Pax5 immunostaining in paraffin-embedded sections of canine non-Hodgkin lymphoma: a novel canine pan pre-B- and B-cell marker.

The Pax5 gene encodes the B-cell specific activator protein (BSAP), a member of the highly conserved paired box (PAX)-domain family of transcription factors and a key regulator in the development and differentiation of B-cells. Pax5 serves as a valuable B-cell marker in the classification of human lymphoma patients as it is restricted to lymphomas of B-cell lineage. In dogs, detection of Pax5 protein in lymphoma tissue has not been reported. Therefore, we have investigated the expression and detection of BSAP using a monoclonal anti-Pax5 antibody (anti-BSAP, clone 24) in canine lymphoma tissue samples to evaluate its diagnostic relevance as a B-cell marker. A series of 25 lymph nodes from 23 canine non-Hodgkin lymphoma patients, a reactive canine lymph node, and a normal non-reactive canine lymph node, were evaluated. All B-cell non-Hodgkin lymphomas (15) were found to express Pax5 protein. In addition, there was a strong correlation between Pax5 and CD79a expression. Three CD3 positive and five CD3 and CD79a positive lymphomas were immunophenotypically negative for anti-Pax5, indicating a T-cell lineage. In conclusion, anti-Pax5 antibody may offer an excellent B-cell marker in canine lymphomas.

The desmopressin stimulation test in dogs with Cushing's syndrome.

Desmopressin is a synthetic analogue of the hypothalamic peptide vasopressin and binds to specific pituitary vasopressin (V3) receptors. The V3-receptor is overexpressed in pituitary corticotrope tumors and the injection of desmopressin induces a marked ACTH and cortisol release in human patients with pituitary- (PDH), but not adrenal tumor (AT) dependent hyperadrenocorticism. In this prospective study, we investigated the effects of desmopressin on serum cortisol levels in 80 dogs suspected of Cushing's syndrome. The aim was to find a sensitive and specific test to exclude AT. According to standard tests the dogs were divided into 3 groups (group 1=other disease, n=27; group 2=PDH, n=46; group 3=AT, n=7). Desmopressin was injected as an i.v. bolus of 4microg and serial blood samples were collected before and after 30, 60 and 90min. Desmopressin significantly stimulated cortisol release in dogs with PDH (median 51%, range -24 to 563%; p<0.0001), whereas no increase was seen in dogs with AT (median -12%, range -44 to 5%; p=0.063) and in controls (median +7%, range -36 to 196%; p=0.131). Using a cut off value of 10% increase over baseline, it was possible to exclude AT in 75% of patients. The results of this study suggest that the desmopressin test could be a useful tool in differentiating pituitary from adrenal dependent Cushing's syndromes. Additional dogs with adrenocortical tumor must be tested in order to recommend its use in clinical practice.

Expression of vascular endothelial growth factor and its receptors in canine lymphoma.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect VEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with diffuse cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGF and VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogeneous lymphoproliferative tumour. Individual differences in VEGF and VEGFR expression must be taken into account when VEGF and VEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs.

Feline Temporal Lobe Epilepsy: Review of the Experimental Literature.

Accumulating evidence suggests that epileptic seizures originating from the temporal lobe (TL) occur in cats. Typically, affected animals have clinically focal seizures with orofacial automatisms including salivation, facial twitching, lip smacking, chewing, licking, and swallowing. Motor arrest and autonomic and behavioral signs also may occur. Many affected cats have magnetic resonance imaging (MRI) changes within the hippocampus or histopathologically confirmed hippocampal sclerosis or necrosis. From the 1950s to the 1980s, cats frequently were used as animal models for neurophysiological experiments and electrophysiological studies, from which important basic knowledge about epilepsy originated, but which has been rarely cited in clinical veterinary studies. These studies were reviewed. Experimental research on cats showed the widespread anatomical connections among TL structures. The ictal clinical signs originating from the hippocampus, amygdala, or lateral temporal cortex are similar, because of their dense interconnections. The ictal signs can be divided into autonomic, somatic, and behavioral. For research purposes, a 6-stage system was established, reflecting the usual sequential progression from focal to generalized seizure: attention response (1), arrest (2), salivation, licking (3), facial twitching (4), head turning or nodding (5), and generalized clonic convulsions (6). Knowledge of this data may help in recognizing low-stage (stage 1 or stage 2) epileptic seizures in clinical practice. Early experimental research data are in accordance with recent clinical observations regarding ictal clinical signs of TL epileptic seizures in cats. Furthermore, the research data supports the idea that TL epilepsy represents a unique clinical entity with a specific seizure type and origin in cats.

Immunophenotype of Peripheral Blood Lymphocytes in Dogs with Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is common in dogs. Despite the known importance of intestinal lymphocytes in its pathogenesis, little is known about the role of peripheral blood lymphocytes (PBLs) in IBD. OBJECTIVES: The aims of this study were (1) comparison of PBLs analyzed by flow cytometry (FCM) in IBD dogs and healthy controls and (2) comparison of PBLs in IBD dogs at the time of diagnosis and in dogs in clinical remission. ANIMALS: Whole blood samples of 19 IBD dogs at the time of diagnosis and blood samples of 6 dogs in clinical remission were collected. Ten healthy dogs served as controls. METHODS: In this prospective observational study, PBLs were analyzed with multicolor FCM by staining with a panel of anticanine and cross-reactive monoclonal antibodies against T- and B-cell differentiation antigens, including CD45, CD3, CD4, CD8α, CD8ß, TCRαß, TCRγδ, CD79αcy, and CD21. RESULTS: The IBD patients' PBLs had significantly decreased percentages of TCRγδ+ T lymphocytes (median: healthy dogs, 3.32; IBD dogs, 0.97; P = 0.03) and CD21+ B cells (median: healthy dogs, 27.61; IBD dogs, 17.26; P = 0.04). There were no significant differences in PBLs between pretreatment and follow-up samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The differences between PBLs in healthy and IBD dogs analyzed by FCM indicate an imbalance of lymphocytes with different immunologic functions and emphasize the potential value of this technique in a larger cohort of dogs. The PBLs did not differ between IBD dogs before treatment and clinically well-controlled dogs after treatment.

Spatial properties of nociceptor sensitization following heat injury of the skin.

A brief noxious heat stimulus applied to half of the receptive field of each cutaneous nociceptor resulted in most cases in the enhanced responsiveness of that half to subsequent heat stimuli (sensitization). No change in responsiveness to heat was observed in the other half of the receptive field, indicating that the effects of heat sensitization did not spread from the site of injury. In certain instances, the receptive field area increased, but the enlargement occurred only within the area of injury. The latter suggests the existence of outlying terminal endings of the parent axon that are normally unresponsive to non-injurious mechanical or heat stimuli but become responsive to these stimuli following injury.

Response properties of high-threshold cutaneous cold receptors in the primate.

Cutaneous high-threshold cold receptors (HCRs) in the monkey were identified as sensitive only to cold temperatures below 27 degrees C and not responsive to mechanical or heat noxious stimulation. Some HCRs had axons conducting in the low A-delta range while others had C fibers. The response properties of HCRs were contrasted with those of mechanothermal nociceptors, the latter believed to contribute to the sense of cold pain. HCRs with A delta fibers may contribute to the sense of innocuous cold below temperatures to which low-threshold cold receptors are maximally responsive.

Changes in axonal impulse conduction correlate with sensory modality in primary afferent fibers in the rat.

Conduction velocity was measured in vivo in single cutaneous afferent fibers of rat sciatic nerve that were characterized by natural stimulation. During sustained electrical stimulation, impulses slowed less and propagated more reliably in cold fibers (both A delta and C) than in nociceptive fibers of similar conduction velocity. Velocity in cold fibers tended to stabilize after an initial decrease rather than decrease throughout the stimulation as for nociceptive fibers. The slowing correlated with axon modality and hence with natural firing pattern, raising the possibility that impulse activity can determine conduction properties of axons.

Mechanoreceptive afferents exhibit functionally-specific activity dependent changes in conduction velocity.

Impulse activity in axons generates aftereffects on membrane excitability that can alter the conduction velocity of subsequently conducted impulses. We used a computerized stimulus pattern (a 1 Hz stimulus period followed by a period of repeated short bursts at 200 Hz) to assess in vivo activity-dependent changes in conduction latency of functionally identified rat cutaneous afferents conducting in the A beta range. Several different parameters of activity dependence were measured: burst supernormality, the average increase in conduction latency following conditioning with a single preceding impulse during high frequency burst stimulation; burst subnormality, the average latency increase during each burst; depression, a long-term increase in latency caused by the high frequency stimulation. The data show that different mechanosensitive A beta afferents with overlapping resting conduction velocities exhibit activity-dependent changes in conduction latency that are characteristic of their particular functions.

Behavioral evidence of thermal hyperalgesia in non-obese diabetic mice with and without insulin-dependent diabetes.

The non-obese diabetic (NOD) mouse, a model of Type 1 diabetes in humans, has proven useful for the study of genetic, immunologic and epidemiologic aspects of inherited diabetes. Behavioral evidence of hyperalgesia may also be present in the NOD mouse but has not been described. This study examined NOD mice with (NOD+) and without (NOD-) insulin-dependent diabetes, and control strain (ILI) mice for evidence of hyperalgesia to a noxious thermal stimulus. Interestingly, both NOD+ and NOD- mice showed reduced mean hindpaw withdrawal latencies when compared with non-diabetic ILI mice. NOD+ and NOD- mice were also abnormal in their general appearance, activity level, posture, gait and muscle bulk when compared with ILI mice. These findings raise the possibility that hyperalgesia in insulin-dependent NOD mice, or insulin-dependent humans with Type 1 diabetes, may be independent of diabetes and due to a primary disturbance within sensory pathways.

Dysmyelinogenesis in animal model of GM1 gangliosidosis.

Magnetic resonance imaging (MRI), pathologic examinations, and biochemical analyses were performed on 2 different canine mutants with GM1 gangliosidosis (i.e., English Springer Spaniel and Portuguese Water Dog) and on age- and sex-matched controls. Serial MRI studies were also performed on a child with infantile-onset GM1 gangliosidosis. The affected dogs had abnormalities on MRI, including a relative increase in gray matter and an abnormal signal intensity of cerebral and cerebellar white matter observed on T2-weighted MRI. White matter changes on MRI were similar to white matter abnormalities observed in a 15-month-old boy with GM1 gangliosidosis. The weight ratio of white to gray matter from the frontal lobe was markedly reduced. Microscopic examination revealed characteristic ballooned neurons which stained lightly with Luxol-fast blue. The central cerebral and cerebellar folia white matter exhibited pallor and gliosis, while the corpus callosum and fornix stained normally with Luxol-fast blue. Axons appeared intact on Bodian staining. Ultrastructural studies revealed fewer myelinated axons in affected puppies. Total gangliosides in gray matter were elevated. Thin-layer chromatography demonstrated GM1 ganglioside as the predominant ganglioside. The amount of cerebrosides and sulfatides was reduced in the gray and white matter when compared to controls but the ratio in gray and white matter remained unchanged. Immunostaining of neutral glycolipids disclosed increased amounts of stage-specific embryonic antigen-1 glycolipid in gray matter. These findings suggest that canine models for GM1 gangliosidosis are associated with abnormal myelin development which may be similar to the human disease.

Acquired cutaneous lymphangiectasis in a dog.

A 3-year-old male mixed-breed dog had swelling of the penile sheath, which developed after the dog was castrated at 1 year of age. Physical examination revealed pitting edema and multiple turgid vesicles on the prepuce and inguinal area. Histologic evaluation of the vesicles revealed thin epidermis elevated by dermal proliferation of dilated lymphatic channels. The diagnosis was acquired cutaneous lymphangiectasia. Clinical signs resolved concurrent with furosemide administration. Preputial swelling without vesicles recurred 1 and 2 years later and partially resolved after furosemide administration.

Phenobarbital-responsive episodic dyscontrol (rage) in dogs.

Episodic dyscontrol (rage) was diagnosed from the clinical history, electroencephalographic findings, and response to oral treatment with phenobarbital in 3 dogs. Clinical features included a mood change heralding aggressive incidents, explosive aggression directed at people or objects, and a postaggressive phase characterized by lethargy and lack of responsiveness. Abnormal electroencephalographic findings included spike activity in the temporal recordings. All 3 dogs responded well to anticonvulsant medication with phenobarbital.

Phenotypic characterization of canine intestinal intraepithelial lymphocytes in dogs with inflammatory bowel disease.

BACKGROUND: Many dogs suffering from inflammatory bowel disease (IBD) are presented to veterinary clinics. These patients are diagnosed based on a history of chronic gastrointestinal signs and biopsy-confirmed histopathologic intestinal inflammation. Intestinal intraepithelial lymphocytes (IEL) are part of the first line of defense in the gastrointestinal immune system. Alterations in IEL subsets may play a role in the pathogenesis of IBD. HYPOTHESIS: The aim of this study was to characterize the phenotypes of IEL in dogs with IBD compared with healthy control dogs. ANIMALS: Intestinal intraepithelial lymphocytes subpopulations of control dogs (n = 5) obtained from endoscopic biopsies (EB) were compared to those obtained from full thickness biopsies (FTB) on the same day. In addition, the phenotypes of IEL from FTB of control dogs (n = 10) were compared with EB of IBD dogs (n = 10). Each participant was scored clinically using the canine inflammatory bowel disease activity index (CIBDAI), and all samples were graded histopathologically. Three-color flow cytometry of isolated IEL was performed using monoclonal antibodies against T- and B-lymphocyte subpopulations. RESULTS: No significant differences in the composition of IEL subpopulations were found in control dogs based on method of biopsy. The IBD dogs had significantly higher CIBDAI and histopathologic scores compared with control dogs and their IEL contained a significantly higher frequency TCRγδ T-cells. CONCLUSIONS AND CLINICAL IMPORTANCE: Endoscopic biopsies provide suitable samples for 3-color flow cytometry when studying canine intestinal IEL and IBD patients show significant changes of major T-cell subsets compared to healthy control dogs.

Suspected limbic encephalitis and seizure in cats associated with voltage-gated potassium channel (VGKC) complex antibody.

BACKGROUND: Treatment-resistant complex partial seizures (CPS) with orofacial involvement recently were reported in cats in association with hippocampal pathology. The features had some similarity to those described in humans with limbic encephalitis and voltage-gated potassium channel (VGKC) complex antibody. HYPOTHESIS/OBJECTIVES: The purpose of this pilot study was to evaluate cats with CPS and orofacial involvement for the presence of VGKC-complex antibody. ANIMALS: Client-owned cats with acute orofacial CPS and control cats were investigated. METHODS: Prospective study. Serum was collected from 14 cats in the acute stage of the disease and compared with 19 controls. VGKC-complex antibodies were determined by routine immunoprecipitation and by binding to leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2), the 2 main targets of VGKC-complex antibodies in humans. RESULTS: Five of the 14 affected cats, but none of the 19 controls, had VGKC-complex antibody concentrations above the cut-off concentration (>100 pmol/L) based on control samples and similar to those found in humans. Antibodies in 4 cats were directed against LGI1, and none were directed against CASPR2. Follow-up sera were available for 5 cats in remission and all antibody concentrations were within the reference range. CONCLUSION AND CLINICAL IMPORTANCE: Our study suggests that an autoimmune limbic encephalitis exists in cats and that VGKC-complex/LGI1 antibodies may play a role in this disorder, as they are thought to in humans.

Blood vitamin levels in dogs with chronic kidney disease.

Chronic kidney disease (CKD) may affect excretion and metabolism of vitamins but data for dogs are limited. In this study, blood vitamin levels were investigated in 19 dogs with chronic renal failure. High performance liquid chromatography was used to quantify retinol, retinyl esters, tocopherol, thiamine, riboflavin, pyridoxal-5'-phosphate, ascorbic acid and 25-hydroxycholecalciferol concentrations, whereas cobalamin, folate, biotin and pantothenic acid were measured by microbiological methods. Levels of retinol, retinyl palmitate, ascorbic acid, and vitamins B1, B2 and B6 were increased compared to healthy dogs. Dogs with CKD showed decreased concentrations of 25-hydroxycholecalciferol and folate. Alpha-tocopherol, biotin, pantothenate and cobalamin levels were not significantly different between controls and dogs with CKD. Whether lower vitamin D and folate concentrations in dogs with CKD justify supplementation has to be evaluated in future studies.

Angiogenic markers in canine lymphoma tissues do not predict survival times in chemotherapy treated dogs.

Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma.

Home-based subcutaneous continuous glucose monitoring in 10 diabetic dogs.

A subcutaneous continuous glucose monitoring system (GlucoDay; Menarini Diagnostics) based on microdialysis was investigated for its clinical applicability in veterinary medicine. Ten diabetic dogs, referred as clinically stable, were equipped with this system and sent home for a maximum observation period of 48 hours. Time of insulin administration, feeding and other events were written in a diary and plotted afterwards in the glucose graph. Implantation of the microdialysis fibre, acceptance of the device and evaluation of individual canine glucose profiles were without complication. Based on the monitoring data, recommended treatment adjustments were given to the referring veterinarians in all 10 dogs; hypoglycaemic or prolonged hyperglycaemic episodes were detected in six dogs.

The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line.

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells. A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 microM sorafenib or with 80-640 microM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs.