Pilot study to evaluate isolation by size of circulating tumour cells in canine oral melanoma.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

Targeting canine mammary neoplastic epithelial cells with a reengineered anthrax toxin: first study.

Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.

The combination of IDO and AHR blockers reduces the migration and clonogenicity of breast cancer cells.

The indoleamine-2,3-dioxygenase (IDO) enzyme causes immunosuppressive consequences in the tumor microenvironment (TME). In addition, the role of aryl hydrocarbon receptor (AHR) in the TME is under discussion. The current study evaluated the role of the IDO and AHR blockers on cell migration, clonogenic, and IDO expression of murine breast cancer cells. The cell migration and clonogenic abilities of breast cancer cells are evaluated by wound­healing assay (cell migration assay) and Colony formation assay (clonogenic assay). Also, flow cytometry analysis was used to detect the IDO-positive breast cancer cells. The results showed that treating cells with a combination of IDO and AHR blockers dramatically reduced breast cancer cells' migration and clonogenic capacities. Treating cells with only AHR blockade suppressed the clonogenic rate. Since both IDO and AHR are involved in their complex molecular networks, blocking both IDO and AHR might cause alterations in their molecular networks resulting in diminishing the migration and clonogenic abilities of breast cancer cells. However, further investigations are required to confirm our findings within in vivo models as a novel therapy for breast cancer.

Effects of Saccharomyces cerevisiae cell wall addition on feed digestibility, fecal fermentation and microbiota and immunological parameters in adult cats.

BACKGROUND: This study aimed to evaluate the effects of increasing dosages of a commercial product composed by Saccharomyces cerevisiae yeast (YAM), with active metabolites, which are beta glucans, nucleotides, organic acids, polyphenols, amino acids, vitamins and minerals (Original XPCtm, Diamond V, IOWA, USA) added to a commercially available dry cat food. Apparent digestibility of dietary nutrients, fecal microbiota, fecal fermentation products and immunological parameters were evaluated. Twenty-seven healthy cats of mixed sexes, with a mean body weight of 4.19 ± 0.83 kg and a mean age of 9.44 ± 5.35 years were distributed by age in an unbalanced randomized block design, consisting of three experimental treatments: CD (control diet), YAM 0.3 (control diet with 0.3% yeast with active metabolites) and YAM 0.6 (control diet with 0.6% yeast with active metabolites). RESULTS: The inclusion of the additive elevated the apparent digestibility of crude fiber (p = 0.013) and ash (p < 0.001) without interfering feed consumption, fecal production and fecal characteristics. Regarding fermentation products present in the feces, prebiotic inclusion increased lactic acid concentration (p = 0.004) while reducing isovaleric acid (p = 0.014), only in the treatment YAM 0.3. No differences were noticed on biogenic amines (BA), fecal pH, ammonia concentration, total and individuals short-chain fatty acids (SCFA) and total and individuals branched-chain fatty acids (BCFA) (except isovaleric acid in YAM 0.3). As regards to fecal microbiota, prebiotic inclusion has resulted in the reduction of Clostridium perfringens (p = 0.023). No differences were found in the immunological parameters evaluated. CONCLUSION: It can be concluded that the additive, at the levels of inclusion assessed shows prebiotic potential and it has effects on fecal fermentation products and microbiota without interfering on crude protein and dry matter digestibility. More studies evaluating grater inclusion levels of the prebiotic are necessary to determine optimal concentration.

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates.

Corticosterone transdermal application in toads (Rhinella icterica): Effects on cellular and humoral immunity and steroid plasma levels.

Previous studies in a variety of vertebrates show stressed animals to exhibit acute increases in glucocorticoid levels, with consequences for immune modulation. Chronically elevated glucocorticoid levels are mostly associated with immunosuppression. However, there is still a paucity of knowledge regarding the amphibian physiological reaction to short- and long-term stress conditions, including glucocorticoid effects on the immune response. In order to better clarify the relationship between glucocorticoids and immune response, newly captured Brazilian toads (Rhinella icterica) (1 week in captivity) were subjected to a daily transdermal application (TA), of corticosterone or vehicle, for 30 consecutive days. Measures were made on the first day (acute stressor) and last day (chronic stressor), at timepoints 1, 6, and 12 hr post TA. A number of variables were analyzed: corticosterone plasma levels (CORT); neutrophil/lymphocyte ratio (N:L); testosterone plasma levels (T); innate immune response, as indicated by bacterial killing ability (BKA); and whole blood phagocytosis. The corticosterone TA only simulated hormonal changes associated with the acute stress response, even after 30 consecutive days of the treatment, with the increased CORT in response after corticosterone TA being evident only 1 hr postapplication and not thereafter. The general responses to corticosterone TA included increased CORT and N:L at first day of the treatment, and increased CORT and phagocytosis on the last day of the treatment. A decrease in T and BKA associated with the time in captivity was also evident, suggesting that captivity may be a chronic stressor for these toads.

Comfrey (Symphytum Officinale. l.) and Experimental Hepatic Carcinogenesis: A Short-term Carcinogenesis Model Study.

Comfrey or Symphytum officinale (L.) (Boraginaceae) is a very popular plant used for therapeutic purposes. Since the 1980s, its effects have been studied in long-term carcinogenesis studies, in which Comfrey extract is administered at high doses during several months and the neoplastic hepatic lesions are evaluated. However, the literature on this topic is very poor considering the studies performed under short-term carcinogenesis protocols, such as the 'resistant hepatocyte model' (RHM). In these studies, it is possible to observe easily the phenomena related to the early phases of tumor development, since pre-neoplastic lesions (PNLs) rise in about 1-2 months of chemical induction. Herein, the effects of chronic oral treatment of rats with 10% Comfrey ethanolic extract were evaluated in a RHM. Wistar rats were sequentially treated with N-nitrosodiethylamine (ip) and 2-acetilaminofluorene (po), and submitted to hepatectomy to induce carcinogenesis promotion. Macroscopic/microscopic quantitative analysis of PNL was performed. Non-parametric statistical tests (Mann-Whitney and χ(2)) were used, and the level of significance was set at P ≤ 0.05. Comfrey treatment reduced the number of pre-neoplastic macroscopic lesions up to 1 mm (P ≤ 0.05), the percentage of oval cells (P = 0.0001) and mitotic figures (P = 0.007), as well as the number of Proliferating Cell Nuclear Antigen (PCNA) positive cells (P = 0.0001) and acidophilic pre-neoplastic nodules (P = 0.05). On the other hand, the percentage of cells presenting megalocytosis (P = 0.0001) and vacuolar degeneration (P = 0.0001) was increased. Scores of fibrosis, glycogen stores and the number of nucleolus organizing regions were not altered. The study indicated that oral treatment of rats with 10% Comfrey alcoholic extract reduced cell proliferation in this model.

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4.

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-alpha. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor.

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study.

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.

Effects of physical and psychological stressors on behavior, macrophage activity, and Ehrlich tumor growth.

The present study analyzed the effects of physical and psychological stressors on behavior, immune function, and serum corticosterone in mice. Adult mice were submitted once daily, for 6 days to one of the following conditions: escapable (ES) or inescapable (IS) footshocks (0.2 mA) signaled by a tone cue or to a psychological stressor (PS) generated through the use of a communication box; in this box, mice received no footshock but were exposed to responses delivered by IS mice. Results showed that IS and PS: (1). decreased locomotor activity observed in an open-field; (2). decreased number of entries into the open arms and decreased time spent in the exploration of the open arms of the plus-maze; (3). decreased macrophage spreading and phagocytosis; (4). increased macrophage H(2)O(2) release; and (5). increased growth of the ascitic form of Ehrlich tumor. Behavioral and/or immunological changes were not observed after ES; this absence of effects, however, might not be attributed solely to footshock controllability since mice of groups ES and IS differed with respect to the psychological setting used and the amount of shock they received. An increase of serum corticosterone concentrations was also observed in the stressed mice of all groups; this increment was higher in animals of group IS. These data provide evidence that inescapable footshock and psychological stressors alter, at the same time and in mice, stress levels, macrophage activity, and Ehrlich tumor growth. They also show that ES and PS induced similarly elevated serum corticosterone concentrations, but significantly differ in the immunological and behavioral outcomes they produced in mice. These findings suggest that another factor besides HPA axis activation might be responsible for behavioral and immunological consequences of IS and PS in mice. It is proposed that the final neural link between behavioral and immunological changes observed after physical and psychological stressors might involve catecholaminergic systems within the central nervous system and/or sympathetic autonomic nerve fibers and also opioid peptides.