Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing's disease

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing's disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P<0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P<0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing's disease.     

The relationship between the anticoccidial effects of clindamycin and the development of immunity in the Eimeria pragensis/mouse model of large intestinal coccidiosis

The therapeutic effect of clindamycin on Eimeria pragensis (E. pragensis) infection in C57BL/6 mice was demonstrated by suppression of oocyst production and the appearance of degenerated endogenous stages of parasite in the intestine. Short-term clindamycin treatment, from 1 to 4 days or 4 to 8 days post infection (pi) at a dose of 800 mg/kg/day was effective to reduce clinical symptoms, oocyst production and schizogonic development. Interestingly, the short-term treatment schedules allowed the development of a measurable degree of protective immunity to challenge infection in the treated mice. In contrast, clindamycin treatment for the full 12 days period, which almost completely inhibited clinical symptoms and oocyst output, prevented the full development of protective immunity in the treated mice. All these data indicate that clindamycin is efficacious as an anti-eimerian agent and that both early and late endogenous developmental stages of E. pragensis exert a deep influence on the development of effective immunity to challenge infection.     

The growth profiles of three types of canine distemper virus on Vero cells expressing canine signaling lymphocyte activation molecule

To know growth profiles of canine distemper virus (CDV) on Vero cells stably expressing canine signaling lymphocyte activation molecule (Vero-DogSLAMtag; Vero-DST cells), the propagation of three strains of CDV was tested in Vero-DST cells in comparison with parental Vero cells. Strain MD77 could grow well in both cell lines, but demonstrated no syncytium formation or indistinguishable rounding cytopathic effects (CPE) in Vero cells. Strains Onderstepoort and KDK-1 also grew well in Vero-DST cells with apparent syncytium CPE, while they grew less or no efficiently, respectively, in Vero cells. All three CDV strains demonstrated the peak titers, in Vero-DST cells before reaching to an extensive CPE and drastic decrease of titers at/after full CPE. Immunohistochemistry revealed that viral antigens of all CDV strains were found exclusively in the syncytia in Vero-DST cells, while in Vero cells, viral antigen was identified in their single cells for strain MD77 but none for other strains. Thus, every strain of CDV could grow well in Vero-DST cells and behaved differently against Vero cells. These results would be of practical value for workers of CDV because 1) In Vero-DST cells, by observation of distinct syncytium CPE, the highest titer or the best growth of virus could be identified; 2) In Vero cells, various CDV strains could be readily classified after propagation in Vero-DST cells.     

Expression of hepatitis C virus core protein associated with malignant lymphoma in transgenic mice

Hepatitis C virus (HCV) is a major causative agent for chronic liver diseases leading to hepatocellular carcinoma (HCC) and has also been suggested to be a possible etiologic factor for different lymphoproliferative diseases, including mixed cryoglobulinemia (MC) and B-cell non-Hodgkin's lymphoma (NHL). To understand the roles of HCV core protein in the pathogenesis of HCV related diseases, we produced two lines of the transgenic mice (HC82310 and HC9053) that express the HCV core transgene. One of the lines, HC9053, developed malignant lymphoma (ML, follicular center cell type) with a high frequency (80%) at the ages over 20 months. Hepatocellular adenoma was also observed in this line of transgenic mouse. We demonstrated expression of HCV core protein and mRNA in the liver of transgenic mice, and also detected the core mRNA in the enlarged lymph nodes of the transgenic mice which developed ML. These results suggest that the core protein may play an important role in the development of ML, and that the HC9053 transgenic mice provide suitable models for understanding the mechanism of HCV-related lymphoproliferative diseases.     

Ganglioglioma in the thalamus of a puppy

A solitary brain mass of a 4-month-old miniature dachshund showing seizure-like neurological signs was examined histopathologically. At necropsy a white tumor mass, replacing the thalamus, approximately 1.5 cm in diameter, was found. There was cystic space filled with yellowish pale fluid in the central area of the tumor mass. Histopathological examination revealed that the mass consisted of irregularly arranged well-differentiated neuronal and glial cells, and multifocal mineral deposits. The neuronal cells had a large clear nucleus and various amount of Nissl substances in the cytoplasm. Some neural cells were bi-nucleated. Neither mitotic figures nor proliferating cell nuclear antigen (PCNA)-positive nuclei was found in the neuronal cells. Immunostaining for glial fibrillary acidic protein (GFAP) revealed diffuse proliferation of GFAP-positive glial cells and their processes, while these glial cells did not show apparent cellular atypism, mitotic activity, or PCNA-immunoreactivity. Accordingly, the present tumor was diagnosed as ganglioglioma, and hamartomatous histogenesis might be possible.     

Age-related histological changes in the canine substantia nigra

Age-related changes in the canine substantia nigra, were examined using immunostaining for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), neurofilament (NF), ubiquitin, single stranded DNA (ssDNA), and alpha-synuclein (alphaSN). Brain sections from 34 necropsied dogs, ranging from 2 months to 18 years old, were used for this study. On general histological examinations, several age-related changes, including lipofuscin deposition, polyglucosan bodies, amorphous basophilic inclusions and eosinophilic crystal inclusions, were found in the aged dogs. Immunohistochemically, TH-positive neurons were located only in the substantia nigra. The number of TH-positive neurons was well preserved in all dogs examined, however, the ratio of TH-positive neurons to GFAP-positive glial cells tended to show slight decrease in aged dogs. By ssDNA immunostaining for apoptotic cells, there were no significant results in the number of ssDNA-positive neurons. The number of ubiquitin- and NF-positive swollen neurites was increased markedly in aged dogs. Ubiquitin immunostaining revealed a small number of basophilic and eosinophilic inclusions, although both types of inclusions were negative for NF. By alphaSN immunostaining, no neurons were immunoreactive and no basophilic or eosinophilic intracytoplasmic inclusions were revealed. These results indicate that in the substantia nigra of aged dogs the dopaminergic neurons are well preserved, but intracytoplasmic inclusions and ubiquitin-positive degenerative neurites are commonly found.     

Lymphocyte subsets and specific IgG antibody levels in clindamycin-treated and untreated dogs experimentally infected with Babesia gibsoni

This study was carried out to clarify the role of lymphocyte subpopulations and Babesia-specific antibody on the treatment of clindamycin in dogs infected with B. gibsoni. Ten beagle dogs were divided into two groups: an untreated group (5 dogs) and a clindamycin-treated group (5 dogs), which was administered clindamycin at 25 mg/ kg body weight, per os, q 12 hr from 7 days to 21 days post-infection (PI). On the acute stage of infection, clindamycin treatment resolved anaemia and other clinical findings. There were no significant differences between treated and untreated dogs either in parasitemia levels or Babesial IgG antibody levels. However, morphological changes that indicated degeneration in the majority of parasites were observed. The numbers of CD4(+) showed a significant increase in treated dogs, especially after treatment. On the chronic stage, CD4(+) cells maintained high level both of the treated and untreated dogs. Although parasitemia maintained low level, their relapses were occurred on the 49th day PI in treated dogs and on the 42nd and 63rd PI in untreated dogs. A rapid humoral antibody response was observed in treated dogs, however, lower humoral antibody responses in untreated dogs after relapses. The antibody levels of treated dogs were significantly higher than those of untreated dogs. These results suggested that clindamycin might not eliminate rapidly parasites from peripheral blood, but damage parasites, which might stimulate efficiently humoral and cellular immunity against Babesia infection, and result in an improvement of clinical conditions.     

A comparative pathological study on granulomatous meningoencephalomyelitis and central malignant histiocytosis in dogs

Histiocytic proliferative disorders in canine central nervous system (CNS) including granulomatous meningoencephalomyelitis (GME) and malignant histiocytosis were compared pathologically. Lesions of GME mainly existed in the white matter of the cerebrum, brainstem and cerebellum and consisted of characteristic perivascular cuffing, parenchymal granuloma and leptomeningeal infiltrates of mononuclear cells. In malignant histiocytosis, there were two histological patterns, diffuse proliferation of neoplastic histiocytes through the leptomeninges and neoplastic nodule formation in the parenchyma. Neoplastic histiocytes exhibited mild to severe cellular atypia and high ability of invasion into the brain parenchyma. Mitotic and phagocytic figures were also observed. Several histiocytic markers, including lysozyme, alpha1-antitrypsin and lectin RCA-1, revealed histiocytic origin of both inflammatory and neoplastic cells, however, those were not determinative for the discrimination between GME and malignant histiocytosis. CD3- and PCNA-positive cells existed in the lesions of both diseases. The number of CD3-positive cells in GME tended to be greater than in malignant histiocytosis, while the difference was not statistically significant.     

Peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC-1 α) expression and the formation of slow-twitch muscle fibers in porcine and bovine skeletal muscles

The peroxisome proliferator‐activated receptor‐γ coactivator‐1 α (PGC‐1 α) induces mitochondria biogenesis in skeletal muscles. To determine the relationships between PGC‐1 α and the muscle fiber types, the expression levels of PGC‐1 α were analyzed in porcine and bovine skeletal muscles. As a first step, the nucleotide sequences of the porcine and bovine PGC‐1 α were determined. The porcine and bovine PGC‐1 α cDNA encoded 796 amino acid sequences and showed 95.1% identity between the two species. The expression levels of the PGC‐1 α mRNA were analyzed in the same 10 skeletal muscles from four pigs and three cattle. The contents of porcine and bovine PGC‐1 α were higher in the tongue, masseter and diaphragm, and lower in the Biceps femoris, semimembranosus, Longissimus thoracis and semitendinosus muscles. The contents of myosin heavy chain slow‐type protein (MyHC‐slow) were also determined in the same muscles by ELISA. The analysis of MyHC‐slow showed results similar to those for the PGC‐1 α contents in all of the muscles except for the tongue. The content of MyHC‐slow in the tongue was the lowest among the porcine muscles, and moderate among the bovine muscles. The results suggest that PGC‐1 α relates to the development of oxidative muscle fibers, but is not the principal factor in determining type I fiber content.     

Correlation of phytoplasma concentration in Hydrangea macrophylla with green-flowering stability

The interaction between phytoplasma concentration and green-flowering stability was studied in hydrangea cultivars. Three green and 18 nongreen cultivars were subjected to polymerase chain reaction (PCR) analysis to determine Japanese hydrangea phyllody (JHP) phytoplasma infection. The results showed that JHP-phytoplasma was detected only in ‘Midori’ plants, which have green sepals. ‘Midori’ plants were propagated, and from 29 rooted cutting plants, they were grouped into three types on the basis of sepal color, that is, green (75.9%), blue-green (13.8%) and blue (10.3%) sepals. To clarify the variability in the sepal color of ‘Midori’ plants, JHP-phytoplasma concentration in the sepals and leaves of green-, blue-green- and blue-flowering plants was determined by PCR analysis. The semiquantitative PCR comparisons of 370 bp DNA fragments showed that the JHP-phytoplasma concentrations in green sepals were 16 times higher than that in blue-green sepals. JHP-phytoplasma could not be identified by PCR analysis in blue sepals and leaves. These results showed that JHP-phytoplasma concentration correlated with green sepal stability in ‘Midori’ plants. A histological observation of sepals showed that epidermal cells of blue and blue-green sepals had a dome shape. Otherwise, green sepals were leaflike with flat epidermal cells, and palisade parenchyma cells with numerous chloroplasts.