Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.     

Flow cytometric analysis of colonic and small intestinal mucosal lymphocytes obtained by endoscopic biopsy in the healthy dog

Flow cytometric analysis of the lymphocyte population of the gut could provide useful information on the immune cells present in the gut that would not be easily obtained in tissue sections. However, little is known of the normal lymphocyte population in the canine gut as determined by flow cytometry, which allows for simultaneous staining of multiple cell surface antigens and identification of specific lymphocytic subsets. Therefore, intraepithelial lymphocytes were obtained from biopsies of the healthy canine proximal small intestine and colon taken with an endoscope, and flow cytometric analysis was used to characterize the lymphocyte subsets present. Endoscopic biopsy of the intestine is a minimally invasive technique commonly used for diagnostic purposes. Although CD3+ lymphocytes were the most abundant subset in both colon and small intestine, CD3+/CD8- lymphocytes predominated in the proximal small intestine, whereas CD3+/CD8+ lymphocytes did in the colon. Canine CD8+ intraepithelial lymphocytes were predominantly CD8alphabeta+ in both small intestine and colon. CD4+ intraepithelial lymphocytes were always much less numerous than CD8+ intraepithelial lymphocytes. As in man, a majority of intraepithelial lymphocytes expressed the T-cell receptor, TCRalphabeta, but TCRgammadelta was expressed by a third of intraepithelial T-cells in the proximal small intestine, and approximately 15% of those in the colon. Very few CD21+ lymphocytes were detected in samples of healthy canine colon and small intestinal intraepithelial cells. We have showed that canine intraepithelial lymphocytes are regionally specialized, and that those from the small intestine are unique in comparison to those of other species such as man and rodents due to the large numbers of CD3+/CD8- intraepithelial lymphocytes. This study provides a baseline for comparison with intraepithelial lymphocytes obtained from canine patients with intestinal disease.     

Expression of cyclooxygenase-2, P-glycoprotein and multi-drug resistance-associated protein in canine transitional cell carcinoma

Cyclooxygenase-2 (COX-2), P-glycoprotein (P-gp) and multi-drug resistance-associated protein (MRP) are considered important tumor-associated proteins in humans and dogs. In the present study, we immunohistochemically evaluated the expression of these proteins in canine patients with transitional cell carcinoma (TCC). Of 52 cases, 30 (57.7%) were positive for COX-2, 40 (76.9%) for P-gp, and only 10 (19.2%) for MRP. In addition, 27 samples (27/52, 51.9%) were positive for two markers, while 3 (5.7%) and 5 (9.6%) cases were positive and negative, respectively, for all three markers. No significant correlations were seen for COX-2 and P-gp on Fisher's exact test and Mann-Whitney's test, but a significance was seen on Spearman's rank correlation analysis using the IHC scoring system (P=0.043). These results suggest that P-gp expression is induced by overexpression of COX-2 in canine patients with TCC.     

Phenotypic analysis of hepatic lymphocytes from healthy dogs

Phenotypes of lymphocytes from laparoscopically biopsied liver tissues of eleven healthy beagle dogs were analyzed. The proportion of CD3(+) lymphocytes (T cells), CD3 (-)CD21(+) lymphocytes (B cells) and CD3 (-)CD21(-) lymphocytes (non-T non-B lymphocytes), and the CD4(+)/CD8(+) ratio in the canine hepatic lymphocytes were 54.8 +/- 11.9%, 4.7 +/- 3.1%, 40.7 +/- 13.2%, and 0.33 +/- 0.12, respectively, while those in peripheral blood lymphocytes were 85.4 +/- 6.5%, 9.3 +/- 6.1%, 5.3 +/- 1.8%, and 1.64 +/- 0.36, respectively. These results indicated that the constitution of hepatic lymphocytes quite differed from that of peripheral blood lymphocytes in dogs, and suggested that the regional immunity in canine liver might be specific.     

The relation between the birth process and the condition of the newborn piglet and calf

Fluxes of the anti-parasitic agents, [³H]-ivermectin, [³H]-selamectin and [³H]-moxidectin were studied across non-transfected and transfected canine kidney epithelial monolayers, MDCK II/wt, MDCK II-MDR1, MDCK II-MRP1 and MDCK II-MRP2. All four lines surprisingly expressed significant levels of P-glycoprotein (P-gp), coded for by MDR1, but MDCK II-MDR1 expressed increased levels compared to the other lines. MDCK II-MRP1 and MDCK II-MRP2 expressed increased levels of MRP1 and MRP2 respectively. Fluxes of [³H]-ivermectin, [³H]-selamectin, [³H]-moxidectin, and the P-gp substrates, rhodamine-123 and DiOC₂, were polarized in the basolateral-to-apical (secretory) direction across the four lines. Selected MRP inhibitors used in relevant pharmacological concentrations did not block the secretory fluxes of either [³H]-ivermectin or [³H]-selamectin in either the non-transfected or MRP-transfected lines. In contrast, secretory fluxes of ivermectin and selamectin were inhibited in all four lines by the P-gp inhibitor, verapamil. These data confirm that ivermectin and selamectin are substrates for P-gp in four additional cell lines, but suggest that they are not significant substrates for MRP1 or MRP2 where there is background expression of P-gp. Since this pattern of expression also pertains on the blood-brain barrier, it is unlikely that MRP1 and MRP2 play a significant role in ivermectin and selamectin blood: brain distribution in vivo.     

Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis

BACKGROUND: Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS: Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS: Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS: Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS: Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.     

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.     

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet [DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.     

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.     

Detection of canine interleukin-2 receptors by flow cytometry.

This study describes a method for detecting canine interleukin-2 receptors (IL-2R) by flow cytometry, using human recombinant IL-2 labeled with phycoerythrin (IL-2-PE). Peripheral blood mononuclear cells from four normal dogs were washed, incubated with IL-2-PE, and then washed to remove any unbound IL-2-PE. Flow cytometric analysis of the cells was performed with a 488 nm argon laser while gating on lymphocytes. Cells expressing the IL-2R were identified by their fluorescence as compared to cells stained with an anti-mouse immunoglobulin-G conjugated to phycoerythrin. The average percentage of resting cells expressing the IL-2R was found to be 21%. The addition of unlabeled human recombinant IL-2 to Day 3 phytohemagglutinin (PHA)-stimulated cells reduced the fluorescence intensity four-fold, thereby demonstrating the specificity of IL-2-PE for canine IL-2R. Following stimulation with optimal concentrations of PHA, the percentage of cells expressing the IL-2R increased daily and reached a maximum on Day 3 (76.4%). IL-2R density, as measured by mean fluorescence intensity, also increased and reached maximal levels on Days 2-3 (twenty-fold greater than resting cells). The binding, inhibition, and kinetic experiments provide evidence that human recombinant IL-2-PE is a useful tool for studying canine IL-2R expression. Thus, a one-step direct method for the flow cytometric detection and quantification of the canine IL-2R is now available.     

Evaluation of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture

OBJECTIVE: To assess expression and function of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture. SAMPLE POPULATION: C2 cells maintained in medium lacking IgE for up to 10 passages before being stored at -80 C. PROCEDURE: Cells were thawed, cultured in medium without IgE for 1 to 3 passages, sensitized for 7 days with IgE-rich serum from dogs naturally sensitized to Ascaris suum, and stimulated with antigen Asc S1 from A suum, goat polyclonal anti-canine IgE, or calcium ionophore and phorbol myristate acetate (PMA). Percentage of intracellular beta-hexosaminidase released and concentration of tumor necrosis factor-alpha (TNF-alpha) synthesized after stimulation were determined. Expression of cell-surface IgE receptors was assessed by use of a flow cytometry. RESULTS: Immunologic stimulation (antigen or anti-IgE) failed to induce release or synthesis of detectable amounts of beta-hexosaminidase or TNF-alpha. In contrast, nonimmunologic stimulation (calcium ionophore and PMA) led to release of beta-hexosaminidase (mean +/- SEM maximum release, 23.95+/-1.96%) and synthesis of TNF-alpha (maximum concentration, 34.34+/-2.34 pg/10(6) cells). As revealed by use of flow cytometry, C2 cells expressed surface IgE receptors that bound canine IgE in vitro. CONCLUSIONS: Continuous culture of the canine mastocytoma cell line C2 in medium without exogenous IgE or cytokines and other growth factors resulted in cell-surface expression of nonfunctional IgE receptors. However, C2 cells maintained in continuous culture may still be a useful tool for the evaluation of mast cell responses to nonimmunologic stimulation and IgE receptor differentiation and maturity.     

The effect of glucocorticoids on canine lymphocyte marker expression and apoptosis

BACKGROUND: Glucocorticoids are commonly administered to dogs for the treatment of inflammatory disorders, autoimmunity and cancers such as lymphoma. Despite evidence of clinical efficacy, understanding of the effects of glucocorticoids on cells of the canine immune system is limited. HYPOTHESIS: Glucocorticoids affect the expression of phenotypic markers on canine lymphocytes and induce apoptosis. ANIMALS: Fifteen healthy mixed breed dogs. METHODS: Prospective randomized study. Prednisone was administered orally for 3 days, and cells aspirated from the popliteal lymph node before prednisone administration, and on days 1, 3, 10, 17, 24, and 38, were labeled with antibodies against canine CD3, CD4, CD8alpha, CD18, CD21, CD45, CD45RA, and CD90 molecules, and analyzed by flow cytometry. Additional samples were cultured in media with prednisolone for 24 hours and analyzed by cytometry for marker expression, and by gel electrophoresis for DNA fragmentation. RESULTS: Treatment of dogs with glucocorticoids resulted in reduced (p < or = .05) proportions of CD3 (days 1, 3, 17, and 24), CD4 (days 3 and 10), CD21 (day 1, 3, and 38), CD45RA (day 17) and CD90 (days 1, 10, and 17) expressing lymphocytes, and reduced intensity of CD18 (day 17) and CD45 (day 17 and 24) molecules on nodal lymphocytes. Culture oflymphocytes with prednisolone for 24 hours caused a significant reduction in the expression of all markers (p < or = .05) and DNA fragmentation. CONCLUSIONS AND CLINICAL IMPORTANCE: Glucocorticoids significantly alter the expression of phenotypic markers on canine lymphocytes, and in vitro induce apoptosis. These findings identify potential mechanisms for clinical immunosuppression from glucocorticoid treatment.     

An immunologic investigation of canine eosinophilic bronchopneumopathy

Immunologic variables in dogs with eosinophilic bronchopneumopathy (EBP) have not been extensively evaluated. The aim of this study was to determine immunoglobulin (Ig) concentrations and to perform phenotypic subtyping of lymphocytes in the bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 12 dogs with EBP at the time of diagnosis (TD) and to compare these data with those obtained in healthy dogs, as well as in EBP dogs after antibiotic therapy (TAB) and during corticosteroid treatment (TM). Matched samples of serum and BALF were used to determine Ig concentrations (IgG, IgM, and IgA) by capture enzyme-linked immunosorbent assay (ELISA), from which a secretory index (SI) was calculated. Lymphocyte subpopulations were studied in the BALF and PB by flow cytometry. Log values of BALF IgM and IgA were significantly higher (0.64+/-0.05 and 1.06+/-0.13, respectively) in EBP dogs at TD than in controls and then tended to decrease at TM (0.55+/-0.03 and 1.02+/-0.17, respectively). A calculated SI for IgA was not significantly increased. In the BALF of dogs with EBg the CD4: CD8 was significantly (P < .05) higher (22.6+/-30.3) than in controls (3.2+/-1.9), due to significantly higher CD4+ T cells and lower CD8+ T cells. At TM, the BALF T-cell percentages returned to normal (2.4+/-0.6). We propose that the influx of eosinophils into the airway of dogs with EBP is at least in part mediated by cytokines derived from CD4+ T cells. Further studies of canine cytokines and chemokines will help determine whether canine EBP involves type I hypersensitivity mechanisms regulated by Th2 lymphocytes.     

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).     

Flow cytometric analysis of cellular immune responses in dogs experimentally infected with a North American isolate of Leishmania infantum

Canine leishmaniasis caused by Leishmania infantum is endemic in the foxhound population in North America. Studies of canine leishmaniasis in the Mediterranean basin indicate a role for both CD4+ and CD8+ lymphocytes with clinical illness and in asymptomatic dogs. Limited information is available on the strain of L. infantum infecting foxhounds in North America. The present study investigated changes in cellular immune responses in dogs experimentally infected with 1x10(7) (low dose, LD; N=4) or 2x10(8) (high dose, HD; N=4) promastigotes of a United States isolate of L. infantum and control dogs (N=2) for 72 weeks. Density gradient separation was used to enrich for peripheral blood lymphocytes from canine blood. Lymphocyte subsets (CD4+ and CD8+) were quantified by flow cytometric analysis. Lymphocyte population expression levels over the course of the present study were compared to clinical status of the dog and antibody responses in infected and control dogs. No significant differences (P>0.05) were observed in either CD4+ or CD8+ lymphocyte expression in of the groups over the experimental period. This study suggests that the cellular immune responses to North American L. infantum in experimentally infected dogs may differ from other strains of L. infantum.     

Determination of Canine Factor VIII-related Antigen Using Commercial Antihuman F VIII Serum

Factor VIII-related antigen (F VIII:AGN) in 21 canine plasma samples was assayed by immunoelectrophoresis using a rabbit anticanine F VIII serum prepared from a canine F VIII concentrate and a commercial rabbit antihuman F VIII serum. A good correlation existed (r value 0.916) between the antigen levels obtained using the two sera. In normal dogs the plasma F VIII:AGN level was 95 +/- 39% (Mean +/- SD) compared to 175 +/- 40% in dogs with severe hemophilia A and 17 +/- 15% in dogs with von Willebrand's disease. It was concluded that there was sufficient cross reactivity between canine F VIII and commercial rabbit antihuman F VIII serum to make the latter useful in the differential diagnosis of F VIII deficiencies in the dog.     

Cellular characterization of multidrug resistance P-glycoprotein, alpha fetoprotein, and neovascular endothelium-associated antigens in canine hepatocellular carcinoma and cirrhotic liver

P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene (MDR-1); alpha fetoprotein (AFP); and vascular endothelium-associated antigens are well-known markers for human and canine hepatic diseases. We obtained liver tissues from 5 dogs with hepatocellular carcinoma (HCC) and 12 dogs with cirrhosis, and we performed histopathologic and immunohistochemical evaluations using anti-P-gp, anti-AFP, anti-CD31, and anti-CD34 antibodies. P-gp was expressed at higher levels in HCC than in cirrhotic livers ( P < .01), and was most commonly localized in biliary canaliculi and small ductuli. AFP was localized mainly in the cytoplasm in HCC ( P < .01) and in a few cases of cirrhosis. In both HCC and cirrhosis, the AFP-positive cells were morphologically similar to normal hepatocytes and showed an even cytoplasmic distribution of AFP. The endothelial markers CD31 and CD34 were used to investigate vascular distribution. CD31 was expressed strongly in the portal area and parenchyma in HCC, but it was rarely observed in the parenchyma in cirrhosis. CD34 expression could not be detected in both HCC and cirrhosis. This study constitutes the first comprehensive study of P-gp, AFP, and endothelial markers in canine HCC and cirrhosis. The importance of these markers in HCC and cirrhosis in dogs was demonstrated and provides a more accurate basis for a definitive diagnosis of HCC and cirrhosis in dogs.     

Flow cytometric analysis of canine umbilical cord blood lymphocytes

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.     

Assessment of antiepileptic drugs as substrates for canine P-glycoprotein

OBJECTIVE: To determine whether antiepileptic drugs (AEDs) are substrates for canine P-glycoprotein (P-gp). Sample Population-OS2.4/Doxo cells (canine osteosarcoma cells induced via exposure to doxorubicin to highly express P-gp). PROCEDURES: Competitive inhibition of rhodamine 123 efflux from OS2.4/Doxo cells was used to determine whether AEDs were substrates for canine P-gp. Flow cytometry was used to quantify mean fluorescence intensity of cells treated with rhodamine alone and in combination with each experimental drug. RESULTS: Known P-gp substrate drugs ivermectin and cyclosporin A altered rhodamine efflux by 90% and 95%, respectively. Experimental drugs altered rhodamine efflux weakly (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or not at all (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide). CONCLUSIONS AND CLINICAL RELEVANCE: At clinically relevant doses, it appeared that AEDs were weak substrates (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or were not substrates (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide) for canine P-gp. Therefore, it seems unlikely that efficacy of these AEDs is affected by P-gp expression at the blood-brain barrier in dogs.