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  • Author or Editor: Yasuhito Fujino x
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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.

Full access
in American Journal of Veterinary Research

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.

Full access
in American Journal of Veterinary Research

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.

Full access
in American Journal of Veterinary Research

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.

Full access
in American Journal of Veterinary Research