Thymic changes due to leishmaniasis in dogs: An immunohistochemical study.

BACKGROUND: The thymus is necessary for the differentiation of T cells, a process that is regulated by the type of antigens found in thymocytes, the environment of surrounding cells and the thymus architecture. There is evidence that infectious diseases may result in morphological changes in this organ, such as premature atrophy and decreased thymocyte proliferation, that can affect the immune response. OBJECTIVES: We characterised the morphology and tissue distribution of haematopoietic and stromal cells in the thymuses of dogs naturally infected with Leishmania infantum, with the aim to determine the changes that may contribute to the pathophysiology of the disease. METHODS: Thymus samples were collected from 15 animals (aged 6 months to 5 years) ELISA-positive for leishmaniasis and from 10 dogs from non-endemic regions for leishmaniasis whose death was not related to infectious causes. The samples were submitted to histological processing and staining with Haematoxylin-Eosin to assess thymic morphometry and histopathological changes. Masson's trichrome staining was used to quantify the connective tissue present (collagen). The immunohistochemical method was used to determine the cellular constitution of the thymus, using antibodies that aimed at marking T lymphocytes (anti-CD3), B lymphocytes (anti-CD79a), macrophages (anti- MAC387), mesenchymal cells (anti-vimentin), epithelial cells (anti-cytokeratin), cells in mitosis (anti-Ki67) and cells in apoptosis (anti-caspase-3). RESULTS: The histopathological evaluation of infected dogs showed more signs consistent with thymus atrophy, including decreased parenchyma, infiltration of adipose and connective tissue near the capsule and between the lobules, lymphoid rarefaction mainly in the cortical region and loss of the cortical-medullary demarcation. In addition, we observed a decrease in the amounts of CD3 + T lymphocytes, macrophages (MAC387) and Ki67-positive cells and an increase in the number of cells positive for cytokeratin and CD79a (B lymphocytes). Finally, the parasite was detected in 46% of infected thymuses and may contribute for the observed changes. CONCLUSIONS: Apparently, leishmaniasis, like other infectious diseases, causes atrophy of the thymus and depletion of thymocytes with a relative increase in thymus epithelial cells. These morphological changes in the normal organisation of the thymus by mechanisms not yet well known may result in the abnormal release of T cells, with consequent damage to the host's immune response.

Influence of serum progesterone levels on the inflammatory response of female dogs with visceral leishmaniosis.

The aim of this study was to evaluate the histopathological changes to the mammary gland that occur in female dogs with visceral leishmaniosis and to correlate the findings with the parasite load, inflammatory cell profile in mammary tissue and serum progesterone levels. For this, 20 adult female dogs that were naturally infected with Leishmania infantum, not spayed, not pregnant and free from mammary tumors were used. They were divided into two groups: G1 (n = 9) with high serum progesterone levels and G2 (n = 11) with low serum progesterone levels. The parasite load and the immunophenotype of leukocytes infiltrated into the mammary tissue (CD3, CD4, CD8 and MCA874) were evaluated using the immunohistochemical technique. In the mammary gland, chronic inflammatory infiltrate was mainly found in G1, sometimes associated with granulomatous inflammation, higher parasite load and higher density of cells immunolabeled for CD3, CD4, CD8 and MCA874. A significant positive correlation (p < 0.05) was observed between the parasite load and the immunolabeled leukocytes. The influence of the serum progesterone level in the mammary gland of infected female dogs can contribute to the maintenance of an immunosuppressive cell profile and favor the persistence of the parasite in this site.

Relationship between the inflammatory tumor microenvironment and different histologic types of canine mammary tumors.

Mammary neoplasms are the tumors with higher incidence in female dogs. Among the factors that contribute for the development of this and other neoplasms, the inflammatory tumor microenvironment plays a crucial role. Several studies reported important roles for lymphocytes, macrophages, plasma cells, neutrophils, eosinophils and mast cells in this context. In the present study, our aim was to evaluate the number of profile cells of inflammatory cells and area of tumor fibrosis and the relation of these features with canine mammary tumors of different histologic and clinical presentation (benign mixed tumor, carcinoma in mixed tumor, solid carcinoma and tubular carcinoma) Counting and staining of inflammatory cells and tumor fibrosis were performed through histochemistry, while counting and staining of CD4+, TCD8+ and FOXP3+ lymphocytes were performed through immunohistochemistry. Statistical analysis of the association between densities of inflammatory cells, tumor fibrosis and histologic types revealed significant difference for plasma cells (p = .035), neutrophils (p = .0113), macrophages (p = .0047), and tumor fibrosis (p = .05). The found data suggest associations between high number of neutrophils and aggressive mammary tumors, between high densities of plasma cells, macrophages and CD8+ cells and between low number of profile cells of CD4+ cells and less aggressive tumors. Larger areas of tumor fibrosis showed relation to more aggressive canine mammary tumors.

Polarized M2 macrophages in dogs with visceral leishmaniasis.

The objective of the present study was to analyze the skin (nasal surface and ear regions), lymph nodes (popliteal and pre-scapular), spleen and liver of dogs with visceral leishmaniasis (VL), in order to investigate the relationship between the parasite load measured as DNA copy number of Alpha gene of DNA polymerase of Leishmania infantum by quantitative PCR and the number of M2 macrophages by immunohistochemistry. A set of 29 naturally infected dogs from an endemic area for VL were sampled and another set of six dogs negative for VL and from a non-endemic area were analyzed as the control group (C). The spleen presented the highest number of Leishmania DNA copies, with significant differences between the groups G1 and G2 (with and without skin lesions, respectively). The M2 phenotype immunostaining predominated among the macrophages in granulomas and inflammatory infiltrates of samples from the skin, lymph nodes and spleens examined. The presence of M2 macrophages in dogs from infected group differed significantly from the control group, in all organs analyzed, excepted liver. The highest proportion of M2 macrophages coincided with the highest parasitism loads found in more susceptible organs of VL dogs, even in the skin, considered a more resistant organ, while the liver showed low parasitism load and low immunostaining for M2 macrophages with no significant differences between infected and negative groups. It was concluded that the predominance of M2 phenotype in VL dogs favored the multiplication of Leishmania infantum in organs of dogs that are more susceptible to Leishmania infection, as skin, lymph nodes and spleen.

Alterations in PTEN, MDM2, TP53 and AR protein and gene expression are associated with canine prostate carcinogenesis.

The PTEN, AR, MDM2 and p53 protein network plays a central role in the development of many human cancers, thus eliciting the development of targeted cancer therapeutics. Dogs spontaneously develop tumours, and they are considered a good model for comparative oncology initiatives. Due to the limited information on these proteins in canine tumours, this study aimed to investigate gene and protein alterations in PTEN, AR, MDM2 and p53 in canine prostate cancer (PC). Protein expression was evaluated by immunohistochemistry (15 normal, 22 proliferative inflammatory atrophy (PIA) and 19 PC samples) and Western blotting (2 normal prostate tissue, 2 BPH, 2 PIA samples and 2 PC samples) and gene expression by RT-qPCR (10 normal, 10 PIA and 15 PC samples) of formalin-fixed tissue. We identified nuclear and cytoplasmic expression of PTEN and p53 in all samples, with only nuclear staining found for MDM2 and AR. Our results revealed high expression of MDM2 in PC and PIA samples compared to normal samples, whereas PTEN, P53 and AR expression was down-regulated in PC compared to normal tissue. All tumour samples (n=19) showed loss of nuclear PTEN expression, and all cancer mimickers showed positive nuclear staining. Therefore, nuclear PTEN staining could be a good diagnostic marker for differentiating between malignant lesions and mimickers. Canine prostate carcinogenesis involves increased expression of MDM2 in association with decreased expression of PTEN, p53 and AR, such as occurs in hormone refractory PC in men. Thus, dogs may be an important model for studying advanced stage PC.

Immunodetection of cells with a CD44+/CD24- phenotype in canine mammary neoplasms.

BACKGROUND: Cancer stem cells (CSCs) are able to self-renew and to form metastases. Using flow cytometry, CSCs were detected in canine mammary tumors as cells CD44(+) and CD24(-). The aim of this study was to detect these CSCs by immunohistochemistry and correlate their frequency with canine mammary neoplasm grade and histopathological type.130 mammary neoplasm samples were selected from tissue blocks at the Department of Pathology at UNESP and classified according to (BJVP 4:153-180, 2011). These samples were composed by adenomas, lymph node metastases, solid carcinomas grades II and III, tubular, papillary and carcinomas in mixed tumor grades I, II and III. Immunohistochemistry was performed with antibodies against CD44 and CD24. Linear regression was performed using Pearson's correlation test. RESULTS: The value at CD44 was positive and CD24 becomes zero was 46.75%. Cells with a CD44(+)/CD24(-) phenotype were detected in 40 out of 130 samples with an advantage of high grade tumors (II and III) and metastases among tubular, papillary and carcinomas in mixed tumors. In these samples, percentages of cells stained by CD44 and CD24 antibodies were 62.2% and 0%, respectively. Published reports usually correlate grade III tumors with the expression of CD44 but not with CD24 expression. Studies using flow cytometry have found CSC frequencies similar to those found in our study. CONCLUSIONS: Immunohistochemistry was found to be a reliable technique for the detection of CSCs in canine mammary neoplasms, and the frequency of these cells positively correlates with grades II and III tumors (poor prognosis).

Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis.

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin.

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi.

The aims of this study were to evaluate the immunomodulatory role of TGF-beta1, IL-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta1, IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta1 was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta1 levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta1 and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.

IL-6 and TNF-alpha production during active canine visceral leishmaniasis.

We investigated the production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) during canine visceral leishmaniasis (VL) to gain a better understanding of the role of such multi-functional cytokines in parasite resistance. IL-6 and TNF-alpha levels were measured by capture ELISA in sera from 8 healthy dogs from a non-endemic area (control group) and in sera from 16 dogs from Araçatuba, SP, Brazil, an area endemic for leishmaniosis. The dogs from the endemic area were selected by positive ELISA serology against total Leishmania chagasi antigen, positive spleen imprints for Leishmania, and the presence of at least three clinical signs associated with active visceral leishmaniasis (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotory difficulty, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). Enhanced systemic IL-6 production was found in sera from dogs with the active disease compared to healthy dogs (t-test, P<0.05). In contrast, TNF-alpha did not differ between the two groups studied. There was no correlation between IL-6 production and anti-leishmanial antibody titers in the sera. Our findings suggest that IL-6 is a good marker of active disease during leishmaniasis, and that other cytokines may be involved in the hypergammaglobulinemia characteristic of canine visceral leishmaniasis.