Arthroscopic evaluation of the synovial membrane and its relationship with histological changes and biomarkers in equine joint disease.

The synovial membrane (SM) presents itself with distinctive characteristics during arthroscopic procedures in cases of osteoarthritis (OA) as well as osteochondritis dissecans (OCD) in horses. Most of the arthroscopic findings of the SM are limited to a description of a nonspecific inflammation state. In the present study, the macroscopic and histological aspects of the SM in OA and OCD horses were compared to those of healthy horses. The expression of interleukin (IL) in SM was also investigated. Besides, the concentrations of ILs and keratan sulfate (KS) in the synovial fluid (SF), and the molecular weights of the SF hyaluronic acid (HA) were also determined and correlated to the macroscopic and histological aspects of SM. This study included 10 healthy horses (control group), 12 horses with OA, and 12 with OCD. Macroscopic scores of the SM were higher in the OA group in comparison to the control and OCD groups. However, histological scores between OA and OCD were not different, and both were higher than the control group. Only in the OA group, there was a correlation between macroscopic and histological aspects of the SM, especially between volume and quantity of villi with perivascular inflammatory cells and synovial proliferation. The OA group has shown decreased expression of IL-10 in the SM, lower IL-10 and KS, and higher IL-1ß and IL-6 in the SF in comparison to the control and OCD groups. There was a significant negative correlation between the macroscopic aspect of the SM and the molecular weights AH in the OA group. There was no correlation between the macroscopic aspect of the SM and all dosages in the OA and OCD group. In the OA joints, the evaluation of the shape of the SM during arthroscopy promotes a better indicator for joint inflammatory or tissue repair processes, while in the osteochondritic joints, investigation of the histological aspects are recommended to rule out an incipient OA development process. Both are helpful and should be considered to guide the postoperative treatment.

The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours.

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

E-cadherin in canine mast cell tumors: decreased expression and altered subcellular localization in Grade 3 tumors.

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells.

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARß by 2.0-fold (quantitative real-time PCR). Our data show that RARß may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARß is epigenetically altered.

Genetically modified animals for use in research and biotechnology.

Transgenic animals are used extensively in the study of in vivo gene function, as models for human diseases and in the production of biopharmaceuticals. The technology behind obtaining these animals involves molecular biology techniques, cell culture and embryo manipulation; the mouse is the species most widely used as an experimental model. In scientific research, diverse models are available as tools for the elucidation of gene function, such as transgenic animals, knockout and conditional knockout animals, knock-in animals, humanized animals, and knockdown animals. We examined the evolution of the science for the development of these animals, as well as the techniques currently used in obtaining these animal models. We review the phenotypic techniques used for elucidation of alterations caused by genetic modification. We also investigated the role of genetically modified animals in the biotechnology industry, where they promise a revolution in obtaining heterologous proteins through natural secretions, such as milk, increasing the scale of production and facilitating purification, thereby lowering the cost of production of hormones, growth factors and enzymes.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of São Paulo, Brazil.

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of São Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of São Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of São Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of São Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM10, can be responsible for this DNA damage.

Immunohistochemical characterization of canine prostatic intraepithelial neoplasia.

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR.

Expression of connexins in normal and neoplastic canine bone tissue.

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and 1 multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

Effect of Cysticercus cellulosae on neutrophil function and death.

Neutrophils, eosinophils and macrophages interact with invading parasites and naive hosts. The initial reaction of leukocytes is the generation of reactive oxygen species (ROS). The cytotoxic effects of extracts derived from intact Cysticercus cellulosae and from the scolex or membrane fractions on neutrophils were examined. DNA fragmentation of neutrophils was observed when cells were incubated with an extract from the intact metacestode; however, the addition of antioxidant enzymes to the incubation medium had a protective effect. The scolex and membrane extracts did not affect DNA fragmentation of neutrophils. Hydrogen peroxide production of neutrophils incubated with metacestode fractions from C. cellulosae increased by 190% (total extract), 120% (scolex) or 44% (membrane). An increase in antioxidant catalase activity (28%) concomitant with the increased production of ROS was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. ROS production by neutrophils in the presence of the intact cysticerci extract did not alter phagocytosis. In contrast, the scolex and membrane fractions increased the phagocytic capacity of neutrophils by 44 and 28%, respectively. The results showed that the extract from intact C. cellulosae was toxic for neutrophils via ROS production, leading to DNA fragmentation and inhibition of phagocytic capacity, but neutrophils are able to protect themselves against oxidative stress by via catalase activity.