Small Molecules Temporarily Induce Neuronal Features in Adult Canine Dermal Fibroblasts.

Several methods have been developed to generate neurons from other cell types for performing regeneration therapy and in vitro studies of central nerve disease. Small molecules (SMs) can efficiently induce neuronal features in human and rodent fibroblasts without transgenes. Although canines have been used as a spontaneous disease model of human central nerve, efficient neuronal reprogramming method of canine cells have not been well established. We aimed to induce neuronal features in adult canine dermal fibroblasts (ACDFs) by SMs and assess the permanency of these changes. ACDFs treated with eight SMs developed a round-shaped cell body with branching processes and expressed neuronal proteins, including ßIII-tubulin, microtubule-associated protein 2 (MAP2), and neurofilament-medium. Transcriptome profiling revealed the upregulation of neuron-related genes, such as SNAP25 and GRIA4, and downregulation of fibroblast-related genes, such as COL12A1 and CCN5. Calcium fluorescent imaging demonstrated an increase in intracellular Ca2+ concentration upon stimulation with glutamate and KCl. Although neuronal features were induced similarly in basement membrane extract droplet culture, they diminished after culturing without SMs or in vivo transplantation into an injured spinal cord. In conclusion, SMs temporarily induce neuronal features in ACDFs. However, the analysis of bottlenecks in the neuronal induction is crucial for optimizing the process.

Identification and characterization of two CD4 alleles in Microminipigs.

BACKGROUND: We previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies. RESULTS: We identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation. CONCLUSIONS: We identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.

Magnetic resonance imaging findings of the primitive neuroectodermal tumour in lumbosacral spinal cord in a cat.

A 5-year-old, castrated, male domestic short-haired cat presented with neurological deficits in the pelvic limbs, back pain and dysuria. Magnetic resonance imaging showed a mass lesion caudal to the L4 vertebrae. In addition, suspected haemorrhage was observed at the cranial aspect of the mass. There was no evidence to support the presence of extravertebral intrusion or vertebral body, osteolysis. Dorsal laminectomy and durotomy were performed to debulk the intraspinal mass. Histopathological and immunohistochemical assessment revealed a primitive neuroectodermal tumour (PNET). To our knowledge, this is the first report to describe the clinical and pathological features and imaging diagnosis of intraspinal PNET without extraspinal invasion in a cat.

Dysuria Associated with Non-Neoplastic Bone Hyperplasia of the Os Penis in a Pug Dog.

A three-year-old male Pug presented with a three-year history of urolithiasis and repeated urethral obstruction. Biochemical analysis, ultrasonography, and retrograde urethrocystography revealed probable portosystemic shunt and incomplete urethral obstruction due to uric acid ammonium calculi. Enhanced computed tomography (CT) revealed portosystemic shunt and proliferation of the osseous tissue of the os penis, which was surgically removed. Histopathologically, the excised osseous tissue comprised bland lamellar bone without atypia or inflammation. Hyperplasia of the os penis was diagnosed based on the image findings and histopathology. The dysuria improved postoperatively. This is the first report of dysuria associated with non-neoplastic bone hyperplasia of the os penis in a dog. Careful evaluation of the os penis by CT is needed for accurate diagnosis in case of repeated penile urethral obstruction.

A case of prostate B-cell lymphoma recognized in the contents of an irreducible perineal hernia in a dog.

We present a case of an intact 14-year-old male dog with a prostate B-cell lymphoma recognized in the contents of an irreducible perineal hernia. The enlarged prostate was replaced after reducing its size by partial excision, and the perineal hernia was repaired using the tunica vaginalis communis. However, the pelvic cavity was largely occupied by the replaced prostate, and urinary retention developed. The prostate was resected on the next day via abdominal median incision, and a bladder-urethra anastomosis was performed. Lymphoma has rarely been demonstrated to develop in the prostate, with the lesion comprising the hernia contents. The present study suggests the necessity of early treatment for perineal hernias and the possibility that lymphoma may be present in the hernia contents.

Three-dimensional culture of keratinocytes and the formation of basement membrane for canine footpad substitute.

A pad equivalent for a dog was prepared as a substitute for the loss of footpad. In addition to the time course of formation on epidermal morphogenesis, we investigated expressions of alpha(6) integrin subunit as adhesive molecule, and laminin and type IV and VII collagens as extracellular matrices of basement membrane components. Epithelium of the pad equivalent was thick enough to be easily confirmed at 5 days at the air-liquid interface, but many creases appeared on it at 7 days, and it shrank at 10 and 14 days. Keratinocytes were increased in 4 to 5 cell layers at 1 day at the air-liquid interface, differentiating into basal cell layer. Granular and corneal cell layers were confirmed until 5 days, and maintained their shape at least until 14 days. Alpha 6 integrin was expressed at almost the same fluorescent intensity as native pad tissue at 1 day at the dermal-epidermal junction. Laminin and type IV collagen were intermittently expressed at 5 and 10 days, respectively, at the dermal-epidermal junction, and at 14 days the fluorescence showed almost the same intensity as native pad tissue. The expression of type VII collagen was discontinuous at 2 days at the dermal-epidermal junction, but remained as it was at 14 days. The present findings suggested that although the formation of anchoring fibrils in basement membrane was incomplete, the pad equivalent in the dog was reconstructed similar to a native pad by epidermal morphogenesis.

Effects of atelocollagen gel containing bone marrow-derived stromal cells on repair of osteochondral defect in a dog.

To clarify the contribution of autologous transplantation of mesenchymal stromal cells (MSCs), an atelocollagen gel containing or not containing fluorescently-labeled canine MSCs was transplanted into an osteochondral defect which did not repair spontaneously and the histological repair of the defect was compared. Although an early repair of the cartilage was not observed in either defect, the reproduction of subchondral bone was remarkable in the MSCs-implanted defect. Moreover, in 2 weeks after operation, the implanted MSCs were located in the deeper regions of the defect, suggesting the differentiation of osteoblasts. There was a possibility that the movement of the implanted MSCs was due to an increase in intra-articular pressure from postoperative inflammation.

Oral Care and Prevention of Pneumonia in Hospitalized Patients With Psychiatric Disorders in Japan.

PURPOSE: Oral care in hospitalized patients with psychiatric disorders is important. However, some patients with psychiatric disorders cannot undergo oral care because of psychiatric symptoms and cognitive decline. The effect of a standardized oral hygiene intervention on the prevention of pneumonia in hospitalized patients with psychiatric disorders was investigated. METHOD: Patients were divided into 2 groups: control group (N = 259), patients without standardized intervention who were enrolled on April 2014 as the time point of baseline, and intervention group (N = 263), patients with standardized intervention who were enrolled on April 2015 as the time point of baseline. Two end points were evaluated: (1) pneumonia onset within 1 year after the enrollment and (2) no pneumonia for 1 year after the enrollment. The following parameters were compared between the groups: sex, age, psychiatric disorders, past history of diseases of the respiratory system, hypertension, diabetes, hyperlipidemia, heart impairment, and pneumonia. RESULTS: No statistically significant differences were found between the 2 groups in the distributions of characteristics except pneumonia by univariate analysis. The presence of pneumonia was significantly associated with age and the absence of the standardized oral hygiene intervention by multivariate logistic regression analysis. CONCLUSIONS: The standardized oral hygiene intervention appears to be effective for preventing pneumonia in patients with psychiatric disorders.

A case of feline sporotrichosis.

We excised surgically a feline granulomatous lesion and performed histopathological, mycological and molecular examinations. As a result, it was diagnosed as sporotrichosis, which was the second recorded case of a cat so afflicted in Japan. After the operation, we recognized another nodule on the lymph node. Histopathological examination was therefore performed, but no fungi were detected. To prevent recurrence, the cat was administered a antimycotic drug, itraconazole. As a result, no recurrence was found. Excision of the lesion is the treatment of choice for feline sporotrichosis.

Inhibitory effect for proliferation of oral bacteria in dogs by tooth brushing and application of toothpaste.

To investigate inhibitory effect for oral bacterial proliferation, we divided 12 dogs into 3 groups; scaling alone (C; control group), brushing (B) and application of toothpaste (P). Before scaling (Pre) and at 0 to 8 weeks after scaling (0-8 w), we collected oral bacteria from the dental surface every week and counted them using a bacterial counter. The results demonstrated a significant reduction in the number of oral bacteria for group B relative to Pre and group C, as well as for group P relative to group C at 5-7 w. Consequently, brushing may inhibit an increase in the number of oral bacteria, and toothpaste may be effective at a certain level, although not more than that of brushing.

Tooth brushing inhibits oral bacteria in dogs.

In this study, scaling, polishing and daily tooth brushing were performed in 20 beagle dogs, and the number of oral bacteria was determined using a bacterial counter. The dogs were randomized into the scaling (S), scaling + polishing (SP), scaling + tooth daily brushing (SB) and scaling + polishing + tooth daily brushing (SPB) groups. Samples were collected from the buccal surface of the maxillary fourth premolars of the dogs immediately after scaling and every week thereafter from weeks 1 to 8. Throughout the study, the number of bacteria was significantly lower in the SB and SPB groups compared with the S group. The findings suggest that daily tooth brushing inhibited oral bacterial growth in the dogs.

Identification of a CD4 variant in Microminipigs not detectable with available anti-CD4 monoclonal antibodies.

The Microminipig is an extra-small sized novel miniature pig developed in Japan. In the process of peripheral blood mononuclear cells analysis by flow cytometry, CD4+ cells could not be detected in some pigs with an anti-pig CD4 antibody (clone 74-12-4), or in some pigs with two other antibodies from different clones (MIL17 and PT90A). In a herd of 178 Microminipigs, 87 pigs (48.9%) were reactive with the anti-CD4 antibody (designated as CD4.A), and 91 pigs (51.1%) were non-reactive (designated as CD4.B). The CD4 types of piglets delivered from parents with CD4.A were CD4.A or CD4.B, and piglets delivered from parents with CD4.B were only CD4.B. This implies that the CD4.A pigs were homozygous for CD4.A or heterozygous for CD4.A and CD4.B, and the CD4.B pigs were homozygous for CD4.B. The CD4.B trait might be recessive. Significant differences could not be found in the percentage of CD3+ and CD8+ cells in whole lymphocytes between CD4.A and CD4.B animals. In the profile of CD4.B pigs, CD4+CD8+ T cells appeared to be detected in the CD4-CD8+ T cell region because the CD8 dull T cell population was observed. Thus, we considered that the CD4 molecules may be expressed on helper T cells, but the CD4 expressing cells could not be detected with the three anti-pig CD4 antibodies. Clinical abnormalities have not been observed in CD4.B pigs. Significant differences were not observed in immunoglobulin concentrations between CD4.A and CD4.B, though lower tendency was observed in plasma IgM concentrations from CD4.B pigs >36-months-old. These results imply that the CD4.B does not affect basic humoral immunity in vivo.

Body and major organ sizes of young mature microminipigs determined by computed tomography.

To understand the anatomical characteristics of microminipigs, one of the smallest miniature pigs, as a large animal model, we measured the body and organ sizes of four-, five-, six-, and seven-month-old microminipigs (n = 4, females) using computed tomography. In addition, the results were compared with those of young mature beagles (10 months old, two males and three females), which have been widely used as a large animal model. The microminipigs at 4-6 months of age were much smaller than the beagles. However, when the microminipigs reached seven months of age, their overall size was similar to that of the beagles. The thoracic cavity volume of the seven-month-old microminipigs was less than half that of the beagles, and the cavity was largely filled by the heart. The liver size of the seven-month-old microminipigs was approximately half of that of the beagles. Moreover, the spleen of the seven-month-old microminipigs was different in morphology, but not different in size from that of the beagles. In addition, although their volumes were the same, the kidneys of the seven-month-old microminipigs, unlike those of the beagles, were flattened in shape. Collectively, the major abdominal organs of the seven-month-old microminipigs were either the same size or smaller than those of the beagles, but the abdominal cavity volume of the seven-month-old microminipigs was larger than that of the beagles. Thus, the abdominal cavity of microminipigs is assumed to be filled with the gastrointestinal tract. The anatomical characteristics of the young mature microminipigs revealed in our study suggest that microminipigs could have great potential as a large animal model for biomedical research.

Enhancement of reactive oxygen species production from canine blood leukocytes by human recombinant interleukin-12.

A novel biological activity of human recombinant interleukin-12 (rhIL-12) on canine peripheral blood mononuclear cells (PBMC) was investigated in vitro. Canine PBMC were cultured in the presence or absence of rhIL-12 for 3 days. The reactive oxygen species (ROS) production induced by opsonized-zymosan (OZ) was then measured by a luminol-dependent chemiluminescense assay and demonstrated that the ROS production was enhanced after culture with rhIL-12. A nitro blue tetrazolium test and flowcytometry analysis revealed that canine lymphocytes, eosinophils, and monocytes were capable of ROS production, but that monocytes had the highest capacity. These results suggest that rhIL-12 enhances ROS production from canine monocytes.

Generation of canine dendritic cells from peripheral blood mononuclear cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.

First case of salivary mucocele originating from the minor salivary gland of the soft palate in a dog.

We found a case of salivary mucocele that originated in the minor salivary gland (palatine gland) of the soft palate in a dog. At first admission, the soft palate swelled remarkably. Computed tomography (CT) revealed cystic radiolucency inside a large quantity of liquid in the soft palate, and most of the airway was occupied. Marsupialization was performed, but since a recurrence was observed one month later, the salivary mucocele was removed. There has been no report of salivary mucocele arising from the minor salivary gland of the soft palate in dogs. To our knowledge, this case is the first. Complete removal, including minor salivary glands surrounding the lesion, is necessary for treatment of salivary mucocele in dogs.

Portal vein aneurysm in a dog.

Portal vein aneurysm (PVA) is a rare abnormal dilatation of the portal vein, which has not been reported in dogs. We describe the findings of ultrasound and computed tomography in a case of PVA in a young male toy poodle, with the final diagnosis established by explorative surgical observation. The dog had an aneurysmal fusiform dilatation in the extrahepatic portal vein with portal hypertension and multiple portsystemic shunts. This is the first report of canine PVA.