Relationship between PD-L1 Expression and Tumor-Infiltrating Lymphocytes in Canine Mammary Tumor

Background: Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors.Materials, Methods & Results: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes (P = 0.034). Survival curves demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1+ and PD-L1– (P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade (P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-L1+/TILs-High vs. PD-L1–/TILs-Low (P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS.Discussion: In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT

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

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3+ showed lower intensity while CD4+ and CD8+ expression were concentrated surrounding of lymphocytic infiltrate tumor region. HSP60+ was observed in the inflammatory and tumor cells.Discussion: Our data are according to a greater risk to the development of breast tumor in old bitches, not castrated and before or after puberty, as well as the use of contraceptives based on progesterone and estrogen. In relation to size of tumor, these findings reinforce that there is a relationship of tumor size with a higher malignancy grade and with a worse prognosis. The predominant tumor location was in the inguinal breasts that is attributed to the high activity of the mammary glands to hormonal stimuli. CMT with low clinical staging are associated with greater overall survival of affected bitches. In relation to tumor microenvironment, it has been reported that heterogeneous populations of the immune system cells often infiltrate the mammary tumors, whose lymphocytes are the main cells. It is suggested that tumor lymphocytosis may be necessary for malignant behavior of the tumor microenvironment. On the other hand, macrophages and neutrophils play an important role that may favor or inhibit the tumor cells development in the tumor microenvironment. In our work, CD4, CD8 and FoxP3 labeling were distributed in peri and intratumoral regions, and consequently, these markers can be used as prognostic for CMT, as well as being a potential target for anticancer therapies. This is the first work that presents results about the participation of HSP60 in CMT, however this data needs further investigation. HSP60 participates as a potent activator of the immune system through its peptides and other HSP types were studied in mammary carcinomas in bitches and presenting results that indicate the association of these proteins with the carcinogenesis process

ACHADOS HEMATOLÓGICOS, BIOQUÍMICOS, CITOLÓGICOS E NECROSCÓPICOS EM GATOS DOMÉSTICOS COM LEISHMANIOSE

Visceral leishmaniasis is a zoonosis caused by the protozoan genus Leishmania spp. It is commonly reported in dogs. However, currently, the number of infected cats has grown currently, being important to study how this disease behaves in these hosts. Clinical evaluation and laboratory tests are essential for the definitive diagnosis of leishmaniasis in felines. Therefore, the present study aimed to evaluate the hematological and cytological alterations in domestic cats with Leishmaniasis. in Two cats from the city of Fortaleza/CE were evaluated with hematological, biochemical, cytological, and necroscopic exams. The main hemogram findings were normochromic nomocytic anemia, erythrocyte rouleaux, platelet aggregates, and, in one of the cases, visualization of the amastigote form. In the biochemical analysis, hypoalbunemia was observed in one case and, in the other case, an increase in serum levels of urea and creatinine was noticed. Hyperproteinemia was observed in both cases. Cytology was used ante-mortem for the diagnosis of one of the cases and post-mortem for both cases to identify amastigote forms in the liver and other lymphoid organs such as lymph nodes, spleen, and bone marrow. Therefore, for cats with nonspecific signs, it is necessary to carry out complementary tests such as blood count, serum biochemistry, cytology of the lesions, and necroscopic examination to obtain a definitive diagnosis.A Leishmaniose Visceral é uma zoonose causada pelo protozoário do gênero Leishmania spp. É mais comumente relatada em cães; mas, atualmente, o número de gatos infectados tem crescido, sendo importante o estudo de casos de como essa doença se comporta nesses hospedeiros. A avaliação clínica e os exames laboratoriais são imprescindíveis para o diagnóstico definitivo de Leishmaniose em felinos. Diante disso, o presente estudo teve como objetivo avaliar as alterações hematológicas e citológicas em gatos domésticos com Leishmaniose em dois gatos, provenientes do município de Fortaleza/CE. Foram avaliados os exames hematológicos, bioquímicos, citológicos e necroscópicos desses pacientes. Os principais achados de hemograma foram anemia normocítica normocrômica, rouleaux eritrocitário, agregados plaquetários e, em um dos casos, a visualização da forma amastigota. Na análise bioquímica, observou-se hipoalbunemia em um caso e, no outro, aumento dos níveis séricos de uréia e creatinina. Em ambos os casos, se observou hiperproteinemia. A citologia foi utilizada ante-mortem, para diagnóstico de um dos casos e post-mortem nos dois casos, a fim de se detectar formas amastigotas no fígado e outros órgãos linfoides, como linfonodos, baço e medula óssea. Portanto, para gatos com sinais inespecíficos, faz-se necessária a realização de exames complementares, como hemograma, bioquímica sérica, citologia das lesões e exame necroscópico, a fim de diagnóstico definitivo

ENVOLVIMENTO DA MATRIZ EXTRACELULAR NO TUMOR MAMÁRIO CANINO

Mammary gland (MG) is a dynamic tissue derived from the epidermis and your development depends on the interaction between mammary cells and stroma. Extracellular matrix (ECM) is the major extracellular content of tissues responsible for supporting connective tissue and basement membrane, and serves as a reservoir for many growth factors. ECM is comprised of insoluble protein fibers as collagens, laminins, fibronectins and soluble polymers as proteoglycans, and glycosaminoglycans. The ECM components are important both during morphogenesis of MG as to maintain this fabric giving support and storage of substrates needed for the growth. ECM disorder in GM may be the progression trigger of the breast tumor. Canine mammary tumor (CMT) is a complex of malignancies that have the participation of several factors for its development, including ECM components. Therefore an investigation of ECM in the diagnosis of CMT becomes important to establish a relationship between componentes of matrix and neoplastic cells, including information on the biological behavior and clinical staging of CMT. The knowledge of ECM molecules participation in the development of CMT may further therapeutic approaches targeting elements of ECM. Thus, this review has a focus on the ECM components participation in the processes that contribute to CMT establishment, which may favor therapeutic approaches targeting elements of ECM.A glândula mamária (GM) é um tecido dinâmico, derivado da epiderme e o seu desenvolvimento depende da interação entre as células mamárias e o estroma. A matriz extracelular (MEC) representa o principal conteúdo extracelular, responsável pela sustentação do tecido conjuntivo, da membrana basal e serve como reservatório para muitos fatores de crescimento. MEC é constituída por fibras proteicas insolúveis, como colágenos, lamininas, fibronectinas, e polímeros solúveis, como proteoglicanos e glicosaminoglicanos. Essas moléculas que compõem a MEC são importantes, tanto durante a morfogênese da GM, como para a sua manutenção conferindo-lhe a sustentação e o armazenamento de substratos necessários para seu crescimento. A desorganização da MEC na GM pode ser um indício necessário para o início e a progressão do tumor de mama. O Tumor mamário canino (TMC) é referido como um complexo de neoplasias que tem a participação de diversos fatores para seu desenvolvimento, incluindo os componentes da MEC. Desta forma, a investigação da MEC no diagnóstico dos TMC torna-se importante, para estabelecer a correlação entre os seus componentes e as células neoplásicas, além de fornecer informações sobre o comportamento biológico e o estadiamento clínico dos TMC. O entendimento da participação dessas moléculas da MEC para o desenvolvimento do TMC pode favorecer abordagens terapêuticas mais específicas, tendo como alvo elementos da MEC. Portanto, esta revisão tem como foco a participação dos componentes da MEC nos processos que contribuem para o estabelecimento do TMC, o que pode favorecer abordagens terapêuticas que visem elementos da MEC

Avaliação da resposta inflamatória em cães naturalmente infectados por Leishmania infantum

A leishmaniose visceral canina (LVC) é manifestada por danos ao organismo dos animais infectados com Leishmania infantum e apresenta-se como doença assintomática ou sintomática. Os diferentes quadros clínicos estão associados a resposta do hospedeiro através da resposta inflamatória e da resposta imunológica específica. Objetivou-se avaliar a resposta inflamatória de cães naturalmente infectados por L. infantum. Cães (n = 12), ambos os sexos, foram divididos em dois grupos: soropositivos (n = 6) e soronegativos (n = 6). Amostras de sangue foram coletadas dos animais para a determinação das contagens de Leucócitos Totais e Neutrófilos e os níveis séricos de proteína C reativa (PCR), fibrinogênio (FIB) e espécies reativas ao ácido tiobarbitúrico (TBARS). Os dados foram expressos em média e desvio padrão e para comparação dos grupos foi utilizado o teste t Student (p < 0,05). Os animais soropositivos apresentaram neutrofilia (p < 0,05), PCR acima dos valores de referência para a espécie (p < 0,05) e níveis elevados de TBARS (p < 0,05) em relação aos animais soronegativos. Pode-se concluir que as proteínas de fase aguda e os mediadores do estresse oxidativo podem ser utilizados como marcadores da resposta inflamatória na LVC.

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

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods &amp; Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (&lt; 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3+ showed lower intensity while CD4+ and CD8+ expression were concentrated surrounding of lymphocytic infiltrate tumor region. HSP60+ was observed in the inflammatory and tumor cells.Discussion: Our data are according to a greater risk to the development of breast tumor in old bitches, not castrated and before or after puberty, as well as the use of contraceptives based on progesterone and estrogen. In relation to size of tumor, these findings reinforce that there is a relationship of tumor size with a higher malignancy grade and with a worse prognosis. The predominant tumor location was in the inguinal breasts that is attributed to the high activity of the mammary glands to hormonal stimuli. CMT with low clinical staging are associated with greater overall survival of affected bitches. In relation to tumor microenvironment, it has been reported that heterogeneous populations of the immune system cells often infiltrate the mammary tumors, whose lymphocytes are the main cells. It is suggested that tumor lymphocytosis may be necessary for malignant behavior of the tumor microenvironment. On the other hand, macrophages and neutrophils play an important role that may favor or inhibit the tumor cells development in the tumor microenvironment. In our work, CD4, CD8 and FoxP3 labeling were distributed in peri and intratumoral regions, and consequently, these markers can be used as prognostic for CMT, as well as being a potential target for anticancer therapies. This is the first work that presents results about the participation of HSP60 in CMT, however this data needs further investigation. HSP60 participates as a potent activator of the immune system through its peptides and other HSP types were studied in mammary carcinomas in bitches and presenting results that indicate the association of these proteins with the carcinogenesis process

Profile of anti-Leishmania antibodies related to clinical picture in canine visceral leishmaniasis.

This research investigated the profile of anti-Leishmania antibodies in different clinical forms of canine visceral leishmaniasis (CVL). Naturally infected dogs were divided into two groups: subclinical dogs (SD, n = 10) and clinical dogs (CD, n = 68). Non-infected dogs (ND, n = 7) comprised the negative control group. The humoral response was evaluated by the profile of total IgG, IgG1, IgG2, IgM, IgA and IgE, determined by ELISA. Infected animals showed increased levels of total IgG, IgA and IgE in addition to IgG1 and IgG2 in groups SD and CD, when compared with group ND. Furthermore, it was observed that IgG2 and IgM were correlated with symptomatology, while total IgG, IgG1 and IgA were negatively correlated and IgE showed no correlation. It follows that serum levels of IgG2 anti-Leishmania are correlated with typical clinical signs of disease. Furthermore the determination of specific anti-Leishmania antibodies could be an important tool in monitoring CVL clinical picture

Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

INTRODUCTION: Canine visceral leishmaniasis (CVL) is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ) were used, selected by immunofluorescence assay (IFA) and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85) were grouped according to clinical signs: negative (CN = 7), subclinical (CS = 10), and clinical (CC = 68). Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%), keratitis (61.8%), and lymphadenopathy (55.9%), and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%), hematocrit (72%), and hemoglobin (62%), as well as leukocytosis (33%), neutropenia (28%), thrombocytopenia (50%), uremia (45%), hyperproteinemia (53%, p<0.05), hypergammaglobulinemia (62%, p<0.01), and hypoalbuminemia (58%). CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported