Transcription profiling of feline mammary carcinomas and derived cell lines reveals biomarkers and drug targets associated with metabolic and cell cycle pathways.

The molecular heterogeneity of feline mammary carcinomas (FMCs) represents a prognostic and therapeutic challenge. RNA-Seq-based comparative transcriptomic profiling serves to identify recurrent and exclusive differentially expressed genes (DEGs) across sample types and molecular subtypes. Using mass-parallel RNA-Seq, we identified DEGs and performed comparative function-based analysis across 15 tumours (four basal-like triple-negative [TN], eight normal-like TN, and three luminal B fHER2 negative [LB fHER2-]), two cell lines (CL, TiHo-0906, and TiHo-1403) isolated from the primary tumours (LB fHER2-) of two cats included in this study, and 13 healthy mammary tissue controls. DEGs in tumours were predominantly upregulated; dysregulation of CLs transcriptome was more extensive, including mostly downregulated genes. Cell-cycle and metabolic-related DEGs were upregulated in both tumours and CLs, including therapeutically-targetable cell cycle regulators (e.g. CCNB1, CCNB2, CDK1, CDK4, GTSE1, MCM4, and MCM5), metabolic-related genes (e.g. FADS2 and SLC16A3), heat-shock proteins (e.g. HSPH1, HSP90B1, and HSPA5), genes controlling centrosome disjunction (e.g. RACGAP1 and NEK2), and collagen molecules (e.g. COL2A1). DEGs specifically upregulated in basal-like TN tumours were involved in antigen processing and presentation, in normal-like TN tumours encoded G protein-coupled receptors (GPCRs), and in LB fHER2- tumours were associated with lysosomes, phagosomes, and endosomes formation. Downregulated DEGs in CLs were associated with structural and signalling cell surface components. Hence, our results suggest that upregulation of genes enhancing proliferation and metabolism is a common feature among FMCs and derived CLs. In contrast, the dissimilarities observed in dysregulation of membrane components highlight CLs' disconnection with the tumour microenvironment. Furthermore, recurrent and exclusive DEGs associated with dysregulated pathways might be useful for the development of prognostically and therapeutically-relevant targeted panels.

Sox9, Hopx, and survivin and tuft cell marker DCLK1 expression in normal canine intestine and in intestinal adenoma and adenocarcinoma.

Self-renewal of the intestinal epithelium originates from stem cells located at the crypt base. Upregulation of various stem cell markers in intestinal epithelial neoplasms indicates a potential role of stem cells in tumorigenesis. In this study, the immunoreactivity of potential intestinal stem cell markers (Sry box transcription factor 9 [Sox9], homeodomain-only protein [Hopx], survivin) and tuft cell marker doublecortin-like kinase 1 (DCLK1) in normal canine intestine and intestinal epithelial neoplasms was investigated. Formalin-fixed paraffin-embedded (FFPE) small and large intestine as well as intestinal neoplasms (55 colorectal adenomas [CRAs], 17 small intestinal adenocarcinomas [SICs], and 12 colorectal adenocarcinomas [CRCs]) were analyzed immunohistologically. Potential stem cell markers Sox9, Hopx, and survivin were detected in the crypts of normal canine small and large intestine. DCLK1+ tuft cells were present in decreasing numbers along the crypt-villus axis of the jejunum and rarely detectable in large intestine. In canine intestinal epithelial tumors, nuclear Sox9 immunoreactivity was detectable in 84.9% (CRA), 80% (CRC), and 77% of epithelial neoplastic cells (SIC). Hopx and survivin were expressed within cytoplasm and nuclei of neoplastic cells in benign and malignant tumors. DCLK1 showed a cytoplasmic reaction within neoplastic cells. The combined score of Hopx, DCLK1, and survivin varied among the examined cases. Overall, malignant tumors showed lower DCLK1 scores but higher Hopx scores in comparison with benign tumors. For survivin, no differences were detectable. In conclusion, stem cell markers Sox9, Hopx, and survivin were detectable at the crypt base and the immunoreactivity of Sox9, DCLK1, survivin, and Hopx was increased in canine intestinal adenomas and adenocarcinomas compared with normal mucosa.

Meningoencephalomyelitis of Unknown Origin in Cats: A Case Series Describing Clinical and Pathological Findings.

Meningoencephalomyelitis of unknown origin (MUO) is an umbrella term describing inflammatory changes of the central nervous system (CNS) with suspected non-infectious etiology. Diagnosis of MUO mostly remains presumed in a clinical setting. Histopathological and immunohistochemical examination of CNS tissue represent additional tools for detection of inflammation and the exclusion of specific infectious agents. While MUO is well-described in canine patients, only little is known about MUO in cats. Previous reports of feline MUO involve either clinical findings or histopathological examination but not both. The present case series is the first report describing both clinical and histopathological findings of feline MUO: Four cats (age: 1.7-17.8 years) showed acute to chronic progressive neurological signs of encephalopathy or myelopathy. Three cats had extraneural signs (hyperthermia, weight loss, hyporexia, leukocytosis). Magnetic resonance imaging (MRI) showed multifocal intraparenchymal lesions in forebrain, brainstem or spinal cord with homogenous contrast enhancement (2/2). Cerebrospinal fluid (CSF) examination was normal or displayed albuminocytologic dissociation. Histopathology revealed a multifocal, lympho-histiocytic meningoencephalitis in three cases and a lympho-histiocytic myelitis in one case. Immunohistochemistry for feline parvovirus, feline coronavirus, feline herpesvirus, tick borne encephalitis virus, Borna disease virus, morbillivirus, rabies virus, suid herpesvirus-1, and Toxoplasma gondii were negative in all cases. ONE SENTENCE SUMMARY: This case series is the first one reporting both clinical and histopathological findings in cats with MUO. Feline MUO incorporates heterogeneous subtypes of sterile CNS inflammation.

Publisher Correction: Analysis of Copy-Number Variations and Feline Mammary Carcinoma Survival.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of six canine prostate adenocarcinoma and three transitional cell carcinoma cell lines derived from primary tumor tissues as well as metastasis.

Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches in vitro. PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment (doxorubicin, carboplatin and meloxicam) were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.

Analysis of Copy-Number Variations and Feline Mammary Carcinoma Survival.

Feline mammary carcinomas (FMCs) are highly malignant. As the disease-free survival (DFS) and overall survival (OS) are short, prognostication is crucial. Copy-number variations (CNVs) analysis by next-generation sequencing serves to identify critical cancer-related genomic regions. Thirty-three female cats with FMCs were followed during two years after surgery. Tumours represented tubulopapillary and solid carcinomas encompassing six molecular subtypes. Regardless of the histopathological diagnosis, molecular subtypes showed important differences in survival. Luminal A tumours exhibited the highest DFS (p = 0.002) and cancer-specific OS (p = 0.001), and the lowest amount of CNVs (p = 0.0001). In contrast, basal-like triple-negative FMCs had the worst outcome (DFS, p < 0.0001; and OS, p < 0.00001) and were the most aberrant (p = 0.05). In the multivariate analysis, copy-number losses (CNLs) in chromosome B1 (1-23 Mb) harbouring several tumour-repressors (e.g. CSMD1, MTUS1, MSR1, DBC2, and TUSC3) negatively influenced DFS. Whereas, copy-number gains (CNGs) in B4 (1-29 Mb) and F2 (64-82.3 Mb) comprising epithelial to mesenchymal transition genes and metastasis-promoting transcription factors (e.g. GATA3, VIM, ZEB1, and MYC) negatively influenced DFS and cancer-specific OS. These data evidence an association between specific CNVs in chromosomes B1, B4 and F2, and poor prognosis in FMCs.

Canine primary jejunal and colonic epithelial cells predominantly express TLR5 and TLR9 but do not change TLR expression pattern after stimulation with certain Toll-like receptor ligands.

The intestinal mucosa is in contact with abundant luminal antigens and coordinates immune responses to differentiate commensals from pathogens. Intestinal epithelial cells (IECs) not only represent a physical barrier but also an immunologically important cell type that recognizes microbe-associated molecular patterns via Toll-like receptors (TLR). The importance of TLR expression has been elucidated for intestinal disorders in humans, mice and dogs. However, as knowledge about canine intestinal TLRs is mainly limited to the transcriptional level, the present study analyzed the protein expression of TLR2, TLR3, TLR4, TLR5 and TLR9 by primary canine IECs in the steady state and after stimulation with TLR ligands. This exhibited TLR5 and TLR9 to be predominantly expressed in canine IECs. TLR stimulation did not result in changes of the TLR expression pattern. Further studies are needed to elucidate whether this implicates hyporesponsiveness of canine IECs towards TLR stimulation under steady state conditions.

TiHo-0906: a new feline mammary cancer cell line with molecular, morphological, and immunocytological characteristics of epithelial to mesenchymal transition.

Feline mammary carcinomas (FMCs) with anaplastic and malignant spindle cells histologically resemble the human metaplastic breast carcinoma (hMBC), spindle-cell subtype. hMBCs display epithelial-to-mesenchymal transition (EMT) characteristics. Herein we report the establishment and characterization of a cell line (TiHoCMglAdcar0906; TiHo-0906) exhibiting EMT-like properties derived from an FMC with anaplastic and malignant spindle cells. Copy-number variations (CNVs) by next-generation sequencing and immunohistochemical characteristics of the cell line and the tumour were compared. The absolute qPCR expression of EMT-related markers HMGA2 and CD44 was determined. The growth, migration, and sensitivity to doxorubicin were assessed. TiHo-0906 CNVs affect several genomic regions harbouring known EMT-, breast cancer-, and hMBCs-associated genes as AKT1, GATA3, CCND2, CDK4, ZEB1, KRAS, HMGA2, ESRP1, MTDH, YWHAZ, and MYC. Most of them were located in amplified regions of feline chromosomes (FCAs) B4 and F2. TiHo-0906 cells displayed an epithelial/mesenchymal phenotype, and high HMGA2 and CD44 expression. Growth and migration remained comparable during subculturing. Low-passaged cells were two-fold more resistant to doxorubicin than high-passaged cells (IC50: 99.97 nM, and 41.22 nM, respectively). The TiHo-0906 cell line was derived from a poorly differentiated cellular subpopulation of the tumour consistently displaying EMT traits. The cell line presents excellent opportunities for studying EMT on FMCs.

Cruciate ligament degeneration and stifle joint synovitis in 56 dogs with intact cranial cruciate ligaments: Correlation of histological findings and numbers and phenotypes of inflammatory cells with age, body weight and breed.

The majority of dogs with ruptured cranial cruciate ligaments (crCLs) have inflammatory changes of the stifle joint suggesting that synovitis is an important factor which is involved in the development of degenerative ligament changes. Detailed information is not available concerning the possible occurrence of inflammatory changes in the stifle joint synovium of dogs with macroscopically intact crCLs and its correlation with presence and severity of degenerative changes of the crCLs. Therefore, the purpose of this study was to examine post mortem tissue samples of 56 dogs with non-ruptured crCLs histologically for the presence of inflammatory and degenerative changes of the stifle joint synovium and cruciate ligaments, respectively. In 25/56 (44.6%) dogs, histology showed that both lymphoplasmacytic synovitis and degenerative alterations of the crCLs were present. In these dogs, there was a significant positive statistical correlation between the severity of synovitis and degenerative crCL lesions. The results suggest that synovitis in the stifle joints of dogs with non-ruptured crCLs is a frequent event and probably is involved in the development of degenerative lesions occurring in canine crCLs. Also, the severity of crCL degeneration in these 25 dogs was significantly correlated with their age and body weight. In 2/56 (3.6%) cases, only lymphoplasmacytic synovitis was found in the absence of degenerative crCL lesions. In 15/56 (26.8%) dogs, only degenerative lesions of the crCLs without synovitis were present. Statistically, a significant correlation was found between the severity of degenerative alterations and age and body weight of these dogs. Phenotyping of inflammatory cells by immunohistochemistry showed that the synovium of dogs which histologically had lymphoplasmacytic synovitis was infiltrated with CD3+ T lymphocytes, CD79+ B lymphocytes, major histocompatibility class II antigen (MHC class II)+ cells and macrophages expressing CD163 or S100/A8/S100A9 (calprotectin), while tartrate-resistant acid phosphatase (TRAP)+ cells were absent. Quantification and statistical evaluation of inflammatory cell types in the inflamed synovium revealed that the numbers of lymphocytic cells and macrophages were significantly correlated with the severity of synovitis. These findings indicate that, besides T and B lymphocytes, both pro- and anti-inflammatory macrophages play a role in the development of degenerative crCL alterations.

Immunohistochemical characterization of gastrointestinal macrophages/phagocytes in dogs with inflammatory bowel disease (IBD) and non-IBD dogs.

Intestinal Mϕ play a pivotal role in the maintenance of gut homeostasis, but can also contribute to inflammation such as inflammatory bowel disease (IBD). In contrast to human tissues, little is known about phenotypes of Mϕ in the canine gastrointestinal tract. Therefore, an immunohistochemical study was performed using Abs against Mϕ-associated molecules (Cluster of differentiation (CD)64, CD163, CD204, ionized calcium-binding adaptor molecule 1, L1 Ag, and MHC II) on stomach, duodenum, jejunum, ileum and colon from non-IBD dogs. In addition, marker-expression in the stomach, duodenum and colon of the non-IBD dogs was compared to that in dogs with IBD. Results revealed predominance of resident Mϕ displaying an anti-inflammatory phenotype represented by expression of CD163 as well as CD204 in the gut of non-IBD dogs with high Mϕ numbers especially present in the small intestinal villus area. Compared to non-IBD tissue counterparts, stomach, duodenum, and colon from dogs with IBD showed reduced Mϕ numbers with the exception of slightly increased numbers of CD64+ Mϕ. Correlation analyses between marker-expression of Mϕ and the Canine Inflammatory Bowel Disease Activity Index as well as histological scores failed to reveal relevant relationships. The present study provides evidence of the canine steady state gastrointestinal tract being dominated by Mϕ with anti-inflammatory properties maintaining gut homeostasis. A significant reduction in these resident Mϕ may reflect disturbances in homeostatic capacity that could contribute to the development of canine IBD. In contrast to human IBD and murine disease models, infiltration of pro-inflammatory Mϕ does not significantly contribute to the inflammatory process of canine IBD, which may illustrate possible species-specific differences in IBD pathogenesis.

Plexiform Vasculopathy in Feline Cervical Lymph Nodes.

Plexiform vasculopathy refers to an endothelial proliferative disorder affecting cervical or inguinal lymph nodes of cats. The cause of this disorder and the origin of the proliferating endothelial cells are still unknown. In 4 cats with a history of a slowly growing, well-demarcated, nonpainful mass adjacent to the thyroid gland, an enlarged dark brown to red lymph node was removed. Histologically, the lymph nodes showed severe loss of lymphoid tissue with accumulations of erythrocytes. In addition, networks of capillary structures with well-differentiated endothelial cells on a collagen-rich stroma were observed, consistent with benign plexiform vasculopathy. Immunohistochemistry revealed the expression of the vascular endothelial markers CD31 and factor VIII-related antigen. In addition, immunolabeling with a Prox-1 antibody indicated a lymphendothelial origin. With respect to our findings, a lymphendothelial origin has to be considered in cases of intranodal vascular neoplasms.

Effects of a Grapevine Shoot Extract Containing Resveratrol and Resveratrol Oligomers on Intestinal Adenoma Development in Mice: In Vitro and In Vivo Studies.

SCOPE: Evidence suggests that the dietary consumption of plant extracts containing polyphenols might help prevent the onset of cancers of the gastrointestinal tract. In the present study, the chemopreventive and antiproliferative efficacy of a grapevine shoot extract (Vineatrol®30) containing resveratrol and resveratrol oligomers is investigated in vivo and in vitro. METHODS AND RESULTS: The in vivo study is performed using ApcMin mice on a high-fat diet, which represents a model of human adenomatous polyposis, while the potential of the extract as well as some of its isolated constituents to inhibit intestinal adenoma cell proliferation in vitro is investigated using APC10.1 cells derived from an ApcMin mouse. Vineatrol®30 at a low (2.3 mg kg-1  diet) or high dose (476 mg kg-1  diet) reduces the adenoma number in male and adenoma volume in female animals. Furthermore, Vineatrol®30 as well as resveratrol and two resveratrol tetramers compromise the expansion of APC10.1 cells by reducing cell number, inducing cell cycle arrest, cellular senescence, and apoptosis. However, except for the extract, none of the isolated resveratrol oligomers is more efficacious than resveratrol in these cells. CONCLUSION: Vineatrol®30 may merit further investigation as a potential dietary gastrointestinal cancer chemopreventive agent in humans.

Zoonotic intestinal helminths interact with the canine immune system by modulating T cell responses and preventing dendritic cell maturation.

Parasite co-evolution alongside the mammalian immune system gave rise to several modulatory strategies by which they prevent exaggerated pathology and facilitate a longer worm survival. As little is known about the immunoregulatory potential of the zoonotic canine parasites Ancylostoma caninum and Toxocara canis in the natural host, the present study aimed to investigate whether their larval excretory-secretory (ES) products can modulate the canine immune system. We demonstrated TcES to increase the frequency of CD4+ Foxp3high T cells, while both AcES and TcES were associated with elevated Helios expression in Foxp3high lymphocytes. ES products were further capable of inducing IL-10 production by lymphocytes, which was mainly attributed to CD8+ T cells. ES treatment of PBMCs prior to mitogen stimulation inhibited polyclonal proliferation of CD4+ and CD8+ T cells. Moreover, monocyte-derived ES-pulsed dendritic cells reduced upregulation of MHC-II and CD80 in response to lipopolysaccharide. The data showed that regulation of the canine immune system by A. caninum and T. canis larvae comprises the modification of antigen-specific and polyclonal T cell responses and dendritic cell maturation.

Heterogeneity of macrophages in canine histiocytic ulcerative colitis.

Histiocytic ulcerative colitis (HUC) is a chronic enteropathy which most notably occurs in Boxer dogs and French bulldogs. The inflamed mucosa is hallmarked by large, foamy, periodic acid-Schiff (PAS)-positive macrophages infiltrating the colonic mucosa. As little is known about their origin and phenotype, an immunohistochemical study was performed using different macrophage markers. Generally, canine colonic macrophages showed high expression of ionised calcium-binding adaptor molecule 1 and MHC class II. In canine HUC, macrophages revealed up-regulation of lysozyme and L1 Ag but decreased CD163 expression compared with controls, suggesting them to be pro-inflammatory cells, whereas the healthy colonic mucosa was characterised by an anti-inflammatory macrophage phenotype. In addition, PAS reaction was used to discriminate macrophage subpopulations. PAS- macrophages displayed higher expression of L1 Ag and CD64, whereas PAS+ cells, which were only present in HUC patients, were characterised by increased expression of lysozyme, inducible nitric oxide synthase and CD204. This indicates PAS+ cells to be mature macrophages contributing to the inflammatory process, which are most likely maintained by differentiation of immature PAS- macrophages continuously recruited from blood monocytes. In summary, macrophage heterogeneity in canine HUC probably illustrates their different maturation states and functions compared with the healthy animals.

Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines.

Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

Cutaneous form of maculopapular mastocytosis in a foal.

BACKGROUND: Cutaneous mastocytosis is a rare benign disease occurring in domestic animals and humans. In previous reports, dermal findings in foals were accompanied by systemic mast cell infiltrations, whereas lesions in human cutaneous mastocytosis, including urticaria pigmentosa and solitary mastocytoma, are usually restricted to the skin. OBJECTIVES: To describe a new variant of equine cutaneous maculopapular mastocytosis lacking systemic involvement. ANIMALS: A 2.5-month-old warmblood foal with multiple skin nodules since birth. METHODS: Clinical examination (including haematology, fine needle biopsy and thoracic radiographs), postmortem examination, histopathology and immunohistochemistry. RESULTS: Clinical examination showed 41 skin nodules that contained numerous mast cells as detected by cytology. Macroscopic examination at postmortem examination revealed intradermal circumscribed lesions ranging from 2 to 5 cm in diameter. Histologically, they were composed of well differentiated mast cells with metachromatic granules stained with toluidine blue accompanied by many eosinophils. Immunohistochemically, mast cells had mast cell growth factor receptor c-KIT predominating at the cell surface and intracytoplasmic expression of tryptase. In other organs similar mast cell infiltrations were not detected. CONCLUSIONS AND CLINICAL IMPORTANCE: The case presented here fulfils the criteria of equine cutaneous maculopapular mastocytosis (ECMM), representing a rare entity in foals that is reported to be associated with spontaneous regression, although the long-term prognosis is not known. Unlike in previous reports, lesions described here were restricted to the skin. This may imply that ECMM is primarily a dermal disease sharing similarities with urticaria pigmentosa in young children.

Prevalence of the Prefoldin Subunit 5 Gene Deletion in Canine Mammary Tumors.

BACKGROUND: A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors. METHODS: Droplet digital PCR for PFDN5 was performed in DNA from 102 malignant, 40 benign mammary tumors/dysplasias, 11 non-neoplastic mammary tissues and each corresponding genomic DNA from leukocytes. The copy number of PFDN5 was normalized to a reference amplicon on canine chromosome 32 (CFA32). Z-scores were calculated, based on Gaussian distributed normalized PFDN5 copy numbers of the leukocyte DNA. Z-scores ≤ -3.0 in tissue were considered as being indicative of the PFDN5 deletion and called as such. The Ki-67 proliferation index was assessed in a subset of 79 tissue samples by immunohistochemistry. RESULTS: The deletion was confirmed in 24% of all malignant tumors, detected in only 7.5% of the benign tumors and was not present in any normal mammary tissue sample. The subgroup of solid carcinomas (n = 9) showed the highest frequency of the deletion (67%) and those malignomas without microscopical high fraction of benign tissue (n = 71) had a 32% frequency (p<0.01 vs. benign samples). The Ki-67 score was found to be significantly higher (p<0.05) in the PFDN5-deleted group compared to malignant tumors without the deletion. CONCLUSIONS: A somatic deletion of the PFDN5 gene is recurrently present in canine mammary cancer, supporting a potential role in carcinogenesis. The association of this deletion with higher Ki-67 indicates an increased proliferation rate and thus a link to tumor aggressiveness can be hypothesized. The confirmation of earlier results warrants further studies on PFDN5 as cancer-driver gene.

Pathology in Captive Wild Felids at German Zoological Gardens.

This retrospective study provides an overview on spontaneous diseases occurring in 38 captive wild felids submitted for necropsy by German zoological gardens between 2004 and 2013. Species included 18 tigers, 8 leopards, 7 lions, 3 cheetahs and 2 cougars with an age ranging from 0.5 to 22 years. Renal lesions, predominantly tubular alterations (intra-tubular concrements, tubular degeneration, necrosis, intra-tubular cellular debris, proteinaceous casts, dilated tubuli) followed by interstitial (lympho-plasmacytic inflammation, fibrosis, metastatic-suppurative inflammation, eosinophilic inflammation) and glomerular lesions (glomerulonephritis, glomerulosclerosis, amyloidosis) were detected in 33 out of 38 animals (87%). Tumors were found in 19 of 38 felids (50%) with 12 animals showing more than one neoplasm. The tumor prevalence increased with age. Neoplasms originated from endocrine (11), genital (8), lympho-hematopoietic (5) and alimentary organs (4) as well as the mesothelium (3). Most common neoplasms comprised uterine/ovarian leiomyomas (5/2), thyroid adenomas/adenocarcinoma (5/1), pleural mesotheliomas (3), hemangiosarcomas (2) and glossal papillomas (2). Inflammatory changes were frequently encountered in the intestine and the lung. Two young animals displayed metastatic mineralization suggestive of a vitamin D- or calcium intoxication. One tiger exhibited degenerative white matter changes consistent with an entity termed large felid leukoencephalomyelopathy. Various hyperplastic, degenerative and inflammatory changes with minor clinical significance were found in several organs. Summarized, renal lesions followed by neoplastic changes as well as inflammatory changes in lung and gastrointestinal tract represent the most frequent findings in captive wild felids living in German zoological gardens.

Canine gut dendritic cells in the steady state and in inflammatory bowel disease.

Alongside the intestinal border, dendritic cells (DCs) sample large amounts of endogenous and potentially pathogenic antigens followed by initiation of protective immune responses or induction of tolerance. Breakdown of oral tolerance towards commensal bacteria is suggested to be crucial for the development of both human and canine inflammatory bowel disease (IBD). The aim of this study was to investigate canine intestinal DCs in the steady state and in dogs with IBD using multicolour immunofluorescence. In the healthy gut, DC-like cells expressed MHC II, CD1a8.2 and CD11c, and, in lower amounts, CD11b, within lamina propria, Peyer's patches (PPs) and mesenteric lymph nodes (MLNs), whereas those expressing CD80 and CD86 were only present in PPs and MLNs. Occasionally, DC-like cells were in contact with the intestinal lumen through transepithelial projections. In canine IBD, CD1a8.2+, CD11b+ and CD11c+ DC-like cells were decreased within the stomach, duodenum and colon, whereas the colonic mucosa revealed elevation of CD86+ DC-like cells. The complex network of DC-like cells in the gut indicates their important role in canine mucosal immunity, including active sampling of luminal antigens. Furthermore, their shift in diseased dogs suggests a pathogenetic significance for canine IBD.

Immunohistochemical investigation of Foxp3 expression in the intestine in healthy and diseased dogs.

Intestinal immune regulation including development of oral tolerance is of great importance for the maintenance of intestinal homeostasis. Concerning this, regulatory T cells (Tregs) occupy a pivotal role in cell-mediated immunosuppression. Dysregulation of mucosal immunology leading to an abnormal interaction with commensal bacteria is suggested to play a key role in the pathogenesis of Inflammatory Bowel Disease (IBD) in men and dogs. The aim of this study was to characterise the expression of Foxp3 in the normal canine gut of 18 dogs (mean age: 6.03 years), in 16 dogs suffering from IBD (mean age: 5.05 years), and of 6 dogs with intestinal nematode infection (mean age: 0.87 years) using immunohistochemistry. In the duodenum, Tregs in healthy dogs declined from villi (median: 10.67/62 500 µm2) to crypts (median: 1.89/62 500 µm2). Tregs were further increased in the villi of middle-aged dogs (median: 18.92/62 500 µm2) in contrast to juvenile (median: 3.50/62 500 µm2) and old (median: 9.56/62 500 µm2) individuals. Compared to healthy controls, animals suffering from IBD revealed reduced numbers of Tregs in duodenal villi (median: 4.13/62 500 µm2). Dogs with intestinal nematode infection displayed increased numbers of Tregs (median: 21.06/62 500 µm2) compared to healthy animals.Age-related changes indicate a progressive establishment of oral tolerance and immunosenescence in the canine elderly. The results further suggest that a defect in Treg homeostasis may be involved in the pathogenesis of canine IBD. In contrast, increased numbers of Tregs in the duodenum may be due to nematode infection.