemistry technique was performed, obtaining 85.2% (23/27) of samples with positive marking. Immunodetection occurred in the lining epithelium of the chorionic villi and was classified according to the degree of intensity of the immunostaining. Immunostaining within the fetal blood vessels was also evidenced in 8.69% (2/23) of the samples. This study demonstrated that the IHQ technique behaved as a valuable tool in the diagnosis of T. gondii infection in placental tissue of naturally infected goats completing the diagnosis in a decisive way and adding greater value to the results obtained in the histopathological and serological analysis.(AU)

Production and evaluation of immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods & Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk.[...]

Production and evaluation of immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods & Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk.[...](AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Abstract Background: The gamma-type phospholipase A2 inhibitor (LI) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A2. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLI identification and characterization. Methods: Bioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLI (SaPLI). The best epitope 151CPVLRLSNRTHEANRNDLIKVA172 was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively. Results: Eighteen IgG anti-PLI mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLI and natural SaPLI. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIs. cDNA sequence alignment of those PLIs from different snake species showed that their epitope segments were highly homologous. Conclusions: The successful preparation of anti-PLImAb is significant for further investigation on the relationship between the structure and function of PLIs, as well as the interaction between PLIs and PLA2s.

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Evaluation of the "shelf life" of nitrocellulose membranes immobilized with Paracoccidioides brasiliensis antigen for performing the immunodiagnosis of paracoccidioidomycosis / Avaliação do "tempo de prateleira" de membranas de nitrocelulose imobilizadas com antígeno de Paracoccidioides brasiliensis para o imunodiagnóstico da paracoccidioidomicose

The immunoblotting reaction for performing the paracoccidioidomycosis (PCM) immunodiagnosis is an in-house methodology; and being a laborious task involving two previous steps, SDS-PAGE and Western blot, we evaluated the shelf life of nitrocellulose membranes containing the immobilized P. brasiliensis antigens, stored at -20 o C for 7, 15, 30, 45, 60 and 90 days. Twenty-eight serum samples were analyzed on two nitrocellulose membranes groups: (a) membranes previously blocked with PBS-5 % non-fat dry milk and (b) the priory non-blocked membranes. No difference was detected in the reactivity pattern in serum samples evaluated in the both membrane groups, especially for those stored for 7, 15, 30, 45 and 60 days. It might be emphasized that a good stability of P. brasiliensis antigens, immobilized on the nitrocellulose membranes, enable them to be stored up to 60 days at -20 o C. This finding contributes to the rapid diagnosis of PCM, and for sending them to other laboratories without adequate infrastructure for carrying out the steps that precede the immunodetection as the antigen production, SDS-PAGE and Western blot techniques. This scheme contributes substantially to improve the quality of PCM serodiagnosis, as it provides reproducible results in the units of the Laboratory Network.(AU)

Considerando-se que o immunoblotting para o imunodiagnóstico da paracoccidioidomicose (PCM) é uma metodologia in house e laboriosa envolvendo duas etapas iniciais, SDS-PAGE e Western blot, neste estudo foi avaliado o tempo de prateleira das membranas de nitrocelulose sensibilizadas com antígeno de P. brasiliensis, armazenadas a -20 o C durante 7, 15, 30, 45, 60 e 90 dias. Vinte e oito amostras de soro foram analisadas em dois grupos de membranas de nitrocelulose (membranas previamente bloqueadas com PBS-leite 5 % e as não-bloqueadas). Não houve diferença no padrão de reatividade quando os soros foram avaliados frente a ambos os grupos, especialmente para membranas armazenadas por 7, 15, 30, 45 e 60 dias. A boa estabilidade do antígeno utilizado para sensibilizar as membranas fez com que estas pudessem ser armazenadas a -20 C até 60 dias. Estas características contribuem para efetuar o diagnóstico rápido da PCM, bem como as perspectivas dessas membranas sensibilizadas serem encaminhadas para os laboratórios, que não possuam infraestrutura necessária para executar as etapas que antecedem a realização de immunoblotting, como a produção de antígeno, as técnicas de SDS PAGE e Western blot. Este procedimento contribui substancialmente para melhorar o diagnóstico sorológico da PCM, pois poderá fornecer resultados reprodutíveis nas unidades componentes da Rede de Laboratórios.(AU)

Influence of apoptosis on liver and spleen resistance in dogs with visceral leishmaniosis / Influência da apoptose na resistência hepática e esplênica de cães com leishmaniose visceral

Abstract The aim of this study was to evaluate apoptosis and parasite load in the liver and spleen of dogs with visceral leishmaniosis (VL), using immunohistochemistry. Liver and spleen samples from 71 dogs with VL were used. The parasite load in the spleen and liver showed significant difference between organs in infected group (P=0.0219). The density of the parasite load in the spleen (median=2.4) was higher than liver (median=0.8). Immunodetection of apoptotic cells was predominant in lymphocytes and differ between the infected and control group in spleen (P=0.0307) and liver (P=0.0346). There was a significant correlation between apoptosis and parasite load (P = 0.0084; r=0.3104) only in the spleen of the infected group, where it was observed that, when increasing the number of apoptotic cells increases the parasitic load. It was concluded that the liver and spleen of infected dogs presented greater numbers of cells undergoing apoptosis (lymphocytes) than the control group, thus suggesting that this process may be contributing towards the survival of Leishmania in these organs, because lymphocyte in apoptosis did not have the ability to present and recognize the antigen, allowing the survival of the parasite.

Resumo O objetivo deste estudo foi avaliar a apoptose e a carga parasitária no fígado e baço de cães com leishmaniose visceral (LV), pela técnica de imuno-histoquímica. Foram utilizadas amostras de fígado e baço de 71 cães com LV. A carga parasitária no baço e fígado mostrou diferença significativa entre os órgãos no grupo infectado (P=0,0219). A densidade da carga de parasita no baço (média=2,4) foi maior do que no fígado (média=0,8). A imunodetecção de células em apoptose foi predominante nos linfócitos, com diferenças entre o grupo infectado e controle no baço (P=0,0307) e fígado (P=0,0346). Houve uma correlação positiva fraca entre apoptose e carga parasitária (P=0,0084; r=0,3104) apenas no baço do grupo infectado, onde observou-se que quando aumentava o número de células em apoptose aumentava a carga parasitária. Concluiu-se que o fígado e o baço de cães infectados apresentam um maior número de células que sofrem apoptose (linfócitos) do que o grupo controle, sugerindo que este processo possa contribuir para a sobrevivência de Leishmania nestes órgãos, pois os linfócitos em apoptose não tiveram a capacidade de apresentar e reconhecer o antígeno, permitindo a sobrevivência do parasita.

Influence of apoptosis on liver and spleen resistance in dogs with visceral leishmaniosis / Influência da apoptose na resistência hepática e esplênica de cães com leishmaniose visceral

Abstract The aim of this study was to evaluate apoptosis and parasite load in the liver and spleen of dogs with visceral leishmaniosis (VL), using immunohistochemistry. Liver and spleen samples from 71 dogs with VL were used. The parasite load in the spleen and liver showed significant difference between organs in infected group (P=0.0219). The density of the parasite load in the spleen (median=2.4) was higher than liver (median=0.8). Immunodetection of apoptotic cells was predominant in lymphocytes and differ between the infected and control group in spleen (P=0.0307) and liver (P=0.0346). There was a significant correlation between apoptosis and parasite load (P = 0.0084; r=0.3104) only in the spleen of the infected group, where it was observed that, when increasing the number of apoptotic cells increases the parasitic load. It was concluded that the liver and spleen of infected dogs presented greater numbers of cells undergoing apoptosis (lymphocytes) than the control group, thus suggesting that this process may be contributing towards the survival of Leishmania in these organs, because lymphocyte in apoptosis did not have the ability to present and recognize the antigen, allowing the survival of the parasite.(AU)

Resumo O objetivo deste estudo foi avaliar a apoptose e a carga parasitária no fígado e baço de cães com leishmaniose visceral (LV), pela técnica de imuno-histoquímica. Foram utilizadas amostras de fígado e baço de 71 cães com LV. A carga parasitária no baço e fígado mostrou diferença significativa entre os órgãos no grupo infectado (P=0,0219). A densidade da carga de parasita no baço (média=2,4) foi maior do que no fígado (média=0,8). A imunodetecção de células em apoptose foi predominante nos linfócitos, com diferenças entre o grupo infectado e controle no baço (P=0,0307) e fígado (P=0,0346). Houve uma correlação positiva fraca entre apoptose e carga parasitária (P=0,0084; r=0,3104) apenas no baço do grupo infectado, onde observou-se que quando aumentava o número de células em apoptose aumentava a carga parasitária. Concluiu-se que o fígado e o baço de cães infectados apresentam um maior número de células que sofrem apoptose (linfócitos) do que o grupo controle, sugerindo que este processo possa contribuir para a sobrevivência de Leishmania nestes órgãos, pois os linfócitos em apoptose não tiveram a capacidade de apresentar e reconhecer o antígeno, permitindo a sobrevivência do parasita.(AU)

IMUNOMARCAÇÃO DE Leishmania sp. E ASPECTOS HISTOLÓGICOS NA TERCEIRA PÁLPEBRA DE CÃES NATURALMENTE INFECTADOS POR Leishmania (leishmania) chagasi

Abstract Canine visceral leishmaniasis (CVL) is a disease with multisystemic injuries, and among the various tissues affected, the third eyelid (TE) is often involved. This ocular adnexa has been studied for the leishmaniasis pathogenesis elucidation and for diagnosis advance. This study aimed to carry out the parasite immunodetection and evaluate histologic lesions in TE of Leishmania (leishmania) chagasi naturally infected dogs. Twenty-six TE samples from infected dogs were submitted to histologic evaluation and to immunohistochemistry for Leishmania sp. The main lesion observed in the third eyelid conjunctiva (TEC) was lymphoplasmacytic inflammation, with Mott cells and parasitized histiocytes. Additionally, loss of epithelial stratification, ulceration, goblet cells thinning or hyperplasia were often found. The same inflammatory pattern was observed in the third eyelid lacrimal gland (TELG), often accompanied by acinar atrophy and secretory ducts dilatation. Immunohistochemistry revealed parasitism in all samples, in different intensities.

Resumo A leishmaniose visceral canina (LVC) é uma doença que envolve lesões multissistêmicas e, dentre os vários tecidos acometidos, a terceira pálpebra está frequentemente envolvida. Este anexo ocular tem sido alvo de estudo tanto para a elucidação da patogênese da doença quanto para o avanço diagnóstico. O presente trabalho teve por objetivo avaliar as alterações histológicas presentes na terceira pálpebra de cães naturalmente infectados por Leishmania chagasi e realizar a imunodetecção do parasita. Vinte e seis amostras de terceira pálpebra de cães sintomáticos foram avaliadas quanto à coloração de HE e à imunoistoquímica com soro de cão positivo para Leishmania sp. A principal alteração observada na conjuntiva da terceira pálpebra foi infiltração inflamatória predominantemente linfoplasmocitária, com células de Mott e histiócitos parasitados permeando a área de exsudação. Adicionalmente, perda de estratificação e ulceração epitelial, rarefação ou hiperplasia de células caliciformes foram achados costumazes. Na glândula lacrimal da terceira pálpebra, o mesmo padrão inflamatório foi observado, acompanhado frequentemente de atrofia acinar e dilatação dos ductos secretórios. A imunoistoquímica revelou parasitismo em todas as amostras, em diferentes intensidades.

IMUNODETECÇÃO DE TOXOPLASMA GONDII EM ÚTERO GESTACIONAL E PLACENTA DE TAYASSUÍDEOS SILVESTRES DA AMAZÔNIA PERUANA

O Toxoplasma gondii é um protozoário cosmopolita e intracelular obrigatório que afeta animais endotérmicos, incluindo o homem. A transmissão pode ocorrer pelas vias horizontal e vertical, esta última, em geral, acaba desencadeando patologias reprodutivas. Com isso, objetivou-se investigar a ocorrência de T. gondii em espécies da Família Tayassuidae de vida livre da Amazônia peruana, pela imunomarcação do protozoário em tecido de útero gestacional e placenta. Para tanto, as amostras de tecido uterino e placentário de 36 Pecari tajacu e 42 Tayassu pecari foram analisadas pelas técnicas de histopatologia e imuno- histoquímica, com os resultados correlacionadospelo teste qui-quadrado. Um total de 12 animais foram positivos (06 P. tajacu e 06 T. pecari). Na IHQ o T. pecari exibiu 7,14% de positividade para T. gondii em útero gestacional e 9,52% em placenta, enquanto o P. tajacuapresentou 11,11% em útero gestacional e 8,33% em placenta. As amostras imunorreativas em IHQ apresentaram intensidade variando de moderada a forte em ambos os órgãos das espécies analisadas, sendo observada as formas parasitárias no epitélio e tecido conjuntivo uterino e placentário, porém sem a observância das lesões características nos tecidos avaliados pelas técnicas de histotopatologia e IHQ. Com isso foi possível determinar a existência da circulação do T. gondii em espécimes T. pecari e P. tajacu de vida livre da Amazônia peruana, possibilitando a continuidade do ciclo deste parasito no ecossistema da região e a transmissão vertical do parasito nestas espécies. Além de ser a primeira pesquisa que relaciona a imunodetecção de T. gondii em espécimes de T. pecari e P. tajacu analisando amostras de útero gestacional e placenta com detecção pela IHQ, que mostrou ser uma técnica altamente sensível.

Toxoplasma gondii is a mandatory cosmopolitan and intracellular protozoan that affects endothermic animals, including humans. Transmission can occur through horizontal and vertical routes, the latter, in general, ends up triggering reproductive pathologies. Thus, the objective was to investigate the occurrence of T. gondii in species of the Tayassuidae Family of free life in the Peruvian Amazon, by immunomarking the protozoan in gestational uterus and placenta tissue. For that, the samples of uterine and placental tissue of 36 Pecari tajacu and 42 Tayassu pecari were analyzed by the techniques of histopathology and immunohistochemistry, with the results correlated by the chi-square test. A total of 12 animals were positive (06 P. tajacu and 06 T. pecari). In IHC, T. pecari exhibited 7.14% positivity for T. gondii in gestational uterus and 9.52% in placenta, while P. tajacu presented 11.11% in gestational uterus and 8.33% in placenta. The immunoreactive samples in IHC showed intensity varying from moderate to strong in both organs of the analyzed species, with parasitic forms being observed in the epithelium and uterine and placental connective tissue, but without observing the characteristic lesions in the tissues evaluated by the histotopathology and IHC techniques. With that, it was possible to determine the existence of the circulation of T. gondii in T. pecari and P. tajacu specimens of free life in the Peruvian Amazon, allowing the continuity of the cycle of this parasite in the region's ecosystem and the vertical transmission of the parasite in these species. In addition to being the first research that links the immunodetection of T. gondii in specimens of T. pecari and P. tajacu, analyzing samples of gestational uterus and placenta with IHC detection, which proved to be a highly sensitive technique.

Immunodetection of myeloid and plasmacytoid dendritic cells in mammary carcinomas of female dogs / Imunodetecção de células dendríticas mieloides e plasmocitoides nos carcinomas mamários em cadelas

Dendritic cells have attracted great interest from researchers as they may be used as targets of tumor immune evasion mechanisms. The main objective of this study was to evaluate the relationship between the dendritic cells (DCs) subpopulation in simple type mammary carcinomas in female dogs. Two groups of samples were used: the control group consisted of 18 samples of mammary tissue without changes and the tumor group with 26 simple type mammary carcinomas. In these groups, we evaluated the immunodetection of immature and mature myeloid DCs, plasmacytoid DCs and MHC-II. In mammary tumor, mature myeloid DCs predominated in the peritumoral region, while immature myeloid DCs and plasmacytoid DCs were evident in the intratumoral region. Immunostaining of MHC-II was visualized in mammary acini (control group), in tumor cells and inflammatory infiltration associated with tumors. The comparison between the control and tumor groups showed a statistically significant difference between immature myeloid DCs, mature myeloid DCs and plasmacytoid DCs. The immunodetection of MHC-II was not significant when comparing the groups. The predominance of immature DCs in the tumor group is possibly related to an inefficient immune response, promoting the development and survival of tumor cells. The presence of plasmacytoid DCs in the same group suggests a worse prognosis for female dogs with mammary tumors. Therefore, the ability of differentiation of canine dendritic cells could be influenced by neoplastic cells and by the tumor microenvironment.(AU)

As células dendríticas têm despertado grande interesse dos pesquisadores, pois podem ser alvo dos mecanismos de evasão imune do tumor. O objetivo principal deste estudo foi avaliar a relação entre as subpopulações de células dendríticas (DCs) nos carcinomas mamários do tipo simples em cadelas. Dois grupos de amostras foram utilizados, o grupo controle composto por 18 amostras de tecido mamário sem alterações e o grupo tumor com 26 carcinomas mamários do tipo simples. Nestes grupos foram avaliadas a imunodetecção de DCs mieloides imaturas e maduras, DCs plasmocitoides e de MHC-II. Nas mamas com tumor, as DCs mieloides maduras predominaram na região peritumoral, enquanto que as DCs mieloides imaturas e as DCs plasmocitoides foram evidentes na região intratumoral. A imunomarcação do MHC-II foi visualizada nos ácinos mamários (grupo controle), nas células tumorais e no infiltrado inflamatório associado aos tumores. Na comparação entre os grupos controle e tumor houve diferença estatística significativa entre as DCs mieloides imaturas, DCs mieloides maduras e DCs plasmocitoides. A imunodetecção de MHC-II não foi significativa na comparação entre os grupos. A predominância de DCs imaturas no grupo tumor, possivelmente, está relacionada com uma resposta imune ineficiente, favorecendo o desenvolvimento e a sobrevivência das células tumorais. A presença das DCs plasmocitoides no mesmo grupo sugere um prognóstico pior para cadelas com tumores de mama. Portanto, a capacidade de diferenciação das células dendríticas caninas poderia ser influenciada pelas células neoplásicas e pelo microambiente tumoral.(AU)

Imunomarcação de Leishmania sp. e aspectos histológicos na terceira pálpebra de cães naturalmente infectados por Leishmania (leishmania) chagasi / Hystopathology and Leishmania sp. Immunostaining in third eyelid of Leishmania (Leishmania) chagasi naturally infected dogs

Canine visceral leishmaniasis (CVL) is a disease with multisystemic injuries, and among the varioustissues affected, the third eyelid (TE) is often involved. This ocular adnexa has been studied for theleishmaniasis pathogenesis elucidation and for diagnosis advance. This study aimed to carry out theparasite immunodetection and evaluate histologic lesions in TE of Leishmania (leishmania) chagasinaturally infected dogs. Twenty-six TE samples from infected dogs were submitted to histologicevaluation and to immunohistochemistry for Leishmania sp. The main lesion observed in the thirdeyelid conjunctiva (TEC) was lymphoplasmacytic inflammation, with Mott cells and parasitizedhistiocytes. Additionally, loss of epithelial stratification, ulceration, goblet cells thinning or hyperplasiawere often found. The same inflammatory pattern was observed in the third eyelid lacrimal gland(TELG), often accompanied by acinar atrophy and secretory ducts dilatation. Immunohistochemistry revealed parasitism in all samples, in different intensities.(AU)

A leishmaniose visceral canina (LVC) é uma doença que envolve lesões multissistêmicas e, dentre osvários tecidos acometidos, a terceira pálpebra está frequentemente envolvida. Este anexo ocular temsido alvo de estudo tanto para a elucidação da patogênese da doença quanto para o avanço diagnóstico.O presente trabalho teve por objetivo avaliar as alterações histológicas presentes na terceira pálpebrade cães naturalmente infectados por Leishmania chagasi e realizar a imunodetecção do parasita. Vintee seis amostras de terceira pálpebra de cães sintomáticos foram avaliadas quanto à coloração de HEe à imunoistoquímica com soro de cão positivo para Leishmania sp. A principal alteração observadana conjuntiva da terceira pálpebra foi infiltração inflamatória predominantemente linfoplasmocitária,com células de Mott e histiócitos parasitados permeando a área de exsudação. Adicionalmente, perdade estratificação e ulceração epitelial, rarefação ou hiperplasia de células caliciformes foram achadoscostumazes. Na glândula lacrimal da terceira pálpebra, o mesmo padrão inflamatório foi observado,acompanhado frequentemente de atrofia acinar e dilatação dos ductos secretórios. A imunoistoquímicarevelou parasitismo em todas as amostras, em diferentes intensidades.(AU)

Imunomarcação de Leishmania sp. e aspectos histológicos na terceira pálpebra de cães naturalmente infectados por Leishmania (leishmania) chagasi / Hystopathology and Leishmania sp. Immunostaining in third eyelid of Leishmania (Leishmania) chagasi naturally infected dogs

Canine visceral leishmaniasis (CVL) is a disease with multisystemic injuries, and among the varioustissues affected, the third eyelid (TE) is often involved. This ocular adnexa has been studied for theleishmaniasis pathogenesis elucidation and for diagnosis advance. This study aimed to carry out theparasite immunodetection and evaluate histologic lesions in TE of Leishmania (leishmania) chagasinaturally infected dogs. Twenty-six TE samples from infected dogs were submitted to histologicevaluation and to immunohistochemistry for Leishmania sp. The main lesion observed in the thirdeyelid conjunctiva (TEC) was lymphoplasmacytic inflammation, with Mott cells and parasitizedhistiocytes. Additionally, loss of epithelial stratification, ulceration, goblet cells thinning or hyperplasiawere often found. The same inflammatory pattern was observed in the third eyelid lacrimal gland(TELG), often accompanied by acinar atrophy and secretory ducts dilatation. Immunohistochemistry revealed parasitism in all samples, in different intensities.

A leishmaniose visceral canina (LVC) é uma doença que envolve lesões multissistêmicas e, dentre osvários tecidos acometidos, a terceira pálpebra está frequentemente envolvida. Este anexo ocular temsido alvo de estudo tanto para a elucidação da patogênese da doença quanto para o avanço diagnóstico.O presente trabalho teve por objetivo avaliar as alterações histológicas presentes na terceira pálpebrade cães naturalmente infectados por Leishmania chagasi e realizar a imunodetecção do parasita. Vintee seis amostras de terceira pálpebra de cães sintomáticos foram avaliadas quanto à coloração de HEe à imunoistoquímica com soro de cão positivo para Leishmania sp. A principal alteração observadana conjuntiva da terceira pálpebra foi infiltração inflamatória predominantemente linfoplasmocitária,com células de Mott e histiócitos parasitados permeando a área de exsudação. Adicionalmente, perdade estratificação e ulceração epitelial, rarefação ou hiperplasia de células caliciformes foram achadoscostumazes. Na glândula lacrimal da terceira pálpebra, o mesmo padrão inflamatório foi observado,acompanhado frequentemente de atrofia acinar e dilatação dos ductos secretórios. A imunoistoquímicarevelou parasitismo em todas as amostras, em diferentes intensidades.

SUSCEPTIBILIDADE DO BAÇO CANINO À INFECÇÃO POR Leishmania infantum

A Leishmaniose Visceral (LV) é uma antropozoonose cosmopolita, cujo mais importante reservatório urbano do parasita Leishmania infantum é o cão. Na LV canina é descrito um quadro de imunossupressão induzida pelo parasita, que pode suprimir a resposta anti-leishmania. O fator de transcrição STAT3 é ativado para a indução da expressão de citocinas associadas a resposta imune Th2, bem como, este fator é uma peça chave para a ligação do STAT6 com genes-alvo na indução da resposta imunossupressora Th2. O baço é o principal órgão linfoide responsável pela ativação de resposta imune sistêmica, no entanto, o mesmo mantém a infecção durante todo o curso da LV. Portanto, o objetivo deste estudo foi avaliar 27 amostras de baços de cães com LV naturalmente infectados e comparar a imunodetecção do STAT3 e do STAT6 com a densidade de macrófagos parasitados (imunomarcação) e a intensidade das lesões esplênicas. Para isto, foram utilizadas amostras de baço de 31 cães (27 amostras de cães infetados e 4 amostras de baço de cães livre de LV) provenientes do arquivo de blocos do Departamento de Patologia Veterinária, da Faculdade de Ciências Agrárias e Veterinárias (FCAV) da Unesp, campus de Jaboticabal. As amostras foram divididas nos grupos de animais controle (GC), assintomático (GA) e sintomático (GS). Os cães do grupo GS, seguidos de GA apresentaram inflamação granulomatosa, com macrófagos parasitados ou não predominantemente em polpa vermelha. Também houve a redução da área da polpa branca, rarefação linfoide e apoptose de linfócitos T. A detecção dos STATs foi observada em linfócitos e macrófagos e foi maior nos grupos infectados, exceto no grupo GS para STAT6. A imunodetecção do parasito mostrou diferenças entre os grupos GS e GA. Portanto conclui-se que STAT3 e STAT6 estavam associadas as lesões esplênicas e a presença do parasito em animais infectados. Estes fatores de transcrição podem estar contribuindo para a susceptibilidade do baço a infecção por Leishmania infantum.

Visceral Leishmaniasis (LV) is a cosmopolitan anthropozoonosis whose largest volume is the urban reservoir of the parasite Leishmania infantum is the dog. A parasite-induced immunosuppression picture, described in LVC, may suppress the response against leishmania. The transcription factor STAT3 is activated to induce the expression of cytokines associated with Th2 immune response, as well as, this factor is a key piece for the binding of STAT6 with target genes in the induction of Th2 immunosuppressive response. The spleen is the major lymphoid organ responsible for the activation of systemic immune response; however, it maintains the infection throughout the course of VL. Therefore, the objective of this study was to evaluate 27 samples from spleens of dogs with naturally infected LV and correlate the immunodetection of STAT3 with the density of parasitized macrophages (immunostaining) and the severity of splenic injuries. For this, spleen samples of 31 (27 samples of infected dogs and 4 samples of spleen of free- LV dogs) from the archive of the Department of Veterinary Pathology, Faculty of Agrarian and Veterinary Sciences (FCAV) of Unesp, Jaboticabal campus, were used. The samples were divided into groups of animals as control (CG), asymptomatic (GA) and symptomatic (GS). The dogs of the GS group, followed by GA, presented granulomatous inflammation, with parasitized or non-parasitized macrophages, reduced white pulp area, linear rarefaction and T lymphocyte apoptosis. The detection of STATs was observed in lymphocytes and macrophages and was higher in infected groups, except in the GS group for STAT6. The immunodetection of the parasite was differentiated between the GS and GA groups. Therefore, it was concluded that STAT3 and STAT6 were associated with splenic lesions and the presence of the parasite in infected animals and may be contributing to the susceptibility of the virus to infection by Leishmania infantum.

AVALIAÇÃO DA IMUNOMARCAÇÃO de COX-2 EM CARCINOMAS INTESTINAIS CANINOS

Devido ao aumento da expectativa de vida dos animais de estimação, o aparecimento de neoplasias tem se tornado uma importante afecção na Medicina Veterinária. As neoplasias gastrointestinais de cães são pouco diagnosticadas e sua etiologia é desconhecida. As localizações mais frequentes são o jejuno, cólon e reto. Objetivou-se avaliar a Cox-2 por meio de imunohistoquímica e a intensidade de PAS positivo nas amostras de intestinos de cães saudáveis (GS) e com neoplasia (GN). As neoplasias foram classificadas por análise histopatológica. As diferenças foram significativas quando P<0.05 (testes não paramétricos). Nas amostras neoplásicas observou-se imunodetecção acentuada de COX-2, quando comparadas aos cães saudáveis, com diferenças significativas entre os grupos. O mesmo ocorreu para a intensidade de PAS, onde se observou diminuição do número de células caliciformes e aumento na produção de muco nas amostras neoplásicas, enquanto nas amostras saudáveis observou-se marcação intensa nas células caliciformes. Com isso pode-se concluir que a COX está envolvida na capacidade do tumor evadir as defesas do sistema imunológico. Apesar da relação entre o processo inflamatório, mais especificamente o papel das prostaglandinas, e o desenvolvimento e propagação tumoral ser bastante claro, ainda muito se têm a ser esclarecido.

Due to the increase in the life expectancy of the pets, the appearance of neoplasias has become an important affection in the Veterinary Medicine. Gastrointestinal neoplasms of dogs are poorly diagnosed and their etiology is unknown. The most frequent locations are jejunum, colon and rectum. The objective of this study was to evaluate Cox-2 by means of immunohistochemistry and the positive PAS intensity in intestinal samples from healthy dogs (GS) and neoplasia (GN). The neoplasms were classified by histopathological analysis. The differences were significant when P <0.05 (non-parametric tests). In the neoplastic samples, marked COX-2 immunodetection was observed when compared to healthy dogs, with significant differences between groups. The same was observed for PAS intensity, where a decrease in the number of goblet cells and an increase in the mucus production were observed in the neoplastic samples, while in the healthy samples intense marking was observed in the goblet cells. With this we can conclude that COX is involved in the ability of the tumor to evade the defenses of the immune system. Although the relationship between the inflammatory process, more specifically the role of prostaglandins, and tumor development and propagation is very clear, much remains to be elucidated.