Differential expression of CD45 on canine microglial cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.     

Effects of bilateral arytenoid cartilage stenting on canine laryngeal resistance ex vivo

Objective: To evaluate the effect of Nitinol stents for bilateral arytenoid lateralization on canine laryngeal resistance. Study Design: Ex vivo experimental study. Animals: Canine cadaver larynges (n=7). Methods: Laryngeal resistance was calculated in all specimens with the epiglottis in open and closed positions. Bilateral arytenoid stenting was performed, rima glottidis width measured, and laryngeal resistance calculated. The effects of stenting on laryngeal resistance were evaluated by repeated measures ANOVA. Results: Calculated laryngeal resistance in the 3 stented groups, 2 cm (0.034±0.059 cmH₂O/L/s), 3 cm (0.034±0.059 cmH₂O/L/s), and 4 cm (0.034±0.059 cm H₂O/L/s), was significantly decreased versus the control (unstented) group (0.947±0.624 cmH₂O/L/s; P=.0098) with an epiglottis in the normal position. Calculated laryngeal resistance in the 3 stented groups, 2 cm (43.407±17.348 cm H₂O/L/s), 3 cm (70.659±34.705 cmH₂O/L/s), and 4 cm (92.637±44.509 cm H₂O/L/s), was significantly increased versus the control (unstented) group (29.561±14.499 cm H₂O/L/s) (P=.0185) with an epiglottis in the closed position. The width of the rima glottidis correlated with the size of the stent (r=0.95, P<.001). Conclusions: Bilateral arytenoid stenting significantly reduced calculated laryngeal resistance with an open epiglottis. Stenting resulted in a significant increase in laryngeal resistance versus the control with a closed epiglottis. Use of bilateral arytenoid stenting in clinical cases of laryngeal paralysis may provide an adequate decrease in open‐epiglottis airway resistance to alleviate clinical signs, while increasing closed‐epiglottis airway resistance. This could potentially lead to a decrease in the risk of postoperative aspiration pneumonia.     

Staphylococccus pseudintermedius surface proteins SpsD and SpsO mediate adherence to ex vivo canine corneocytes

The Gram-positive bacterium Staphylococcus pseudintermedius is regarded as the major cause of canine bacterial pyoderma. Despite its clinical importance, there is only very limited knowledge about the pathogenesis of S. pseudintermedius infection and the specific bacterial virulence factors involved in causing disease. Using a whole-genome approach, we have previously identified 18 predicted cell-wall-anchored surface proteins representing possible virulence factors in a clinical isolate of S. pseudintermedius (strain ED99). They were designated S. pseudintermedius surface proteins A-R (SpsA-SpsR). The present study tested three of the putative Sps proteins (SpsD, SpsL and SpsO) for their ability to mediate adherence of bacteria to canine corneocytes. The three proteins were expressed on the surface of the nonpathogenic surrogate host Lactococcus lactis, a Gram-positive bacterium that does not adhere to canine corneocytes. Adherence assays were performed using corneocytes from different healthy canine donors (n = 5), and bacterial cells were quantified using computerized image analysis. Two of the proteins, SpsD and SpsO, mediated adherence of L. lactis to canine corneocytes, suggesting that they contribute to S. pseudintermedius pathogenesis and may represent novel therapeutic targets to combat infection.     

The role of rituximab in the treatment of canine lymphoma: an ex vivo evaluation

Targeting the CD20 receptor that is common to many B-cell Non-Hodgkin's Lymphoma subtypes in people, rituximab is a chimeric monoclonal antibody which has significantly improved disease-free survival rates compared with the use of cytotoxic agents alone. This study evaluated ex vivo canine B cell binding and depletion by rituximab with flow cytometric technique as possible proof of concept for treatment of canine lymphoma. Despite immunohistochemistry supporting CD20 expression, rituximab did not bind or deplete canine B cells and it is unlikely that it will be added to the armamentarium of treatment options for canine lymphoma.     

Characterization of canine adenovirus type 1 isolated from American black bears

Viruses recovered from tissues taken at necropsy from American black bears were examined by use of immunofluorescence with polyclonal and monoclonal antibodies, virus neutralization with monoclonal antibodies, and restriction endonuclease analyses of the viral genomes. With these techniques, viruses were determined to be canine adenovirus type 1. Seronegative dogs that were inoculated with the virus had clinical signs typical of infectious canine hepatitis, suggesting that the virus, which was virulent for bears, was not a vaccinal strain, but a wild strain of canine adenovirus type 1.     

Characterization of neuron-like cells derived from canine bone marrow stromal cells

Regenerative therapy using bone marrow stromal cells (BMSCs) has begun to be clinically applied in humans and dogs for neurological disorders such as spinal cord injury. Under appropriate conditions in vitro, BMSCs differentiate into neuronal cells, which may improve the effects of regenerative therapy. In this study, we evaluated canine neuron-like cells (NLCs) derived from BMSCs. We speculated on their suitability for neuro-transplantation from the point of view of their morphological features, long-term viability, abundant availability, and ability to be subcultured. Canine NLCs were differentiated as follows: third-passage BMSCs were maintained in pre-induction medium containing 2-mercaptoethanol and dimethylsulfoxide for 5 h, and then cells were transferred to neuronal induction medium containing fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, dibutyryl cyclic AMP, and isobutylmethylxanthine for 7 or 14 days. Canine NLCs fulfilled the transplantation criteria and expressed markers of both immature neurons (nestin, 84.7 %) and mature neuronal cells (microtubule-associated protein-2, 95.7 %; βIII-tubulin protein, 12.9 %; glial fibrillary acidic protein, 9.2 %). These results suggest that canine BMSCs can be induced to differentiate into neuronal cells and may be suitable for neuro-transplantation. This study may provide information for improving cellular therapy for neurological diseases.     

Chlortetracycline modulates acute phase response of ex vivo perfused pig livers, and inhibits TNF-alpha secretion by isolated Kupffer cells

Tetracyclines have been shown to have anti-inflammatory effects in addition to their antimicrobial action. We investigated the effects of in vivo administration of chlortetracycline (CTC) on ex vivo perfused pig livers. The retention and clearance of Salmonella choleraesuis, production of acute phase proteins C-reactive protein (CRP), and haptoglobin (HPG) by whole livers were studied. The in vitro modulation by CTC of TNF-alpha secretion by pig Kupffer cells (KC) was also studied. Pigs were dosed orally with CTC for three days, and given injections of Salmonella LPS 24 h before removal of the liver. Salmonella retention and clearance by livers of pigs given CTC was lower than by control livers (p < 0.01 and p < 0.05, respectively). We demonstrated an increase of CRP and HPG by livers from control pigs after a three-hour perfusion while pigs from CTC pretreated pigs varied in this response. Further, CTC decreased the secretion of TNF-alpha by cultured KC incubated in vitro with LPS. Modulation of TNF-alpha production by CTC suggests a potential for attenuating the inflammatory response. However, this possible beneficial action of CTC was accompanied by a significant decline in the antimicrobial effect of the liver.     

In vitro and ex vivo inhibition of canine cyclooxygenase isoforms by robenacoxib: A comparative study

In vitro whole blood canine assays were used to quantify the inhibitory actions of the novel non-steroidal anti-inflammatory drug (NSAID) robenacoxib on the cyclooxygenase (COX) isoenzymes, COX-1 and COX-2, in comparison with other drugs of the NSAID class. COX-1 activity was determined by measuring serum thromboxane (Tx)B₂ synthesis in blood samples allowed to clot at 37 °C for 1 h. COX-2 activity was determined by measuring prostaglandin (PG)E₂ synthesis in blood samples incubated at 37 °C for 24 h in the presence of lipopolysaccharide. The rank order of selectivity for inhibition of COX-2 versus COX-1 (IC₅₀ COX-1:IC₅₀ COX-2) for veterinary drugs was highest with robenacoxib (128.8) compared to deracoxib (48.5), nimesulide (29.2), S+ carprofen (17.6), meloxicam (7.3), etodolac (6.6), R? carprofen (5.8) and ketoprofen (0.88). Selectivity expressed as the clinically relevant ratio IC₂₀ COX-1:IC₈₀ COX-2 was highest for robenacoxib (19.8) compared to deracoxib (2.3), S+ carprofen (2.5), R? carprofen (2.1), nimesulide (1.8), etodolac (0.76), meloxicam (0.46) and ketoprofen (0.21).An in vivo pharmacokinetic ex vivo pharmacodynamic study in the dog established dosage and concentration–effect relationships for single oral doses of robenacoxib over the dosage range 0.5–8.0 mg/kg. Values of C_max and AUC were linearly related to dosage over the tested range. Robenacoxib did not inhibit serum TxB₂ synthesis (COX-1) ex vivo at dosages of 0.5–4.0 mg/kg and produced only transient inhibition (at the 1 h and 2 h sampling times) at the 8 mg/kg dosage. All dosages of robenacoxib (0.5–8 mg/kg) produced marked, significant and dose related inhibition of PGE₂ synthesis (COX-2) ex vivo.The data demonstrate that in the dog robenacoxib is a highly selective inhibitor of the COX-2 isoform of COX, and significantly inhibits COX-2 and spares COX-1 in vivo when administered orally over the dosage range 0.5–4.0 mg/kg.     

The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies

Even though intra‐articular injections play an important role in the treatment of joint‐related lameness in horses, little is known about pharmacokinetic properties of substances used. Therefore, an ex vivo model for pharmacokinetic studies was developed using distal forelimbs of slaughtered horses. The extremity was perfused with gassed Tyrode solution for up to 8 h. Tissue viability was confirmed by measurements of glucose consumption, lactate production, and lactate dehydrogenase activity in the perfusate. Standard criteria for tissue viability had been determined in preliminary experiments (n = 11), which also included histological examinations of the joint capsule. As the model's first implementation, the articular efflux rate of betamethasone (BM), administered as BM disodium phosphate intra‐articularly to the fetlock joint (4 mg BM/joint), was investigated. The concentration of BM in the venous perfusate of the radial vein was measured by means of high‐performance liquid chromatography. The average BM efflux rate per minute was calculated to be 5.1 μg/min with values ranging from 9 μg/min to 2.9 μg/min. 7.5 h after i.a. application, 2.3 mg BM had left the joint via the radial vein. Using this inexpensive setup, the presented model allows studying a variety of pharmacological topics without the ethical limitations of animal studies.     

Predicting likelihood of in vivo chemotherapy response in canine lymphoma using ex vivo drug sensitivity and immunophenotyping data in a machine learning model

We report a precision medicine platform that evaluates the probability of chemotherapy drug efficacy for canine lymphoma by combining ex vivo chemosensitivity and immunophenotyping assays with computational modelling. We isolated live cancer cells from fresh fine needle aspirates of affected lymph nodes and collected post‐treatment clinical responses in 261 canine lymphoma patients scheduled to receive at least 1 of 5 common chemotherapy agents (doxorubicin, vincristine, cyclophosphamide, lomustine and rabacfosadine). We used flow cytometry analysis for immunophenotyping and ex vivo chemosensitivity testing. For each drug, 70% of treated patients were randomly selected to train a random forest model to predict the probability of positive Veterinary Cooperative Oncology Group (VCOG) clinical response based on input variables including antigen expression profiles and treatment sensitivity readouts for each patient's cancer cells. The remaining 30% of patients were used to test model performance. Most models showed a test set ROC‐AUC > 0.65, and all models had overall ROC‐AUC > 0.95. Predicted response scores significantly distinguished (P < .001) positive responses from negative responses in B‐cell and T‐cell disease and newly diagnosed and relapsed patients. Patient groups with predicted response scores >50% showed a statistically significant reduction (log‐rank P < .05) in time to complete response when compared to the groups with scores <50%. The computational models developed in this study enabled the conversion of ex vivo cell‐based chemosensitivity assay results into a predicted probability of in vivo therapeutic efficacy, which may help improve treatment outcomes of individual canine lymphoma patients by providing predictive estimates of positive treatment response.     

Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture

A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 microM indomethacin, 10 microg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental). demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 +/- 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.     

Characterization of stages of Hepatozoon americanum and of parasitized canine host cells

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in "onion skin" cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a "tail" in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.     

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.