Clinical, cytological, histopathological and immunohistochemical features of a T-Zone lymphoma in a mixed-breed dog

Background: Non-Hodgkin lymphoma are a group of lymphoid neoplasms originating from the proliferation of precursors or mature, T, B and/or NK lymphocytes. T-Zone lymphoma (TZL) is characterized as an indolent lymphoma due to its slow progression and poor chemotherapy’s response. Dogs affected by this neoplasm may live for many years without clinical signs and are often underdiagnosed. The aim of this study was to report a TZL case in a 9-year-old male mixed breed dog, submitted to clinical follow-up and chemotherapy. Case: A 9-year-old male mixed-breed dog was presented due to the observation of an increased left mandibular lymph node. The previous cytological examination was suggestive of reactive hyperplasia and histopathological examination, by incisional biopsy, compatible with lymphocytic low-grade lymphoma. Physical examination revealed enlarged and firm left mandibular lymph node and adequate physical condition. A cytological examination was performed on the mandibular and both popliteal lymph nodes and revealed many small lymphocytes with hyperchromatic chromatin, rarely evident nucleolus and whose cytoplasm often projected in the form of a “hand-mirror” or “comet tail”, compatible with lymphocytic lymphoma (low grade) and suggestive of TZL, in the three assessed lymph nodes. The histopathological and immunohistochemical examination, of the mandibular lymph node were chosen to confirm the diagnosis. In histopathology it was observed that 40% of the sample contained a monotonous cell population, composed by small lymphocytes, with some presenting “hand-mirror” morphology. Two mitotic figures were evidenced per field of high magnification (40x), inferring a low-grade disease. Immunohistochemical analysis revealed neoplastic proliferation with immunolabeling of CD3 lymphocytes and positivity for Ki-67 in 48% of neoplastic cells, but negative for CD20, CD79a, CD45, MUM-1 and...

Clinical, cytological, histopathological and immunohistochemical features of a T-Zone lymphoma in a mixed-breed dog

Background: Non-Hodgkin lymphoma are a group of lymphoid neoplasms originating from the proliferation of precursors or mature, T, B and/or NK lymphocytes. T-Zone lymphoma (TZL) is characterized as an indolent lymphoma due to its slow progression and poor chemotherapys response. Dogs affected by this neoplasm may live for many years without clinical signs and are often underdiagnosed. The aim of this study was to report a TZL case in a 9-year-old male mixed breed dog, submitted to clinical follow-up and chemotherapy. Case: A 9-year-old male mixed-breed dog was presented due to the observation of an increased left mandibular lymph node. The previous cytological examination was suggestive of reactive hyperplasia and histopathological examination, by incisional biopsy, compatible with lymphocytic low-grade lymphoma. Physical examination revealed enlarged and firm left mandibular lymph node and adequate physical condition. A cytological examination was performed on the mandibular and both popliteal lymph nodes and revealed many small lymphocytes with hyperchromatic chromatin, rarely evident nucleolus and whose cytoplasm often projected in the form of a “hand-mirror” or “comet tail”, compatible with lymphocytic lymphoma (low grade) and suggestive of TZL, in the three assessed lymph nodes. The histopathological and immunohistochemical examination, of the mandibular lymph node were chosen to confirm the diagnosis. In histopathology it was observed that 40% of the sample contained a monotonous cell population, composed by small lymphocytes, with some presenting “hand-mirror” morphology. Two mitotic figures were evidenced per field of high magnification (40x), inferring a low-grade disease. Immunohistochemical analysis revealed neoplastic proliferation with immunolabeling of CD3 lymphocytes and positivity for Ki-67 in 48% of neoplastic cells, but negative for CD20, CD79a, CD45, MUM-1 and...(AU)

Histopathological and immunophenotypical characterization of canine multicentric lymphoma in Brazil: a study of 203 cases / Caracterização histopatológica e imunofenotípica do linfoma multicêntrico canino no Brasil: um estudo de 203 casos

The immunophenotype is regarded as an independent prognostic factor in high-grade lymphomas, seeing that lymphomas of T-cell origin are associated with shorter survival time. Although a number of studies have evaluated the immunophenotypical profile of lymphoma in the USA and Europe, Brazilian research on the matter remains scarce. Exact characterization of the histopathological type is crucial to establish proper treatment and prognosis. This study evaluated the database of immunohistochemistry laboratories that perform immunophenotyping of canine lymphoma in Brazil. A total of 203 cases of multicentric lymphoma were classified according to the WHO classification. Immunophenotyping was able to identify 71.4% lymphomas of B-cell line, 27.1% of T-cell line and 1.5% of non-B cells and non-T cell lines. Diffuse large B-cell lymphoma was the most common with 59.1% of the cases. Among T-cell lymphomas, lymphoblastic was the most common (11.33% of the cases). Even though canine lymphomas tend to be high-grade, indolent lymphomas comprised 11.82% of the cases and T-zone lymphoma was the most prevalent (8.86%). The immunophenotype of multicentric lymphoma in Brazil is similar to those in other parts of the world, which suggests similar etiologic factors to the development of this disease.(AU)

O imunofenótipo é considerado um fator prognóstico independente em linfomas de alto grau, visto que os linfomas de origem de células T estão associados a menor tempo de sobrevida. Apesar de vários estudos terem avaliado o perfil imunofenotípico do linfoma nos EUA e na Europa, a pesquisa brasileira sobre o assunto ainda é escassa. A caracterização exata do tipo histopatológico é crucial para estabelecer o tratamento e o prognóstico adequados. Este estudo avaliou a base de dados de laboratórios de imuno-histoquímica que realizam imunofenotipagem do linfoma canino no Brasil. Um total de 203 casos de linfoma multicêntrico foi classificado de acordo com a classificação da OMS. A imunofenotipagem foi capaz de identificar 71,4% dos linfomas da linhagem de células B, 27,1% da linhagem de células T e 1,5% das linhagens de células não B e não T. O linfoma difuso de grandes células B foi o mais comum em 59,1% dos casos. Entre os linfomas de células T, o linfoblástico foi o mais comum (11, 33% dos casos). Embora os linfomas caninos tendam a ser de alto grau, os linfomas indolentes representaram 11,82% dos casos e o linfoma da zona T foi o mais prevalente (8,86%). O imunofenótipo do linfoma multicêntrico no Brasil é semelhante ao de outras partes do mundo, o que sugere fatores etiológicos semelhantes ao desenvolvimento dessa doença.(AU)

Histopathological and immunophenotypical characterization of canine multicentric lymphoma in Brazil: a study of 203 cases / Caracterização histopatológica e imunofenotípica do linfoma multicêntrico canino no Brasil: um estudo de 203 casos

The immunophenotype is regarded as an independent prognostic factor in high-grade lymphomas, seeing that lymphomas of T-cell origin are associated with shorter survival time. Although a number of studies have evaluated the immunophenotypical profile of lymphoma in the USA and Europe, Brazilian research on the matter remains scarce. Exact characterization of the histopathological type is crucial to establish proper treatment and prognosis. This study evaluated the database of immunohistochemistry laboratories that perform immunophenotyping of canine lymphoma in Brazil. A total of 203 cases of multicentric lymphoma were classified according to the WHO classification. Immunophenotyping was able to identify 71.4% lymphomas of B-cell line, 27.1% of T-cell line and 1.5% of non-B cells and non-T cell lines. Diffuse large B-cell lymphoma was the most common with 59.1% of the cases. Among T-cell lymphomas, lymphoblastic was the most common (11.33% of the cases). Even though canine lymphomas tend to be high-grade, indolent lymphomas comprised 11.82% of the cases and T-zone lymphoma was the most prevalent (8.86%). The immunophenotype of multicentric lymphoma in Brazil is similar to those in other parts of the world, which suggests similar etiologic factors to the development of this disease.(AU)

O imunofenótipo é considerado um fator prognóstico independente em linfomas de alto grau, visto que os linfomas de origem de células T estão associados a menor tempo de sobrevida. Apesar de vários estudos terem avaliado o perfil imunofenotípico do linfoma nos EUA e na Europa, a pesquisa brasileira sobre o assunto ainda é escassa. A caracterização exata do tipo histopatológico é crucial para estabelecer o tratamento e o prognóstico adequados. Este estudo avaliou a base de dados de laboratórios de imuno-histoquímica que realizam imunofenotipagem do linfoma canino no Brasil. Um total de 203 casos de linfoma multicêntrico foi classificado de acordo com a classificação da OMS. A imunofenotipagem foi capaz de identificar 71,4% dos linfomas da linhagem de células B, 27,1% da linhagem de células T e 1,5% das linhagens de células não B e não T. O linfoma difuso de grandes células B foi o mais comum em 59,1% dos casos. Entre os linfomas de células T, o linfoblástico foi o mais comum (11, 33% dos casos). Embora os linfomas caninos tendam a ser de alto grau, os linfomas indolentes representaram 11,82% dos casos e o linfoma da zona T foi o mais prevalente (8,86%). O imunofenótipo do linfoma multicêntrico no Brasil é semelhante ao de outras partes do mundo, o que sugere fatores etiológicos semelhantes ao desenvolvimento dessa doença.(AU)

Differential expression of T helper cytokines in the liver during early pregnancy in sheep

Liver plays important roles in the innate and adaptive immunity, and contributes to the maternal immune adjustments during pregnancy in mice and rats. T helper 1 (Th1) and Th2 cytokines are related to immune response. However, expression of Th1 and Th2 cytokines in maternal livers is unclear during early pregnancy in sheep. In this study, livers were collected on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to analyze the expression of Th1 and Th2 cytokines in the livers. Our results showed that interferon-gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6 and IL-10 were downregulated, and IL-5 was upregulated in the livers during early pregnancy. Furthermore, there was no effect for early pregnancy on expression of TNF-β in the livers, and the IFN-γ protein was limited to the endothelial cells of the proper hepatic arteries and portal veins. In conclusion, early pregnancy exerted its effect on the liver to regulate the Th cytokines expression, but there was no evident shift from Th1 to Th2 cytokines, which may be necessary for the maternal hepatic immune adjustments during early pregnancy in sheep.

Differential expression of T helper cytokines in the liver during early pregnancy in sheep

Liver plays important roles in the innate and adaptive immunity, and contributes to the maternal immune adjustments during pregnancy in mice and rats. T helper 1 (Th1) and Th2 cytokines are related to immune response. However, expression of Th1 and Th2 cytokines in maternal livers is unclear during early pregnancy in sheep. In this study, livers were collected on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to analyze the expression of Th1 and Th2 cytokines in the livers. Our results showed that interferon-gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6 and IL-10 were downregulated, and IL-5 was upregulated in the livers during early pregnancy. Furthermore, there was no effect for early pregnancy on expression of TNF-β in the livers, and the IFN-γ protein was limited to the endothelial cells of the proper hepatic arteries and portal veins. In conclusion, early pregnancy exerted its effect on the liver to regulate the Th cytokines expression, but there was no evident shift from Th1 to Th2 cytokines, which may be necessary for the maternal hepatic immune adjustments during early pregnancy in sheep.(AU)

LINFOMA ALIMENTAR EM FELINOS DOMÉSTICOS: CARACTERIZAÇÃO CLÍNICO-PATOLÓGICA, HISTOLÓGICA E IMUNOFENOTÍPICA E ASSOCIAÇÃO COM FIV E FELV

O linfoma alimentar (LA) é uma doença emergente na clínica de felinos domésticos, com aumento importante de casos na última década. Os vírus da leucemia felina (FeLV) e da imunodeficiência felina (FIV) são agentes com potencial oncogênico conhecido e podem estar envolvidos no desenvolvimento das enteropatias crônicas. Este estudo teve como objetivos diagnosticar felinos com LA, estimar sua frequência na população de estudo e caracterizar clínico-patológica, histológica e fenotipicamente os casos, assim como buscar uma associação das enteropatias crônicas com as infecções por FIV e FeLV através da investigação dos agentes por ensaios imunoenzimáticos e imuno-histoquímicos. Gatos com enteropatia crônica foram avaliados no período de setembro de 2020 a maio de 2021, totalizando 29 animais; destes 48,2% (7/14) foram selecionados e realizaram o procedimento de biópsia intestinal, sendo 50% (7/14) diagnosticados com LA, e os demais (50%, 7/14), com doença inflamatória intestinal idiopática (DII). Dos animais com LA, 85,7% (6/7) eram fêmeas e 14,2% (1/7) machos, 28,5% (2/7) eram adultos maduros (idade média de 9,5 anos), e 71,4% (5/7) seniores, com idade média de 12,2 anos. O tipo morfológico predominante foi o de pequenas células (85,7%, 6/7), com apenas um caso composto por células intermediárias (14,2%, 1/7). Em todos os casos o fenótipo encontrado foi o de células T, com 42,8% (3/7) classificados em enteropatia associada ao linfoma de células T tipo 1 (EATL-1) e 57,1% (4/7), EATL-2. Dos 14 pacientes com enterite avaliados, apenas um foi positivo para FeLV e um para FIV na sorologia, com diagnóstico final de LA e DII, respectivamente. Na análise imuno-histoquímica das amostras intestinais, FeLV foi detectado em apenas um caso (7,1%, 1/14), este igualmente positivo na sorologia para o retrovírus; FIV foi detectado em 28,5% (4/14) dos pacientes, três deles com LA e um com DII, sendo apenas um igualmente positivo no teste imunoenzimático, indicando uma possível associação deste retrovírus nas enterites de causa inflamatória e neoplásica. Mais estudos envolvendo FIV são necessários, através do uso de técnicas mais sensíveis, como as de PCR, a fim de encontrar um possível papel deste retrovírus no desenvolvimento das enteropatias crônicas felinas.

Alimentary lymphoma (AL) is an emerging disease in domestic feline clinics, with a significant increase in cases in the last decade. Feline leukemia (FeLV) and feline immunodeficiency (FIV) viruses are agents with known oncogenic potential and may be involved in the development of chronic enteropathies. This study aimed to diagnose cats with AL, estimate its frequency and characterize clinicopathologically, histologically and phenotypically, as well as to seek an association of enteritis with FIV and FeLV infections, through the investigation of agents in immunoenzymatic and immunohistochemical assays. Cats with chronic enteropathy were evaluated from september 2020 to may 2021, totaling 29 animals; of these, 48.2% (7/14) underwent the intestinal biopsy procedure, 50% (7/14) diagnosed with LA, and the others (50%, 7/14), idiopathic inflammatory enteritis. Of the animals with LA, 85.7% (6/7) were female and 14.2% (1/7) male, 28.5% (2/7) were mature adults (mean age 9.5 years), and 71.4% seniors (5/7), with an average age of 12.2 years. The predominant morphological type was small cells (85.7%, 6/7), with only one case composed of intermediate cells (14.2%, 1/7). In all cases, the phenotype found was that of T-cells, with 42.8% (3/7) classified as type 1 T-cell lymphoma-associated enteropathy (EATL-1) and 57.1% (4/7), EATL-2. Of the 14 patients with enteritis, only one was positive for FeLV and one for FIV in serology, with a diagnosis of LA and IBD, respectively. In the immunohistochemistry of intestinal samples, FeLV was detected in only one case (7.1%, 1/14), this one equally positive in serology; FIV was detected in 28.5% (4/14) of patients, three of them with LA and one with IBD, with only one positive in the immunoenzymatic test, indicating a possible association of this retrovirus in inflammatory and neoplastic enteritis. More studies involving FIV are needed, using more sensitive techniques, such as PCR, to find a possible role for this retrovirus in the development of feline chronic enteropathies.

Classificação de 86 casos de linfoma em bovinos de acordo com a Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage e a Revised European-American Classification of Lymphoid Neoplasms (REAL) / Classification of 86 bovine lymphomas cases according to the Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage and The Revised European-American Classification of Lymphoid Neoplasms (REAL)

Por meio de um estudo retrospectivo, realizou-se avaliação fenotípica (histologia) e imunofenotípica (imuno-histoquímica [IHQ]) de 86 casos de linfoma bovino. Quanto ao padrão de distribuição, todos os linfomas foram incluídos como difusos. Em relação à dimensão dos linfócitos neoplásicos, 83,8% dos linfomas foram considerados como de grandes células e 11,6% como de pequenas células. Linfomas mistos, ou seja, constituídos por grandes e pequenas células, representaram apenas 4,6% dos casos. Quanto ao número de mitoses, 84,9% dos linfomas foram incluídos como de grau intermediário, 10,5% como de baixo grau e 4,6% como de alto grau. No que se refere à morfologia do núcleo, linfomas em que predominavam linfócitos não clivados (58,2%) ou linfócitos clivados (37,2%) foram mais frequentes do que aqueles em que havia uma mistura igualmente proporcional de linfócitos clivados e não clivados (4,6%). Com base nestes resultados, os 86 linfomas foram assim distribuídos utilizando-se a classificação proposta pela Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage: difuso de grandes células não clivadas (46,5%), difuso de grandes células clivadas (33,7%), difuso de pequenas e grandes células (4,6%), difuso de pequenas células - tipo plasmocitoide (7%), imunoblástico (3,5%), difuso de pequenas células - tipo intermediário (2,3%), difuso de pequenas células não clivadas (1,2%) e difuso de pequenas células não clivadas - tipo Burkitt (1,2%). Na imuno-histoquímica, 27 dos 86 (31,4%) linfomas foram positivos para o anticorpo monoclonal CD79αcy, utilizado para detecção de linfócitos B, e nenhum caso foi positivo para o anticorpo policlonal CD3, utilizado para detecção de linfócitos T. Com base nestes resultados, os 27 linfomas B foram assim distribuídos utilizando-se a Revised European-American Classification of Lymphoid Neoplasms (REAL): linfoma difuso de grandes células B (81,5%), linfomas imunoblásticos de grandes células (11,1%) e linfomas [...](AU)

A retrospective study of 86 cases of bovine lymphoma classified as with diffuse pattern of distribution and verified by phenotypic (histology) and immunophenotypic (immunohistochemistry [IHC]) is presented. Regarding the size of the neoplastic lymphocytes, 83.8% was classified as large cells lymphoma and 11.6% as small cells lymphoma. Mixed lymphomas, i.e., formed by large and small cells simultaneously represented only 4.6% of all cases. Regarding their mitotic index, 84.9% of lymphomas was included in the intermediate-grade, 10.5% as low-grade and 4.6% as high-grade. Regarding the nucleus morphology, lymphomas with mostly non-cleaved cells (58.2%) or cleaved cells (37.2%) were the more frequent than those with a balanced mixed proportion of cleaved and non-cleaved cells (4.6%). Based on these results, the 86 lymphoma cases were classified by the Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage as: diffuse large non-cleaved cell (46.5%), diffuse large cleaved cell (33.7%), diffuse mixed small and large cell (4.6%), diffuse small cell - plasmacytoid (7%), immunoblastic (3.5%), diffuse small cell - intermediate (2.3%), diffuse small non-cleaved cell (1.2%), and diffuse small non-cleaved cell Burkitt's (1.2%). According to the IHC, 27 out of 86 (31.4%) lymphomas were positive to monoclonal antibody CD79αcy, used to detect B cells, and none were positive for polyclonal antibody CD3, used to detect T cells. Based on this, the 27 B-cell type lymphomas were distributed as follows: diffuse large B-cell lymphoma (81.5%), large cell immunoblastic lymphoma (11.1%), and lymphoplasmacytoid lymphoma (7.4%), according the Revised European-American Classification of Lymphoid Neoplasms (REAL). The results of this retrospective study, similar to what has been described in other parts of the world, allow us to conclude that bovine lymphomas are basically diffuse and predominantly made of intermediate-grade, large cells, with cleaved or non-cleaved [...](AU)

Cell localization of ACTH, dopamine, and GnRH receptors and PPARΓ in bovine corpora lutea during diestrus

Background: Corpora lutea (CL) are transient organs, essentials for the pregnancy to be successful, that results from the ovulatory follicle rupture. If pregnancy fails to occur, the CL undergo luteolysis, a dynamic regression process that ends with their complete functional and structural demise. The life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotropic and luteolytic mediators. The life-span of bovine CL is regulated by a multifactorial system that includes various hormonal regulators that up to date are not yet well studied, therefore the aim of the present study was to evaluate the immunohistochemical presence of receptors for adrenocorticotropic hormone (melanocortin-2 receptor, MC2R), dopamine (DR1-5), gonadotropin-releasing hormone (GnRHR), and peroxisome proliferators-activated receptor γ (PPARγ) in early, mid, late and regressive CL during diestrous cycle of bovine. Materials, Methods & Results: Ovaries from 24 clinically healthy cyclic cows were collected from licensed abattoirs. The animals had undergone a general veterinary assessment of their reproductive tract and ovaries before slaughter to exclude any apparent abnormalities and early pregnancy. Corpora lutea were separated by blunt dissection from the surrounding ovarian tissues and classified into four groups, covering the entire diestrous cycle length: early, mid, late and regressive. Tissue specimens were immediately processed for immunohistochemical investigation. The slides were incubated with the following primary antibodies: rabbit polyclonal anti-GnRHR, rabbit polyclonal anti-MC2R, rabbit polyclonal anti-DRD1-DR5, and mouse monoclonal anti-PPARγ. The slides were incubated with the specific biotinylated secondary antibody exposed to avidin-biotin complex and the peroxidase activity sites were visualized using the DAB kit as chromogen. Tissue sections in which the primary antibody was omitted or substituted by the specific IgG were used as negative controls of non-specific staining. The intensity of receptor immunostaining in CL was assessed and comparated microdensitometrically. The data of the densitometric analysis were examined by Levene's test and one-way ANOVA followed by Student-Newman-Keuls t-test. Differences were considered significant at P < 0.01. GnRHR, PPARγ, and DR2 immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. The immunostaining of MC2R was strong in the cytoplasm and nucleus of luteal cells during early, mid and late CL, and decreased (P < 0.01) in regressive ones. PPARγ immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. DR2 immunosignals were only found in early, mid and late stages, whereas DR5 was evidenced strongly in the cytoplasm of early and mid CL, and weakly (P < 0.01) in late and regressive ones. DR1, -3, and -4 were immunonegative in all CL types. Discussion: The present study showed that in bovine CL GnRHR, MC2R, PPARγ, DR2 and DR5 are variously present throughout the different luteal phases of the diestrous cycle. These preliminary data confirm the multifactorial complexity of the bovine CL life span regulation, in particular the fine tuning of the mediators that balance the CL luteotropic and luteolytic condition.

CAUSAS DE MORTES EM GATOS NO SUL DO BRASIL

A intensificação da criação de felinos em todo o mundo demandam uma ampliação e aprofundamento dos conhecimentos da clínica e patologia de doenças infecciosas que acometem os gatos. Entretanto, existe uma escassez de informações sobre doenças infecciosas que acometem os gatos do Brasil, especialmente da região sul. Esta tese de doutorado teve como objetivo realizar um levantamento das principais causas de morte em gatos, além da caracterização anatomopatológica e identificação de agentes infecciosos em tecidos e células de gatos através da técnica de imuno-histoquímica (IHQ) e imunocitoquímica (ICQ). O primeiro artigo teve como objetivo determinar as principais causas de morte em gatos na região Sul do Brasil, através de um estudo retrospectivo, de 2000 a 2015. Foram realizadas um total de 1753 necropsias de felinos domésticos, destas 1364 (77,8%) foram conclusivas e 389 foram inconclusivos (22,2%). As categorias mais prevalentes foram: neoplasmas (20%), doenças infecciosas/parasitárias (17,8%), doenças do sistema digestório (14,1%), traumatismos (13,4%), doenças do sistema urinário (11,4%), doenças do sistema cardiovascular (6,9%), doenças do sistema respiratório (5,8%) e outras (8,5%). As causas de morte mais frequentes incluíram: politraumatismo (13,4%), linfoma (8,8%) e peritonite infecciosa felina (7,9%). O segundo artigo teve como objetivo fazer a identificação por ICQ dos antígenos do coronavírus felino (FCoV) em macrófagos e monócitos fixados em formol e embebidos em parafina a partir de derrame e sangue como alternativa diagnóstica ante mortem da peritonite infecciosa felina. Foram selecionados para o estudo gatos com pelo menos um dos seguintes sinais clínicos: derrame em cavidades abdominal e/ou torácica e/ou pericárdica, temperatura retal acima de 40oC, icterícia, linfoadenomegalia, e sinais clínicos oftálmicos e neurológicos. Foram recebidas 25 amostras provenientes de derrames. Dessas, 16 tiveram marcação positiva no interior de macrófagos. Nove desses casos, foram confirmados pela necropsia. De 17 amostras de sangue total, 3 amostras tiveram marcação positiva no interior de monócitos; dois desses casos foram confirmadas na necropsia. O teste de ICQ anti-FCoV foi sensível e específico como método diagnóstico ante mortem da PIF. O terceiro artigo teve como objetivo descrever cinco casos de gatos apresentando cardiomiopatia hipertrófica e miocardite associadas ao vírus da imunodeficiência felina (FIV). Na necropsia, os cinco gatos apresentaram o coração acentuadamente aumentado de tamanho, por hipertrofia ventricular esquerda, e múltiplos focos brancacentos coalescentes no miocárdio e no epicárdico. Microscopicamente, no miocárdio, havia infiltrado multifocal acentuado composto por linfócitos, alguns macrófagos, neutrófilos e plasmócitos. Na IHQ para FIV houve intensa imunomarcação no citoplasma e no núcleo, principalmente de linfócitos e no citoplasma de ocasionais macrófagos no miocárdio. O infiltrado inflamatório caracterizou-se por linfócitos T e macrófagos, o que foi evidenciado por imunomarcação específica para essas células. Este trabalho demonstra a importância das doenças infecciosas/parasitárias, consideradas como causa de morte em gatos, demonstra a utilidade da técnica técnica ocorrência de infecção pelo do vírus da imunodeficiência felina em células inflamatórias no miocárdio de gatos com miocardite e cardiomiopatia hipertrófica.

The increase of cat breeding worldwide demands an expansion in qualified information on clinic clinical and pathological aspects of feline infectious diseases. There is, however, a lack of information on the infectious diseases that affect cats in Brazil, especially in the Southern region. This doctoral thesis aimed to survey of the main causes of death caused in cats, as well as the anatomopathological characterization of tissue response and identification of infectious agents in the tissues and cells through immunohistochemistry (IHC) and immunocytochemistry (ICH). Three articles were written. The first one aimed to determine the main causes of death in cats in southern Brazil, through a retrospective study, from 2000 to 2015. A total of 1,753 domestic feline necropsies were reviewed, from which 1,364 (77.8%) had a conclusive diagnosis and 389 were inconclusive (22.2%). The most prevalent categories were neoplasms (20%), infectious/parasitic diseases (17.8%), diseases of the digestive system (14.1%), traumatisms (13.4%), diseases of the urinary system (6.9%), diseases of the respiratory system (5.8%) and others (8.5%). The most frequent causes of death included multiple trauma (13.4%), lymphoma (8.8%) and feline infectious peritonitis (7.9%). lymphoma (8.8%) and feline infectious peritonitis (7.9%). The second article aimed to tests and alternative antemortem diagnostic method to identified cats with feline infectious peritonitis feline coronavirus (FCoV) antigens; the method consists of applying ICH to macrophages and monocytes sampled from effusion and blood, fixed in formalin and embedded in paraffin. Cats with at least one of the following clinical signs were selected for being tested: cavitary (abdominal and/or thoracic and/or pericardial) effusion, rectal temperature above 40 ° C, jaundice, enlargement of lymph nodes, clinical ophthalmic and neurological signs. Twenty-five samples from effusions were received, of which 16 had positive marking within macrophages, 9 of which were confirmed by necropsy examination. Seventeen samples consisted of whole blood, from these, 3 samples had positive staining within monocytes and two cases were confirmed by postmortem examination. The anti-FCoV ICQ test was sensitive and specific as a PIF antemortem diagnostic method. The second article aimed to identify by ICQ the feline coronavirus (FCoV) antigens in macrophages and monocytes fixed in formalin and embedded in paraffin from effusion and blood as a diagnostic alternative antemortem of feline infectious peritonitis. Cats with at least one of the following clinical signs were selected: cavitary (abdominal and/or thoracic and/or pericardial) effusion, rectal temperature above 40C, jaundice, enlargement of lymph nodes, clinical ophthalmic and neurological signs. Twenty-five samples from effusions were received, of which 16 had positive marking inside macrophages, 9 of which were confirmed by necropsy examination. 17 samples were also received from whole blood, these 3 samples had positive staining within monocytes and in 2 cases confirmed by postmortem examination. The anti-FCoV ICQ test was sensitive and specific as a PIF antemortem diagnostic method. The third article aimed to describe five cases of cats presenting hypertrophic cardiomyopathy and myocarditis associated with feline immunodeficiency virus (FIV). At necropsy, the five cats presented a markedly enlarged heart, marked left ventricular hypertrophy, multiple myocardial and epicardial whitening foci. Microscopically, in the myocardium, there was marked multifocal infiltrate composed of lymphocytes, macrophages, neutrophils and plasma cells. In the IHC for FIV there was intense immunostaining in the cytoplasm and nucleus, mainly of lymphocytes and occasional macrophages cytoplasm in the myocardium. The inflammatory infiltrate was predominantly composed of T lymphocytes and macrophages, evidenced by immunostaining. This work demonstrates the importance of infectious/parasitic diseases, as the cause of death in cats, demonstrate the use of the IHC technique as an alternative antemortem diagnostic tool for FIP and the occurrence of feline immunodeficiency virus infection in inflammatory cells in the myocardium of cats with myocarditis and hypertrophic cardiomyopathy.

CD279 (PD-1) e seus ligantes CD273 (PD-L1) e CD274 (PD-L2) regulam a linfoproliferação, a produção de IL-10 e do óxido nítrico na leishmaniose visceral canina

PD-1 ligado a PD-L1 ou PD-L2, desencadeia sinais incapacitantes às células T, podendo estar envolvidos na supressão das respostas imunes na LVC. Investigamos a expressão de PD-1 e seus ligantes no baço na LVC, e sua correlação com a carga parasitária. Para confirmar o seu papel regulador na leishmaniose, estes receptores foram avaliados após infecção in vitro por L. infantum. Em cães infectados, foi avaliado o bloqueio destes receptores na produção de citocinas e seus efeitos no sobrenadante da cultura, de linfoproliferação com antígeno solúvel (AtgS) de L. infantum. Foram selecionados 23 cães saudáveis e 54 com LV. PD-1 e seus ligantes foram determinados por imunohistoquímica em tecidos esplênicos. A carga parasitária de cães infectados foi avaliada por PCR em tempo real. As citocinas nos sobrenadantes de cultura foram determinados por ELISA de captura e o NO por Griess. A Linfoproliferação das células do linfonodo e expressão de PD-1 e seus ligantes após infecção in vitro por L. infantum foram avaliadas por citometria de fluxo. Observou-se que a infecção por L. infantum, aumenta a expressão de PD-1 e seus ligantes. Uma correlação positiva foi observada entre a expressão de PD-1 e PD-L1 em linfócitos B e o aumento da carga parasitária. O bloqueio destes receptores restaura a proliferação de linfócitos, aumenta a produção de NO e diminui a IL-4 e IL-10. L. infantum modula a expressão de PD-1 e seus ligantes na LVC, suprimi a linfoproliferação, alterando a produção de citocinas, podendo facilitar a persistência dos parasitas nos macrófagos, e podem ser direcionados para o desenvolvimento de drogas que estimulem a resposta imune na LVC.

PD-1 when bound to PD-L1 or PD-L2, triggers signals disabling T cells, and they may be involved in the suppression of cellular immune responses in CVL. We investigated the expression of PD-1 and its ligands in spleen in CVL, and its correlation with parasite burden in this tissue, to confirm its regulatory role in leishmaniasis. These receptors were evaluated after in vitro infection by L. infantum. In infected dogs, we evaluated the blocking of these receptors in the production of cytokines in the effects of supernatant of cultured cells, and antigen specific lymphoproliferation. We selected 23 healthy dogs and 54 with VL. PD-1 and its ligands were determined by immunohistochemistry in splenic tissues. Parasitic load of infected dogs was evaluated by RT-PCR. Cytokines in culture supernatants were determined by capture ELISA and NO by Griess. Lymphoproliferation lymph nodes and expression of PD-1 and its ligand after in vitro infection by L. infantum was evaluated by flow cytometry. We observed that L. infantum infection increases expression of PD-1 and its ligands. A positive correlation was observed between the expression of PD-1 and PD-L1 B lymphocytes and an increase in parasite load. Blockade these receptors restores lymphocyte proliferation, increases the production of NO and decreases IL -4 and IL-10. In conclusion, L. infantum modulates expression of PD-1 and its ligands in CVL, suppressing lymphoproliferative response and altering cytokine production, which in turn can facilitate parasitic persistence in the macrophages. PD-1 and its ligands can be targeted for the development of drugs stimulating the immune response in CVL.

AVALIAÇÃO DA RESPOSTA IMUNOLÓGICA NO TRATO GASTRINTESTINAL DE FRANGOS DESAFIADOS COM Salmonella enterica: USO DE ADITIVOS FITOGÊNICOS E ÁCIDOS ORGÂNICOS.

Salmonella é uma bactéria responsável por perdas econômicas na cadeia avícola e por toxinfecções alimentares em pessoas. A bactéria pode ser sistêmica como Salmonella Pullorum e Salmonella Gallinarum, causando septicemia e mortalidade ou não sistêmica, como Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg e Salmonella Minnesota que desenvolvem poucos sinais clínicos nos animais. A presente Tese está dividida em 6 capítulos. O capítulo 1 refere-se à Introdução, abordando aspectos básicos da epidemiologia da Salmonella e o objetivo geral do trabalho. No capítulo 2 é apresentada uma revisão sobre a patogenicidade da Salmonella, abordando seus principais fatores de virulência na célula hospedeira. Além disso, também são abordadas as principais alterações na dinâmica celular do trato gastrintestinal ocasionada pela Salmonella e as principais medidas de controle da enfermidade em plantéis avícolas. Essas medidas de controle vão desde medidas básicas de biosseguridade até aditivos como ácidos orgânicos, aditivos fitogênicos e produtos que proporcionam exclusão competitiva. No capítulo 3 foi demonstrado que o extrato de sanguinarina na água de bebida é eficiente em reduzir a contagem de Salmonella Enteritidis no papo e ceco de frangos, além de reduzir a expressão de células caliciformes e CD3+ no duodeno e jejuno e aumentar a expressão de células imunológicas no sangue periférico quando comparados ao grupo que não recebeu sanguinarina. No capítulo 4 foi avaliada a patogenia da Salmonella Minnesota em frangos de corte e avaliou-se o efeito in vitro e in vivo de diferentes ácidos orgânicos demonstrando a baixa eficiência destes no controle da Salmonella Minnesota. No capítulo 5 foi realizada a análise retrospectiva de resultados de microbiologia e dinâmica da imunidade celular do trato gastrintestinal de frangos desafiados com Salmonella enterica sorovares Enteritidis, Minnesota e Heidelberg, onde se observou que a quantidade de salmonela no ceco e a mobilização de células imunológicas no trato gastrintestinal de frangos desafiados é dependente do sorovar avaliado. No capítulo 6, em trabalho realizado em parceria com a Universidade da Geórgia, foi avaliada a contribuição de genes da Salmonella Typhimurium de função desconhecida na colonização do trato gastrointestinal de frangos. Estes estudos demonstram a interferência dos diferentes sorovares de salmonelas paratíficas na dinâmica celular do trato gastrointestinal de aves e algumas medidas de controle desta enfermidade. Palavras-chave: Salmonella enterica, imunoistoquímica, células caliciformes, sistema imunológico, linfócitos T, aditivos.

Salmonella is a bacterium responsible for economic losses in the poultry chain and food poisoning in humans. This bacterium can be systemic in birds such as Salmonella Pullorum and Salmonella Gallinarum causing septicemia and mortality or non-sistemic such as Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Minnesota that present few clinical signs in animals. This Thesis is divided into six chapters. Chapter 1 refers to Introduction, addressing basic aspects of Salmonella epidemiology and the overall objective of the thesis. Chapter 2 presents a review of Salmonella pathogenicity, addressing its major virulence factors into the host cell. It is also addressed the major changes in cellular dynamics of the gastrointestinal tract caused by Salmonella and the main forms of disease control in poultry flocks. These forms of control range from basic biosecurity measures to additives such as organic acids, phytogenic additives and products that provide competitive exclusion. Chapter 3 shows that sanguinarine extract in drinking water is effective in reducing Salmonella Enteritidis count in crop and cecum of chickens, while reducing the expression of goblet cells and CD3+ cells in duodenum and jejunum and increasing immune cell expression in the peripheral blood when compared to the group that did not receive sanguinarine. Chapter 4 presents the evaluation of Salmonella Minnesota pathogenesis in broilers where the effect in vitro and in vivo of different organic acids was evaluated showing their low efficiency of Salmonella Minnesota control. Chapter 5 demonstrates a retrospective analysis of results of microbiology and immune cellular dynamics of gastrointestinal tract of challenged chickens with Salmonella enterica serovar Enteritidis, Minnesota and Heidelberg, where it was observed that the number of Salmonella in the cecum and the mobilization of immunological cells in gastroint estinal tract of challenged chickens is dependent on the assessed serovar. Chapter 6 presents the study performed in partnership with the University of Georgia where it was evaluated Salmonella Typhimurium gene contribution of unknown function in colonizing the gastrointestinal tract of chickens. These studies demonstrate the interference of different paratyphoid Salmonella serovars in cellular dynamics of poultry gastrointestinal tract and some control measurements of this disease.

AVALIAÇÃO DE MACRÓFAGOS ASSOCIADOS AO TUMOR (TAM) E FATOR DE CRESCIMENTO ENDOTELIAL VASCULAR (VEGF) EM LINFOMAS MULTICÊNTRICOS DE CÃES

Acredita-se que, no microambiente tumoral, os macrófagos estimulem a proliferação das células malignas, mediante produção de fatores de crescimento que favorecem a angiogênese, como o fator de crescimento endotelial vascular (VEGF). Esta pesquisa teve como objetivo avaliar a marcação de TAM e VEGF em linfomas multicêntricos mediante técnica de imuno-histoquímica. Foram selecionadas amostras de linfonodo provenientes de 15 cães com linfoma multicêntrico, conservadas em blocos parafinados, histologicamente classificados como linfoma difuso de grandes células B (LDGCB) e seis amostras de linfonodos de cães saudáveis para grupo controle. A expressão do VEGF e TAM foi determinada pela técnica de imuno-histoquímica utilizando os anticorpos anti-VEGF (VG-1, Abcam) e anti-macrófago (MAC 387, DBS). Para a quantificação de macrófagos, avaliou-se o número absoluto de células positivas que se apresentaram entremeadas ao tumor. Para a determinação do VEGF, foi realizada a soma dos escores dos percentuais de células tumorais coradas cujo percentual de marcação variou de um a quatro, sendo 1: até 25% das células positivas, 2: 25 a 50% das células positivas, 3: 50 a 75% das células positivas e 4: mais de 75% das células positivas. Para comparar as médias de reatividade dos TAM entre o grupo LDGCB e o grupo controle, aplicou-se o teste t de Student. Em relação ao VEGF, utilizou-se o teste Mann-Whitney para dados não paramétricos. Consideraram-se os resultados significativos quando p<0,05. Na avaliação da contagem de TAM, o grupo controle apresentou média (6,167±4,27) superior em relação à média do grupo dos cães com linfoma (3,55±1,82).Contudo, esta diferença não foi significativa (p>0,05). Em relação ao VEGF apenas dois animais apresentaram grau 2 (25% a 50% de células marcadas), enquanto os demais apresentaram grau 1 (até 25% de células marcadas).Com isso, foi possível oncluir que a imunomarcação de macrófagos e VEGF não influenciou no tipo neoplásico estudado, já que a semelhança destas amostras com o grupo controle foi observada.

It is believed that the tumor microenvironment, the macrophages stimulate the proliferation of malignant cells through production of growth factors that promote angiogenesis, such as vascular endothelial growth factor (VEGF). This research aimed to evaluate the marking of TAM and VEGF in multicenter lymphomas by immunohistochemistry technique. 15 samples of canine multicentric lymphoma, preserved in paraffin blocks, histologically classified as diffuse large B-cell lymphoma (DLBCL) and six samples of lymph nodes without cancer from healthy dogs for the control group were selected. The expression of VEGF and TAM was determined by immunohistochemical technique using the anti-VEGF antibody (VG-1, Abcam), anti-macrophage (MAC 387, DBS). Quantification of macrophages was evaluated by the absolute number of positive cells that appeared within tumor. For the determination of VEGF was performed the sum of the scores of the percentage of tumor cells stained marker whose percentage varied from one to four, 1: up to 25% of cells positive, 2: 25 to 50% positive cells 3: 50 to 75% of cells positive, 4: more than 75% positive cells. To compare mean reactivity of TAM between DLBCL group and the control group, we used the Student t test. Compared to VEGF, we used the Mann-Whitney test for nonparametric data. The significant results were considered when p <0.05. In assessing the TAM count, the control group had a mean (± 6.167 4.271) higher compared to the average of the group of dogs with lymphoma (3.55 ± 1.828). However, this difference was not significant (p> 0.05). Thus, it was concluded that the immunostaining of macrophages and VEGF did not influence in tumor type studied, since the similarity of these samples with the control group was observed.

AVALIAÇÃO DO FATOR DE CRESCIMENTO ENDOTELIAL VASCULAR (VEGF) E MACRÓFAGOS ASSOCIADOS AO TUMOR (TAM) EM CARCINOMAS MAMÁRIOS DE CADELAS

No microambiente tumoral, os macrófagos podem estimular a proliferação das células malignas, mediante produção de fatores de crescimento que favorecem a angiogênese. Embora a relação entre angiogênese e macrófagos tenha sido reconhecida em diversas pesquisas envolvendo tumores mamários de mulheres, esta relação na espécie canina permanece sob investigação. O objetivo deste estudo foi avaliar a marcação de macrófagos associados ao tumor (TAM) e do fator de crescimento endotelial vascular (VEGF) em carcinomas mamários de cadelas mediante técnica de imuno-histoquímica. Foram selecionados do setor de patologia do Hospital Veterinário da Universidade de Franca/SP (UNIFRAN), 99 blocos de parafina de carcinomas mamários de cadelas. As amostras foram submetidas a reações de imuno-histoquímica com os anticorpos anti-VEGF (VG-1, Abcam) e anti-macrófago (MAC 387, DBS). Para a determinação do VEGF, foi realizada a soma dos escores dos percentuais de células tumorais coradas cujo percentual de marcação variou de um a quatro, sendo 1: até 25% das células positivas, 2: 25 a 50% das células positivas, 3: 50 a 75% das células positivas e 4: mais de 75% das células positivas. Para a quantificação de macrófagos, avaliou-se o número absoluto de células positivas que se apresentaram entremeadas ao tumor. Os resultados foram avaliados,por análise de variância seguida pelo teste de Tukey, Kruskal-Wallis, seguido pelo teste de Dunns e teste t de Student, considerando-se nível de significância de 5%. Comparando-se os escores de marcação do VEGF, observou-se que carcinomas tubulares, papilares e carcinomas em tumor misto apresentaram médias de escores superiores ao carcinoma sólido (p<0,001). Avaliando-se as contagens de macrófagos entre os tipos histológicos, os carcinomas tubulares apresentaram média (13,52±16,38) superior aos carcinomas sólidos (2,77±3,59) (p<0,01). Contudo, não houve diferença na contagem de VEGF e de macrófagos entre os graus histológicos I, II e III (p>0,05). Quando categorizados em dois grupos de arcação de VEGF (50% e >50%), a média de TAM foi maior nos animais com marcação acima de 50% de VEGF (p=0,002). Conclui-se que, apesar do VEGF e de macrófagos não terem se apresentado aumentados nos tumores considerados agressivos, ambos demonstraram estar relacionados, uma vez que marcações de VEGF acima de 50% revelaram maiores contagens de macrófagos nas neoplasias mamárias caninas.

Mammary gland tumors are the most common malignancies in dogs. In the tumor 8 microenvironment, macrophages can stimulate the proliferation of malignant cells through production of growth factors that promote angiogenesis. Although the 10 relationship between angiogenesis and macrophages has been recognized in several studies involving mammary tumors in women, this relationship remains under 12 investigation in dogs. The objective of this study was to evaluate the immunostaining of 13 tumor-associated macrophages (TAM) and vascular endothelial growth factor (VEGF) in 14 mammary carcinomas of female dogs by means of immunohistochemistry technique. Ninety-nine 99 paraffin blocks of mammary carcinomas of female dogs were selected 16 from Veterinary Hospital of the University of Franca / SP (UNIFRAN). The samples under went immunohistochemical reactions with antibodies anti-macrophage (MAC 387, 18 DBS) and anti-VEGF (VG-1, Abcam). macrophages quantification was evaluated by the absolute number of positive cells around the tumor. VEGF analysis was performed by 20 percentage scores as follow: 1: up to 25% of positive cells, 2: 25 to 50% of positive cells 21 3: 50-75% of positive cells and 4: more than 75% of the positive cells. Analysis of 22 variance plus Tukey's test or Kruskal-Wallis plus Dunn's test was performed considering 23 5% significance level. Macrophages counts between histologic types showed higher 24 mean of tubular carcinomas (13,52 ± 16,38) that the solid carcinomas (2,77 ± 3,59) 25 (p<0,01). When comparing VEGF scores, it was observed that tubular, papillary and 26 mixed tumor carcinomas showed higher mean scorethat the solid carcinoma (p<0,001). 27 However, there was no difference to macrophages count or VEGF scores means 28 between tumor grades I, II and III (p> 0,05). When classified into two groups VEGF 29 labeling (50% and> 50%), the TAM mean was higher in animals with VEGF labelling 30 above 50% (p = 0,002). In conclusion, although VEGF and macrophages were not 31 increased in aggressive tumors, both markers could be related, since VEGF labeling over 50% showed higher macrophages density in canine mammary tumors.

Aspectos epidemiológicos, clínicos, anatomopatológicos e imuno-histoquímicos do linfoma em bovinos (1965-2014)

Foi realizado um estudo retrospectivo dos arquivos do Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM) com base nos casos de necropsia e materiais de necropsia de bovinos dos últimos 50 anos (1965-2014). Foram incluídos nesse estudo somente os laudos que possuíam o diagnóstico de linfoma (n=128). Dos laudos que informavam o sexo (n=111), 84,7% correspondiam a fêmeas e 15,3% a machos. Dos laudos em que constava a raça (n=108), a mais prevalente foi a holandesa (63%). Em relação à idade (n=107), houve uma variação entre um e 14 anos. A maioria dos bovinos era adulta (80,4%) e a maior concentração dos casos ocorreu entre 5-8 anos (57,9%). Clinicamente (n=89), linfadenomegalia foi o achado mais frequentemente observado (74,1%). Na necropsia (n=125), 71,2% dos bovinos apresentavam aumento de volume dos linfonodos; essa linfadenomegalia foi classificada como localizada em 89,6% dos casos e generalizada em 10,3% dos casos. Realizou-se também a avaliação fenotípica (histologia) e imunofenotípica (imuno-histoquímica [IHQ]) de 86 casos de linfoma bovino. Com base nestes resultados, os linfomas foram assim distribuídos utilizando-se a classificação proposta pela Working Formulation (WF) of Non-Hodgkins Lymphomas for Clinical Usage: difuso de grandes células não clivadas (46,5%), difuso de grandes células clivadas (33,7%), difuso de pequenas e grandes células (4,6%), difuso de pequenas células tipo plasmocitoide (7%), imunoblástico (3,5%), difuso de pequenas células tipo intermediário (2,3%), difuso de pequenas células não clivadas (1,2%) e difuso de pequenas células não clivadas tipo Burkitt (1,2%). Na IHQ, 27 dos 86 (31,4%) linfomas foram positivos para o anticorpo monoclonal CD79cy, utilizado para detecção de linfócitos B, e nenhum caso foi positivo para o anticorpo policlonal CD3, utilizado para detecção de linfócitos T. Com base nestes resultados, os linfomas B foram assim distribuídos utilizando-se a Revised European-American Classification of Lymphoid Neoplasms (REAL): linfoma difuso de grandes células B (81,5%), linfomas imunoblásticos de grandes células (11,1%) e linfomas linfoplasmocíticos (7,4%). Os resultados aqui apresentados permitem concluir que à semelhança do que vem sendo descrito em outras partes do mundo, os linfomas bovinos são basicamente difusos e predominantemente de grau intermediário, de grandes células, com núcleos clivados ou não clivados. Esses linfomas são decorrentes da proliferação neoplásica de linfócitos B e correspondem, em sua quase totalidade (92,6%), ao que atualmente é incluído na REAL como linfoma difuso de grandes células B.

The epidemiological, clinical and pathological aspects of 128 cases of bovine lymphoma are retrospectively described in this study (1965-2014).Out of the cases were the gender was informed (n=111), 84.7% of affected animals were females and 15.3% were males. Out of the cases were breed was informed (n=108), 63% of affected animals were Holstein cows. The age of affected cows (n=107) varied from 1 to 14 years, with most animals being adults (80.4%) with 5 to 8 years of age (57.9%). The most common clinical sign (n=89) was lymphadenomegaly (74.1%). Gross findings (n=125) included enlargement of lymph nodes in 71.2% of the cases; this finding was classified as localized in 89.6% of the cases and generalized in 10.3% of the cases. A retrospective study of 86 cases of bovine lymphoma classified as with diffuse pattern of distribution and verified by phenotypic (histology) and immunophenotypic (immunohistochemistry [IHC]) is presented. Based on these results, the lymphoma cases were classified by the Working Formulation (WF) of Non-Hodgkins Lymphomas for Clinical Usage as: diffuse large non-cleaved cell (46.5%), diffuse large cleaved cell (33.7%), diffuse mixed small and large cell (4.6%), diffuse small cell plasmacytoid (7%), immunoblastic (3.5%), diffuse small cell intermediate (2.3%), diffuse small non-cleaved cell (1.2%), and diffuse small non-cleaved cell Burkitts (1.2%). According to the IHC, 27 out of 86 (31.4%) lymphomas were positive to monoclonal antibody CD79cy, used to detect B cells, and none were positive for polyclonal antibody CD3, used to detect T cells. Based on this, the B-cell type lymphomas were distributed as follows: diffuse large B-cell lymphoma (81.5%), large cell immunoblastic lymphoma (11.1%), and lymphoplasmacytoid lymphoma (7.4%), according the Revised European-American Classification of Lymphoid Neoplasms (REAL). The results of this retrospective study, similar to what has been described in other parts of the world, allow us to conclude that bovine lymphomas are basically diffuse and predominantly made of intermediate-grade, large cells, with cleaved or non-cleaved nuclei. These lymphomas are due to neoplastic proliferation of B cells and correspond to almost all (92.6%) to what is currently classified as diffuse large B-cell lymphoma by REAL.

Identificação imuno-histoquimica de Helicobacter spp e avaliação do grau de inflamação na região da margo plicatus do estômago equino

Existem diversos fatores envolvidos na etinopatogenia das ulcerações gástricas em equinos destacando-se: alimentação, jejum e confinamento em baias, exercícios intensos, estresse, uso de antiinflamatórios não esteroidais, presença de parasitas e presença de Helicobater spp. O gênero Helicobacter é o agente mais comum de infecção gástrica crônica em humanos e, possivelmente, também nos animais de companhia, colonizando a mucosa gástrica. A colonização por Helicobacter sp. resulta na indução de resposta inflamatória, caracterizada pela migração de neutrófilos, células mononucleares e CD4 T- helper. A região da margo plicatus possuí alta prevalência de alterações histopatológicas tanto em potros quanto em animais adultos. O objetivo desse trabalho foi identificar por meio da técnica de imuno-histoquímica a presença de Helicobater spp e avaliar o grau de inflamação da margo plicatus do estômago equino. Foram utilizados 38 estômagos de equinos machos e fêmeas, com idade entre 3 a 20 anos, sem raça pré definida, previamente abatidos em abatedouro comercial. Para o exame histopatológico foram utilizados fragmentos de mucosa e submucosa da mucosa da margo plicatus. Os cortes histológicos foram avaliadas quanto à presença de Helicobater sp. por meio da imuno-histoquímica com anticorpo anti-H.pylori e classificadas como positivas quando a bactéria corou em vermelho e negativas. Dos 38 animais avaliados foram observados 10,52% (4/38) com inflamação leve, 31,57% com inflamação moderada e 57,89% de inflamação grave. Para identificação e quantificação de Helicobacter spp foram avaliadas 31 amostras com os seguintes resultados: 19,35% das amostras (6/31) apresentaram grau 0, 9,67% (3/31) grau 1 e grau 2 e 61,29% (19/31) grau 3. Equinos destinados ao abatedouro comercial são naturalmente infectados por Helicobacter spp. A região da margo plicatus demonstra grande susceptibilidade para infecção por Helicobacter spp no estômago de equinos. Há correlação entre o grau de infecção por Helicobacter spp e o grau de inflamação no estômago equino. Há correlação entre o grau de infecção por Helicobacter spp e os infiltrados linfocítico e eosinofílico no estômago dos equinos. Não há correlação entre o grau de infecção por Helicobacter spp e os infiltrados plasmocítico e neutrofílico no estômago de equinos.

There are several factors involved in etiopathogeny of gastric ulcers in horses highlighting: food, fasting and confinement in stalls, intense exercise, stress, use of nonsteroidal anti-inflammatory drugs, the presence of parasites and presence of Helicobacter spp. Helicobacter genus is the most common agent of chronic gastric infection in humans and possibly also in pets, colonizing the gastric mucosa. The colonization by Helicobacter sp. results in the induction of inflammatory response, characterized by the migration of neutrophils, mononuclear cells and CD4 + T-helper. The region of plicatus margo possess high prevalence of pathological changes in both foals and adults animals. The aim of this study was to identify by immunohistochemical technique the presence of Helicobacter spp and assess the degree of inflammation of the margo plicatus the equine stomach. Were used 38 horses stomachs of males and females, aged 3-20 years, with no predefined race, previously slaughtered in commercial abattoir. For histopathology were used mucosa and submucosa fragments of mucosa plicatus margo. The histological sections were evaluated for the presence of Helicobacter sp. by immunohistochemistry with anti- H.pylori antibody and classified as positive when the bacterium stained in red and negative. Of the 38 animals evaluated were observed 10.52% (4/38) with mild inflammation, 31.57% with moderate inflammation and 57.89% of severe inflammation. For identification and quantification of Helicobacter spp were evaluated 31 samples with the following results: 19.35% of the samples (6/31) had grade "0", 9.67% (3/31) grade "1" and grade "2 "and 61.29% (19/31) level" 3 ". Equines intended for commercial slaughterhouse are naturally infected by Helicobacter spp. The region of plicatus margo shows great susceptibility to infection by Helicobacter spp in equine stomach. There is a correlation between the degree of infection by Helicobacter spp, and the degree of inflammation in equine stomach. There is a correlation between the degree of infection by Helicobacter spp and lymphocytic and eosinophilic infiltrates in the stomach of horses. There is no correlation between the degree of infection by Helicobacter spp and plasmacytic and neutrophilic infiltrates in the equine stomach.

T-2 mycotoxin intoxication in piglets: a systematic pathological approach and apoptotic immunohistochemical studies

An experimental study was performed in order to analyze the histopathological findings of gastrointestinal mucosa and regional lymph-nodes in piglets intoxicated with T-2 toxin as well as the mechanism of T-2 induced apoptosis in the intestinal crypt epithelia. Sixteen piglets were divided into 4 groups receiving a single oral administration of T-2 toxin. Group A: 2.5 mg T-2/kg b.w.; group B: 2 mg T-2/kg b.w.; group C: 1.5 mg T-2/kg b.w.; group D: control group without toxin. Organ lesions were classified in grades 0 to 4 according with severity and were statistically analyzed. Apoptosis was examined in intestinal sections only in groups A and D. Group A piglets showed clinical signs and pathological changes related with those seen in circulatory shock. Microscopic lesions were observed only in pigs from groups A and B. In stomach, lesions consisted of congestion, hemorrhages and necrotic cells in the isthmus and neck regions. Submucosal edema and necrotic crypt epithelial cells were seen in duodenum, jejunum, ileum, cecum and colon. The greatest grade of lesions was observed in colon. In lymph node, grade of lymphocytes depletion and necrosis was significantly higher in cortex than paracortex. Apoptotic bodies were present in intestinal crypt cells, lymphoid cells from lamina propria and ileal Peyer's patches. Apoptosis seems to be the mechanism involved in the development of intestinal lesions.

Resposta imune celular e humoral em aves (Gallus gallus) vacinadas, antes e após o desafio com Salmonella enteritidis

Salmonella Enteritidis (SE) causa doença transmitida por alimentos (DTA) em humanos. Carne de frango e ovos frequentemente estão associados a esses casos. O controle da infecção em aves, baseia-se em medidas de biossegurança, incluindo-se a vacinação. Tem sido comum a utilização de vacinas vivas (VV) e inativadas (Bacterinas - BA), porém pouco se sabe sobre os mecanismos imunes desencadeados pelas vacinas contra SE. Neste estudo, utilizaram-se quatroprogramas vacinais (VV; VV+VV; BA; VV+BA) em galinhas leves de variedade branca para postura de ovos de mesa, vacinadas com 5 e/ou 25 dias de vida, e desafiadas aos 45 dias de vida com SE. No dia anterior à infecção (1 DAI) e 1, 6 e 9 dias pós-infecção (DPI), cinco aves/grupo foram sacrificadas para amostragem. A população de linfócitos CD4+ e CD8+ foi avaliada por imuno-histoquímica em tonsilas cecais e fígado; citocinas foram quantificadas por RT-qPCR em tempo real em tonsilas cecais e baço; níveis de IgG e IgM foram mensurados por ELISA no soro e IgA em lavado intestinal. Os níveis de imunoglobulina (IgG, IgM e IgA) estavam significativamente mais altos em aves vacinadas com BA, do 1 DAI ao 9 DPI, em comparação aos grupos vacinados somente com VV. Os níveis de IFN-?, na tonsila cecal, eram similares em todos os grupos após o desafio. Antes do desafio (1 DAI), IL-10 foi altamente expressa em baço de aves que receberam somente BA (25 dias de vida), sugerindo o desenvolvimento de resposta por linfócitos T CD4+ auxiliar 2 (Ta2), reforçado pelos altos níveis de IgG encontrados neste grupo (p<0.05). Os níveis de TGF-?4 e das citocinas pró-inflamatórias IL-6 e TNFSF15 eram bastante elevados no grupo que recebeu VV+VV. A vacinação por via oral com VV aumentou significativamente o fluxo de linfócitos T CD8+ para as tonsilas cecais após o desafio. Isso poderia...

Salmonella Enteritidis (SE) causes foodborne infection in humans. Poultry meat and eggs are frequently associated with these cases. The control of this bacterium is based on sanitary measures and mainly, vaccination of chicken flocks. Vaccine programs are used worldwide to control SE in poultry flocks. Live (LV) and killed vaccines (KV) are often combined, although few studies regarding immune mechanisms developed by SE vaccines are available. In this work, four vaccine programs were studied (LV; LV+LV; KV; LV+KV) in white layer-hens vaccinated at 5 and/or 25 days-old and challenged at 45 days-old with SE. At 1 day before (dbi) and 1, 6 and 9 days post-infection (DPI), five birds/group were sacrificed for sampling. The population of CD4+ and CD8+ T cells was evaluated by immunohistochemistry in caecal tonsils and liver, cytokines were quantified by real time RT-qPCR in caecal tonsils and spleen; IgG, IgM and IgA levels were measured by ELISA in serum and the latter in intestinal washes. The immunoglobulin levels (IgG, IgM and IgA) were significantly higher in birds vaccinated with KV, from 1 dbi to 9 DPI, than in groups that received only LV. In caecal tonsils, IFN-? levels were similar in all groups after challenge. Before challenge (1 dbi), IL-10 was highly expressed in spleen of birds that received only KV (25 days-old), suggesting the development of the T CD4+ helper 2 (Th2) type of immune response, reinforced by high IgG levels against SE seen in this group (p<0.05). TGF-?4 and the proinflammatory cytokines IL-6 and TNFSF15 were higher in caecal tonsils in the group that received LV+LV. Vaccination by oral route with LV clearly increased the influx of CD8+ T cells in caecal tonsils after challenge. This could be correlated with the better control of SE noticed in groups that received at least one dose of LV, including...

Expressão espaço-temporal do sistema VEGF e do EG-VEGF no timo de cães / Spatio-temporal expression of the vascular endothelial growth factor (VEGF system and EG-VEGF in the thymus of dogs

O objetivo deste trabalho foi avaliar a expressão de fatores de crescimento responsáveis pelo processo vasculogênico durante o desenvolvimento e involução do timo em cães. Para este propósito, timos de fetos de cães (30, 40 50 e 60 dias de desenvolvimento), cães jovens (6 meses de idade) e cães adultos (1 ano de idade) foram coletados. Os tecidos foram analisados estereológicamente e submetidos à imunohistoquímica, PCR tempo real e western blot para os componentes do sistema VEGF (VEGF-A, VEGF-C, Flt-1, KDR e Flt-4) e endocrine gland-derived (EG)-VEGF. Pela estereologia, observamos um aumento no número dos vasos durante o desenvolvimento do órgão e uma diminuição durante a involução (p<0.05). Todos os componentes do sistema VEGF e do EG-VEGF foram detectados em todos os estágios durante o desenvolvimento e involução. As proteínas correspondentes foram localizadas nas células endoteliais e células epiteliais da região cortical e medular, sendo que o EG-VEGF foi observado apenas nas células epiteliais da região medular. A expressão do RNAm do sistema VEGF e do EG-VEGF apresentou um perfil tempo-dependente durante o desenvolvimento e involução. O sistema VEGF apresentou uma expressão constante durante o desenvolvimento e um aumento progressivo durante a involução, com exceção do KDR que se mostrou crescente durante todo o período de desenvolvimento e involução e do VEGF-C que apresentou alta expressão durante a involução. A expressão do RNAm do EG-VEGF foi crescente durante as fases estudadas, já a expressão da sua proteína foi alta no início do desenvolvimento do timo e diminui na involução, onde permanece praticamente constante durante todo o período. Nossos resultados sugerem que o sistema VEGF e o EG-VEGF possuem atividades biológicas similares nas glândulas endócrinas e diferentes papéis durante o desenvolvimento e involução, o qual pode ser especificado em estudos futuros. Possíveis funções incluem um efeito modulatório na vasculogênese tímica e microambiente, influenciando na diferenciação e proliferação de timócitos, maturação de células T, interação célula-célula e secreção de hormônios tímicos

The aim this study was therefore to evaluate the expression of growth factors responsible for the vasculogenic process throughout development and initial involution of the canine thymus. For that purpose, thymuses from fetuses (30, 40, 50 and 60 days), juvenile (6 months) and adult (1 year) dogs were collected. The tissues were examined stereologically and subjected to immunohistochemistry, Real Time PCR and western blot for components of the VEGF-system (VEGF-A, VEGF-C, Flt-1, KDR and Flt-4) and endocrine gland-derived (EG)-VEGF. By means of stereology, the total number of blood vessels increased during development and decreased during involution. These changes were statistically significant (p<0.05). All components of the VEGF-system and EG-VEGF were detected in all stages during development and involution. The corresponding proteins were localized in endothelial and epithelial cells of cortical and medullar regions, and EG-VEGF was detected only in epithelial cells of medular region. The VEGF-system and EG-VEGF protein and mRNA expression showed a specific time-dependent profile during development and involution. The VEGF system showed a constant expression during development and a progressive increase during involution, except KDR that showed increased during all development and involution and VEGF-C that showed high expression during involution. The EG-VEGF RNAm expression was increased during development and involution, but protein expression was high in early thymus development and decreased in involution, and remains constant during all period. Our results suggest that VEGF-system and EG-VEGF have similar biological activities in endocrine glands and different roles during thymus development and involution which have to be specified in further studies. Possible functions include a modulatory effect on thymic vasculogenesis and microenvironment, influencing thymocyte proliferation and differentiation, T-cell maturation, cell-cell interaction and thymic hormonal secretion

Reatividade de tecidos neoplásicos caninos à proteína associada à resistência a múltiplas drogas-1 (MRP1), à glutationa-s-transferase pi (GSTpi) e à proteína p53

Tendo em vista a expressão da proteína associada à resistência a múltiplas drogas (MRP1), da enzima glutationa-S-transferase pi (GSTpi) e da proteína p53 com o desenvolvimento da resistência a múltiplas drogas (RMD) nas células neoplásicas, objetivou-se neste estudo avaliar a expressão desses três marcadores, pela imunoistoquímica, em 68 espécimes de neoplasias caninas, incluindo tumores venéreos transmissíveis (TVTC), mastocitomas, carcinomas mamários e de glândula hepatóides e linfomas. Os espécimes foram subdivididos em: TVT, Tumor venéreo transmissível (n=9); TVTR, TVTs resistentes à quimioterapia (n=5); MASTI, mastocitomas cutâneos grau I (n=8); MASTIII, mastocitomas cutâneos grau III (n=8); CARM, carcinomas mamários (n=14); CARH, carcinomas de glândulas hepatóides (n=8); LINFB, linfomas de células B (n=9); LINFT, linfomas de células T (n=7). Resultados mostraram que as expressões da MRP1, GSTpi e p53 foram observadas em 38 (55%), 43 (62,3%) e 50 (72,4%) espécimes, respectivamente. Em 27 (39,1%) espécimes houve coexpressão dos três marcadores. A expressão da MRP1, GSTpi e p53 isoladas ou associadas pôde ser observada em todos os grupos experimentais, exceto o grupo TVTC que não expressou a MRP1. A localização da marcação nas células tumorais foi citoplasmática para MRP1 e nuclear e/ou citoplasmática para a GSTpi e p53. Não foi observada diferença na expressão dos marcadores de resistência a quimioterapia em relação à resistência a quimioterapia (TVT e TVTR), gradação histológica (MASTI e III) e imunofenótipo (LINFB e T). Há relação direta entre o aumento da expressão da MRP1 e da GSTpi nos linfomas T

Multidrug resistance in tumors involves the expression of multidrug resistance protein-1 MRP1, enzyme glutathione-S-transferase pi (GSTpi), and p53 protein. Therefore, this study aimed at evaluating the expression of these three markers, by immunohistochemistry, in neoplasic cells. Sixty-eight canine tumor samples, including transmissible venereal tumor (TVTC), cutaneous mast cell tumor, mammary carcinoma, hepatoid gland carcinoma, and lymphoma were studied. Samples were assigned into one of the following subgroups: TVT, transmissible venereal tumor (n=9); TVTR, chemoresistant TVT (n=5), MASTI, grade-I cutaneous mast cell tumor (n=8); MASTIII, grade-III cutaneous mast cell tumor (n=8); CARM, mammary carcinoma (n=14); CARH, hepatoid gland carcinoma (n=8); LINFB, B-cell lymphoma (n=9); LINFT, T-cell lymphoma (n=7). We observed that 38 (55%), 43 (62.3%), and 50 (72.4%) samples expressed MRP1, GSTpi, and p53, respectively. Co-expression of the three markers was present in 27 (39.1%) samples. Expression of MRP1, GSTpi, and p53 alone or associated could be observed in all experimental groups, except for TVT group which did not express MRP1. Staining in the tumor cells was cytoplasmatic to MRP1, and both nuclear and cytoplasmatic to GSTpi and p53. No significant difference in the expression of the markers could be observed with relation to chemoresistance (TVT and TVTR), histological grade (MASTI and MASTIII) and immunophenotype (LINFB and LINFT). A direct relation was present between the raise of the expression of MRP1 and GSTpi in T cells lymphoma