Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function.

BACKGROUND: Monocytes/macrophages are likely key cells in immune modulation in dogs with osteosarcoma (OSA). Increased peripheral monocyte counts are negatively correlated with shorter disease-free intervals in dogs with OSA. Understanding the monocyte/macrophage's modulatory role in dogs with OSA can direct further studies in immunotherapy development for OSA. HYPOTHESIS/OBJECTIVES: That OSA evades the immune response by down-regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses. ANIMALS: Eighteen dogs with OSA that have not received definitive treatment and 14 healthy age-matched controls METHODS: Clinical study-expression of peripheral blood monocyte cell surface receptors, monocyte mRNA expression and cytokine secretion, monocyte chemotaxis, and survival were compared between clinical dogs with OSA and healthy control dogs. RESULTS: Cell surface expression of multiple chemokine receptors is significantly down-regulated in peripheral blood monocytes of dogs with OSA. The percentage expression of CCR2 (median 58%, range 2-94%) and CXCR2 expression (median 54%, range 2-92%) was higher in control dogs compared to dogs with OSA (CCR2 median 29%, range 3-45%, P = 0.0006; CXCR2 median 23%, range 0.2-52%, P = 0.0007). Prostaglandin E2 (PGE2 ) (OSA, median 347.36 pg/mL, range 103.4-1268.5; control, 136.23 pg/mL, range 69.93-542.6, P = .04) and tumor necrosis factor-alpha (TNF-α) (P = .02) levels are increased in OSA monocyte culture supernatants compared to controls. Peripheral blood monocytes of dogs with OSA exhibit decreased chemotactic function when compared to control dogs (OSA, median 1.2 directed to random migration, range 0.8-1.25; control, 1.6, range of 0.9-1.8, P = .018). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with OSA have decreased monocyte chemokine receptor expression and monocyte chemotaxis, potential mechanisms by which OSA might evade the immune response. Reversal of monocyte dysfunction using immunotherapy could improve survival in dogs with OSA.

Expression of Apoptosis-regulating Proteins Bcl-2 and Bax in Lymph Node Aspirates from Dogs with Lymphoma.

BACKGROUND: Dysregulated apoptosis is a hallmark of tumorigenesis, and is also involved in resistance to cytotoxic treatment, and might be relevant in lymphoma in dogs. HYPOTHESIS/OBJECTIVES: That Bcl-2/Bax expression patterns differ between lymphoma immunophenotypes, and that Bcl-2/Bax ratio is correlated with prognosis. ANIMALS: Fifty-five client-owned dogs with multicentric lymphoma and 5 healthy dogs. METHODS: Prospective, case-control study. We compared 3 methods (flow cytometry, qRT-PCR, Western blot) for Bcl-2 and Bax quantification in a subset of dogs. The effect of time on Bcl-2/Bax ratios measured by flow cytometry was assessed in lymphoma cell lines. Immunophenotype and Bcl-2/Bax expression by flow cytometry were determined in LN aspirates from all dogs with multicentric lymphoma compared to healthy dogs. Progression-free survival (PFS) was retrospectively evaluated in a group of dogs all receiving similar treatment. RESULTS: Bcl-2/Bax ratios remain consistent for at least 5 days after sample collection. Bcl-2/Bax ratio was higher in dogs with T-cell lymphoma (TCL; median 0.97, range 0.37-1.36) compared to B-cell lymphoma (BCL; median 0.36, range 0.07-1.45) (P < .0001) and normal dogs (median 0.36, range 0.21-0.48) (P = .0006), respectively. Dogs with Bcl-2/Bax ratios higher than the median of the group experienced a median PFS of 101 days and dogs with ratios equal and lower than the median had PFS of 130 days (P = .19). CONCLUSIONS AND CLINICAL IMPORTANCE: Higher intrinsic resistance to apoptosis following cytotoxic treatment might contribute to the less favorable prognosis associated with multicentric TCL in dogs. Whether Bcl-2/Bax will be helpful to identify canine BCL and TCL with more aggressive and more indolent behavior, respectively, should be evaluated in larger prospective clinical studies.

CD45+ and CD45- lymphocyte populations identified by flow cytometry from dogs with lymphoma exhibit similar morphology and the same clonal (B cell or T cell) lineage.

Flow cytometric analysis of canine lymphoma sometimes demonstrates a mixed population of CD45+ and CD45- lymphocytes. Recently, indolent forms of canine lymphoma have been described which are associated with the loss of CD45 expression, warranting further investigation of the role of CD45 in canine lymphoma. The purpose of this study was to compare morphology and assess clonal origin between CD45+ and CD45- lymphocyte populations identified by flow cytometry in confirmed cases of canine B- and T-cell lymphoma. Our hypothesis was that the CD45- population of lymphocytes represented a phenotypic variant of the CD45+ population. Fifteen client-owned dogs with lymphoma and distinct CD45+ and CD45- lymphocyte populations identified by flow cytometry were identified for a blinded, prospective assessment of morphology and clonal origin (B cell or T cell) between populations of sorted CD45+ and CD45- cells. Lymphocytes were isolated from 11 dogs for paired cytologic evaluation. In 10/11 dogs, the CD45+ and CD45- samples were similar (95% C.I., 0.301-1.00). DNA was harvested from sorted populations of CD45+ and CD45- cells from 12/15 dogs and PARR analysis produced amplicons of identical size from both populations, indicating that 100% (12/12) were of the same lineage, B cell or T cell (95% C.I., 0.757-1.00). Collectively, our data suggests that the CD45- population identified in dogs with lymphoma represents a phenotypic variant of the CD45+ population.

Lymphoma immunophenotype of dogs determined by immunohistochemistry, flow cytometry, and polymerase chain reaction for antigen receptor rearrangements.

BACKGROUND: Immunohistochemistry (IHC), flow cytometry (FC), and PCR for antigen receptor rearrangements (PARR) are 3 widely utilized tests to determine immunophenotype in dogs with lymphoma (LSA). OBJECTIVES: This study evaluated the ability of FC and PARR to correctly predict immunophenotype as defined by IHC and to determine the level of agreement among the 3 tests. ANIMALS: Sixty-two dogs with lymphoma. METHODS: Retrospective study. Medical records were searched to identify dogs with LSA that had concurrent IHC, FC, and PARR performed. Immunophenotype results were categorized as B-cell, T-cell, dual immunophenotype (B- and T-cell), or indeterminate. The results of FC and PARR were evaluated for correctly classifying B- and T-cell LSA as compared with IHC. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were evaluated in addition to concordance between each test. RESULTS: The sensitivity of FC was significantly higher than PARR for both B-cell (91% versus 67%; P < 0.0072) and T-cell (100% versus 75%; P < 0.0312) LSA. The percent agreement between FC and IHC was 94%, between PARR and IHC was 69%, between FC and PARR was 63%, and among all 3 tests was 63%. CONCLUSIONS AND CLINICAL IMPORTANCE: Flow cytometry is superior to PARR in correctly predicting immunophenotype when evaluating lymph nodes from dogs already diagnosed with B- or T-cell LSA. If fresh samples are not available for FC, PARR is an acceptable assay for determination of immunophenotype given its high specificity.

Fozivudine tidoxil as single-agent therapy decreases plasma and cell-associated viremia during acute feline immunodeficiency virus infection.

BACKGROUND: Feline immunodeficiency virus (FIV) is a lentivirus that infects domestic and wild felidae and the course of disease is similar to that of human immunodeficiency virus infection. The thymidine nucleoside analog fozivudine (FZD) tidoxil is a lipid-zidovudine (ZDV) conjugate and member of the family of nucleoside reverse transcriptase (RT) inhibitors (NRTIs). HYPOTHESIS: FZD administration to cats during acute FIV infection produces antiviral activity with fewer adverse effects than its parent compound ZDV (AZT). ANIMALS: Male, neutered cats approximately 7 months of age (n = 12). METHODS: FZD (45 mg/kg q12h, n = 6) or placebo (n = 6) was administered PO in a nonblinded trial for 6 weeks to cats infected with the NCSU(1) isolate of FIV. Peripheral blood was collected preinfection and at 2, 4, and 6 weeks postinfection for CBC, evaluation of CD4(+) and CD8(+) cell counts by flow cytometry, and quantification of plasma and cell-associated viremia by real time RT-PCR. RESULTS: Treatment of cats with FZD during the acute stage of FIV infection decreased plasma and cell-associated viremia during the first 2 weeks of infection, but was not protective against FIV, as all cats were infected by 6 weeks. CONCLUSIONS: At the dosage used in this study, treatment with FZD results in a short-term decrease in viral load with no adverse effects. Further investigation of FZD is warranted to assess pharmacokinetics, optimal dosage, and to directly compare the antiviral activity of FZD to ZDV in naturally infected cats.