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- Author or Editor: Koichi Ohno x
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Abstract
OBJCTIVE To investigate the effects of dietary lipid overload on bile acid metabolism and gallbladder motility in healthy dogs.
ANIMALS 7 healthy Beagles.
PROCEDURES In a crossover study, dogs were fed a high-fat–high-cholesterol diet (HFCD) or a low-fat diet (LFD) for a period of 2 weeks. After a 4-month washout period, dogs were fed the other diet for 2 weeks. Before and at the end of each feeding period, the concentrations of each of the gallbladder bile acids, cholecystokinin (CCK)-induced gallbladder motility, and bile acid metabolism–related hepatic gene expression were examined in all dogs.
RESULTS The HFCD significantly increased plasma total cholesterol concentrations. The HFCD also increased the concentration of taurochenodeoxycholic acid and decreased the concentration of taurocholic acid in bile and reduced gallbladder contractility, whereas the LFD significantly decreased the concentration of taurodeoxycholic acid in bile. Gene expression analysis revealed significant elevation of cholesterol 7α-hydroxylase mRNA expression after feeding the HFCD for 2 weeks, but the expression of other genes was unchanged.
CONCLUSIONS AND CLINICAL RELEVANCE Feeding the HFCD and LFD for 2 weeks induced changes in gallbladder bile acid composition and gallbladder motility in dogs. In particular, feeding the HFCD caused an increase in plasma total cholesterol concentration, an increase of hydrophobic bile acid concentration in bile, and a decrease in gallbladder sensitivity to CCK. These results suggested that similar bile acid compositional changes and gallbladder hypomotility might be evident in dogs with hyperlipidemia.
Abstract
OBJECTIVE To examine bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge.
ANIMALS 18 dogs with gallbladder mucocele (GBM group), 8 dogs with immobile biliary sludge (i-BS group), 17 dogs with mobile biliary sludge (m-BS group), and 14 healthy dogs (control group).
PROCEDURES Samples of gallbladder contents were obtained by use of percutaneous ultrasound-guided cholecystocentesis or during cholecystectomy or necropsy. Concentrations of 15 bile acids were determined by use of highperformance liquid chromatography, and a bile acid compositional ratio was calculated for each group.
RESULTS Concentrations of most bile acids in the GBM group were significantly lower than those in the control and m-BS groups. Compositional ratio of taurodeoxycholic acid, which is 1 of 3 major bile acids in dogs, was significantly lower in the GBM and i-BS groups, compared with ratios for the control and m-BS groups. The compositional ratio of taurocholic acid was significantly higher and that of taurochenodeoxycholic acid significantly lower in the i-BS group than in the control group.
CONCLUSIONS AND CLINICAL RELEVANCE In this study, concentrations and fractions of bile acids in gallbladder contents were significantly different in dogs with gallbladder mucocele or immobile biliary sludge, compared with results for healthy control dogs. Studies are needed to determine whether changes in bile acid composition are primary or secondary events of gallbladder abnormalities.
Abstract
OBJECTIVE
To examine effects of a common mutation (2-base insertion in exon 5) of the TP53 gene on biological function of p53 protein in canine histiocytic sarcoma cells.
SAMPLE
Canine histiocytic tumor cell lines DH82 with deletion of TP53 and CHS-3 with the wild-type TP53 and canine wild-type and mutant TP53 fragments.
PROCEDURES
Wild-type or mutant TP53 with a polyprotein peptide tag at the N-terminus was transduced into DH82 and CHS-3 cells. Expression of p53 protein, changes in function as a transcription factor, and susceptibility to doxorubicin and nimustine were compared.
RESULTS
Transduced p53 protein was detected in wild-type TP53–transduced DH82 and CHS-3 cells, whereas expression was not detected in mutant TP53–transduced cells. There were significant increases in expression of target genes of p53 protein, including p21 and MDM2, in wild-type TP53–transduced cells, compared with results for native and mock-transfected cells, but not in mutant TP53–transduced cells. There was no significant difference in drug susceptibilities among native and derivative cells of CHS-3. However, cell viabilities of wild-type TP53–transduced DH82 cells incubated with doxorubicin were significantly lower than viabilities of native, mock-transfected, and AT insertion mutation–TP53–transduced DH82 cells; susceptibility to nimustine did not differ significantly among cells.
CONCLUSIONS AND CLINICAL RELEVANCE
Expression of p53 protein and its function as a transcription factor were lost after addition of a 2-base insertion in the TP53 gene in canine histiocytic tumor cells. Additional studies are needed to investigate the clinical relevance of this mutation in histiocytic sarcomas of dogs.
Abstract
Objective—To compare composition and colony formation of bone marrow mononuclear cells (BMMCs) harvested from dogs by means of a new perfusion method and the conventional aspiration method.
Animals—7 healthy adult Beagles.
Procedures—BMMCs were collected from the humeri and femurs of Beagles via perfusion and aspiration methods. Flow cytometric analysis was performed to quantify the presence of contaminant cells from the peripheral blood and the percentage of CD34+ progenitor cells in the BMMCs. A CFU assay was conducted to determine the number of progenitor cells in the BMMCs.
Results—The perfusion method was safely performed in all 7 dogs. Flow cytometric analysis revealed that the percentages of contaminant CD3+CD4+, CD3+CD8+, and CD21 + lymphocytes in BMMCs obtained via perfusion were significantly lower than percentages obtained via aspiration. The percentage of CD34+ cells obtained via perfusion was significantly higher than that obtained via aspiration. In addition, perfusion yielded a significantly higher CFU count than did aspiration.
Conclusions and Clinical Relevance—The perfusion method used in this study can minimize the contamination of bone marrow samples with peripheral blood and was a more efficient means for collecting canine bone marrow progenitor cells than the conventional aspiration method. Therefore, the perfusion method can be more suitable than aspiration for harvesting bone marrow cells for transplantation in dogs.
Abstract
Objective—To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1).
Sample Population—Madin-Darby canine kidney (MDCK) epithelial cell line.
Procedures—The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus–based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays.
Results—P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1.
Conclusions and Clinical Relevance—Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.
Abstract
OBJECTIVE To determine the prognostic value of CD44 variant isoform expression in dogs with multicentric high-grade B-cell lymphoma (BCL).
ANIMALS 45 dogs with multicentric BCL and 10 healthy control Beagles.
PROCEDURES The medical record database of a veterinary teaching hospital was searched to identify dogs with BCL that were treated between November 2005 and April 2015. Information regarding overall response to chemotherapy, progression-free survival (PFS) time, and overall survival time was extracted from each record. Archived lymph node aspirate specimens from dogs with BCL and lymph node aspirate specimens from the 10 control dogs underwent real-time PCR analysis to determine mRNA expression of CD44 variant isoforms of exons 3, 6, and 7 and the CD44 whole isoform. For each isoform, mRNA expression was compared between dogs with BCL and control dogs. The mean relative expression of each isoform was used to classify dogs with BCL into either a high- or low-expression group, and overall response rate, PFS time, and overall survival time (ie, indices of prognosis) were compared between the 2 groups.
RESULTS For all isoforms evaluated, mean relative mRNA expression for dogs with BCL was numerically lower than that for control dogs. Dogs with BCL and high CD44 isoform expression had a lower overall response rate, median PFS time, and median overall survival time, compared with dogs with BCL and low CD44 isoform expression.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for dogs with BCL, high expression of exons 3, 6, and 7 was associated with a poor prognosis.
Abstract
OBJECTIVE To investigate effects of prednisolone administration on gallbladder emptying rate and gallbladder bile composition in dogs.
ANIMALS 6 healthy Beagles.
PROCEDURES Prednisolone was administered (2 mg/kg, SC, once daily for 2 weeks) to each dog and tapered over 2 weeks. Gallbladder emptying rate and bile composition were evaluated before and after administration of prednisolone for 2 weeks as well as 1 week after cessation of prednisolone administration.
RESULTS Gallbladder emptying rate decreased significantly after prednisolone administration (median, 27%; range, 0% to 38%), compared with rate before administration (median, 59%; range, 29% to 68%), but then increased 1 week after cessation of administration (median, 45%; range, 23% to 48%). Gallbladder bile mucin concentration decreased significantly after prednisolone administration (median, 8.8 mg/dL; range, 6.2 to 11.3 mg/dL), compared with concentration before administration (median, 13.1 mg/dL; range, 10.7 to 21.7 mg/dL), but then increased 1 week after cessation of administration (median, 14.3 mg/dL; range, 9.6 to 26.7 mg/dL). Gallbladder taurochenodeoxycholic acid concentration decreased significantly after prednisolone administration (8.1 mmol/L; range, 6.8 to 15.2 mmol/L), compared with concentration before administration (median, 27.2 mmol/L; range, 22.0 to 31.9 mmol/L), but then increased 1 week after cessation of administration (median, 26.4 mmol/L; range, 15.1 to 31.5 mmol/L).
CONCLUSIONS AND CLINICAL RELEVANCE A lower gallbladder emptying rate caused by prednisolone administration may be involved in the pathogenesis of gallbladder disease in dogs. Further studies are required to determine the clinical importance of lower gallbladder bile mucin concentrations caused by glucocorticoid administration in the pathogenesis of gallbladder disease in dogs.
Abstract
Objective—To examine the DNA methylation status of the ABCB1 gene in tumor cells of dogs with lymphoma.
Animals—27 dogs with multicentric B-cell high-grade lymphoma (19 chemotherapy-sensitive dogs and 8 chemotherapy-resistant dogs).
Procedures—The DNA methylation profile of the CpG island of the ABCB1 gene was analyzed by use of bisulphite sequencing and real-time methylation-specific PCR assay in lymphoma cells. Quantitative reverse transcriptase PCR assay of the ABCB1 gene was conducted to measure the amount of mRNA. Correlation between the amount of ABCB1 mRNA and the methylation rate was examined.
Results—The CpG island of the ABCB1 gene was hypomethylated in most dogs in both the chemotherapy-sensitive and -resistant groups. No significant difference was detected in the methylation rate between the 2 groups, and no significant correlation was detected between the methylation rate and the mRNA expression level.
Conclusions and Clinical Relevance—Expression of the ABCB1 gene was not suppressed by hypermethylation of its CpG island in most dogs with lymphoma regardless of their chemotherapy sensitivity status.
Abstract
Objective—To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors.
Sample Population—27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs.
Procedure—Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay.
Results—Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean ± SEM, 14.5 ± 0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens.
Conclusion and Clinical Relevance—Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors. (Am J Vet Res 2001; 62:1539–1543)
Abstract
Objective—To generate an adenoviral vector that expressed the canine p53 gene and investigate its growth-inhibiting effect on canine osteosarcoma and mammary adenocarcinoma cell lines.
Sample Population—2 canine osteosarcoma cell lines (HOS, OOS) and 3 canine mammary adenocarcinoma cell lines (CHMp, CIPm, and CNMm).
Procedure—An adenoviral vector that expressed the canine p53 gene (AxCA-cp53) was generated. p53 gene expression was examined by use of reverse transcription (RT)-polymerase chain reaction (PCR) assay and immunohistochemistry. Susceptibility of cell lines to the adenoviral vector was determined by infection with an adenoviral vector that expresses β-galactosidase (AxCA-LacZ) and 3-indolyl-β-D-galactopyranoside staining. Growth inhibitory effects were examined by monitoring the numbers of cells after infection with mock (PBS) solution, AxCA-LacZ, or AxCA-cp53. The DNA contents per cell were measured by flow cytometry analysis. Apoptotic DNA fragmentation was detected by use of a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay.
Results—AxCA-cp53-derived p53 gene mRNA and P53 protein were detected by RT-PCR analysis and immunohistochemistry, respectively. Multiplicity of infection at which 50% of cells had positive 3-indolyl- β-D-galactopyranoside staining results ranged from 10 to 50. AxCA-cp53 induced growth inhibition in a dosedependent manner. Arrest of the G1-phase population and apoptotic DNA fragmentation were observed in cells infected with AxCA-cp53.
Conclusions and Clinical Relevance—AxCA-cp53 inhibits cell growth via induction of cell cycle arrest and apoptosis in canine osteosarcoma and mammary adenocarcinoma cell lines that lack a functional p53 gene. AxCA-cp53 may be useful to target the p53 gene in the treatment of dogs with tumors. (Am J Vet Res 2003;64:880–888)
