Effects of fibroblasts derived from the olfactory bulb and nasal olfactory mucosa on proliferation of olfactory ensheathing cells harvested from the olfactory bulb

Olfactory ensheathing cells (OECs) have been reported to promote axonal regeneration when transplanted to rodent spinal cord injury models. OECs are available from the olfactory bulb (OB) and olfactory mucosa (OM). Although harvesting OECs from the OM is less traumatic, OECs originating from the OM are less proliferative than those from the OB (OB-OECs). One possible reason for this difference is coexisting fibroblasts. Here, we examined the effect of coculturing either fibroblasts from the OB (OB-Fibs) or fibroblasts from the OM (OM-Fibs) on the proliferation of OB-OECs. Proliferation of OB-OECs was significantly higher in 5:5 coculture with OB-Fibs and in 7:3 and 5:5 cocultures with OM-Fibs than without fibroblasts. These results indicated that coculture with both OB-Fibs and OM-Fibs promoted the proliferation of OB-OECs.     

Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44⁺CD24⁻ population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10⁴ sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

Comparison of rectal mucosal cultures and fecal cultures in detecting Salmonella infection in horses and cattle

Bacteriologic cultures of 65 rectal mucosal samples and 335 fecal samples from 53 horses and 5 cattle shedding Salmonella were performed. Salmonella spp were isolated from 34 (52%) rectal mucosal samples, 21 (32%) concurrent fecal samples, and 150 (45%) total fecal samples. The use of rectal mucosal samples when compared with concurrently obtained fecal samples significantly (P less than 0.025) improved the ability to isolate Salmonella spp. Concurrent bacteriologic culture of rectal mucosal samples and fecal samples resulted in 39 (60%) isolations. Compared with a series of fecal samples, Salmonella was isolated significantly more often when rectal mucosa and feces were cultured concurrently. Salmonella was isolated from rectal mucosal samples when it was not isolated from feces.     

Virus-induced interferon production in canine lymphoid cell cultures

Virus-induced interferon (IFN) production in canine lymphoid cells was studied, using Newcastle disease virus as principal inducer. It was found that spleen cells at a concentration of 5 X 10(6) cells/ml with Newcastle disease virus at a multiplicity of infection of 1, incubated at 37 C in 5% CO2 for 24 hours, produced highest titers of IFN. Among the lymphoid cells from different tissues, IFN production was in the order of spleen = bone marrow greater than thymus greater than mesenteric lymph node greater than or equal to peripheral blood lymphocytes. Macrophages did not produce IFN, and virus-induced IFN production in spleen cells did not depend on the presence of phagocytic mononuclear cells. These optimal conditions and macrophage independence are different from those of mitogen-induced IFN production in canine lymphoid cells.     

Comparison of growth and differentiation of normal and neoplastic canine keratinocyte cultures

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.     

Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.     

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-β (TGF-β) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1β, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.     

A modified bilateral transfrontal sinus approach to the canine frontal lobe and olfactory bulb: surgical technique and five cases

Five adult dogs presented for an acute onset of seizure activity. Magnetic resonance imaging revealed lesions in the olfactory bulbs, frontal lobes of the cerebrum, or both. A modified bilateral transfrontal sinus craniotomy was performed on each patient. The goal of removing the lesion was to relieve clinical signs and to provide tissue for histopathological diagnosis. In each instance, excision of the lesion was possible using this approach. No postoperative complications were observed. The modified bilateral transfrontal sinus craniotomy provides excellent access to the canine olfactory bulbs and frontal lobes.     

Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles. One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age). Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells. Muscles were digested after the removal of all visible connective tissue from the excised muscle. One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein. The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6. Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures. Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures. The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures. Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures. These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes.     

Passage of duck hepatitis virus in cell cultures derived from avian embryos of different species

An egg-attenuated strain of duck hepatitis virus was successfully passaged through cell cultures of avian embryos derived from goose, turkey, guinea fowl, Japanese quail, pheasant and chicken. Two field strains of the virus were passaged in a more limited range of species.     

Establishment and characterization of a cell line, MCO-Y4, derived from canine mammary gland osteosarcoma

A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39+/-4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.     

Variation of virus replication in primary bovine kidney cell cultures derived from 68 three-day-old calves

Primary kidney cell cultures were prepared from 68 three-day-old calves. Complete monolayers of these cultures were infected separately with viral diarrhea, infectious bovine rhinotracheitis, parainfluenza, and adenovirus 7 viruses. The yield of virus from all infected cultures was calculated by plaque titer assay after 2 to 4 days' incubation. The variation of virus yield was substantial between individual cultures.     

Parvalbumin‐immunoreactive cells in the olfactory bulb of the pigeon: Comparison with the rat

The olfactory bulb (OB) shows special characteristics in its phylogenetic cortical structure and synaptic pattern. In the OB, gamma‐aminobutyric acid (GABA), as an inhibitory neurotransmitter, is secreted from GABAergic neurons which contain parvalbumin (a calcium‐binding protein). Many studies on the distribution of parvalbumin‐immunoreactive neurons in the rodent OB have been published but poorly reported in the avian OB. Therefore, in this study, we compared the structure of the OB and distribution of parvalbumin‐immunoreactive neurons in the OB between the rat and pigeon using cresyl violet staining and immunohistochemistry for parvalbumin, respectively. Fundamentally, the pigeon OB showed layers like those of the rat OB; however, some layers were not clear like in the rat OB. Parvalbumin‐immunoreactive neurons in the pigeon OB were predominantly distributed in the external plexiform layer like that in the rat OB; however, the neurons did not have long processes like those in the rat. Furthermore, parvalbumin‐immunoreactive fibres were abundant in some layers; this finding was not shown in the rat OB. In brief, parvalbumin‐immunoreactive neurons were found like those in the rat OB; however, parvalbumin‐immunoreactive fibres were significantly abundant in the pigeon OB compared to those in the rat OB.