Genomic hypomethylation in neoplastic cells from dogs with malignant lymphoproliferative disorders

DNA methylation is an epigenetic modification in which a methyl group is added usually to the fifth carbon position of a cytosine residue. Dysregulation of this process is an important molecular event which has been shown to be associated with neoplastic transformation and tumour progression in humans and mice. Features of methylation dysregulation in many different types of neoplasms include general genomic hypomethylation, focal hypermethylation, and altered expression of genes which encode a series of DNA (cytosine-5) methyltransferases. Interestingly, many types of neoplasia that are recognised in humans also develop spontaneously in the dog. By comparing the restriction patterns of MspI and HpaII, this study demonstrates that as in human, genomic hypomethylation is a feature of neoplastic cells in the majority of canine lymphoma cases and approximately one-third of canine leukemia cases confirming that dysregulation of the DNA methylating machinery is implicated in malignant transformation of lymphoid cells in some dogs.     

Cephalosporin-induced changes in the ultrastructure of canine bone marrow

Fourteen healthy dogs were given 540 to 840 mg/kg of cefazedone (Refosporen) intravenously for up to 4 months or until peripheral blood cell count were depressed. Within 6 to 10 weeks treated dogs developed pancytopenia (5/14), thrombocytopenia (11/14), moderate to severe neutropenia (8/14), and/or normocytic anemia with erythroblastemia (8/14). Ultrastructural changes in bone marrow of severely cytopenic dogs included mitochondrial damage in hematopoietic and nonhematopoietic cells, thickening of endosteal bone lining layers, increased adventitial coverage of vascular sinuses, and an increased number of active macrophages. Swollen, ruptured mitochondria were in erythroid, granulocytic, and megakaryocytic cells, and, to a lesser extent, in macrophages, reticular endothelial, and bone lining cells. Maturation arrest was evident in both erythroid and granulocytic cell lines. There was also evidence of ineffective erythropoiesis and granulopoiesis. None of these changes were observed in bone marrow of controls, treated dogs that did not develop cytopenia, or dogs allowed to recover after cessation of dosing.     

Evaluation of proliferative disorders in canine bone marrow by use of flow cytometric scatter plots and monoclonal antibodies

The combination of flow cytometric scatterplot analysis and specific monoclonal antibodies was used to evaluate the lineage of cells from six dogs with proliferative disorders of bone marrow. Scatterplot analysis was used to identify mature and immature myeloid and erythroid cells. The immunophenotype of cells in the immature myeloid gate was determined by labeling cells with four monoclonal antibodies. These results were compared to results of cytologic and cytochemical evaluation. The immunophenotype of a dog with a diagnosis of myelogenous leukemia was a cluster of differentiation-18 (CD-18) positive, CD-14 negative, Thy-1 negative, and a major histocompatibility complex (MHC) class II negative. The immunophenotype of a dog with a diagnosis of myelomonocytic leukemia was CD-18 positive, CD-14 positive, Thy-1 positive, and MHC class II positive. Although this phenotype clearly differentiated myelomonocytic leukemia from myelogenous leukemia, it was similar to the immunophenotype of dogs with a diagnosis of malignant histiocytosis or hemophagocytic syndrome. The immunophenotype of two dogs with myelodysplastic syndrome was CD-18 positive and CD-14 negative. Results for Thy-1 and MHC class II were variable. As additional lineage-specific monoclonal antibodies become available, immunophenotyping should become a valuable tool for determination of the lineage of cells in canine myeloproliferative disorders.     

Isolation and characterization of hematopoietic progenitor cells in canine bone marrow

For ultimate diagnoses of canine leukemia or malignant lymphoma, we sought to isolate hematopoietic progenitor cells (HPCs) from canine bone marrow (BM) using physiological phenotypes. Canine BM cells were separated by equilibrium discontinued density centrifugation, and HPCs, detected by in vitro colony formation, were significantly enriched in the relatively low density (LD) fraction. In flow cytometry, many CD34 or MHC class II expressing cells were detected in the LD fraction, but these were not significantly enriched. When the LD cells were separated, using a cell-sorting method, into cells with high affinity of wheat germ agglutinin (WGAhigh) and cells with WGAlow, almost all multipotent HPCs (MHPCs) and HPCs committed to myeloid lineage were found in the WGAhigh population. When the WGAhigh population was further stained for rhodamin 123, almost all MHPCs were included in the dull population (Rhlow), but not in the bright one (Rhhigh). Morphologically, most Rhlow cells were round, blastic cells containing a large nucleus with nucleoli and narrow cytoplasm. Based on these results, we suggest that all of the MHPCs in canine BM show the Rhlow WGAhigh LD phenotype, and may contain hematopoietic stem cells, which are the primitive HPCs.     

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.     

The morphological effects of vincristine sulfate on canine bone marrow cells

This study documents the morphologic changes observed in the bone marrow aspirate biopsies from dogs 6 and 24 hours after receiving a single therapeutic dose (0.025 mg/kg) of vincristine sulfate (Oncovin: Eli Lilly & Co., Indianapolis, Ind.) intravenously. The most striking cytologic changes were observed in the erythroid cell line. Abnormalities included increased numbers of mitotic figures, abnormal nuclear configurations, and fragmented nuclei. Erythroid cells in metaphase were prominent in marrow samples collected 6 hours post-vincristine, accounting for a mean of 27% of all erythroid precursors. Fragmented nuclei and atypical nuclear configurations were seen in low numbers (mean = 7%) of erythroid cells from these animals. In contrast, marrow collected from dogs 24 hours post-vincristine exhibited low numbers (mean = 1%) of erythroid cells in metaphase, but erythroid cells with atypical nuclear configurations and fragmented nuctei accounted for a mean of 41% of the erythroid cells present. Less dramatic increases in the number of mitotic non-erythroid cells were seen 6 hours post-vincristine (mean = 5% of non-erythroid cells) and 24 hours post-vincristine (mean = 1% of non-erythroid cells). Only rare nuclear fragmentation was observed in these cell lines. Significant alterations in megakaryocytes and myeloid to erythroid (M:E) ratios were not observed in samples taken 6 hours post-vincristine; however, M:E ratios were considerably higher in three of the four samples taken from dogs 24 hours post-vincristine. Similar time-related changes were observed in four clinical cases in which bone marrow aspirates were performed after vincristine administration.     

In vitro generation of mature neutrophils from canine Lin- bone marrow cells

The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (Lin-) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from Lin- cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells.     

Matrix metalloproteinases (MMPs) activity in cultured canine bone marrow stromal cells (BMSCs)

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1β while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.     

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.     

Effect of incorporation of serum from dogs with renal impairment on canine erythroid bone marrow cultures

Serum from dogs with surgically induced renal impairment was incorporated into the medium for erythroid bone marrow cultures. A significant correlation was found between serum activities of erythropoietin and numbers of erythroid colony-forming units grown in culture. Serum creatinine concentrations had no correlation, and serum parathyroid hormone activities had a negative correlation with numbers of erythroid colony-forming units that was below the level of significance. Purified 1-84 parathyroid hormone added to bone marrow cultures was found to be stimulatory to erythroid colony-forming unit growth in higher concentrations, but decreased the number of burst-forming units. Unmeasured substances in the canine serum appeared to have a greater effect on the canine erythroid bone marrow cultures than did creatinine or parathyroid hormone values.     

Histopathology and immunohistochemistry of canine distemper virus-induced footpad hyperkeratosis (hard Pad disease) in dogs with natural canine distemper

Hard pad disease represents an uncommon manifestation of canine distemper virus (CDV) infection with a still uncertain pathogenesis. To study the pathogenesis of this uncommon, virally induced cutaneous lesion, the footpads of 19 dogs with naturally occurring distemper were investigated for histologic changes and distribution pattern of CDV antigen. All dogs displayed clinical signs of distemper, which had lasted from 10 to 75 days. Overt digital hyperkeratosis was observed in 12 animals (group A), whereas the footpads of the remaining seven dogs appeared normal macroscopically (group B). Orthokeratotic hyperkeratosis (12/12; 100%), irregular acanthosis (11/12; 92%), thickened rete ridges (10/12; 83%), and mild mononuclear perivascular (10/ 12; 83%) and periadnexal (7/12; 58%) dermatitis were the most common findings in dogs with hard pad disease. Surprisingly, orthokeratotic hyperkeratosis (5/7; 71%), irregular acanthosis (5/7; 71%), and thickened rete ridges (4/7; 57%) were also seen in the dogs without clinical evidence of digital hyperkeratosis. CDV-specific inclusion bodies and ballooning degeneration were not observed in the footpad epidermis of the 19 dogs. Immunohis-tochemistry revealed that CDV antigen was most frequently found in the stratum spinosum and granulosum and in the epithelial cells of the eccrine sweat glands and only rarely in the basal layer. Fibroblasts, pericytes, endothelial cells, and hair follicles were also positive in some animals. Despite the obvious difference regarding the macroscopic picture, the microscopic changes were less prominent between the animal groups. The selective infection of keratinocytes in the stratum spinosum might be the key event for the development of hard pad disease in the dog.     

Antibodies in carrier wildebeest to the lymphoproliferative herpesvirus of malignant catarrhal fever

Six types of antibody to malignant catarrhal fever virus (MCFV) were measured in 132 sera collected from Wildebeest in Kenya Masailand. The titre of all types of antibody declined slowly with increasing age of the wildebeest. A significantly greater proportion of wildebeest calves had higher titres of antibodies to MCFV early antigens, IgM antibodies to MCFV late antigens and complement-fixing antibodies, than did older animals. One seronegative calf, reared in isolation without colostrum, became seropositive 4 1/2 weeks after birth but did not show any clinical signs indicative of MCFV infection. Similarities between MCFV infection of wildebeest calves and other inapparent infections with lymphoproliferative herpesviruses are discussed.     

Use of memantine in treatment of canine compulsive disorders

Medication plays an important role in the treatment of canine compulsive disorders (CCD). Not all cases can be managed with the current medications, and new pharmacological options are needed. This case series assesses the efficacy of the NMDA receptor blocker memantine as a possible new treatment option for CCD. Thirteen dogs with different manifestations of CCD were enrolled in the study, and data from 11 dogs were available for analysis. A behavioral and clinical history was obtained for each dog. Further medical tests were performed if deemed necessary.The main presenting complaints in the included cases were light/shadow chasing, spinning/circling, and tail chasing. All dogs were either treated with memantine alone or memantine was added to ongoing fluoxetine treatment. All owners of dogs included in the study were provided with a specific behavior modification plan in addition to the pharmacological treatment. Memantine was administered orally twice a day at a starting dose of 0.3-0.5 mg/kg. The dose was increased over time if necessary, and side effects permitting, to a dose not higher than 1 mg/kg. The owners were asked to use a clinical global impression scale (CGI) on a daily basis to assess the severity of their dog's compulsive behaviors. This score was obtained daily for up to 4 consecutive weeks, and average weekly scores were calculated.Seven (64%) of the dogs included in the study showed a reduction in the severity of CCD, with CGI scores reduced by the second week of treatment. Only 1 out of 11 dogs showed a side effect (increased frequency of urination) that was potentially related to the medication. The results suggest that memantine may be an effective, well-tolerated option for the treatment of CCD either as a sole treatment or as an add-on to fluoxetine. Further studies are needed to investigate the efficacy of memantine in the treatment of CCD.     

Cytology of bone marrow.

Cytologic examination of bone marrow aspirates can provide a wealth of diagnostic information. Practitioners should not hesitate to perform bone marrow aspirates when indicated. This article is designed to assist the practitioner in the evaluation of bone marrow aspiration biopsies. The indications for marrow evaluation, methods of sample collection, sample preparation, and cytologic examination of bone marrow are discussed. Cases are provided to demonstrate accurate interpretation of bone marrow aspirates.     

Associations between canine benign and malignant neoplasms

Associations between benign and malignant neoplasms of the dog were quantitatively evaluated. Using histologically confirmed submissions and population-based methodology, canine benign tumors were significantly associated with both the unordered and subsequent development of additional distinct malignancies in the same dogs. A detailed examination of the methods indicated that these results are not likely due to methodologic artifact. The implication is that benign and malignant tumor development are associated and that the occurence of a benign neoplasm is an indicator of a parallel predisposition to malignancy.     

Influence of an autologous serum-supplemented medium on the proliferation and differentiation into neurons of canine bone marrow stromal cells

We investigated the influence of autologous serum (AS)-supplemented medium on the proliferation and differentiation into neurons of canine bone marrow stromal cells (BMSCs). Canine BMSCs were cultured using α-MEM only, α-MEM with 10% fetal bovine serum (FBS), and 5, 10 and 20% AS-supplemented α-MEM. Growth of canine BMSCs was observed in all AS groups. The proliferation capacity of canine BMSCs in the AS groups was similar to that in the FBS group. No significant differences between the FBS and AS groups were observed in the percentage of the cells that changed to the neuron-like morphology and neuron-specific enolase-positive ratio after neuronal differentiation. Canine BMSCs cultured using AS-supplemented medium were able to proliferate and showed neuronal differentiation potency.     

Metabolic correlates of tumour hypoxia in malignant canine mammary carcinoma

Given its importance in human and canine tumour biology, a profound understanding of tumour hypoxia is of paramount importance. Therefore, the aim of this work was to investigate the relationship between tumour hypoxia and the expression of a number of hypoxia-induced proteins that play a role in tumour metabolism. The hypoxia marker pimonidazole was administered to dogs affected by spontaneous mammary carcinoma and compared with immunohistochemical staining for GLUT1 and 3, HK 2 and CA IX. A statistically significant correlation was found between pimonidazole staining and GLUT1-expression (R = 0.607; p = 0.001). These results indicate a strong interaction between tumour hypoxia and tumour metabolism by the induction of proteins essential to maintain a stable tumour microenvironment.     

Proliferation capacity, neuronal differentiation potency and microstructures after the differentiation of canine bone marrow stromal cells into neurons

We examined the proliferation capacity and neuronal differentiation potency of canine bone marrow stromal cells (BMSCs). In addition, the microstructures of neuron-like cells after neuronal differentiation were observed under a scanning electron microscope. Canine BMSCs grew to confluency at 10.0 ± 2.5 days, and 3.8 ± 2.1 × 10(6) BMSCs were collected in one passage. Approximately 65% of canine BMSCs changed to neuron-like morphology after neuronal differentiation, and nearly all neuron-like cells stained positive against neuron-specific enolase. In addition, microstructures such as the cellular organelles, filaments and growth cones of these cells bore a close resemblance to those of the original mature neurons. These results suggested that canine BMSCs might be capable of differentiating into neurons.