Strategies for studying immune and non-immune human and canine mammary gland cancer tumour infiltrate.

The tumour microenvironment (TME) is usually defined as a cell environment associated with tumours or cancerous stem cells where conditions are established affecting tumour development and progression through malignant cell interaction with non-malignant cells. The TME is made up of endothelial, immune and non-immune cells, extracellular matrix (ECM) components and signalling molecules acting specifically on tumour and non-tumour cells. Breast cancer (BC) is the commonest malignant neoplasm worldwide and the main cause of mortality in women globally; advances regarding BC study and understanding it are relevant for acquiring novel, personalised therapeutic tools. Studying canine mammary gland tumours (CMGT) is one of the most relevant options for understanding BC using animal models as they share common epidemiological, clinical, pathological, biological, environmental, genetic and molecular characteristics with human BC. In-depth, detailed investigation regarding knowledge of human BC-related TME and in its canine model is considered extremely relevant for understanding changes in TME composition during tumour development. This review addresses important aspects concerned with different methods used for studying BC- and CMGT-related TME that are important for developing new and more effective therapeutic strategies for attacking a tumour during specific evolutionary stages.

Cutaneous lymphoma of the tarsus in a cat - case study / Linfoma cutáneo tarsal en gato - estudio de caso

ABSTRACT Lymphoma is the most common lymphoproliferative disorder in cats. Cutaneous lymphoma, however, is a rare form of extranodal lymphoma. Recently, several cutaneous lymphomas at the tarsal region have been reported in cats. As it differs clinically and histopathologically from the common cutaneous lymphoma, it was denominated cutaneous tarsal lymphoma. The present study describes the case of a 13-year-old male domestic longhair cat that presented with a subcutaneous mass, of 30-days evolution, at the tarsal region of the right pelvic limb. Histopathology analysis showed malignant neoplasia of round cells, morphologically suggestive of large cell lymphoma. Immunohistochemistry confirmed the diagnosis of B-immunoblastic lymphoma. Patient was treated with lomustine, and prednisolone with an overall survival time of 2.1 months. The aggressiveness of this feline lymphoma reinforces the need for further studies to understand better the disease progression and to establish improved therapeutic protocols that can increase survival time and improve quality of life of these patients.

RESUMEN El linfoma representa la enfermedad linfoproliferativa más frecuente en gatos. Sin embargo, el linfoma cutáneo es una rara forma de linfoma extranodal. Recientemente, el linfoma cutáneo se reportó localizado en el tarso. Tanto clínica como histopatológicamente, esta forma difiere de la forma cutánea típica, y se denominó linfoma tarsal felino. Este estudio describe el caso de un gato, doméstico de pelo largo, 13 años de edad con una masa subcutánea, de 30 días de evolución en la región tarsal del miembro pélvico derecho. La histopatologia reveló neoplasia de células redondas, sugestiva de linfoma de células grandes. La inmunohistoquímica confirmó el diagnóstico de linfoma imunoblástico de células B. El tratamiento realizado fue lomustina y prednisolona con tiempo de sobrevida de 2,1 meses. La agresividad de este linfoma, refuerza la necesidad de estudios para entender su curso y mejorar protocolos terapéuticos que incrementen tanto la sobrevida como la calidad de vida para estos pacientes.

Molecular detection of Anaplasma species in dogs in Colombia.

Anaplasma platys and A. phagocytophilum are tick-borne pathogens that parasitize platelets and neutrophils, respectively, of humans and animals. The former is the etiological agent of canine cyclic thrombocytopenia, while the latter is that of canine granulocytic anaplasmosis. This work involved the detection and identification of Anaplasma species in blood samples from dogs in Colombia, using molecular techniques. Between December 2008 and April 2009, blood samples were drawn from the cephalic vein of 91 dogs in the central-western region of Colombia (cities of Bogota, Villavicencio and Bucaramanga) and stored in tubes containing EDTA. These samples were used in 16S rRNA-Anaplasma spp. nPCR and the preparation of blood smears. One (1.1%) of the 91 sampled dogs showed inclusions suggestive of Anaplasmataceae agents in the cytoplasm of platelets. Based on PCR followed by sequencing and phylogenetic analysis, A. platys and Anaplasma sp. closed related to A. phagocytophilum were detected in two and one dog, respectively. Interestingly, all the samples were negative for specific msp-2-A. phagocytophilum real-time qPCR, suggesting the circulation of an Anaplasma species phylogenetically related to A. phagocytophilum in dogs in the aforementioned region. Hence, Anaplasma spp. circulates among dogs in Colombia, albeit with low frequency. To the best of authors' knowledge, this is the first molecular detection of Anaplasma spp. in dogs in Colombia.

Molecular detection of Anaplasma species in dogs in Colombia / Detecção molecular de espécies de Anaplasma em cães na Colômbia

Abstract Anaplasma platys and A. phagocytophilum are tick-borne pathogens that parasitize platelets and neutrophils, respectively, of humans and animals. The former is the etiological agent of canine cyclic thrombocytopenia, while the latter is that of canine granulocytic anaplasmosis. This work involved the detection and identification of Anaplasma species in blood samples from dogs in Colombia, using molecular techniques. Between December 2008 and April 2009, blood samples were drawn from the cephalic vein of 91 dogs in the central-western region of Colombia (cities of Bogota, Villavicencio and Bucaramanga) and stored in tubes containing EDTA. These samples were used in 16S rRNA-Anaplasma spp. nPCR and the preparation of blood smears. One (1.1%) of the 91 sampled dogs showed inclusions suggestive of Anaplasmataceae agents in the cytoplasm of platelets. Based on PCR followed by sequencing and phylogenetic analysis, A. platys and Anaplasma sp. closed related to A. phagocytophilum were detected in two and one dog, respectively. Interestingly, all the samples were negative for specific msp-2-A. phagocytophilum real-time qPCR, suggesting the circulation of an Anaplasma species phylogenetically related to A. phagocytophilum in dogs in the aforementioned region. Hence, Anaplasma spp. circulates among dogs in Colombia, albeit with low frequency. To the best of authors' knowledge, this is the first molecular detection of Anaplasma spp. in dogs in Colombia.

Resumo Anaplasma platys e A. phagocytophilum são patógenos transmitidos por carrapatos que parasitam plaquetas e neutrófilos, respectivamente, de humanos e animais. Enquanto o primeiro é agente etiológico da trombocitopenia cíclica canina, o último é o agente responsável pela anaplasmose granulocítica canina. O presente trabalho objetivou detectar e caracterizar, utiizando técnicas moleculares, a presença de espécies de Anaplasma em amostras de sague de cães na Colômbia. Entre Dezembro de 2008 e Abril de 2009, amostras de sangue colhidas em tubos contendo EDTA da veia cefálica de 91 cães da região centro-oeste da Colômbia (cidades de Bogotá, Villavicencio e Bucaramanga). As amostras de sangue foram utilizadas em reações de nPCR para 16S rRNA-Anaplasma spp. e confecção de esfregaços sanguíneos. Um (1,1%) dos 91 cães amostrados mostraram inclusões sugestivas de agentes Anaplasmataceae no citoplasma de plaquetas. Baseada na PCR seguida de sequenciamento e análise filogenética, A. platys and Anaplasma sp. relacionado a A. phagocytophilum foram detectados em dois e um cão, respectivamente. Interessantemente, todas as amostras mostraram-se negativas na PCR em tempo real quantitativa específica para msp-2-A. phagocytophilum, sugerindo a circulação de uma espécie de Anaplasma sp. filogeneticamente relacionado ao A. phagocytophilum em cães na região estudada. Embora em uma baixa freqüência, Anaplasma spp. circula entre cães na Colômbia. O presente trabalho representa a primeira detecção de Anaplasma spp. em cães na Colômbia.

Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis.

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P<0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P<0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase.

Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: effect on the pathogenesis of the disease.

The aim of this study was to evaluate nitric oxide levels, lipid peroxidation, protein oxidation and glutathione reductase activity in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups were used: negative control (n=5) and infected by E. canis (n=5). The concentration of nitrite/nitrate (NOx), lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and glutathione reductase (GR) activity in sera were evaluated. Samples were collected on days 0, 3, 6, 18 and 30 post-infection (PI). NOx and TBARS levels were significantly (P<0.05) higher in the infected group at 18 and 30 days PI, as well as AOPP levels at 30 days PI when compared to samples from control group. The GR activity was significant (P<0.05) increased in serum of dogs infected by E. canis on days 18 and 30 PI. Based on the increased levels of NOx, TBARS, AOPP and GR activity we concluded that dogs experimentally infected by E. canis develop a state of redox imbalance and that these changes might be involved in the pathophysiology of the disease.

Experimental Ehrlichia canis infection changes acute-phase proteins.

Early diagnosis of canine ehrlichiosis favors prompt institution of treatment and improves the prognosis for the animal, since this disease causes mortality among dogs. Studies have shown that determining the concentration of acute-phase proteins (APPs) may contribute towards early detection of disease and aid in predicting the prognosis. This study aimed to evaluate the APP profile in dogs experimentally infected with Ehrlichia canis, at the start of the infection and after treatment. It also investigated whether any correlation between APP levels and the clinical and laboratory alterations over the course of the disease would be possible. The results obtained showed abnormal levels of all the APPs on the third day after infection (D3), with the highest levels being reached on D18, with the exception of ceruloplasmin and acid glycoprotein, which presented their peaks on D6 and D12 respectively. We concluded that assessment of APP levels could contribute towards establishing an early diagnosis of canine ehrlichiosis, particularly regarding acid glycoprotein and ceruloplasmin, since these proteins were detected at increased levels even before the onset of clinical and laboratory findings of the disease.

Experimental Ehrlichia canis infection changes acute-phase proteins / A infecção experimental por Ehrlichia canis altera as proteínas de fase aguda

Early diagnosis of canine ehrlichiosis favors prompt institution of treatment and improves the prognosis for the animal, since this disease causes mortality among dogs. Studies have shown that determining the concentration of acute-phase proteins (APPs) may contribute towards early detection of disease and aid in predicting the prognosis. This study aimed to evaluate the APP profile in dogs experimentally infected with Ehrlichia canis, at the start of the infection and after treatment. It also investigated whether any correlation between APP levels and the clinical and laboratory alterations over the course of the disease would be possible. The results obtained showed abnormal levels of all the APPs on the third day after infection (D3), with the highest levels being reached on D18, with the exception of ceruloplasmin and acid glycoprotein, which presented their peaks on D6 and D12 respectively. We concluded that assessment of APP levels could contribute towards establishing an early diagnosis of canine ehrlichiosis, particularly regarding acid glycoprotein and ceruloplasmin, since these proteins were detected at increased levels even before the onset of clinical and laboratory findings of the disease.

O diagnóstico precoce da erliquiose canina favorece a pronta instituição do tratamento e melhora o prognóstico do animal, pois se trata de uma doença de alta mortalidade em cães. Estudos têm apontado que a determinação da concentração de proteínas de fase aguda (PFA) pode contribuir para detecção precoce de doenças e auxiliar na predição do prognóstico. O presente estudo objetivou avaliar o perfil de proteínas de fase aguda (PFA) em cães experimentalmente infectados com Ehrlichia canis, no início da infecção e após o tratamento. Além disso, se seria possível associá-las com as alterações clínico-laboratoriais durante o curso da doença. Os resultados obtidos evidenciaram que todas as PFA estudadas alteraram suas concentrações em D3 (dia 3), comparadas ao D0, atingindo concentrações máximas em D18, com exceção da ceruloplasmina e da glicoproteína ácida, cujos picos foram observados em D6 e D12, respectivamente. Concluímos que a avaliação das concentrações de PFA poderiam contribuir para o diagnóstico precoce da erliquiose canina, principalmente com relação à ceruloplasmina e glicoproteína ácida, pois seus aumentos foram anteriores ao aparecimento dos sinais clínicos e das alterações laboratoriais da doença.

Molecular characterization of Hepatozoon canis in dogs from Colombia.

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogotá, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.