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Background: Invasive urothelial carcinoma (iUC) is a major cause of death in humans, and approximately 165,000 individuals succumb to this cancer annually worldwide. Comparative oncology using relevant animal models is necessary to improve our understanding of progression, diagnosis, and treatment of iUC. Companion canines are a preferred animal model of iUC due to spontaneous tumor development and similarity to human disease in terms of histopathology, metastatic behavior, and treatment response. However, the comprehensive molecular characterization of canine iUC is not well documented. In this study, we performed transcriptome analysis of tissue samples from canine iUC and normal bladders using an RNA sequencing (RNA-Seq) approach to identify key molecular pathways in canine iUC.Methods: Total RNA was extracted from bladder tissues of 11 dogs with iUC and five healthy dogs, and RNA-Seq was conducted. Ingenuity Pathway Analysis (IPA) was used to assign differentially expressed genes to known upstream regulators and functional networks.Results: Differential gene expression analysis of the RNA-Seq data revealed 2531 differentially expressed genes, comprising 1007 upregulated and 1524 downregulated genes, in canine iUC. IPA revealed that the most activated upstream regulator was PTGER2 (encoding the prostaglandin E2 receptor EP2), which is consistent with the therapeutic efficiency of cyclooxygenase inhibitors in canine iUC. Similar to human iUC, canine iUC exhibited upregulated ERBB2 and downregulated TP53 pathways. Biological functions associated with cancer, cell proliferation, and leukocyte migration were predicted to be activated, while muscle functions were predicted to be inhibited, indicating muscle-invasive tumor property.Conclusions: Our data confirmed similarities in gene expression patterns between canine and human iUC and identified potential therapeutic targets (PTGER2, ERBB2, CCND1, Vegf, and EGFR), suggesting the value of naturally occurring canine iUC as a relevant animal model for human iUC. [ABSTRACT FROM AUTHOR]

Abstract: Although decreased intestinal IgA expression has been reported in dogs with inflammatory bowel disease (IBD), the mechanism underlying this decrease is unknown. Transmembrane activator and calcium-modulating cyclophilin–ligand interactor (TACI) and B cell-activating factor of the TNF family (BAFF) receptor (BAFF-R) are key receptors for T cell-independent IgA class switching by the binding of IgA-inducing cytokine a proliferation-inducing ligand (APRIL) and BAFF. Here we show decreased TACI and BAFF-R mRNA expression and hypermethylation of their corresponding genes TNFRSF13B and TNFRSF13C, respectively in the duodenal mucosa of dogs with IBD. To examine whether DNA methylation of the TNFRSF13B and TNFRSF13C influences the mRNA expression of TACI and BAFF-R, respectively, we first analyzed methylation and mRNA expression levels in vitro using 2 canine B lymphoid cell lines, GL-1 and CLBL-1. Methylation profiles in the cells were examined by bisulfite sequencing and methylation-specific PCR (MSP) with primer pairs specific to methylated or unmethylated sequences. These methylation analyses revealed hypermethylation of the CpG islands of both TNFRSF13B and TNFRSF13C in GL-1, but not in CLBL-1 cells. The mRNA expression levels of TACI and BAFF-R were significantly lower in GL-1 than in CLBL-1 cells. Treatment with 5-aza-2′-deoxycytidine significantly increased TACI and BAFF-R mRNA expression in GL-1 cells through demethylation of TNFRSF13B and TNFRSF13C, respectively. These results suggest that the mRNA expression of TACI and BAFF-R is regulated through methylation of their genes in canine B cells. Quantitative real-time MSP showed significant hypermethylation of the CpG islands of TNFRSF13B and TNFRSF13C in the duodenal mucosa of dogs with IBD. Furthermore, duodenal mRNA expression levels of TACI and BAFF-R were significantly lower in dogs with IBD than in healthy controls. The mRNA expression levels of TACI positively correlated with intestinal IgA expression, whereas the methylation level of its gene (TNFRSF13B) negatively correlated with IgA expression. The present results suggest the role of TACI in the regulation of mucosal IgA expression through epigenetic modifications. [Copyright &y& Elsevier]

Abstract: Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca2+ concentration was measured using the Ca2+-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P <0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P <0.01), thymus and activation regulated chemokine (TARC/CCL17, P <0.01), and interleukin 8 (IL-8/CXCL8, P <0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P <0.05), GM-CSF (P <0.05), TARC/CCL17 (P <0.05), and IL-8/CXCL8 (P <0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca2+ concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis. [Copyright &y& Elsevier]

Abstract: The interaction between fractalkine/CX3CL1 and its receptor CX3CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX3CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX3CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX3CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX3CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX3CR1+ PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX3CR1+ PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX3CR1+ PBMCs. These results suggest that the fractalkine-CX3CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs. [Copyright &y& Elsevier]

Abstract: The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9+ cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. [Copyright &y& Elsevier]

Abstract: Fractalkine, also known as CX3CL1, is a unique chemokine that mediates inflammatory responses and is involved in the pathogenesis of several inflammatory disorders, including inflammatory bowel disease (IBD) in humans. In this study, we isolated cDNAs encoding canine fractalkine and its receptor CX3CR1, and assessed the biological activity of these molecules. The deduced amino acid sequence of the canine fractalkine cDNA showed 66% and 57% identity to human and mouse homologs, respectively. The N-terminal chemokine domain of the canine fractalkine showed 68% and 65% identity to human and mouse counterparts, respectively. The canine CX3CR1 amino acid sequence showed close homology to its human (83% identity) and mouse (81% identity) counterparts. Fractalkine and CX3CR1 mRNA were detected in all tissues in this study. Relatively higher expression levels of fractalkine mRNA were observed in the brain, medulla spinalis, small intestine, and mesenteric lymph nodes (MLNs), whereas higher expression levels of CX3CR1 mRNA were observed in the medulla spinalis, brain, liver, small intestine, and MLNs. The cross-reactivities of anti-human fractalkine antibody and anti-rat CX3CR1 antibody to canine proteins were confirmed using recombinant canine fractalkine and a cell line overexpressing canine CX3CR1, respectively. A transwell chemotaxis assay showed that the recombinant canine fractalkine induced migration in canine lymphoid cells expressing CX3CR1. The present study will be useful in understanding the canine immune system and the immunopathogenesis of canine inflammatory diseases. [Copyright &y& Elsevier]

Abstract: House dust mite (HDM) allergens are the most common allergens involved in the induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD); however, direct stimulation of canine keratinocytes by mite allergens has not been well investigated. In the present study, we investigated the effects of Der f 1, a major allergen of Dermatophagoides farinae, on cytokine and chemokine gene expression in a canine keratinocyte cell line, CPEK. CPEK constitutively expressed mRNA for TNF-α, IL-12p35, IL-18, GM-CSF, TGF-β, IL-8/CXCL8, TARC/CCL17, CTACK/CCL27 and MEC/CCL28. Of all the cytokines and chemokines investigated in CPEK, transcription levels of GM-CSF, IL-8/CXCL8 and TNF-α mRNA were significantly enhanced by stimulation with Der f 1. The present results suggest that Der f 1 can directly augment inflammatory cytokine and chemokine production from keratinocytes, and may initiate allergic inflammation independently of Type-I hypersensitivity. [Copyright &y& Elsevier]

Simple Summary: 5-aminolevulinic acid (5-ALA) is a natural amino acid that is metabolized in the mitochondria and used in the synthesis of heme. In human medicine, the fluorescence property of 5-ALA has been used for photodynamic tumor diagnosis. Nevertheless, research on 5-ALA in veterinary medicine is very limited. In this study, we aimed to evaluate the efficacy of 5-ALA to detect canine urothelial carcinoma and to clarify its diagnostic accuracy. The addition of 5-ALA to tumor cells from the urine of patients with urothelial carcinoma and canine urothelial carcinoma cell lines caused red fluorescence, and the amount of fluorescence was significantly higher than that in healthy dogs. Additionally, cases with high fluorescence intensity had more tumor invasion and metastasis. This study showed that 5-ALA can be used to detect canine urothelial carcinoma cells in urine with relatively high diagnostic accuracy. This study aimed to establish a method to detect canine urothelial carcinoma cells in urine using 5-aminolevulinic acid (5-ALA) and to evaluate its diagnostic accuracy. Urine samples were collected from 21 dogs diagnosed with urothelial carcinoma and three urothelial carcinoma cell lines were used. Urine samples obtained from seven healthy dogs were used as controls. Cells in the urine sediment, or urothelial carcinoma cell lines, were cultured with 5-ALA and then observed under a fluorescence microscope. Moreover, we examined the relationship between fluorescence intensity and the presence of metastasis as well as tumor invasion into the bladder wall in cases of urothelial carcinoma. Urine-derived cells from urothelial carcinoma and urothelial carcinoma cell lines showed clearer red fluorescence with the addition of 5-ALA compared to that exhibited by the cells from healthy dogs. The sensitivity and specificity of the diagnosis of urothelial carcinoma were 90% and 86%, respectively. Significant associations were found between fluorescence intensity and tumor metastasis and bladder wall invasion. This study showed that 5-ALA can be used to detect urothelial carcinoma cells in dogs with relatively high diagnostic accuracy. Further, the fluorescence intensity of tumor cells caused by 5-ALA correlated with the clinical condition of urothelial carcinoma cases, which suggested that 5-ALA could be used as a prognostic marker for canine urothelial carcinoma. [ABSTRACT FROM AUTHOR]

Although definitive diagnosis of chronic enteropathy (CE) and gastrointestinal (GI) lymphoma requires histopathological evaluation of the GI tract, these conditions are often still difficult to differentiate from each other. Polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) has been applied recently as an adjunctive for diagnosis of lymphoid tumors; however, its clinical value in canine CE and GI lymphoma remains unclear. The purpose of this study was to investigate the relationship between PARR and histopathological diagnosis, degree of enteritis or lymphoma, and long-term prognosis in dogs, in order to evaluate the clinical significance of PARR. Endoscopic biopsy specimens obtained from 96 dogs with chronic enteritis (mild, n = 14; moderate, n = 20; marked, n = 62) and 21 dogs with GI lymphoma were used. Clonality was observed in 51% of the animals with chronic enteritis; interestingly, it was found in 29% of those with only mild enteritis. In dogs with marked enteritis, the rate of PARR was higher in those with lymphocyte epitheliotropism than in those without epitheliotropism. The sensitivity of PARR in animals with GI lymphoma was 76%. There was no significant prognostic difference between chronic enteritis with or without clonal rearrangements. In contrast, dogs histopathologically diagnosed with marked enteritis had a significantly shorter survival time than did those with mild or moderate enteritis. While the significance of PARR in the diagnosis of GI lymphoma remains uncertain, the pathological roles of clonally expanding lymphocytes in canine CE should be investigated further. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction (PCR) amplification to detect immunoglobulin heavy chain (IgH) and T cell receptor γ-chain (TCRγ) gene rearrangements has recently become widely used as part of the diagnostic strategy for lymphoid tumors in dogs. In this study, we constructed a multicolor GeneScan analytical system to improve the sensitivity and resolution of the clonality analysis of antigen receptor gene rearrangements in dogs. We used 7 reactions per sample, with 2 PCR conditions, to amplify IgH/TCRγ and control genes. By using multicolor-labeled primers, these 7 PCR products could be combined into 3 tubes before capillary electrophoresis. Clonal rearrangement of the IgH/TCRγ genes was detected in 93.3% of dogs with multicentric lymphoma and 84.6% of dogs with gastrointestinal lymphoma. Detection sensitivity of the clonally expanded cells in the background of normal peripheral blood mononuclear cells was 1–10%. The multicolor GeneScan analytical system developed here may prove to be helpful for the diagnosis of lymphoid tumors in dogs. [ABSTRACT FROM AUTHOR]