Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.     

Flow Cytometric Assessment of Acrosomal Status and Viability of Dog Spermatozoa

Contents
A procedure was attempted to simultaneously evaluate viability and acrosomal integrity of dog spermatozoa by flow cytometry and the dual staining technique using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide (PI). Three ejaculates were obtained from three dogs; each of which was divided into five aliquots and increasing concentrations (0–288 μmol/l) of lysophosphatidylcholine (LPC) were added to each one to artificially induce the acrosome reaction in different proportions of spermatozoa. Data obtained by flow cytometric analysis of each sample were compared with those obtained by microscopic evaluation under epifluorescence illumination and by light microscopy evaluation of smears stained with Spermac^® staining. Regression analysis was used to compare the flow cytometric assay with the epifluorescence and light microscopic techniques, and the results indicated that flow cytometry was highly correlated with the Spermac^® staining whereas the correlation with the epifluorescence microscopy was lower. In comparison with the Spermac^® staining, the results from this study validate flow cytometry as a precise method for evaluating the acrosomal integrity of canine spermatozoa.     

Th1/Th2 balance in canine peripheral blood lymphocytes--a flow cytometric study

The canine immune system undergoes continuous remodeling with advancing age. We measured the Th1/Th2 balance in peripheral blood lymphocytes (PBLs) obtained from 23 Beagles ranging in age from 0.5 to 11.8 years by flow cytometric analysis using intracellular cytokine staining. The percentage of CD4 cells producing interferon-gamma (Th1) increased with age. The percentage of CD4 cells producing interleukin-4 (Th2) increased but much less so. In conclusion, we demonstrated that the Th1/Th2 balance in canine peripheral blood could be measured by this flow cytometry technique and the Th1/Th2 balance inclined to dominance of the Th1 subpopulation in PBLs as the dog matured.     

Sperm membrane functionality in the dog assessed by flow cytometry

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.     

CFSE标记法分析番鸭呼肠孤病毒感染对番鸭回肠淋巴细胞归巢的影响

旨在建立一种可靠的体外活番鸭淋巴细胞的荧光标记方法,并用于分析番鸭呼肠孤病毒感染对雏番鸭回肠淋巴细胞归巢的影响。选择活细胞荧光染剂5（6）-羧基二乙酸荧光素琥珀酰亚胺酯（CFDA-SE,CFSE）对分离的番鸭淋巴细胞进行标记和利用流式细胞术对体外标记的淋巴细胞体内示踪检测分析,并利用该方法对MDRV感染雏番鸭回肠组织的淋巴细胞数量进行流式细胞术和石蜡切片免疫荧光检测分析;结果最终确定CFSE体外标记番鸭淋巴细胞的条件为以PBS为孵育液,终浓度10 μmol·L^-1,37℃ 30 min;试验结果表明番鸭外周血内的CFSE⁺淋巴细胞率基本稳定在2%~5%,CFSE⁺淋巴细胞峰值出现顺序依次为脾、空肠、回肠、盲肠、十二指肠、法氏囊和胸腺;此外,感染MDRV后1~10 d MDRV组中CFSE⁺淋巴细胞率极显著（P<0.01）高于MOCK组,该结果与α4⁺淋巴细胞率和石蜡切片免疫荧光检测结果一致。结果表明本试验CFSE标记的淋巴细胞可用于体内淋巴细胞示踪,初步应用结果提示MDRV感染促进番鸭淋巴细胞向回肠归巢,为进一步阐明MDRV感染的肠道组织致病机制奠定了基础。     

Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa

OBJECTIVE: To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. SAMPLE POPULATION: Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. PROCEDURE: Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. RESULTS: Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. CONCLUSIONS AND CLINICAL RELEVANCE: Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

使用荧光染色和流式细胞术检测山羊精子

分别使用PI(碘化丙碇),FITC-PSA(异硫氰酸荧光素络合豌豆凝集素)和RH123(罗丹明)对山羊冷冻精子进行染色,再使用流式细胞仪进行检测、分析。试验发现,对于山羊的冷冻精子,PI染色阴性的为质膜完整的精子;FITC-PSA染色阴性的可能为顶体完整的精子,染色FITC-PSA+和FITC-PSA++,可能分别为顶体反应中的精子和顶体破损精子;RH123+应为线粒体无活性精子,有绿色荧光可能是因为染料残留在细胞膜上导致,而RH123++可能应为线粒体有活性的活精子。不同个体羊的精子对于低温敏感度不一样。不同个体羊之间的冻精顶体完整率、质膜完整率、线粒体有活性的精子比率之间差异显著,据此应选择合适的山羊个体进行精液冷冻。     

Evaluation of cell replication by bovine T cells in polyclonally activated cultures using carboxyfluorescein succinimidyl ester (CFSE) loading and flow cytometric analysis

Studies reported here demonstrated that carboxyfluorescein succinimidyl ester (CFSE) loading of lymphocytes and flow cytometric analysis is a powerful assay to assess the kinetics and extent of cellular replication by bovine T-cell subpopulations in heterogeneous cultures of peripheral blood mononuclear cells (PBMC) where subpopulation interactions can occur. As CFSE analysis allows determination of the proportion of lymphocytes that divided, as well as the number of cell divisions each cell underwent, distinctions in responses among mitogen-stimulated cultures could be made even when(3)H-thymidine incorporation was equivalent. When combined with surface staining for detection of differentiation antigens, differences among T-cell subpopulations with regard to the number of divisions their members had undergone, were found. Anti-CD3 mAb stimulated both CD8(+)and CD4(+)T cells to undergo several cell divisions in 72 hours, while there was essentially no division by gamma delta T cells. In contrast, in concanavalin A-stimulated cultures, all T-cell subpopulations had divided.     

Optimization of coagulometric tests that incorporate human plasma for determination of coagulation factor activities in canine plasma

OBJECTIVE: To optimize methods used to measure coagulation factor activities in canine plasma, define reference ranges in dogs, and compare activities between canine and human plasma. SAMPLE POPULATION: Human plasma samples (n = 5) and plasma from healthy dogs (140) and dogs with low factor V activity (7), high factor V activity (7), and low factor VIII:C activity (6). PROCEDURE: Coagulometric tests incorporated human plasma deficient in a single coagulation factor (human deficient plasma). Standard curves were generated with pooled plasma from 100 healthy dogs. Effect of sample dilution was evaluated, using plasma from dogs with high or low factor V activity and low factor VIII:C activity. Reference ranges for healthy dogs were established. Activities in human plasma were determined by comparison with standard curves obtained with canine plasma. RESULTS: Activities of factors V and VIII:C in samples diluted < or = 1:20 influenced results of tests for other coagulation factors. Activities of factors V and VIII:C in human plasma were significantly less than in canine plasma. For the other coagulation factors, significant differences in human plasma-to-canine plasma activity ratios were detected among different sample dilutions. CONCLUSIONS AND CLINICAL RELEVANCE: Accurate measurement of coagulation factor activities in canine plasma, using human deficient plasma, requires higher sample dilutions (ie, > 1:20) than typically used for human plasma. Differences in activities between human and canine plasma and nonparallelism of the standard curves emphasize the necessity for use of species-specific standard curves for accurate determination of coagulation factor activity.     

Characterization of canine microglial cells isolated ex vivo

Microglia are the principal immune effector elements of the brain sharing immunophenotypic and functional characteristics of macrophages as well as of antigen presenting cells (APCs). The purpose of this study was to isolate canine microglial cells and make them available for ex vivo characterizations of their functions and immunophenotype. After isolation, carried out by density gradient centrifugation, microglial cells accumulated on distinct interfaces of 1.077 and 1.066 g/ml of a Percoll gradient. Identification of microglial cells in other species is realized by their specific immunophenotype of CD11b/c+ and CD45low. Our results indicate, that expression of CD45 is very low or even absent in canine microglial cells. In addition, they expressed CD18 and CD11b/c+, as determined by flow cytometry and immunohistochemistry. Fourteen additional monoclonal antibodies (mAbs) were used to characterize and compare canine microglial cells with monocytes. Microglia and monocytes can be clearly distinguished by their differential expression intensity of surface antigens (CD45, CD44, CD14). Functional characterization was assessed by a reactive oxygen species (ROS)-generation test and phagocytosis assay using flow cytometry. In conclusion, ex vivo examination of microglia is possible in dogs and most probably reflects the conditions in vivo. The measurement of tissue culture artifacts can be largely avoided using this method.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs

The detection of red blood cell (RBC)-bound immunoglobulins in case of anaemia with the direct agglutination test (DAT or Coombs test) has been reported to be of low sensitivity. We therefore tested the applicability of flow cytometry for the detection of canine IgG on RBC using two different IgG-specific secondary reagents: goat-anti-dog IgG (GalphaD-IgG) and rabbit-anti-dog IgG (RalphaD-IgG). Membrane staining RBC samples were performed at 4 degrees C. Comparisons of agglutination test at 37 degrees C and 4 degrees C showed, that binding of the secondary antibodies at 4 degrees C was more sensitive compared to agglutination at 37 degrees C and the two antisera differed considerable in their agglutination activity. Binding of GalphaD-IgG and RalphaD-IgG to RBC of healthy dogs (n=15) was low and mean fluorescence intensities were taken to calculate thresholds above which RBC of patients were judged positive. As in agglutination tests, both secondary antisera displayed considerable differences (concentration-dependent binding and histogram profiles) after flow cytometric analysis. Using flow cytometry, with GalphaD-IgG 8 of 17 agglutination-negative patients were positive and RalphaD-IgG was positive with 3 of 3 agglutination-negative RBC samples. Thus, flow cytometric analysis of proved to be a sensitive technique, detecting RBC-bound canine IgG of DAT-negative patients. The results of both techniques, however, are significantly influenced by the used IgG-specific polyclonal reagents.     

Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody

BACKGROUND: Flow cytometry may be used to determine immunophenotype or lineage of leukemic cells, but few antibodies are available that are specific for cells of monocytic and granulocytic lineage. OBJECTIVE: The purpose of this study was to evaluate the flow cytometric staining patterns of 3 commercial monoclonal antibodies for monocytes and granulocytes in clinically healthy dogs and in dogs with acute myeloid leukemia (AML). METHODS: Mouse antihuman macrophage antibody (MAC387), mouse anti-human myeloperoxidase (MPO), and a canine neutrophil-specific antibody (NSA) were evaluated using flow cytometry on blood from 6 clinically healthy control dogs, and on blood (n = 7) and/or bone marrow (n = 2) from 8 dogs with AML. A diagnosis of acute leukemia was confirmed by >30% blasts in bone marrow or >30% blasts in peripheral blood, together with bi- or pancytopenia, circulating CD34-positive blast cells, and clinical signs of disease. Leukemic samples also were evaluated using a wide panel of monoclonal antibodies. RESULTS: MAC387 stained neutrophils and monocytes from control dogs, although the staining profiles for the 2 cell types differed. MPO and NSA resulted in strong positive staining of neutrophils; MPO also stained monocytes weakly. Lymphocytes did not stain with any of the antibodies. One case was classified as AML of granulocytic lineage (AML-M1), 6 cases were classified as acute monocytic leukemia (AML-M5), and 1 case was classified as acute myelomonocytic leukemia (AML-M4). Neoplastic myeloblasts in the dog with granulocytic AML were positive for MPO, NSA, MAC387, and CD4. All monoblasts from the dogs with AML-M5 were positive for CD14, 5 of 6 were positive for MAC387, and 2 were positive for MPO. NSA staining was negative in the 2 dogs with AML-M5 in which it was evaluated. In the dog with AML-M4 variable percentages of blast cells were positive for CD14, MPO, MAC387, CD4, and NSA. CONCLUSIONS: Antigens identified by antibodies to MAC387, MPO, and NSA were expressed not just by normal mature neutrophils and monocytes, but also by neoplastic myeloblasts and monoblasts. These 3 antibodies may be useful as part of a wider panel for immunophenotyping AML in dogs.     

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.     

Biological activity of dihydroartemisinin in canine osteosarcoma cell lines

OBJECTIVE: To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro. SAMPLE POPULATION: 4 canine osteosarcoma cell lines. PROCEDURES: Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100 microM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2',7'-dihydrofluorescein diacetate and flow cytometry. RESULTS: The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6 microM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.     

Pathophysiologic and immunologic changes in a canine endotoxemia over a period of 24 hours

In this study, the pathophysiologic and immunologic parameters from a 24-hr of canine endotoxemia model by lipopolysaccharide (LPS) infusion were evaluated. For that, twelve healthy beagles received a continuous 24-hr IV infusion of low dose LPS (10 μg/kg/h, from Escherichia coli serotype O111:B4) dissolved in saline. Complete blood counts and serum biochemical analysis as well as histopathologic examination were performed to assess pathophysiologic changes such as neutrophil migration and organ injury. To evaluate immunologic parameters, the concentrations of plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were determined, and neutrophil activation was also evaluated based on cell surface expression of CD11b using flow cytometry analysis. As results, systemic signs of endotoxemia including fever, vomiting, and hemorrhagic diarrhea were observed within short time after LPS infusion. Severe leukopenia and increased fluorescent intensity of CD11b on neutrophils were observed at 3 hr while percent positive of CD11b was the maximum at 12 hr during the experiment. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and creatinine phosphokinase (CPK) concentrations increased markedly, and organ damage was confirmed on histopathologic examination. Plasma TNF-α peaked at 3 hr and decreased rapidly, while the concentrations of IL-6 and IL-10 increased gradually until 6 hr and decreased thereafter. Using this canine endotoxemia model, we were able to determine the kinetics of pathophysiologic and immunologic parameters over a period of 24 hr. This study will enhance our understanding of their mechanisms underlying canine sepsis.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.