Ko Nakashima

DECREASED GALLBLADDER EMPTYING IN DOGS WITH BILIARY SLUDGE OR GALLBLADDER MUCOCELE

Biliary sludge in dogs is dismissed commonly as an incidental finding. On the other hand, gallbladder mucocele is reported increasingly in dogs and can lead to biliary obstruction or gallbladder rupture. Cholestasis is suspected to play a role in development of sludge and mucoceles, though there are no data in dogs to support this. We investigated gallbladder emptying, a key factor in biliary flow, in dogs with mobile sludge, immobile sludge, or gallbladder mucocele and in healthy controls. Gallbladder ejection fraction estimated by ultrasonography was used as the index of gallbladder emptying. The ejection fraction at 60 min after eating was significantly decreased in all three abnormal groups. Moreover, all dogs with sludge or a mucocele had gallbladder distension. These changes were the greatest in the mucocele group. Thus, biliary stasis occurs not only in dogs with gallbladder mucocele but also in dogs with biliary sludge. Cholestasis may play a role in the pathogenesis or progression of these diseases in dogs.

CT characteristics of primary hepatic mass lesions in dogs.

Little information is available on the relationship between computed tomography (CT) imaging findings and the pathologic diagnosis of canine hepatic tumors. Our purpose was to clarify the characteristic features of CT findings in liver tumors in dogs. Data from 33 dogs with either a hepatocellular carcinoma, n = 14, hepatocellular adenoma, n = 14, or nodular hyperplasia, n = 5 were summarized from medical records. CT features for each histologic diagnosis were characterized and analyzed statistically. Common findings in hepatocellular carcinoma included central (79%, P = 0.0030) and marginal enhancement (93%, P = 0.00043) in the arterial phase, cyst-like lesions (93%), capsule formation (93%), and hypoattenuation in the portal (86%), and equilibrium phases (93%). Hepatic adenoma was characterized by a characteristic diffuse enhancement pattern during the arterial phase (57%, P = 0.013), which was also found in nodular hyperplasia (60%), but never in hepatocellular carcinoma. Nodular hyperplasia was less likely to have a capsule structure (20%, P = 0.0087). Mass size was significantly smaller in nodular hyperplasia than in hepatocellular carcinoma and hepatic adenoma (P = 0.0033 and 0.038, respectively). Hyperattenuation in the arterial and the portal phase i.e. contrast retention, was more frequent in hepatic adenoma than in the other groups (P = 0.037 and 0.037, respectively). Nodular hyperplasia was more frequently isoattenuating in the equilibrium phase (P = 0.043).

Decreased Immunoglobulin A Concentrations in Feces, Duodenum, and Peripheral Blood Mononuclear Cells of Dogs with Inflammatory Bowel Disease

BACKGROUND Although immunoglobulin A (IgA) plays a key role in regulating gut homeostasis, its role in canine inflammatory bowel disease (IBD) is unknown. HYPOTHESIS IgA expression may be altered in dogs with IBD, unlike that observed in healthy dogs and dogs with other gastrointestinal diseases. ANIMALS Thirty-seven dogs with IBD, 10 dogs with intestinal lymphoma, and 20 healthy dogs. METHODS Prospective study. IgA and IgG concentrations in serum, feces, and duodenal samples were measured by ELISA. IgA(+) cells in duodenal lamina propria and IgA(+) CD21(+) peripheral blood mononuclear cells (PBMCs) were examined by immunohistochemistry and flow cytometry, respectively. Duodenal expression of the IgA-inducing cytokine transforming growth factor β (TGF-β), B cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL) was quantified by real-time RT-PCR. RESULTS Compared to healthy dogs, dogs with IBD had significantly decreased concentrations of IgA in fecal and duodenal samples. The number of IgA(+) CD21(+) PBMCs and IgA(+) cells in duodenal lamina propria was significantly lower in dogs with IBD than in healthy dogs or dogs with intestinal lymphoma. Duodenal BAFF and APRIL mRNA expression was significantly higher in IBD dogs than in the healthy controls. Duodenal TGF-β mRNA expression was significantly lower in dogs with IBD than in healthy dogs and dogs with intestinal lymphoma. CONCLUSIONS AND CLINICAL IMPORTANCE IBD dogs have decreased IgA concentrations in feces and duodenum and fewer IgA(+) PBMCs, which might contribute to development of chronic enteritis in dogs with IBD.

GeneScan analysis to detect clonality of T-cell receptor γ gene rearrangement in feline lymphoid neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity.

Quantification of chemokine and chemokine receptor gene expression in duodenal mucosa of dogs with inflammatory bowel disease

Although chemokines and their receptors play an integral role in the regulation of the immune response, there is very little information about their involvement in canine inflammatory bowel disease (IBD). The objective of this study was to evaluate the mRNA expression of 9 selected chemokines and 6 chemokine receptors by real-time reverse transcription PCR in the duodenal mucosa from 21 dogs with IBD and 25 control dogs. The transcription levels of monocyte chemotactic protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-3 alpha (MIP-3α)/CCL20, thymus-expressed chemokine (TECK)/CCL25, mucosae-associated epithelial chemokine (MEC)/CCL28 and IL-8/CXCL8 mRNA in IBD dogs were significantly higher than the corresponding levels in control dogs, but there was no significant difference in the mRNA levels of the chemokine receptors between the 2 groups. In addition, the CCL2 and CXCL8 mRNA levels were significantly higher in the high clinical severity score group than in the low clinical severity score group. However, there was no correlation between chemokine or chemokine receptor mRNA expressions and histopathological severity score. The present results suggest that several chemokines may play important roles in the pathogenesis of canine IBD.

Serum amyloid A as a prognostic marker in cats with various diseases

Serum amyloid A (SAA) is reported not only as a marker for the presence of inflammation but also as a prognostic indicator in human beings. In cats, however, there is no report on the association between SAA concentration and prognosis. The objective of the current study was to evaluate SAA concentration as a prognostic marker in diseased cats. A total of 175 cats with neoplastic diseases, inflammatory diseases, and other diseases were retrospectively recruited, and the medical records of these cats, including follow-up data on mortality, were reviewed. Cats were divided into 2 groups according to SAA concentration, and differences in survival between each group were assessed. Median survival time of cats in the elevated SAA (>0.82 mg/l) group was significantly shorter than that in the nonelevated SAA (≤0.82 mg/l) group (P < 0.001). Furthermore, by multivariate analysis, SAA concentration was shown as a significant and independent prognostic marker in cats with various diseases (P = 0.015). Serum amyloid A concentration in diseased cats is a useful predictive indicator of prognosis regardless of diagnosis.

Prognostic factors in dogs with protein-losing enteropathy

Canine protein-losing enteropathy (PLE) is associated with severe gastrointestinal disorders and has a guarded to poor prognosis although little information is available regarding factors affecting prognosis. The purpose of this study was to identify the prognostic factors for survival of dogs with PLE. Ninety-two dogs diagnosed with PLE from 2006 to 2011 were included in a retrospective cohort study. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Variables recorded at the time of diagnosis were statistically analysed for possible prognostic factors in a univariate and multivariate Cox proportional hazard model. In the multivariate analysis, the predictors for mortality in dogs with PLE were more highly scored in terms of canine inflammatory bowel disease activity index (CIBDAI) (P = 0.0003), clonal rearrangement of lymphocyte antigen receptor genes (P = 0.003), and elevation of blood urea nitrogen (BUN) (P = 0.03). Using histopathological diagnosis, both small- and large-cell lymphomas were associated with significantly shorter survival times than chronic enteritis (CE) and intestinal lymphangiectasia (IL). Normalization of CIBDAI and plasma albumin concentration within 50 days of initial treatment was associated with a longer survival time. In conclusion, CIBDAI, clonal rearrangement of lymphocyte antigen receptor genes, histopathological diagnosis, and response to initial treatments would be valuable in separating the underlying causes and could be important in predicting prognosis in dogs with PLE.

Expression of Fibrosis-Related Genes in Canine Chronic Hepatitis

Molecular regulation of fibrosis in chronic canine hepatitis is poorly understood. The authors employed quantitative polymerase chain reaction (PCR) to determine the expression levels of genes reported to be related to fibrosis in other species (human, mouse, and rat) and to elucidate the relationship of these genes with the degree of fibrosis and the presence or absence of ascites and/or jaundice in dogs with hepatitis. Nine fibrosis-related genes were assayed: PDGFB, PDGFD, MMP2, TIMP1, THBS1, COL1A1, COL3A1, TGFB1, and TGFB2. Liver samples of 15 dogs with chronic hepatitis and 4 healthy control dogs were obtained via laparoscopic biopsy and subjected to histologic and quantitative PCR analyses. The expression of all 9 genes showed significant positive correlation (P < .01, r > .70) with the degree of fibrosis. Furthermore, the expression levels of all genes except TGFB1 were significantly higher (P < .05) in dogs with hepatic failure–related symptoms (ascites/jaundice). Results suggest that these 9 genes are integral to the development of fibrosis in canine chronic hepatitis.

Increased expression of fractalkine and its receptor CX3CR1 in canine inflammatory bowel disease and their possible role in recruitment of intraepithelial lymphocytes

The interaction between fractalkine/CX(3)CL1 and its receptor CX(3)CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX(3)CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX(3)CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX(3)CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX(3)CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX(3)CR1(+) PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX(3)CR1(+) PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX(3)CR1(+) PBMCs. These results suggest that the fractalkine-CX(3)CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs.

Efficacy of leflunomide for treatment of refractory inflammatory colorectal polyps in 15 Miniature Dachshunds.

Inflammatory colorectal polyp (ICRP), common in miniature dachshunds, presents with hematochezia, tenesmus and mucoid feces. Although an 80% response rate has been reported when treated with prednisolone and cyclosporine, effective treatment is needed for the remaining 20% of ICRP dogs. Leflunomide is an immunosuppressive drug reported as effective in several immune-mediated diseases. In the present study, we retrospectively evaluated the efficacy and adverse effects of leflunomide in 15 ICRP dogs that were refractory to treatment with prednisolone and cyclosporine. Treatment efficacy was assessed by endoscopy, clinical symptoms and rectal palpation. Adverse effects were determined by clinical symptoms and blood testing during follow-up. The leflunomide treatment response rate was 93.3%. The median dosage of leflunomide and the median response time were 3 mg/kg (range: 1.7–4.0 mg/kg) and 35 days (range: 20–119 days), respectively. Adverse effects observed included lethargy (3 dogs), anorexia (1 dog), respiratory symptoms (1 dog), leukocytopenia (2 dogs), thrombocytopenia (1 dog), anemia (1 dog) and liver enzyme elevation (8 dogs). Most of the adverse effects improved with symptomatic treatment and leflunomide discontinuation or dosage reduction. In conclusion, leflunomide treatment is effective in ICRP dogs refractory to treatment with prednisolone and cyclosporine. Because several adverse effects were observed, close monitoring is needed during leflunomide treatment follow-up.