Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes

OBJECTIVE: To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes. SAMPLE POPULATION: Nodal lymphocytes from 21 clinically normal dogs. PROCEDURES: Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors. RESULTS: Expression of P-gp was (mean +/- SEM) 50.62 +/- 13.39 (n = 21) and that of MRP was 2.16 +/- 0.25 (13). Functional activity was 1.27 +/- 0.06 (n = 21) for P-gp and both inhibitors and 21.85 +/- 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated. CONCLUSIONS AND CLINICAL RELEVANCE: Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.     

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.     

Selamectin is a potent substrate and inhibitor of human and canine P-glycoprotein

The transport of the antiparasitic agents, ivermectin, selamectin and moxidectin was studied in human intestinal epithelial cell monolayers (Caco-2) and canine peripheral blood lymphocytes (PBL). Both models expressed the mdr1-coded 170 kDa ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp). Fluxes of the P-gp substrate rhodamine-123 (Rh-123) across Caco-2 monolayers showed that ivermectin and selamectin acted as potent P-gp inhibitors with IC50 values of 0.1 microm. In contrast, moxidectin was a weaker P-gp inhibitor with an IC50 of 10 microm. The transport of radiolabelled ivermectin, selamectin and moxidectin through Caco-2 monolayers showed that ivermectin, selamectin and moxidectin were P-gp substrates with secretory/absorptive ratios of 7.5, 4.7 and 2.6 respectively. Secretory transport of [3H]-ivermectin and [3H]-selamectin was blocked by the P-gp inhibitor, verapamil. Ivermectin and selamectin inhibited the efflux of Rh-123 from PBL and the concentration of inhibition was similar to that of verapamil. In contrast, moxidectin did not have a significant effect on Rh-123 efflux from PBL. The data suggest that ivermectin and selamectin are potent P-gp substrates, while moxidectin is a weak one.     

Canine epilepsy: what can we learn from human seizure disorders?

Epilepsy is a common neurological disorder in both dogs and humans. It is refractory to therapy in approximately one-third of canine patients, and even with the advent of new antiepileptic drugs for humans, appropriate treatment options in dogs remain limited. The pathogenesis and pathophysiology of epilepsy is being studied extensively in both human patients and rodent models of experimental epilepsy at the cellular and molecular level, but very little is known about the aetiologies of epilepsies in dogs. In this review, canine epilepsy will be discussed with reference to the human epilepsies and experimental epilepsy research. There is much work to be done in order to classify canine seizure types and breed-specific epileptic syndromes, particularly with reference to electroencephalographic abnormalities and possible genetic abnormalities. The review considers the appropriate use of antiepileptic drugs: phenobarbitone and potassium bromide are effective in most canine patients, although dosing regimes need to be carefully tailored to the individual, with serum concentration measurement. However, a significant proportion of patients remains refractory to these drugs. Work is currently underway to test the efficacy of newer antiepileptic drugs in the treatment of canine epilepsy, and preliminary data suggest that human drugs such as levetiracetam and gabapentin are of benefit in dogs with refractory epilepsy.     

Pharmacokinetics of the cytochrome P-450 substrates phenytoin, theophylline, and diazepam in healthy Greyhound dogs

The purpose of this study was to determine the pharmacokinetics of phenytoin, theophylline, and diazepam in six healthy Greyhound dogs. Additionally, the pharmacokinetics of the diazepam metabolites, oxazepam and nordiazepam, after diazepam administration was determined. Phenytoin sodium (12 mg/kg), aminophylline (10 mg/kg), and diazepam (0.5 mg/kg) were administered IV on separate occasions, and blood was collected at predetermined time points for the quantification of plasma drug concentrations by fluorescence polarization immunoassay (phenytoin, theophylline) or mass spectrometry (diazepam, oxazepam, and nordiazepam). The terminal half-life was 4.9, 9.2, and 1.0 h, respectively, for phenytoin, theophylline, and diazepam, and 6.2 and 2.4 h for oxazepam and nordiazepam after IV diazepam. The clearance was of 2.37, 0.935, and 27.9 mL · min/kg, respectively, for phenytoin, theophylline, and diazepam. The C(MAX) was 44.7 and 305.2 ng/mL for oxazepam and nordiazepam, respectively, after diazepam administration. Temazepam was not detected above 5 ng/mL in any sample after IV diazepam.     

Therapeutic implications of the MDR-1 gene

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. P-glycoprotein (P-gp), the product of the MDR1 (ABCB1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. A number of clinically relevant, structurally and functionally unrelated drugs are substrates for P-gp. P-gp is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs. A number of MDR1 polymorphisms have been described in human patients, some of which result in altered drug pharmacokinetics and susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and others. An MDR1 polymorphism in herding breed dogs, including collies and Australian shepherds, has been demonstrated to be the cause of ivermectin sensitivity in these breeds. Recent evidence suggests that this polymorphism, a 4-bp deletion mutation, results in increased susceptibility to the toxicity of several drugs in addition to ivermectin. Furthermore, data in rodent models suggest that P-gp may play an important role in regulating the hypothalamic-pituitary-adrenal axis.     

Fumagillin, a new P-glycoprotein-interfering agent able to modulate moxidectin efflux in rat hepatocytes

We have tested the ability of two compounds licensed in veterinary medicine: fumagillin and diminazene diaceturate to increase intracellular moxidectin quantity in rat hepatocytes. These compounds significantly increased the quantity of 14C-moxidectin (expressed as area under the time curve concentrations) in cultured rat hepatocytes by 44% and 65% for diminazene and fumagillin treatments respectively. In addition, we have tested these drugs for their interference with P-glycoprotein (P-gp) function in porcine kidney epithelial cells transfected with murine mdr1a (Mdr1a-LLCPK1). We examined the intracellular accumulation of rhodamine 123 (Rho 123) as a functional test to evaluate the effects of these two drugs on P-gp activity. In this model, only fumagillin led to a marked intracellular accumulation of Rho 123. After transforming the data to express the results as a percentage of the accumulation in the presence of the P-gp inhibitor valspodar (VSP), the maximal Rho 123 accumulation was 47% of that with VSP for 100 microm fumagillin. The EC50, the concentration needed to determine 50% of the maximal effect was 34 microm. Fumagillin interacts with P-gp function and appears as a promising compound among registered drugs available, which may optimize the therapeutic use of macrocyclic lactones (MLs).     

P-glycoprotein-mediated transport of oxytetracycline in the Caco-2 cell model

ATP-dependent drug transporters such as P-glycoprotein (P-gp), multi-drug resistance associated protein (MRP2) and breast cancer resistant protein (BCRP) are expressed at the brush border membrane of enterocytes. These efflux transporters excrete their substrates, among other various classes of antibiotics, into the lumen thus reducing net absorption as indicated by a low bioavailability after oral administration. Oxytetracycline (OTC) has been used for decennia in veterinary medicine for its extensive spectrum of antimicrobial activity. A major limitation has been, and still remains, its low bioavailability following oral administration. The present study aimed to investigate to what extent this low bioavailability is attributable to the fact that OTC is a substrate for one or more efflux transporters. As an experimental model to study the transmembrane transport of OTC, differentiated Caco-2 cells grown as monolayers on permeable supports were used. With this model it was shown that the secretion of OTC is slightly higher than its absorption. PSC833, a potent inhibitor of P-gp, decreased the secretion of OTC without affecting its absorption, while the MRP-inhibitor MK571 did not exert any effect. These data indicate that OTC is a substrate for P-gp. The affinity of OTC to these transporters seems to be rather low, as suggested by the low efflux ratio of 1:1.3. In competition experiments, OTC decreased the effluxes of other P-gp substrates such as Rhodamine123 and ivermectin. These findings are of clinical relevance, as they clearly indicate potential drug-drug interactions at the level of P-gp-mediated drug transport.     

Establishment of a cell line for assessing drugs as canine P‐glycoprotein substrates: proof of principle

P‐glycoprotein (P‐gp), encoded by the ABCB1 (MDR1) gene, dramatically impacts drug disposition. P‐gp is expressed in the intestines, biliary canaliculi, renal tubules, and brain capillaries where it functions to efflux substrate drugs. In this capacity, P‐gp restricts oral absorption, enhances biliary and renal excretion, and inhibits central nervous system entry of substrate drugs. Many drugs commonly used in veterinary medicine are known substrates for canine P‐gp (vincristine, loperamide, ivermectin, others). Because these drugs have a narrow therapeutic index, defective P‐gp function can cause serious adverse drug reactions due to enhanced brain penetration and/or decreased clearance. P‐gp dysfunction in dogs can be intrinsic (dogs harboring ABCB1‐1Δ) or acquired (drug interactions between a P‐gp inhibitor and P‐gp substrate). New human drug candidates are required to undergo assessment for P‐gp interactions according to FDA and EMA regulations to avoid adverse drug reactions and drug–drug interactions. Similar information regarding canine P‐gp could prevent adverse drug reactions in dogs. Because differences in P‐gp substrates have been documented between species, one should not presume that human or murine P‐gp substrates are necessarily canine P‐gp substrates. Thus, our goal was to develop a cell line for assessing drugs as canine P‐gp substrates.     

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

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.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.     

Pharmacokinetics of single-dose intravenous levetiracetam administration in normal dogs

Objective: To determine plasma pharmacokinetics of levetiracetam after a single intravenous dose (60 mg/kg) in normal dogs using a high‐performance liquid chromatography assay validated for canine plasma. Design: Pharmacokinetic study. Setting: A university‐based canine research facility. Animals: Six healthy adult dogs. Interventions: Intravenous drug administration, multiple blood sample procurement. Measurements and main results: There were no obvious adverse effects associated with the intravenous (IV) bolus administration of levetiracetam in any of the dogs. Plasma levetiracetam concentrations remained above or within the reported therapeutic range for humans (5–45 μg/mL) for all dogs, for all time periods evaluated. Mean and median (in parentheses) values for pharmacokinetic parameters included the following: maximum plasma concentration, 254 μg/mL (254 μg/mL); half‐life, 4.0 hours (4.0 hours); volume of distribution at steady state, 0.48 L/kg (0.48 L/kg); clearance, 1.4 mL/kg/min (1.5 mL/kg/min); and median residence time, 6.0 hours (6.0 hours). Conclusions: In normal dogs, a 60 mg/kg IV bolus dose of levetiracetam is well tolerated and achieves plasma drug concentrations within or above the therapeutic range reported for humans for at least 8 hours after administration. Based on the favorable pharmacokinetics and tolerability demonstrated for IV levetiracetam in this study, in addition to previously demonstrated efficacy of oral levetiracetam, IV levetiracetam may be a useful treatment option for emergency management of canine seizure activity.     

Canine ABCB1 and macrocyclic lactones: heartworm prevention and pharmacogenetics

The impact of drug transporters on drug pharmacokinetics and pharmacodynamics has been increasingly recognized in recent years. P-glycoprotein (P-gp), the product of the ABCB1 (formerly MDR1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. P-gp is expressed by a variety of normal tissues, including the intestines, brain capillary endothelial cells, renal tubular cells, and biliary canalicular cells, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs and enhances their excretion from the body. Many drugs used in veterinary medicine are substrates for P-gp, including many chemotherapeutic agents and macrocyclic lactones (avermectins and milbemycin). A 4-base pair deletion mutation in the ABCB1 gene occurs in many herding breed dogs, including collies, Australian shepherds, and Shetland sheepdogs. The mutation (ABCB1-1Delta) renders affected animals extremely susceptible to toxicosis induced by substrate drugs, such as the macrocyclic lactones at doses well below those tolerated by dogs with the wild-type ABCB1 gene. However, at the manufacturer's recommended dose, all FDA-approved heartworm preventive products marketed in the United States are safe, even for dogs with the ABCB1 mutant/mutant genotype.     

Expression of P-glycoprotein and breast cancer resistance protein in three cases of canine lymphoma showing drug resistance

In canine lymphoma, drug resistance is the major factor hindering treatment. In this study, we performed immunohistochemical examination of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which are considered as transporters related to multidrug resistance in three recurrent canine lymphomas. All cases were negative for both transporters before anticancer drug administration, but became positive after this administration. The expression was confirmed in capillary endothelial cells, such as in brain capillaries acting as the blood-brain barrier (BBB). It is suggested that both transporters expressed on capillary endothelial cells in lymphoma tissue may inhibit the spread of anticancer drugs into tumor tissues from blood, the same as the BBB. Therefore, capillary endothelial cells could act as a blood-tumor barrier, which might be involved in drug resistance in canine lymphoma.     

Intestinal drug transport: ex vivo evaluation of the interactions between ABC transporters and anthelmintic molecules

The family of ATP‐binding cassette (ABC) transporters is composed of several transmembrane proteins that are involved in the efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in human and livestock antiparasitic therapy. The aim of the work reported here was to assess the interaction between three different anthelmintic drugs with substrates of the P‐glycoprotein (P‐gp) and the breast cancer resistance protein (BCRP). The ability of ivermectin (IVM), moxidectin (MOX) and closantel (CST) to modulate the intestinal transport of both rhodamine 123 (Rho 123), a P‐gp substrate, and danofloxacin (DFX), a BCRP substrate, across rat ileum was studied by performing the Ussing chamber technique. Compared to the controls, Rho 123 efflux was significantly reduced by IVM (69%), CST (51%) and the positive control PSC833 (65%), whereas no significant differences were observed in the presence of MOX (30%). In addition, DFX efflux was reduced between 59% and 72% by all the assayed drug molecules, showing a higher potency than that observed in the presence of the specific BCRP inhibitor pantoprazole (PTZ) (52%). An ex vivo intestinal transport approach based on the diffusion chambers technique may offer a complementary tool to study potential drug interactions with efflux transporters such as P‐gp and BCRP.     

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.     

The in vitro effects of piroxicam and meloxicam on canine cell lines

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.     

Aetiology and long-term outcome of juvenile epilepsy in 136 dogs

The aetiology and outcome of dogs with juvenile-onset seizures were investigated. One hundred and thirty-six dogs whose first seizure occurred before the age of one year were investigated. One hundred and two dogs were diagnosed with idiopathic epilepsy (IE), 23 with symptomatic epilepsy (SE), nine with reactive seizures (RS) and two with probable symptomatic epilepsy (pSE). The outcome was known in 114 dogs; 37 per cent died or were euthanased as a consequence of seizures. The mean survival time of this population of dogs was 7.1 years. Factors that were significantly associated with survival outcome included the diagnosis of SE and the number of antiepileptic drugs (AEDs) used before investigation. The use of one AED before investigation and a diagnosis of SE were associated with a negative outcome, whereas receiving no AED medications before referral was associated with a longer survival. For dogs with IE, survival time was shortened if the dog was a border collie or with a history of status epilepticus;receiving no AEDs before referral in the IE group was associated with a positive outcome. Seizure-free status was achieved in 22 per cent of dogs diagnosed with IE. While the survival times were longer than previously reported in canine epilepsy, similar remission rates to those reported in childhood epilepsy, where a 70 per cent remission rate is documented, were not seen in the canine juvenile population.     

Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells

BackgroundCancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function.MethodsThese studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines.ResultsWe found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells.ConclusionThese results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.