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.

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 tumo...

RESUMO:Objetivou-se investigar o efeito do extrato acetato de etila de Cocos nucifera (EAECN) sobre parâmetros fisiológicos e sobre a inflamação tópica induzida por xileno. EAECN foi obtido a partir da água da fibra da casca do coco verde e o teste fitoquímico indicou a presença de taninos condensados, flavononas, flavonóis, flavononóis, xantonas e esteróides. EAECN foi administrado aos camundongos Swiss por via oral em dose única diária de 10, 30, 100 e 250 mg Kg -1 por cinco dias consecutivos para os protocolos de toxicidade e inflamação tópica. No ensaio de toxicidade foram observadas as freqüências cardíacas e respiratórias, a presença de diarréia, analgesia e apatia e realizada a contagem total dos leucócitos do sangue periférico, avaliação macroscópica dos órgãos e peso relativo do rim, fígado, timo e baço. O efeito do EAECN sobre a inflamação tópica foi realizado utilizando-se grupos testes com as diferentes concentrações de EAECN e grupos controles positivos que receberam, pela mesma via nas mesmas condições, NaCl 0,9% ou DMSO a 5% ou o antiinflamatório padrão, Dexametasona (6 mg Kg -1 ). Todos os animais receberam o agente flogístico (25 L) nas partes interna e externa da orelha duas horas após o último tratamento, enquanto os animais do grupo controle negativo não receberam qualquer tratamento. Após 50 minutos da aplicação do xileno, os animais foram sacrificados, e uma porção de cada orelha foi retirada e pesada. A diferença de peso entre as orelhas representa o efeito induzido pelos tratamentos. EAECN não desenvolveu toxicidade, não alterou a contagem total de leucócitos, não alterou o peso e nem o peso relativo dos órgãos dos animais tratados em relação aos controles. EAECN não inibiu a inflamação provocada pelo xileno, apresentando efeito pró-inflamatório dependente da dose. Conclui-se que EAECN nos protocolos utilizados não é tóxico e não possui atividade antiinflamatória tópica. Palavras-chave:Cocus nucifera, xileno, Inflamação tópica, toxicidade, parâmetros fisiológicos ABSTRACT: Toxicological evaluation and effect of ethyl acetate extract of the fiber of Cocos nucifera L. (Palmae) on inflammatory response in vivo. This study aimed to investigate the effect of ethyl acetate extract of Cocos nucifera (EAECN) on physiological parameters and xylene-induced topic inflammation. EAECN was obtained from the water of the green coconut husk fiber and the phytochemical test indicated the presence of condensed tannins, flavanones, flavonols, xanthones and steroids. EAECN was orally administered to Swiss mice at a single daily dose of 10, 30, 100 and 250 mg Kg -1 during five consecutive days for toxicity and topic inflammation protocols. Toxicity experiments included the observation of heart and respiratory frequencies, diarrhea, analgesia and apathy, besides total leukocyte count in peripheral blood, macroscopic evaluation of the organs, and relative weight of kidneys, liver, thymus and spleen. The effect of EAECN on topic inflammation was assessed using test groups with different EAECN concentrations and...

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.

Background: Canine leishmaniasis (CanL) is caused by an obligatory intracellular parasite of Leishmania genus that  affects organs and tissues. Several studies evaluate the role of reactive oxygen species (ROS) in the pathogenesis of many diseases. The overproduction of ROS on infectious diseases can induce an imbalance between oxidants and antioxidants at cellular or systemic level. Thus, the aim of this study was to evaluate the activity of antioxidant enzymes in CanL.Materials, Methods & Results: Females (n = 17) and males (n = 10), at different ages and with different weight, were selected for this study. Dogs were divided into two groups according classical clinical signs and sorological test to CanL. Animals were considered infected based on indirect immunofluorescent assay and ELISA titration ≥ 1:40.  Group B (n = 15) composed by positive dogs to CanL from Zoonosis Control Center of Fortaleza (Ceará, Brazil) and group A (n = 12) was composed by dogs from private kennel that were serologically negative to L. infantum and had absence of clinical signs to CanL. Blood sample were collected for evaluation of hematological and biochemical parameters and glutathione peroxidase (GPx) and catalase (CAT) enzymatic activity. Data were analyzed by Student’s t-test and Pearson correlation coefficient (P < 0.05). Total proteins (TP, mg/dL) and alkaline phosphatase (ALP, U/L) were increased (P < 0.05) on group B (8.2 ± 1.2; 165.4 ± 46.4) when compared to group A (6.5 ± 1.1; 109.1 ± 38.3), respectively. Hemoglobin (Hb; g/dL) and hematocrit (Hct; %) were decreased (P < 0.05) on Group B (14.7 ± 1.8; 48.2 ± 5.7) when compared to group A (16.5 ± 1.3; 52.1 ± 2.4), respectively. Group B presented CAT (U/g Hb) and GPx (mU/mg Hb) lower (189.4 ± 90.4; 3,609.6 ± 1,569.1) than group A (326.6 ± 104.5; 5,055.6 ±1,569.1), respectively (P < 0.001). Positive correlation was observed between RBC and CAT; however, it was not significant.Discussion: Organisms require a good defense system in order to revert the overproduction of free radicals and consequently the injuries caused by them. This is possible through the production of antioxidant agents, which act on oxidative prevention and on tissue and cellular regeneration, by taking the reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes in the frontline. Erythrocyte changes promoted by CanL suggest possible correlation between anemia and the appearance of clinical signs, which in many cases is not seen. Erythrocytes contain SOD, CAT and GPx enzymes, thus, changes in these cells will reflect on the activity of these enzymes. In our results only CAT showed positive correlation with erythrocyte count, however it was not significant. GPx activity was lower (P < 0.001) in infected dogs than control group. This result agrees with another study, which showed a decrease in GPx levels in CanL, although it was not significant. However, it was found a positive correlation (P < 0.001) between erythrocytes and GPx activity and between hemoglobin and GPx activity in animals with leishmaniasis. These results suggest that the reduction in detoxification activity can be related to the decrease in erythrocyte count and that the GPx activity depends on the control mechanism of the antioxidant system in CanL. Furthermore, this result could be associated with decrease of blood cell count in animals with CanL, once GPx is an erythrocyte enzyme, which plays an important role in hemoglobin protection against oxidative damage. This study was carried out in naturally infected dogs with L. infantum. In conclusion, CAT and GPx activities are relate to oxidative stress induced by L. infantum infection and can be used as biomarkers on CanL.