Hoxa-10 and Cyclin D3 Overexpression in the Decidual Reaction in a Superovulation Protocol in Young Adult C57BL/6J Mice.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

Influence of hepatocyte-rich liver cell mixture and liver fibroblasts on prolonging graft islet survival in rats without immunosuppressive drugs.

BACKGROUND: We noted that a liver cell suspension, made up of a mixture of several kinds of hepatic cells, affected allogenic islet survival when it was transplanted into the liver, mixed with the islets or separately. AIM: To study if this effect was related to a liver cell mixture rich in hepatocytes (hp) or to liver fibroblasts (fb). METHODS: We studied 14 groups of rats: (A) a sham group with saline; (B) a group receiving transplantation with hepatic cells alone; (C) a control group, with islets alone via the portal vein, without hepatic cells (hp or fb). For the other groups, we used a different ratio of cells/islets (100 : 1, 150 : 1 and 200 : 1) and different co-transplantation techniques with both types of cells. For the D, E, J groups, a mixture of hepatocytes (hepatocyte-rich liver cell mixture) or fibroblasts with islets was injected into the portal vein. For the other groups, we used a sequential procedure with a 15 minute interval between a first injection of hp or fb into the portal vein or into the vena cava, and a second injection of islets always into the portal vein; thus, it was a sequential portal/portal procedure with hepatocyte-rich liver cell mixture (hp) (F, G) or fibroblasts (K, L) and a sequential cava/portal with hp (H, I) or fibroblasts (M, N). RESULTS: Most of the co-transplantation groups showed functional islets (blood glucose < 250 mg/dl) on the first or second day of transplantation; after several days they once again had high glucose levels, though not as high as pre-transplantation. There was statistical significance (p < 0.001) between the presence or not of hepatic cells to obtain prolongation of graft survival (blood glucose < 250 mg/dl). Statistical significance (p < 0.001) was found for several sequential groups with hp (F, I) and fb (K, L). It was also remarkable that 3 rats (37.5 %) from the I group (sequential cava/portal with hp/islets 200 : 1) were euglycemic (blood glucose < 150 mg/dl) for more than 3 months. ANOVA showed a large interaction between the type of transplant performed and the cellular ratio used, with a significance of p < 0.001. Histological studies in rats with prolonged euglycemia, showed insulin-producing cell aggregates in the liver, while there was a remarkable decrease in insulin-producing cells in the remaining islets of pancreatic tissue. CONCLUSION: The results showed a marginal prolongation of islet graft survival when they are co-transplanted with a hepatocyte-rich liver cell mixture or with liver fibroblasts. The mechanism does not seem to be a cellular interaction between different hepatic cells and islets, but some kind of cellular interaction or released factor from either two cell types on the immune system, blocking or modulating it, at least temporarily.

Epithelioid malignant peripheral nerve sheath tumour in a dog.

An unusual morphological variant of malignant peripheral nerve sheath tumour, in which the cells exhibited a predominantly epithelial morphology, was diagnosed in a dog. The animal had a history of progressive ataxia. Post-mortem examination revealed an infiltrative growth in the sublumbar region, encroaching on the L1 and L2 vertebral bodies; additional masses were observed in the kidneys, liver and lungs. Histologically, the subcutaneous mass was composed of a dense population of spindle-shaped cells arranged in fascicles. The internal masses were composed of epithelioid cells arranged in a nodular pattern. Immunohistochemically, the spindle-shaped cells of the subcutaneous mass were positive for S-100, and the epithelioid cells of the internal growths exhibited weak S-100 immunoreactivity.

Immunohistochemical expression of p53, fibroblast growth factor-b, and transforming growth factor-alpha in feline vaccine-associated sarcomas.

Fifty feline sarcomas associated with vaccine-site injection were evaluated to determine the immunohistochemical expression of p53 protein, basic fibroblast growth factor (FGF-b), and transforming growth factor-alpha (TGF-alpha). Forty-one tumors (82%) were fibrosarcomas (FS), eight (16%) were malignant fibrous histiocytomas (MFH), and one (2%) was a chondrosarcoma (CS). Overexpression of p53 protein was observed in the nuclei of tumor cells in 28 (56%) sarcomas; FGF-b expression was found in the cytoplasm of tumor cells in 40 (80%) feline sarcomas, but the staining was more intense in the spindle-shaped cells of FS than in polygonal or round cells of MFH. The single CS faintly expressed FGF-b. The majority of feline vaccine-associated sarcomas (43 of 50, 86%) expressed moderate or intense staining for TGF-alpha in the cytoplasm of tumor cells. Heterogeneous immunolabeling for p53, FGF-b, and TGF-alpha was present in neoplastic, multinucleated giant cells. Intense expression of FGF-b was statistically associated with younger cats (P < 0.01) and with tumors with nodular growth patterns (P = 0.02). In addition, sarcomas negative for p53 protein expressed FGF-b more frequently than did p53-positive tumors (P = 0.04). The frequency of FGF-b immunostaining was significantly higher in sarcomas with intense expression of TGF-alpha (P = 0.05). Immunohistochemical detection of p53 protein, FGF-b, and TGF-alpha suggests that these growth-regulating proteins may play different roles in the development of sarcomas associated with vaccine sites.

A comparison between disk diffusion and microdilution for susceptibility testing of Mycobacterium fortuitum complex.

We evaluated a disk diffusion method using Mueller-Hinton agar for susceptibility testing of Mycobacterium fortuitum complex organisms. Ninety-five strains were tested both by broth microdilution and disk diffusion. Global results showed good correlation for all antimicrobials except for clarithromycin and erythromycin. However, when the results were analyzed according to species, correlation was poor except for a few antimicrobials. The analysis of the resistant/susceptible results was good for all the antimicrobials tested except azithromycin and erythromycin. In conclusion, the disk diffusion technique could be useful as a screening technique for some antibiotics, but the results must be confirmed by using an accepted reference technique.

Disseminated mycoses in a dog by Paecilomyces sp.

We describe a case of canine mycoses initially diagnosed by clinical signs and enzyme-linked immunosorbent assay anti-fungal test, and later confirmed by the isolation of Paecilomyces sp. during the post-mortem examination. The fungus was isolated from lesions in the kidneys, mitral valve, abdominal aorta and vertebral discs. In this kind of process, it is important to identify the responsible agent early in order to make a study of anti-fungal susceptibility and establish effective treatment.

Intraspinal synovial cyst in a dog.

An eight-year-old, male Siberian husky cross was referred with a history of an acute onset of pelvic-limb ataxia and paraparesis. Radiography and subsequent myelography of the spine revealed an extradural compression of the spinal cord at the level of the 13th thoracic (T13) to first lumbar (L1) vertebrae. Hemilaminectomy resulted in the successful removal of an extradural cystic lesion. The morphological diagnosis based on histopathology was a synovial cyst with chondromatosis. There were no postoperative complications, and the dog's condition improved markedly. At two years postoperatively, the animal remains normal on both physical and neurological examination. To the authors' knowledge, this article is the first report of an intraspinal synovial cyst in a dog.

Ultrasonographic appearance of ovarian tumors in 10 dogs.

Signalment, clinical history, physical examination, clinicopathologic, radiographic and ultrasonographic findings of 10 female dogs with histologically confirmed ovarian neoplasms were reviewed. Ultrasonographic images and reports were reviewed for (1) location, size, outer margins, and echogenicity of the mass(es), (2) presence of free abdominal fluid, (3) evidence of uterine abnormalities, and (4) signs of metastatic disease. The masses were classified according to their ultrasonographic pattern in solid, solid with cystic component, and cystic. The masses were ultrasonographically reported as being of ovarian origin in eight dogs, and this origin was included in the list of differentials in the remaining two dogs. When present, abdominal effusions and uterine abnormalities were diagnosed by means of ultrasound.

Antibody response in dogs experimentally infected with Leishmania infantum: infection course antigen markers.

Five-month-old beagle dogs were experimentally infected with amastigotes of Leishmania infantum and kept for 14 months. Infection course was monitored by clinical examination, serum protein variations, and levels of specific antibodies against Leishmania estimated by indirect immunofluorescence test and Western blotting (WB). Infected animals developed notable changes in serum protein levels reaching maximum protein concentrations 2-3 months postinfection (p.i.) related to the gamma-globulin fraction. Specific antibody titers were in good agreement with the serum protein rise, reaching immunofluorescence values of over 1:800 3 months p.i. Serial Western blotting analysis with L. infantum promastigotes protein showed a strong response against immunodominant antigens of 50-57, 42, and 29 kDa during most of the studied period with immunofluorescence titers of over 1:100 and in addition the response was remarkably homogeneous among the infected dogs. Immunoreactivity patterns displayed time-related variations; the response against 29 and 50-57 kDa was seen very early, followed by the reaction around 42, 76, and 86 kDa. In addition the recognition of peptides around 34-35.4 and 26 kDa was restricted to the acute phase of the experimental infection. Preliminary results obtained in naturally infected dogs seem to support the predictive value of the WB.

Erosive colitis in experimental canine Leishmaniasis.

Six 4 month-old beagles were inoculated with Leishmania infantum, three of them intraperitoneally (Group A) and the other three intravenously (Group B). The animals from Group A were killed at 109, 433 and 592 days after inoculation and animals from Group B 109, 171 and 334 days after inoculation. At 350 days (Group A) and 275 days (Group B) after inoculation, dogs started to develop a chronic large bowel diarrhoea. At necropsy a diffuse colonic and rectal wall thickening of gradually increasing severity was observed. Histological examination of the colon showed a diffuse inflammatory infiltration of the mucosa and submucosa by macrophages with amastigotes, lymphocytes, plasma cells and some neutrophils and eosinophils. The surface epithelium developed increasingly extensive degeneration, which caused the development of erosions on the mucosal surface. The crypts of Lieberkühn decreased in number and showed degeneration of the crypt epithelium.

Amyloidosis in adrenal glands of hamsters experimentally infected with Leishmania infantum.

Thirty 10-week-old hamsters were inoculated intraperitoneally with leishmania infantum amastigotes and were serially killed after 15, 30, 45, 60, 75 and 89-90 days. The adrenal glands of each of them were examined by means of light and electron microscopy. The cortex and medulla of the adrenal gland showed a progressive deposition of amyloid, selectively identified by both optical and ultrastructural techniques. It has been suggested that adrenal amyloidosis during visceral leishmaniasis is directly related to a stimulation of the phagocitary mononuclear system due to the persistence of the antigenic stimulation of the parasite. In addition to these deposits, the presence of inflammatory infiltrates containing lymphocytes, plasmocytes and macrophages with leishmanias confirmed the establishment of leishmaniasis. The deposition of the amyloid fibrils finally leads to the partial destruction of the adrenal parenchyma.

Structural and ultrastructural hepatic changes in experimental canine leishmaniasis.

Six 4 month-old beagles were inoculated with Leishmania donovani infantum, three of them intraperitoneally (Group A) and the other three intravenously (Group B). The animals from Group A were killed 109, 433 and 592 days after inoculation and animals from Group B 109, 171 and 334 after inoculation. The liver of each of them was examined by means of light and electron microscopy. The lesions observed in both groups were very similar, but developed more rapidly in Group B. A chronic hepatitis appeared due to infection, characterized by the presence of multiple intralobular granulomas and portal inflammatory infiltrates consisting of lymphocytes, plasmocytes and macrophages with a variable number of amastigotes. The Kupffer cells were hyperplastic and contained parasites in their cytoplasm. Gradually the hepatocytes developed a progressive cellular swelling, which during the end-stages of the process showed itself with severe nuclear degeneration, disintegration of cytoplasmic organelles, enlargement of the cytoplasmic matrix and disruption of the plasma membranes, leading to cytolysis.