Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA.

Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.

Diameter of the shunt vessel in dogs with incidental extrahepatic portosystemic shunts.

Clinical signs of extrahepatic portosystemic shunts (EHPSS) depend on the amount of blood shunted. In this study, dogs with EHPSS without noticeable clinical signs including 34 left gastro-phrenic, 3 left gastro-azygos and 2 left spleno-gonadal shunts were evaluated. In dogs with EHPSS without noticeable clinical signs, the median maximum diameter of the shunt vessel was significantly smaller compared to PV (p < 0.005). Whenever the diameter of the EHPSS is small in relation to the diameter of the PV, it seems likely that no obvious clinical signs of EHPSS are observed by the owners.

Factors contributing to the swimmer puppy syndrome found in Labrador retrievers.

BACKGROUND: Swimmer puppy syndrome is a disease found in neonatal puppies mainly characterized by the inability to stand, but its direct cause is unknown. Since swimmer puppies were observed infrequently but continuously among the Labrador retriever colony at the Hokkaido Guide Dogs for the Blind Association in Japan, based on their birth record and pedigree, factors related to the onset of swimmer puppy syndrome in Labrador retrievers were examined. RESULTS: The total number of offspring over seven years was 436, of which 16 were swimmer puppies. Most of the affected puppies except one recovered steadily. As for the swimmer puppies, the litter size was significantly lower, and the body weights on the 10th and 28th day after delivery were significantly higher than the non-symptomatic puppies. These results suggested that the onset may be related to weight gain in the neonatal stages due to a small litter size. According to the genetic analysis, 26 ancestors common to the affected individuals were confirmed, but the causative individual could not be identified with the inbreeding coefficient. The heritability of the swimmer-puppy onset trait was 0.80, and the heritability for the the 10th-day body-weight trait was equally high at 0.78, both of which strongly suggest genetic involvement. CONCLUSIONS: In this study, the onset of swimmer puppy syndrome in the Labrador retrievers was associated with litter size and early weight gain, and result of study suggests that genetic influence might be involved.

Effect of Stem Positioning on Biomechanical Performance of a Novel Cementless Short-Stem Canine Total Hip Implant.

OBJECTIVE: The aim of this study was to evaluate the effect of stem positioning on the biomechanical performance of a novel, collared, short-stem total hip implant under compression and torsion ex vivo. STUDY DESIGN: Six canine cadaveric femurs were implanted with a collared short-stem femoral implant. Canal flare index (CFI), stem angle, absolute and relative cut heights and relative size were measured radiographically and used as independent variables. Biomechanical performance of the construct was evaluated using physiologic loading (loading) and supraphysiologic loading (failure) protocols. RESULTS: During loading protocols, compressive stiffness was influenced by absolute cut height (p = 0.018). During failure protocols, peak torque was influenced by CFI (p = 0.004) and craniocaudal relative size (p = 0.005). Peak load and torsional stiffness were not impacted by any of the radiographic variables (p > 0.05). Three of six femurs developed longitudinal fractures originating at the medial calcar at the time of failure. CONCLUSION: The biomechanical performance of the collared short-stem implant was positively impacted by preserving more of the femoral neck, having a higher CFI and using a smaller implant size relative to the femoral neck isthmus.

Corneal protective effects of novel tear substitutes containing sodium hyaluronate and dodecahydrosqualene, squalane, in a porcine dry eye model.

To develop a novel tear substitute (TS) containing sodium hyaluronate (SH) and dodecahydrosqualene (DHS, squalane), we improved the prescription of a previously developed TS containing saline, 0.5% SH and 1% castor oil (CO), which had corneal protective effects against 60-min desiccation in a porcine dry eye model and viscosity of 106.8 mPa·S. Fresh porcine eyes were treated with a TS containing saline, 0.1%, 0.25%, 0.3% or 0.5% SH, and 1% CO or 1%, 2.5% or 5% DHS, and TS-treated eyes were desiccated for up to 180 min. The corneal damage was evaluated by the staining score of methylene blue (MB), absorbance of MB extracted from the cornea, the staining density of lissamine green (LG) and histopathology. The viscosities of the examined TS were also measured. A saline/0.5% SH/1% DHS solution had corneal protective effects for 90 min under desiccation and a viscosity of 110.0 mPa·s. A TS with saline, 0.1%, 0.25% or 0.3% SH and 1% or 2.5% DHS did not have better protective effects than a saline/0.5% SH/1% DHS solution, although a saline/0.3% SH/5% DHS solution exhibited greater corneal protection against 180-min desiccation on MB and LG staining and histopathological examination, and its viscosity was 34.5 mPa·s, which was similar to the 29.5 mPa·s of 0.3% SH. The saline/0.3% SH/5% DHS solution is available as a novel 3-hr long-lasting TS containing mucinomimetic and liquid oil components to treat and relieve dry eye symptoms in animals and humans.

Tafenoquine Is a Promising Drug Candidate for the Treatment of Babesiosis.

Due to drug resistance, commonly used anti-Babesia drugs have limited efficacy against babesiosis and inflict severe side effects. Tafenoquine (TAF) was approved by the U.S. Food and Drug Administration in 2018 for the radical cure of Plasmodium vivax infection and for malaria prophylaxis. Here, we evaluated the efficacy of TAF for the treatment of Babesia infection and elucidated the suspected mechanisms of TAF activity against Babesia parasites. Parasitemia and survival rates of Babesia rodhaini-infected BALB/c and SCID mice were used to explore the role of the immune response in Babesia infection after TAF treatment. Parasitemia, survival rates, body weight, vital signs, complete blood count, and blood biochemistry of B. gibsoni-infected splenectomized dogs were determined to evaluate the anti-Babesia activity and side effects of TAF. Then, to understand the mechanism of TAF activity, hydrogen peroxide was used as an oxidizer for short-term B. rodhaini incubation in vitro, and the expression levels of antioxidant enzymes were confirmed using B. microti-infected mice by reverse transcription-quantitative PCR (qRT-PCR). Acute B. rodhaini and B. gibsoni infections were rapidly eliminated with TAF administration. Repeated administration of TAF or a combination therapy with other antibabesial agents is still needed to avoid a potentially fatal recurrence for immunocompromised hosts. Caution about hyperkalemia should be taken during TAF treatment for Babesia infection. TAF possesses a babesicidal effect that may be related to drug-induced oxidative stress. Considering the lower frequency of glucose-6-phosphate dehydrogenase deficiency in animals compared to that in humans, TAF use on Babesia-infected farm animals and pets is eagerly anticipated.

Intramedullary spinal nephroblastoma in a mixed breed dog.

A 1-year-old male mixed breed dog presented for the evaluation of progressive hindlimb paresis. Neurological examination indicated a spinal cord lesion between the 3rd thoracic and 3rd lumbar vertebrae. Magnetic resonance imaging (MRI) revealed an intramedullary spinal cord lesion located at the level of the 1st and 2nd lumbar vertebrae. Following cytoreductive surgery of the mass, palliative radiation therapy was administered. A diagnosis of nephroblastoma was made based on histological examination. After radiation therapy, the disappearance of the spinal lesion was confirmed by MRI. The dog was improved from gait abnormality and alive at 16 months postoperatively, with slight signs of neurological dysfunction.

Quantitative analysis of the BRAF V595E mutation in plasma cell-free DNA from dogs with urothelial carcinoma.

Circulating tumor DNA (ctDNA), which carries tumor-specific mutations, is an emerging candidate biomarker for malignancies and for monitoring disease status in various human tumors. Recently, BRAF V595E mutation has been reported in 80% of dogs with urothelial carcinoma. This study investigates the BRAF V595E allele concentration in circulating cell-free DNA (cfDNA) and assesses the clinical significance of BRAF-mutated ctDNA levels in canines with urothelial carcinoma. A total of 15 dogs with urothelial carcinoma were included. cfDNA concentration was measured using a real-time polymerase chain reaction (PCR) of the LINE-1 gene. To measure the concentration of the mutated BRAF gene in cfDNA, allele-specific real-time PCR with a locked nucleic acid probe was performed. BRAF mutations were detected in 11 (73%) of the 15 tested tumor samples. BRAF-mutated ctDNA concentrations were significantly higher in dogs with the BRAF mutation (14.05 ± 13.51 ng/ml) than in wild-type dogs (0.21 ± 0.41 ng/ml) (p = 0.031). The amount of BRAF-mutated ctDNA in plasma increased with disease progression and responded to treatment. Our results show that BRAF-mutated ctDNA can be detected using allele-specific real-time PCR in plasma samples of canines with urothelial carcinoma with the BRAF V595E mutation. This ctDNA analysis may be a potentially useful tool for monitoring the progression of urothelial carcinoma and its response to treatment.

Three-legged radiographic view for evaluating cranioventral lung region in standing calves with bovine respiratory disease.

This study proposed a novel radiographic positioning in order to image the cranioventral lung region using a portable X-ray unit and digital radiography system. In the novel position, calves were restrained in a chute and a unilateral forelimb was pulled cranially with the contralateral forelimb tied to the chute; the forelimbs were then spread cranio-caudally as in a scissor position (Three-legged view: TL view). In a preliminary study, we applied the TL view for imaging of 14 clinically healthy calves. In a clinical study, accuracy in detecting cranioventral lung lesions was compared between the standard standing view and the TL view for 19 calves, which were culled from herd; the results of postmortem examination were used as gold standard. Seven evaluators independently interpreted the images. The median (range) number of trials and the time for obtaining optimal position were 2 (1-7) and 263 sec (105-488), respectively in 14 healthy calves. Calves thicker than approximately 40 cm were not considered candidates for TL view in this setting because of difficulty in restraint and the low output of the portable X-ray unit. The TL view improved the detection of consolidation in the cranioventral lung region, compared with the standard view. The TL view was considered an optional view when the cranioventral lung region was an area of interest, because this view was relatively easy to perform and required a small number of personnel, even for large calves.

Gene and protein expression of a soluble form of CTLA-4 in a healthy dog.

Cytotoxic T lymphocyte associated gene-4 (CTLA-4) is a costimulatory molecule, expressed on the surface of activated T cells that negatively regulates T cell activation. In humans, alternative splicing of the CTLA-4 gene generates two major isoforms of mRNA, and a soluble form of CTLA-4 (sCTLA-4) was detected in normal human serum. We describe alternatively spliced mRNA expressed in peripheral blood mononuclear cells obtained from a healthy dog lacking the transmembrane domain coded by exon 3 of the CTLA-4 gene. Immunoprecipitation and western blotting of dog serum revealed a band of approximately 23-kDa, which is consistent with the predicted size, based on the amino acid sequence of the canine sCTLA-4 obtained in this study.

Influence of transfusion of lymphokine-activated T killer cells on inflammatory responses in dogs after laparotomy.

The influence of transfusion of lymphokine-activated T killer cells (T-LAK) on inflammatory responses was examined in dogs after laparotomy. Plasma C-reactive protein (CRP) level, cell numbers of peripheral blood lymphocytes (PBLs) and T lymphocyte subsets (CD3(+), CD4(+) and CD8(+)) and mRNA expression levels of cytokines including interleukin (IL)-2, IL-12, IL-4, IL-10 and transforming growth factor (TGF)-ß in peripheral blood mononuclear cells (PBMCs) were measured in dogs with (T-LAK group) or without (control group) a single T-LAK administration immediately after laparotomy. The plasma CRP level initially increased and then decreased to the normal range at 7 days after laparotomy in the T-LAK group, which was earlier than in the control group. The expression level of IL-10 mRNA showed a marked postoperative increase and was significantly higher than the preoperative level on day 7 (P<0.05), whereas the level in the control group showed no clear change after laparotomy. A significant increase in IL-2 mRNA expression level in the T-LAK group was observed on day 14, which was two weeks earlier than in the control group (P<0.05). These results suggest that T-LAK therapy in dogs after laparotomy leads to earlier resolution of postoperative inflammation by production of an anti-inflammatory cytokine (IL-10) in the early phase of the postoperative period and earlier restoration of cell-mediated immunity related to cytokine production by PBMCs.

A comparison of the immunological effects of propofol and isoflurane for maintenance of anesthesia in healthy dogs.

Most anesthetics have an immuno-suppressive effect on cellular and neurohumoral immunity, and research shows that total intravenous anesthesia (TIVA) with propofol has a greater immuno-protective effect than inhalational anesthesia in human medicine. However, in veterinary clinics, these effects remain ambiguous. To clarify the details, we focused on propofol and isoflurane, investigating clinical blood hematology and immunological profiles drawn from healthy dogs under and after two anesthesia techniques. Twelve healthy adult beagles were included in this study, randomly assigned to the propofol anesthesia group (group P: n=6) or the isoflurane anesthesia group (group I: n=6). In both groups, the number of lymphocytes in peripheral blood decreased after 2 hr of anesthesia (2 hr), but group P showed significantly less decrease than group I. For T-lymphocyte subsets examined by flowcytometry, the ratio of CD3+, CD4+ and CD8+ lymphocytes in the peripheral blood mononuclear cell (PBMC) of group P at 2 hr also exhibited a high level compared to group I. Moreover, for mRNA expression of cytokines measured by real-time PCR, the IL2 (pro-inflammatory cytokine) of group P showed no decrease like group I. The IL10 (anti-inflammatory cytokine) of group P also showed no increase like group I, while both cytokines maintained nearly the same level until 2 hr. These results suggest that, compared to propofol, isoflurane had more strongly immuno-suppression caused by anesthesia, and propofol itself might have some immuno-protective effects. Thus, TIVA with propofol might benefit immunological support in the perioperative period of dogs.

The DC-HIL ligand syndecan-4 is a negative regulator of T-cell allo-reactivity responsible for graft-versus-host disease.

Acute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo-reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen-presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan-4 (SD-4) on effector T cells mediates the inhibitory function of DC-HIL on APC, we proposed that SD-4 negatively regulates the T-cell response to allo-stimulation in acute GVHD, using SD-4 knockout mice. Although not different from their wild-type counterparts in responsiveness to anti-CD3 stimulation, SD-4(-/-) T cells lost the capacity to mediate the inhibitory function of DC-HIL and were hyper-reactive to allogeneic APC. Moreover, infusion of SD-4(-/-) T cells into sub-lethally γ-irradiated allogeneic mice worsened mortality, with hyper-proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD-4 deletion had no deleterious effect on T-cell-suppressive activity compared with SD-4(+/+) regulatory T cells. We conclude that SD-4, as the T-cell ligand of DC-HIL, is a potent inhibitor of allo-reactive T cells responsible for GVHD and a potentially useful target for treating this disease.

DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells.

DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA-transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL-knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8(+) T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL-knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy.

Depleting syndecan-4+ T lymphocytes using toxin-bearing dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand: a new opportunity for treating activated T cell-driven disease.

Because syndecan-4 (SD-4) is expressed by some (but not all) T cells following activation and serves as the exclusive ligand of dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL), we envisioned the DC-HIL/SD-4 pathway to be a therapeutic target for conditions mediated by selectively activated T cells. We conjugated soluble DC-HIL receptor with the toxin saporin (SAP; DC-HIL-SAP) and showed it to bind activated (but not resting) T cells and become internalized by and deplete SD-4(+) T cells. In hapten-sensitized mice, DC-HIL-SAP injected i.v. prior to hapten challenge led to markedly suppressed contact hypersensitivity responses that lasted 3 wk and were restricted to the hapten to which the mice were originally sensitized. Such suppression was not observed when DC-HIL-SAP was applied during sensitization. Moreover, the same infusion of DC-HIL-SAP produced almost complete disappearance of SD-4(+) cells in haptenated skin and a 40% reduction of such cells within draining lymph nodes. Our results provide a strong rationale for exploring use of toxin-conjugated DC-HIL to treat activated T cell-driven disease in humans.

Binding of DC-HIL to dermatophytic fungi induces tyrosine phosphorylation and potentiates antigen presenting cell function.

APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1beta, and TNF-alpha. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.

The DC-HIL/syndecan-4 pathway inhibits human allogeneic T-cell responses.

T-cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC-HIL on APC to syndecan-4 (SD-4) on activated T cells potently inhibits T-cell activation. In humans, we now show that DC-HIL also binds to SD-4 on activated T cells through recognition of its heparinase-sensitive saccharide moiety. DC-HIL blocks anti-CD3-induced T-cell responses, reducing secretion of pro-inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC-HIL phosphorylates SD-4's intracellular tyrosine and serine residues. Anti-SD-4 Ab mimics the ability of DC-HIL to attenuate anti-CD3 response more potently than Ab directed against other inhibitory receptors (CTLA-4 or programmed cell death-1). Among leukocytes, DC-HIL is expressed highest by CD14(+) monocytes and this expression can be upregulated markedly by TGF-beta. Among APC, DC-HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC-HIL expression on CD14(+) monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF-beta reduced this capacity, whereas knocking down the DC-HIL gene augmented it. Our findings indicate that the DC-HIL/SD-4 pathway can be manipulated to treat T-cell-driven disorders in humans.

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion.

Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel-17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal-sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary-cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by alpha-melanocyte-stimulating hormone (MSH) (but not beta-MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN-gamma and TNF-alpha. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD-dependent manner. These results indicate that Gpnmb is a melanosome-associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.

In vitro differentiation of canine celiac adipose tissue-derived stromal cells into neuronal cells.

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.