Association between Degree of Anaplasia and Degree of Inflammation with the Expression of COX-2 in Feline Injection Site Sarcomas.

Feline injection site sarcomas (FISSs) are mesenchymal neoplasms that develop at the sites of delivery of vaccines or other injectable products. Vaccine adjuvants can trigger an intense and persistent inflammatory response that may lead to neoplastic transformation. The proinflammatory role of cyclo-oxygenase (COX)-2 is well known and its overexpression has prognostic value in multiple neoplastic processes. One hundred and seventeen FISSs were evaluated for the degree of inflammation and anaplasia. Immunohistochemistry was used to determine the expression of COX-2 in these sarcomas. There was a significant association between the degree of inflammation and the expression of COX-2 by neoplastic cells. COX-2 expression was lower in tumours with higher degrees of anaplasia. These findings may be useful in predicting the sensitivity of FISSs to treatment with COX-2 inhibitors. The potential therapeutic use of such agents could then be restricted to tumours with lower degrees of anaplasia.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Immunohistochemical expression of growth factors in the follicular wall of normal and cystic ovaries of sows.

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF-I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF-I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF-II was very similar to pattern noted in IGF-I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF-II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.

Protective effects of intranasal immunization with recombinant glycoprotein d in pregnant BALB/c mice challenged with different strains of equine herpesvirus 1.

Equine herpesvirus (EHV)-1 induces respiratory infection, neurological disorders and abortion in horses. Most of the currently available attenuated or inactivated vaccines against this infection are administered intramuscularly and only provide partial protection against the respiratory disease. The present study examines the effect of intranasal immunization with purified EHV-1 recombinant glycoprotein D (gD) in BALB/c mice followed by challenge with three different EHV-1 strains during early to mid-pregnancy. The induced viral infection was evaluated by virus isolation, DNA detection by polymerase chain reaction, histopathology and immunohistochemical localization of antigen in the lung, placenta and uterus. Non-immunized mice showed clinical signs of infection, positive virus isolation from lungs and uteri, and abortion induced by one of the virus strains. Endometrial lesions developed in some of these animals that have been described previously only in horses. Immunized mice and their offspring had no viral infection or typical lesions. Intranasally administered gD therefore induced partial or complete protection against three different EHV-1 strains in BALB/c mice.

Lectin-binding pattern in ovarian structures of rats with experimental polycystic ovaries.

Numerous experimental models in different species have been developed for the study of polycystic ovarian syndrome. In this study, we used a model of induction of polycystic ovaries (PO) in rats by exposure to constant light to study the distribution and variations of glycosylated residues present in the different ovarian structures. Seven biotinylated lectins were used (Con-A, WGA, DBA, SBA, PNA, RCA and UEA-I) on tissue sections, and detection was performed using the streptavidin/peroxidase method. In tissue sections was observed an increase in affinity for Con-A in the granulosa and theca interna of growing follicles and cysts in animals with PO in relation to the control group. Follicular cysts showed higher affinity for WGA and RCA-I which growing follicles in the same group, and there was a decrease in affinity for PNA in the cysts in relation to the growth of follicles in both groups. Atretic follicles in both groups showed greater labelling with lectins PNA, SBA and RCA-I in relation to healthy follicles. It could also be noted that the zona pellucida of cystic follicles lost the affinity for the lectin Con-A. There was no staining on follicles in any category with the lectins DBA and UEA-I, although it was staining in the corpus luteum (control group) and in the mesothelium and interstitial glands of both groups with DBA. These observations probably reflect changes in the glycosaminoglycans present in the different ovarian compartments or in the glycosylation of cellular components essential for proper follicular dynamics.

Lectin-binding sites on the normal and pathologic uterus of sows.

The lectin-binding pattern was compared in the normal and pathological uterus of sows during the ovarian cycle. The following biotinylated lectins were used: Con A, DBA, SBA, PNA, RCA-I, UEA-I and WGA. Glycoconjugate labelling showed differences between phases of ovarian cycle and presence of morphologic lesions. Cystic endometrial hyperplasia increased the RCA-I reaction in the apical region of the glandular epithelium. There was higher intensity of labelling of WGA in the glandular epithelium in uteri with endometritis. In addition, increased Con A binding in the glandular epithelium and mild reduction of UEA-I reactivity in the glycocalyx of the glandular epithelium were detected in the cases of endometritis. The results of this study show that morphologic alterations modify the sugar pattern in the porcine uterus. These modifications in glycoconjugates may be one of the reasons for decreased fertility in sows.

Heat shock protein 70 and sex steroid receptors in the follicular structures of induced ovarian cysts.

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERalpha than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERalpha in the treated group. An increase in total ERalpha protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERbeta in animals with COD, and the total protein expression of ERbeta was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.

Efeito da Mannheimia granulomatis sobre cultivo de fibroblastos / Effect of Mannheimia granulomatis on fibroblastos cultures

Primary cultures of Mannheimia granulomatis were established in chicken embryos to assess their capacity to stimulate fibroblast proliferation. The capacity of the bacterium-activated macrophages to stimulate cytokine and enzyme proliferation was assessed in a mouse peritoneum macrophage culture. To evaluate the bacteria infection on fibroblasts and their growth within 48h in relation to the active macrophages, cultures were washed and trypsinized and the cells counted. Results showed no significant differences when the bacteria-infected fibroblasts were mixed with bacterial extract (P=0.9682). The treatment using just products of macrophages resulted similar to the negative control. Significant differences on cell proliferation were established (P=0,0039) when the products of M. granulomatis-activated macrophages were used, meaning that bacterial components were unable to promote fibroblast increase. Further research is needed to elucidate the effect of M. granulomatis on the macrophages.

Histological and lectin binding changes in the genital tract of mice infected with Tritrichomonas foetus.

An experimental murine model of bovine genital tritrichomonosis is described. Female mice were inoculated per vaginam with Tritrichomonas foetus and a sample of the study population was killed every 3 days up to 60 days post-infection. Microscopical changes in the reproductive organs were assessed and immunohistochemistry was used to detect T. foetus within these tissues. Lectin histochemistry was used to determine changes in the expression of carbohydrates within the reproductive mucosa. A range of microscopical changes were detected in the uterine endometrium by 10 days post-inoculation and these were associated with the presence of the protozoan. The endometrial changes included endometritis and ulceration, mucosal atrophy and glandular metaplasia, and were similar to those reported in naturally infected cows. Changes in lectin binding were recognized first in the vagina where there was increased binding of Ulex europaeus agglutinin-1 (UEA-1) which was maximal on day 16 post-inoculation. Within the uterus, there was increased binding of soy bean agglutinin (SBA) which was maximal on day 19 post-inoculation, and of peanut agglutinin (PNA) which was maximal on day 16 post-inoculation. These changes in carbohydrate expression parallel the infection kinetics, since they appeared first in the vagina and later in the uterus. The changes may reflect either a host reaction against the infection or the production of enzymes by T. foetus, which act to enhance adhesion and colonization of the genital organs by the organism. The kinetics and pathogenesis of this murine infection are similar to those of the natural bovine disease, suggesting that this model system may be valuable for further studies of this disease.

Estrogen receptors alpha and beta and progesterone receptors in normal bovine ovarian follicles and cystic ovarian disease.

The purpose of this study was to determine by immunohistochemistry the expression of estrogen and progesterone receptors in ovarian follicular structures from cows with cystic ovarian disease (COD) and to compare these with normal ovarian structures. Secondary, tertiary, atretic, and cystic follicles were evaluated. The follicular cysts of animals with COD presented a significantly higher expression of estrogen receptor alpha in all follicular layers than secondary, tertiary, and atretic follicles in both groups (P < .05). The intensity of estrogen receptor beta in the granulosa cell layer was stronger in tertiary than in secondary and atretic follicles in normal animals (P < .05) and in growing and cystic follicles in animals with COD (P < .05). Theca cells were scarcely stained in the 2 groups. Growing follicles and cysts from COD animals were less stained than tertiary follicles from normal animals (P < .05). Differences did not exist between the 2 groups with regard to the progesterone receptor. Ovaries of animals with COD exhibited altered estrogen receptors expression compared with that in normal animals.

Characterization of cytoskeletal proteins in follicular structures of cows with cystic ovarian disease.

The distribution of intermediate filaments (vimentin, cytokeratins, desmin) and microfilaments (alpha-smooth muscle actin and muscle specific actin) was studied immunohistochemically in bovine ovaries, with and without cystic ovarian disease. The immunohistochemically stained area (IHCSA), was quantified by image analysis, to evaluate the expression of these cytoskeletal proteins in the follicular wall of healthy antral, atretic, and cystic follicles. The granulosa cell layer of cystic follicles and atretic follicles had a significantly larger IHCSA for vimentin than did healthy antral follicles. Cytokeratins reacted lightly in the granulosa cells of antral follicles of normal ovaries, whereas granulosa cells of atretic and cystic follicles showed significantly higher IHCSA values. Immunohistochemical localization of desmin, muscle specific actin, and alpha-smooth muscle actin was restricted to the theca externa. This study supports earlier suggestions that strongly positive reactions with vimentin and cytokeratin antibodies observed in the granulosa cells of cystic follicles are due to the reorganization that occurs in the follicle during the process of cystic development, and are associated with changes in the expression of cytoskeletal proteins that are essential to proper cellular functioning.

Intraovarian localization of growth factors in induced cystic ovaries in rats.

We hypothesized that the special hormonal environment present in animals with cystic ovarian disease (COD) interferes with cellular production of growth factors (GFs). The objective of the present study was to characterize the expression of insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in induced COD using immunohistochemistry. We used an experimental model based on the exposure to constant light of adult rats during 15 weeks. We quantified the expression of GFs in cystic and normal ovaries by the Immunohistochemical Stained Area (IHCSA). In animals with COD, a significant reduction in the IHCSA of IGF-I in the follicular fluid, theca and granulosa layers of cysts occurred; and an increase in the interstitial tissue with regard to the control group. We found moderate immunoreactivity of FGF-2 in granulosa and theca layers of secondary and tertiary follicles and lower expression in the granulosa and theca interna layers of cystic follicles. Immunoexpression of VEGF was found in granulosa and theca cells of secondary and tertiary follicles. This study shows changes in the ovarian expression of IGF-I, FGF-2 and VEGF in induced COD. We can propose that an alteration in the control of the follicular dynamic, through the GFs, added to other features, could be involved in the ovarian cyst pathogenesis.

Macrophages and giant cell proliferation associated with bone protein synthesis and calcification in the trachea and bronchi of rabbits intoxicated with Solanum glaucophyllum.

A histopathologic, immunohistochemical, and ultrastructural study of the trachea and the bronchi of 6 rabbits experimentally intoxicated with the calcinogenic plant Solanum glaucophyllum was performed. Histologically, infiltration of the mucosa and the submucosa of the trachea and the bronchi by macrophages, multinucleated giant cells, a few lymphocytes and mast cells, and calcium deposits in the basal lamina of the epithelium and in elastic fibers were observed. Expression of osteocalcin, osteonectin, and osteopontin was detected in the mucosa, lamina propria, and epithelium. Electron microscopic study of the corresponding areas showed numerous macrophages in the process of fusion to form multinucleated giant cells, activated mesenchymal cells, and calcium precipitation in the basal lamina of epithelium and in elastic fibers. It is suggested that the high levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the plant induces macrophage proliferation, multinucleated giant-cell formation, mesenchymal cell activation, bone-protein synthesis, and calcification. In addition, the synthesis of 1,25(OH)2D3 by local macrophages may have contributed to the calcification.

Glicogenose hereditária em bovinos Brahman no Brasil / Inherited glycogenosis in Brahman cattle in Brazil

Relata-se uma enfermidade hereditária em bovinos caracterizada por acúmulo lisossomal de glicogênio em diversos órgãos. A doença foi diagnosticada em um rebanho da raça Brahman, no município de Porto Lucena, Rio Grande do Sul, Brasil. Os animais afetados, a partir de 1 mês de idade, apresentavam dificuldade em acompanhar a mãe e crescimento retardado, desenvolviam fraqueza e tremores musculares, letargia e perda de condição corporal progressivos. Todos os bezerros eram descendentes do mesmo touro. Foi realizada necropsia em três bezerros doentes; palidez muscular do tronco e membros foi a única alteração macroscópica encontrada. Vacuolização citoplasmática de diversos órgãos foi a principal alteração histológica observada. Os vacúolos citoplasmáticos eram mais evidentes na musculatura esquelética, miocárdio, especialmente nas fibras de Purkinje e em neurônios do Sistema Nervoso Central (SNC). Nos tecidos mais afetados foi observada grande quantidade de grânulos ácido periódico de Schiff (PAS), positivos e negativos quando o tecido era tratado previamente com diastase. Uma mutação no gene da glicosidase alfa ácida, causadora da glicogenose generalizada em bovinos Brahman, a 1057?TA, foi detectada pela técnica de reação em cadeia de polimerase (PCR) em tecidos dos animais necropsiados. Também foi detectada a presença dessa mutação em amostras de sangue de animais parentes dos bezerros doentes. Os achados clínicos, patológicos e moleculares são semelhante ás descrições de glicogenose tipo II em bovinos da raça Brahman descritos na Austrália. Não foram encontrados relatos anteriores em revistas indexadas sobre glicogenose hereditária em bovinos Brahman no Brasil.

Lectin histochemistry of foam cells in tissues of cattle grazing Brachiaria spp.

Brachiaria decumbens and B. brizantha (signal grass), which occupy millions of acres in Brazil, are an important source of fodder for ruminants. Sporadic outbreaks of photosensitization in ruminants grazing on signal grass have been reported. Intoxicated animals showed the presence of foamy cells in the liver, spleen, intestinal submucosa and lymph nodes. These foamy cells are macrophages. They are very difficult to distinguish with haematoxylin and eosin stain, especially in the case of isolated cells. The purpose of the present study was to detect specific carbohydrate residues of storage material in the foamy cells in tissues of cattle exposed to Brachiaria spp. The characterization of glycoconjugates provides clues to the pathogenesis of these cells. Besides, the lectin peanut agglutinin was found to be an excellent marker to differentiate and quantify the foam cells, and could be used as a specific marker.

Cyclic related and pathological changes in the lectin-binding sites on the swine oviduct.

This study was carried out to compare the lectin-binding pattern in the normal and pathological oviduct of sows during the ovarian cycle. Lectin-binding patterns showed differences between segments, phases of ovarian cycle and presence of morphologic lesions. In the infundibulum, it was observed that the cysts, in the follicular phase, reduced Ricinus communis-I (RCA-I) and Ulex europaeus-I (UEA-I) binding. Furthermore, in the pathological oviducts of the luteal-phase group, there is a reduction of Concanavalia ensiformis (Con-A) reactivity in this segment of the tube having wall cysts, adenomyosis and diverticulus. The Arachis hypogaea (PNA) binding in the infundibulum, during the luteal phase, decreased in the tube having adenomyosis. In animals with wall cysts, the Con-A, Triticum vulgaris (WGA) and RCA-I reactivity was minor in the glycocalyx of the isthmus epithelium during follicular phase. Con-A and Dolichos biflorus (DBA) binding pattern was minor in the luteal-phase isthmus of the tube having wall cysts, adenomyosis and diverticulus. In the ampulla, the wall cysts impaired the Con-A reaction only in the basal region of the epithelium, in the follicular phase. Binding with Con-A was decreased in the ampulla of animals in the luteal phase in the tube lesions with cysts and diverticulus. In addition, the diverticulus observed in the ampulla, during the luteal phase, reduced the PNA tubaric binding. The results of this study showed that the morphologic alterations modify the sugar pattern in the oviduct of sows. These modifications in glycoconjugates may be one of the reasons for the failure of fertility in sows.

Papillary adenocarcinoma of the extrahepatic bile duct in a Holstein cow.

A 15-year-old female Holstein cow was presented with a history of anorexia and weight loss. Abnormal physical examination findings included poor body condition, dehydration, icterus, and photodermatitis on nonpigmented areas of the skin of the dorsum. At necropsy, the common bile duct was greatly enlarged, with thickened walls, and tightly adhered to the hepatic capsular surface and serosa of adjacent loops of the small intestine. Two sessile, yellowish, friable, well-circumscribed, cauliflower-like, bulging masses (14 and 8 cm in diameter) were attached to the inner surface of the common bile duct blocking its lumen. Microscopically, the tumor was composed of well-differentiated, columnar epithelial cells with oval or round, vesicular, basal nuclei arranged in papillary projections. Periodic acid-Schiff-positive material accumulated in the apical cytoplasm of the neoplastic cells. Immunostaining of the tumor cells was positive for cytokeratin (CK) of wide-spectrum screening and for CK 7. The diagnosis of papillary adenocarcinoma of the extrahepatic bile duct was based on the morphologic features of the neoplasm and evidence of local invasion. To the best of our knowledge, extrahepatic bile duct carcinomas have not been described in cattle.

Lectin binding patterns in hyperplastic and metaplastic bullock prostate tissues after diethylstilbestrol administration.

Hyperplasia and squamous metaplasia of the prostatic epithelium are conditions induced by oestrogens. Diethylstilbestrol (DES) has been banned from cattle used for beef production because of the health risks. The potential use of molecular markers for the detection of illegal oestrogen administration was evaluated by taking samples of prostatic tissue from control bullocks, bullocks which had been treated with oestrogens, and bullocks sacrificed 21 and 90 days after a single dose of DES. The expression of the glycoconjugates was examined by lectinhistochemistry and the lectin binding pattern was characterised in epithelium and connective tissue. In the animals sacrificed after 21 days there was an increase in the binding of one lectin (JAC) and there was an increase in the binding of one of the other lectins (DBA) in the animals sacrificed after 90 days. An increase in SWGA lectin staining was observed in the bullocks that had probably been treated with oestrogen and in the animals sacrificed 90 days after the inoculation with DES. There were also differences between the binding of SWGA in the control bullocks and the other groups.

Cutaneous lymphangiosarcoma in a young dog: clinical, anatomopathological and lectinhistochemical description.

Lymphangiosarcoma is a rare tumour in domestic animals arising from lymphatic endothelial cells. Occasionally, microscopic differentiation with haemangiosarcoma may be difficult. The aim of the present study was to describe a lymphangiosarcoma in a 1-year-old female Doberman Pinscher dog and to characterize its lectinhistochemical binding pattern as compared with that of haemangiosarcoma. The dog was presented because of a cutaneous painful swelling located in the left axilla. Histological diagnosis confirmed lymphangiosarcoma. The dog was killed. Necropsy revealed mediastinal lymph nodes' involvement. Twenty lectins were tested in tissue sections of this case as well as in four haemangiosarcomas from other dogs. Staining intensity was issued upon optical density determinations. Percentage of lectinhistochemical staining area was also conducted. RCA-I showed the most intense and wide distributed labelling pattern for lymphangiosarcoma. PHA-E was the counterpart for haemangiosarcoma. Should similar results be obtained in further studies, such differences could aid in the differential diagnosis between lymphangiosarcoma and haemangiosarcoma when histological pictures were not conclusive.

Effects of high molybdenum intake on 1.2 - dimethylhydrazine-induced intestinal tumors in rats

Wistar male rats, 3 months of age were given ad-libitum a nutritionally adequate diet and demineralized drinking water. The Molybdenum (Mo) and Tungsten (W) were provided in the drinking water at 200 ppm concentration. Intestinal tumors were induced by 1,2-dimethylhydrazine (DMH) given subcutaneously as 16 weekly doses at 20 mg/kg body weight. Mo in the form of (NH4)6 Mo7O24 4H2O or W in the form of (Na2WO4) were provided in the drinking water two months before the first DMH treatment and were continued during 4 months more until the last DMH treatment. Three months after the last carcinogen injection, all animals were sacrificed and examined for intestinal tumors. The number, size and location of the tumors were recorded and the pathology was examined. The addition of Mo to the drinking water induced an increase of hepatic Mo content. At the end of the second month, the hepatic content of Mo was 5.61 ppm, compared with control and W groups (2.18 and 0.96 ppm, respectively). A significantly lower incidence of tumors was observed in the Mo group (47), compared with the control group given DMH alone (105) and W group (113). On the other hand, the Mo group showed a significant decrease in the numbers of multiple tumors per rat.