Intraoperative hypertensive crisis in a dog with functional paraganglioma of the gall bladder.

A 15-year-old spayed female mongrel presented with anorexia and an abdominal mass. The mass originated from the gall bladder and was surgically resected along with divisionectomy of the central hepatic division. Paroxysmal hypertension and tachycardia were noted during manipulation of the mass. Following resection, arterial blood pressure decreased significantly. Histopathological analysis confirmed a diagnosis of neuroendocrine neoplasm. Immunohistochemical staining for synaptophysin and chromogranin A yielded diffuse and strong positive results, while gastrin was positive in only 10% of the cells. The preoperative elevated concentrations of catecholamine in the urinalysis showed a marked decrease after surgery. Based on these findings, the tumour was diagnosed as a functional paraganglioma of the gall bladder. The patient has undergone regular thoracic radiographs and ultrasound examinations and, until 431 days after surgery, has shown no signs of metastases or recurrences. Based on our literature search, we report the first case of functional paraganglioma of the gall bladder in a dog.

Clinical relationship between histopathological necrotic/partial necrotic findings and disease condition of gallbladder mucoceles in dogs.

Gallbladder mucocele (GM) is a common extrahepatic biliary disease recognized in dogs and is defined as the expansion and extension of the gallbladder by an accumulation of semi-solid bile or bile acid. Histopathological diagnosis of necrotizing cholecystitis and transmural coagulative necrosis of the gallbladder wall shows poor prognosis. Conversely, histopathological diagnosis with partial necrotic findings is often achieved. We hypothesized that histopathological partial necrosis of the gallbladder wall is the primary lesion of necrotic cholecystitis or transmural ischemic necrosis. Therefore, we investigated the relationship between histopathological necrosis/ partial necrosis findings and their clinical conditions. We retrospectively analyzed 55 dogs diagnosed with GM that had undergone cholecystectomy at the Yamaguchi University Animal Medical Center. The group with histopathological necrosis/partial necrosis of the gallbladder wall showed elevated levels of preoperative white blood cells, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase, total bilirubin, and C-reactive protein compared to the non-necrotic group. Partial necrosis of the gallbladder wall may affect the progression of the disease and hematological abnormalities. Additionally, all death cases until 2 weeks were included in the histopathological necrosis/partial necrosis group. In this study, we found that poor prognosis factors were associated with partial necrosis of the gallbladder wall. Furthermore, these cases of partial necrosis showed elevated levels of blood test parameters. These results suggest that necrosis of the gallbladder wall is associated with poor prognosis and poor pathophysiological conditions.

Oncolytic reovirus therapy: Pilot study in dogs with spontaneously occurring tumours.

Oncolytic virotherapy is a novel treatment involving replication-competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 108 to 5.0 × 109 TCID50 given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti-reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post-treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well-tolerated and can be given safely to tumour-bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours.

Radioresistance of cancer stem-like cell derived from canine tumours.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

Role of hyperfine coupling in magnetic and quadrupolar ordering of Pr3Pd20Si6.

We study the ternary clathrate Pr3Pd20Si6 in specific heat and ac susceptibility measurements on a high-quality single crystal, distinguishing antiferromagnetic and antiferroquadrupolar ordering, as well as a hitherto unknown magnetic low-temperature transition. The specific heat shows the direct involvement of nuclear spin degrees of freedom in the antiferromagnetic ordering, which is well supported by our calculation of the hyperfine level scheme without adjustable parameters. Pr3Pd20Si6 is, therefore, one of the rare materials where the nuclear moments are involved in the formation of the magnetic ground state.

Magnetic phase diagram of clathrate compound Ce3Pd20Si6 with quadrupolar ordering.

We present results of specific heat measurements on a Ce3Pd20Si6 single crystal and construct the magnetic phase diagram for the three cubic principal directions [100], [110] and [111]. The highly anisotropic phase diagram is discussed and can be qualitatively explained by the Zeeman splitting at the 8c-site. For B â€– [100], the present study found two different quadrupolar ordered phases, which meet the paramagnetic phase at a tri-critical point and establish the new phase boundaries.

Involvement of geranylgeranylation of Rho and Rac GTPases in adipogenic and RANKL expression, which was inhibited by simvastatin.

Simvastatin suppresses myoblast differentiation via inhibition of Rac GTPase, which is involved in the mevalonic acid pathway that produces cholesterol. Statins also inhibit adipogenic differentiation and receptor activator of NFκB ligand (RANKL) expression, possibly through the mevalonic acid pathway, although the involvement of that pathway and effector proteins in these cellular events has not been fully clarified. In the present study, we aimed to elucidate the mechanism of the effects of simvastatin on adipogenic differentiation and calcitriol-induced RANKL expression in bone marrow stromal ST2 cells. Adipogenesis and mRNA up-regulation of peroxisome proliferator-activated receptor γ and adipocyte fatty acid-binding protein were induced by troglitazone, and those events were efficiently inhibited by simvastatin. In addition, RANKL expression induced by calcitriol was abrogated by simvastatin in ST2 cells. The inhibitory effects of simvastatin were adequately compensated by the addition of either mevalonic acid or an intermediate of the mevalonic acid pathway, geranylgeranyl pyrophosphate, but not by another intermediate, farnesyl pyrophosphate. These findings suggest that protein geranylgeranylation is related to cellular differentiation in those two directions. Furthermore, inhibitor analysis demonstrated that Rac GTPase is involved in adipogenic differentiation, whereas Rho GTPase was found to be involved in RANKL expression. Taken together, the present findings suggest that geranylgeranylation of Rho family GTPase is involved in both adipogenesis and RANKL expression of stromal cells, while Rac GTPase is involved in adipogenesis and Rho GTPase in RANKL expression.

Detection of Merkel cell polyomavirus with a tumour-specific signature in non-small cell lung cancer.

BACKGROUND: We searched for a viral aetiology for non-small cell lung cancer (NSCLC), focusing on Merkel cell polyomavirus (MCPyV). METHODS: We analysed 112 Japanese cases of NSCLC for the presence of the MCPyV genome and the expressions of RNA transcripts and MCPyV-encoded antigen. We also conducted the first analysis of the molecular features of MCPyV in lung cancers. RESULTS: PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas (ACs), 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed large T antigen (LT) RNA transcripts. Immunohistochemistry showed that MCPyV LT antigen was expressed in the tumour cells. The viral integration sites were identified in one SCC and one AC. One had both episomal and integrated/truncated forms. The other carried an integrated MCPyV genome with frameshift mutations in the LT gene. CONCLUSION: We have demonstrated the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumour-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumour-specific molecular signatures support the possibility that MCPyV is partly associated with the pathogenesis of NSCLC in a subset of patients.

Propeptide processing and proteolytic activity of proenzymes of the staphylococcal and enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser_1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn.1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.

Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line.

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.

Gene silencing of STAT6 with siRNA ameliorates contact hypersensitivity and allergic rhinitis.

BACKGROUND: Silencing of genes using small interfering RNA (siRNA) is a recently developed strategy to regulate the synthesis of target molecules. Signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity. METHODS: To elucidate the therapeutic potential of using siRNA to inhibit STAT6 in allergic reactions, we determined the nucleotide sequences of siRNA specific for STAT6. RESULTS: The selected sequences of STAT6 siRNA specifically inhibited the generation of STAT6 synthesis in dermal fibroblasts and eotaxin (CCL11) production in response to IL-4/TNF-α in vitro. Local administration of STAT6 siRNA in vivo alleviated contact hypersensitivity responses to chemical haptens. This was accompanied by reduced local production of IL-4, IL-13, eotaxin (CCL11), TARC (CCL17) and MDC (CCL22). Similarly, consecutive intranasal instillation of STAT6 siRNA markedly inhibited inflammatory cellular infiltration of mucosal tissues in allergic rhinitis responses in association with reduced IL-4 and IL-5 production from regional lymph node cells. Immediate responses, such as sneezing and nasal rubbing behaviors, were also improved by STAT6 siRNA. CONCLUSIONS: Local administration of STAT6 siRNA is thus a promising therapeutic strategy for both Th2-mediated cutaneous diseases and allergic rhinitis.

Relationship of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children.

OBJECTIVES: The aim of this study was to assess the relationships of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers with the colonization of mutans streptococci (MS) in plaque and caries status in their 2.5-year-old children. Furthermore, the dynamics of caries status in the children was evaluated in a 2-year follow-up survey. METHODS: After oral examination of 54 mother-and-child pairs, the saliva samples from the mothers and the plaque samples from the children were collected. The levels (log DNA copies/ml saliva) of S. mutans and S. sobrinus were quantified using real-time polymerase chain reaction (PCR) assays, while MS in the plaque samples were detected using a cultivation method. In addition, 50 of the 54 children participated in a 2-year follow-up survey of caries prevalence. RESULTS: In the 2.5-year-old children, the percentage of dft-positive subjects and mean number of dft were significantly higher in the MS(+) group when compared with the MS(-) group. Findings from the 2-year follow-up survey indicated that MS(+) subjects had a persistently higher mean number of dft at 4.5 years. The 2.5-year-old children were divided into three groups based on the quantitative levels of salivary S. mutans and S. sobrinus in their mothers: those whose mothers had low levels of S. mutans (<4 log DNA copies/ml) and S. sobrinus (<2) (group 1); those whose mothers had a high level of S. mutans (> or = 4) and low level of S. sobrinus (<2) (group 2); and those whose mothers had high levels of both (> or = 4 and > or = 2, respectively) (group 3). Among the three groups, the percentages of MS(+) and dft-positive children were highest in group 3 and lowest in group 1. Furthermore, multiple logistic regression analyses revealed that grouping the mothers based on salivary level of S. mutans and S. sobrinus was an efficient means to predict both MS colonization (OR = 2.96) and prevalence of dental caries (OR = 9.39) in children at 2.5 years of age. CONCLUSIONS: In the 54 mother-and-child pairs tested, the maternal salivary levels of S. mutans and S. sobrinus determined by real-time PCR were significantly related to MS colonization in plaque as well as dental caries in their children at 2.5 years of age. Thus, determination of maternal levels of both organisms using the present cut-off values is proposed as an efficient method to indicate the risks of maternal transmission of MS and childhood dental caries.

Occurrence of staphylococci in the oral cavities of healthy adults and nasal oral trafficking of the bacteria.

To investigate a possible peroral route of infective endocarditis (IE), the occurrence of staphylococci in the oral cavity was examined using saliva and supragingival plaque specimens from 56 systemically and periodontally healthy adults aged 22-43 years old (27.1+/-5.3). Nine Staphylococcus species and 334 isolates were identified. In saliva, the total occurrence rate was 83.9 % and the total number of bacteria was 10(2)-10(4) c.f.u. ml(-1). Staphylococcus aureus was the most frequent species (46.4 %), followed by Staphylococcus epidermidis (41.1 %) and others (Staphylococcus hominis, Staphylococcus warneri, Staphylococcus intermedius, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus gallinarum, isolation frequencies ranging in order from 12.5 to 1.8 %). A similar isolation tendency was observed in supragingival plaque, with a total occurrence rate of 73.2 % and amounts of bacteria ranging from 10(2) to 10(5) c.f.u. g(-1). Four common Staphylococcus species (S. aureus, S. epidermidis, S. lugdunensis and S. hominis) were isolated from nasal swab samples taken from the oral staphylococci-positive subjects. Genotyping of all 18 combinations of oral- and nasal-derived isolates by PFGE indicated that identical clones or close relatives were commonly distributed in these two cavities. Since the provision of micro-organisms from the nasal cavity was shown and occurrence rates in the oral cavity were adequate, these results suggest a possible peroral route of staphylococcal IE, as in cases of viridans streptococcal IE.

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis.

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.

Anti-MuSK-positive myasthenia gravis: neuromuscular transmission failure in facial and limb muscles.

The presence of antibodies against muscle-specific receptor tyrosine kinase (MuSK) appears to define a subgroup of patients with myasthenia gravis (MG) characterized by weakness predominant in bulbar, facial and neck muscles compared with anti-acetylcholine receptor (AChR) antibody-positive MG. To investigate the patterns and severity of neuromuscular transmission failure in different muscles in MuSK-positive MG, we performed single fiber electromyography (SFEMG) in the facial (frontalis) and limb (extensor digitorum communis, EDC) muscles in three anti-Musk-positive patients, and compared results with those of 11 anti-AChR-positive patients. Only one of the three MuSK-positive patients had abnormal jitter in EDC, but all the three showed clearly increased jitter in the frontalis. By contrast, the AChR-positive patients showed similarly abnormal jitter for the two muscles. These results suggest that when the diagnosis of anti-MuSK-positive MG is suspected, SFEMG should be performed in most prominently affected muscles.

FTY720 suppresses CD4+CD44highCD62L- effector memory T cell-mediated colitis.

FTY720, a sphingosine-derived immunomodulator, causes immunosuppression via enhancement of lymphocyte sequestration into secondary lymphoid organs, thereby preventing their antigen-activated T cell egress to sites of inflammation. FTY720 is highly effective in inhibiting autoimmunity in various animal models. However, there is little known about how FTY720 controls the migration property of memory T cells. Here, we demonstrated that FTY720 prevents the development of colitis induced by the adoptive transfer of lamina propria (LP) colitogenic effector memory CD4+ T cells (TEM cells; CD45RB(low)CD44(high)CD62L-) into severe combined immunodeficiency (SCID) mice and suppresses interferon-gamma, interleukin-2, and tumor necrosis factor-alpha production by LP CD4+ T cells. The numbers of spleen, peripheral blood, mesenteric lymph node, and LP CD4+ T cells in FTY720-treated mice were significantly reduced compared with those in control mice. Notably, LP CD4+ TEM cells as well as splenic CD4+CD45RBhigh T cells expressed several spingosine-1-phosphate receptors that are targets for FTY720. Furthermore, FTY720 also prevented the development of colitis induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells into SCID mice. Collectively, the present data indicate that FTY720 treatment may offer the potential not only to prevent the onset of disease but also to treat memory T cell-mediated autoimmune diseases including inflammatory bowel diseases.

Naturally arising CD4+CD25+ regulatory T cells suppress the expansion of colitogenic CD4+CD44highCD62L- effector memory T cells.

Naturally arising CD4+CD25+ regulatory T (T(R)) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (T(EM)) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4+CD44(high)CD62L(-) lamina propria (LP) T cells obtained from colitic CD4+CD45RB(high) T cell-transferred mice, we have shown in the present study that CD4+CD25+ T(R) cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ T(EM) cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ T(R) cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ T(EM) cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ T(R) cells and the their suppressive activity on colitogenic LP CD4+ T(EM) cells.

Development of microporous covered stents: geometrical design of the luminal surface.

To reduce in-stent restenosis rates we have developed newly designed covered stents, in which a stent strut is buried into a microporous elastomeric cover film to provide a physical barrier against tissue ingrowth and a pharmacological reservoir for drug-eluting. The covered stents were prepared by dip-coating balloon expandable stents mounted on a stainless steel rod in a segmented polyurethane (SPU) solution, and were subsequently subjected to laser-processed microporing (pore diameter, 100 microm; interpore distance, 200 microm). The covered stents, which possessed flat luminal surfaces and micropores that were homogeneously arranged on the whole surface of the covering film, were deployed into the bilateral common carotid arteries of normal New Zealand white rabbits. Angiography after one month of implantation showed all stents were patent with little thrombus formation. The mean thickness of the formed neointimal layers was 292 +/- 177 microm (n=8), which was close to the size in non-covered bare stent (231 +/- 58 microm, n=7), but markedly decreased (about 2/3) from that in the previously developed wrapping-type covered stents (415 +/- 173 microm, P<0.01, n=8).

Association between neuroleptic drug-induced extrapyramidal symptoms and dopamine D2-receptor polymorphisms in Japanese schizophrenic patients.

The aim of the present study is to examine the relationship between dopamine D2-receptor gene (DRD2) polymorphisms (Taq1A, Taq1B, -141C Ins/Del) and the risk of extrapyramidal adverse effects (EPS), assessed according to the Drug-Induced Extra-Pyramidal Symptoms Scale (DIEPSS), or the maintenance dose of antipsychotics in schizophrenic patients. The DIEPSS score was significantly higher in patients bearing the -141C Del allele than in those without it. Taq1A and Taq1B restriction fragment length polymorphisms (RFLPs) did not significantly affect the DIEPSS score. On the other hand, maintenance doses of neuroleptics and antiparkinsonian drugs were significantly higher in patients with the B1 allele of Taq1B RFLP than in those without it, while the Taq1A RFLP and -141C Ins/Del polymorphisms were not significantly related to the maintenance doses. In conclusion, the risk of EPS may be increased in patients with the -141C Del allele of the DRD2 gene. In these patients, antipsychotics should be administered with caution.