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Expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role, by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB, at normoxia, upregulated the expression of cell cyclins and increased cell survival while it prevented apoptosis in MCF7 cells. Also, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, loss of MB enhanced partial epithelial to mesenchymal transition, thus augmenting the migratory and invasive cell behavior. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Made available in DSpace on 2022-04-28T19:52:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-03-01 Fundação para a Ciência e a Tecnologia Cancer registries are fundamental tools for collecting epidemiological cancer data and developing cancer prevention and control strategies. While cancer registration is common in the human medical field, many attempts to develop animal cancer registries have been launched over time, but most have been discontinued. A pivotal aspect of cancer registration is the availability of cancer coding systems, as provided by the International Classification of Diseases for Oncology (ICD-O). Within the Global Initiative for Veterinary Cancer Surveillance (GIVCS), established to foster and coordinate animal cancer registration worldwide, a group of veterinary pathologists and epidemiologists developed a comparative coding system for canine neoplasms. Vet-ICD-O-canine-1 is compatible with the human ICD-O-3.2 and is consistent with the currently recognized classification schemes for canine tumors. It comprises 335 topography codes and 534 morphology codes. The same code as in ICD-O-3.2 was used for the majority of canine tumors showing a high level of similarity to their human counterparts (n = 408). De novo codes (n = 152) were created for specific canine tumor entities (n = 126) and topographic sites (n = 26). The Vet-ICD-O-canine-1 coding system represents a user-friendly, easily accessible, and comprehensive resource for developing a canine cancer registration system that will enable studies within the One Health space. Departamento de Estudo de Populações Vet-OncoNet ICBAS Instituto de Ciências Biomédicas Abel Salazar Universidade do Porto EPIUnit—Instituto de Saúde Pública Universidade do Porto Laboratório Para a Investigação Integrativa e Translacional em Saúde Populacional (ITR) Department of Veterinary Medicine and Animal Production University of Naples Federico II Institute of Pathology School of Medicine Technical University of Munich Associate Laboratory for Animal and Veterinary Science-AL4AnimalS Animal and Veterinary Research Centre (CECAV) University of Trás-os-Montes and Alto Douro (UTAD) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), SP Division of Neurological Sciences DCR-VPH Vetsuisse Faculty University of Bern Department of Veterinary Sciences University of Turin National Disease Registration Service NHS Digital International Agency for Research on Cancer IARC Institute of Veterinary Pathology Vetsuisse Faculty University of Zurich School of Veterinary Science Gatton Campus The University of Queensland School of Veterinary Medicine and Animal Science University of São Paulo, SP School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), SP Fundação para a Ciência e a Tecnologia: UIDB/CVT/00772/2020

A limited number of species, including men and dogs, spontaneously develop prostate cancer (PC). The histological and molecular relevance of canine PC as a model for the disease in men remains controversial. To address this challenge, this study aimed to assess the histomorphology and expression of basal cell, urothelial and neuroendocrine markers [p63, high molecular weight cytokeratin (HMWCK), Uroplakin 3 (UPIII), neuron-specific enolase (NSE)] in canine PC (n��=��41). Based on histomorphology, 10/41 (24%), 21/41 (51%) and 9/41 (22%) were classified as adenocarcinoma (AC), urothelial carcinoma (UC), and mixed carcinoma, respectively. Tumour inflammation was common, frequently severe [20/41 (49%)], and associated with neutering (p���

A 9-year-old cat was referred with multiple, raised, ulcerative and non-ulcerative nodules in the periocular area, sclera and ear-base region, and on the ventral aspect of the tongue. In addition, a progressive ulcerative skin nodule on the tail was observed. Fine-needle aspirations of multiple nodules from the eyelid and sclera revealed the presence of histiocytes with numerous acid-fast intracellular bacilli. The replication of slowly growing mycobacteria in liquid media was detected from biopsied nodules after three months of incubation. The molecular characterization of the isolate identified Mycobacterium (M.) lepraemurium as the cause of the infection. The cat was treated with a combination of surgical excision and a four-week course of antimicrobial therapy including rifampicin combined with clarithromycin. This is an unusual manifestation of feline leprosy and the first molecularly confirmed M. lepraemurium infection in a cat with ocular involvement in Europe. The successful combination of a surgical and antimycobacterial treatment regimen is reported.

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species., Neoplasia, 23 (4), ISSN:1522-8002, ISSN:1476-5586

Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity., Chlamydia suis intestinal infection of gnotobiotic piglets is associated with intestinal NF-κB activation, which appears to be important in the early innate unprimed porcine immune response to C. suis.

Bovine abortion is a worldwide problem, but despite extensive histopathologic and molecular investigations, the cause of abortion remains unclear in about 70% of cases. Cellular debris is a commonly observed histopathologic finding in the fetal placenta and is often interpreted as necrosis. In this study, the nature of this cellular debris was characterized, and histologic changes in the normal fetal placenta during pregnancy and after delivery were assessed. In addition, the presence of the most common abortifacient pathogens in Switzerland ( Chlamydiaceae, Coxiella burnetii, Neospora caninum) was tested by polymerase chain reaction. We collected 51 placentomes and 235 cotyledons from 41 and from 50 cows, respectively. In total, cellular debris was present in 48 of 51 (94%) placentomes and in 225 of 235 (96%) cotyledons, inflammation occurred in 1 of 51 (2%) placentomes and in 46 of 235 (20%) cotyledons, vasculitis was seen in 1 of 51 (2%) placentomes and 46 of 235 (20%) cotyledons, and 18 of 51 (35%) placentomes and 181 of 235 (77%) cotyledons had mineralization. The amount of cellular debris correlated with areas of positive signals for cleaved caspase 3 and lamin A. Therefore, this finding was interpreted as an apoptotic process. In total, 10 of 50 cotyledons (20%) were positive for C. burnetii DNA, most likely representing subclinical infections. The results of our study indicate that histologic features in the fetal placenta such as cellular debris, inflammation, vasculitis, and mineralization must be considered physiological processes during pregnancy and after delivery. Therefore, their presence in placentae of aborted fetuses must be interpreted with caution and might not be necessarily linked to an infectious cause of abortion.

Canine urothelial carcinoma (UC) is the most common type of cancer of the lower urinary tract and tends to affect elderly neutered female dogs, with a high predisposition for Scottish terriers. Tumour stroma, inflammation and necrosis are poorly characterized in canine UC and their role as prognostic factors is unknown. The aims of this study were to (1) assess histologically 381 canine UCs, with emphasis on myxoid tumour stroma, inflammation and necrosis and (2) assess possible associations between these features and the available epidemiological data as well as bladder wall muscle invasion. In 103 of 381 (27%) cases, the stroma was mixed collagenous and myxoid (fibromyxoid), which was strongly associated with invasive growth of muscle (P

Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80-90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.

Breast cancer today is typically seen as malignancy with longer survival due to early-detection diagnostics, yet the cancer remains by far the most common tumor type in adult females. Recently, our group co-discovered myoglobin (MB) to be expressed in luminal cells of healthy and cancerous breast epithelia of human and mice. In human patients, MB positivity emerged as hallmark of the luminal subtype and was directly correlated with estrogen receptor alpha positivity and hence a significantly better prognosis. Cancerous cells’ MB also appears to interact with the known tumor suppressor p53. However, if and how myoglobin itself restricts mammary tumorigenesis is completely unclear. To understand how MB exerts its alleged tumor-suppressive effects and if it impacts response of mammary tumors to chemo- and/or radiotherapy, we examine the molecular role of MB in breast tumor formation and progression through a) in vitro studies (MCF7 breast cancer BrCa cell clones; wildtype (wt) controls versus CRISPR/Cas9-engineered clones of a MB and/or p53 knockout (MBko/p53ko), b) in vivo mouse models to obtain spontaneously forming breast tumors, with or without MB, in a p53 deficient background. Comparing MCF7 BrCa MBko and p53ko clones vs. their corresponding wildtype counterpart supports our hypothesis of a feed forward loop between MB and p53 proteins, besides interfering with estrogen receptor expression. In addition to that, loss of MB was noticed to exert p53-independent upregulation on cell cyclins with more S phase cell population. That was reflected by a significant increase in survival of the MBko cells, in presence or absence of p53. Hypoxic MBko cells exhibited a partial epithelial to mesenchymal transition and hence a more migratory phenotype, relative to MBwt controls. Additionally, MB seems to stimulate apoptosis of the normoxic cancer cells. On the other hand, murine tumors were found to phenocopy human BrCa in the extent of their MB expression. Interestingly, MB-devoid tumors showed a significantly higher proliferation index and fat accumulation. Further studies will look into the clinical significance of that. In conclusion, MB seems to occupy unknown roles in cancer cells beyond or different from its classical O2 storage/transport functions. Unraveling these novel roles in governing tumor development and virulence will hopefully provide innovative strategies for future breast cancer interventions. Citation Format: Mostafa A. Aboouf, Julia Armbruster, Franco Guscetti, Markus Thiersch, Max Gassmann, Thomas A. Gorr. The role of myoglobin in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-17.

Allogeneic hematopoietic cell transplantation (allo-HCT) not only is an effective treatment for several hematologic malignancies but can also result in potentially life-threatening graft-versus-host disease (GvHD). GvHD is caused by T cells within the allograft attacking nonmalignant host tissues; however, these same T cells mediate the therapeutic graft-versus-leukemia (GvL) response. Thus, there is an urgent need to understand how to mechanistically uncouple GvL from GvHD. Using preclinical models of full and partial MHC-mismatched HCT, we here show that the granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by allogeneic T cells distinguishes between the two processes. GM-CSF drives GvHD pathology by licensing donor-derived phagocytes to produce inflammatory mediators such as interleukin-1β and reactive oxygen species. In contrast, GM-CSF did not affect allogeneic T cells or their capacity to eliminate leukemic cells, retaining undiminished GvL responses. Last, tissue biopsies and peripheral blood mononuclear cells from patients with grade IV GvHD showed an elevation of GM-CSF-producing T cells, suggesting that GM-CSF neutralization has translational potential in allo-HCT.

Data collected in animal cancer registries comprise extensive and valuable information, even more so when evaluated in context with precise population data. The authors evaluated 11 740 canine skin tumors collected in the Swiss Canine Cancer Registry from 2008–2013, considering data on breed, sex, age, and anatomic locations. Their incidence rate (IR) per 100 000 dogs/year in the Swiss dog population was calculated based on data from the official and mandatory Swiss dog registration database ANIS. The most common tumor types were mast cell tumors (16.35%; IR, 60.3), lipomas (12.47%; IR, 46.0), hair follicle tumors (12.34%; IR, 45.5), histiocytomas (12.10%; IR, 44.6), soft tissue sarcomas (10.86%; IR, 40.1), and melanocytic tumors (8.63%; IR, 31.8) with >1000 tumors per type. The average IR of all tumor types across the 227 registered breeds was 372.2. The highest tumor incidence was found in the Giant Schnauzer (IR, 1616.3), the Standard Schnauzer (IR, 1545.4), the Magyar Vizsla (IR, 1534.6), the Rhodesian Ridgeback (IR, 1445.0), the Nova Scotia Duck Tolling Retriever (IR, 1351.7), and the Boxer (IR, 1350.0). Mixed-breed dogs (IR, 979.4) had an increased IR compared to the average of all breeds. Previously reported breed predispositions for most tumor types were confirmed. Nevertheless, the data also showed an increased IR for mast cell tumors and melanocytic tumors in the Nova Scotia Duck Tolling Retriever and for histiocytomas in the Flat Coated Retriever. The results from this study can be taken into consideration when selecting purebred dogs for breeding to improve a breed’s health.

Feline injection site sarcomas (FISS) were first described in the early 1990s. Despite extensive research, the pathogenesis of these tumours has not been elucidated conclusively. Their appearance and the marked increase in their incidence has been mainly connected to the injection of vaccines, and it is assumed that a chronic inflammatory reaction at the injection site triggers subsequent malignant transformation. The role of alum-based adjuvants has been discussed, but is controversial. The present study of the Swiss Feline Cancer Registry (SFCR) with data from 2009 to 2014 revealed a marked decrease of the incidence of fibrosarcomas compared with the previous observation period. Notably, this drop occurred after a non-adjuvanted feline leukaemia virus vaccine was introduced in Switzerland in 2007. This observation, together with the previous findings of the SFCR, further supports the notion that alum-adjuvanted vaccines are involved in the genesis of FISS and that non-adjuvanted vaccines might be safer for cats.

Diagnostic records are a key feature of any cancer epidemiology, prevention or control strategy for man and animals. Therefore, the information stored in human and animal cancer registries is essential for undertaking comparative epidemiological, pathogenic and therapeutic research. This study presents the Swiss Canine Cancer Registry, containing case data compiled between 1955 and 2008. The data consist of pathology diagnostic records issued by three veterinary diagnostic laboratories in Switzerland. The tumours were classified according to the guidelines of the International Classification of Oncology for Humans on the basis of tumour type, malignancy and body location. The dogs were classified according to breed, age, sex, neuter status and place of residence. The diagnostic data were correlated with data on the Swiss general dog population and the incidence of cancer in dogs was thus investigated. A total of 67,943 tumours were diagnosed in 121,963 dogs and 47.07% of these were malignant. The most common tumour location was the skin (37.05%), followed by mammary glands (23.55%) and soft tissue (13.66%). The most common tumour diagnoses were epithelial (38.45%), mesenchymal (35.10%) and lymphoid tumours (13.23%). The results are compared with data in other canine registries and similarities in tumour distribution and incidence are noted. It is hoped that this study will mark the beginning of continuous registration of dog tumours in Switzerland, which, in turn, will serve as a reference for research in the fields of animal and human oncology.

Objective: CXL penetration depth is an important variable influencing clinical treatment effect and safety. The purposes of this study were to determine the penetration depth of CXL in rabbit and equine corneas in epithelium-on and epithelium-off procedures and to assess an ex vivo fluorescent biomarker staining assay for objective assessment of CXL penetration depth. Procedures: CXL treatment was performed according to a standardized protocol on 21 and 17 rabbit eyes and on 12 and 10 equine eyes with and without debridement, respectively. Control corneas were treated similarly, but not exposed to CXL. Hemicorneas were stained with either phalloidin and DAPI to visualize intracellular F-actin and nuclei, or with hematoxylin and eosin. Loss of actin staining was measured and compared between groups. Results: Epithelium-off CXL caused a median actin cytoskeleton loss with a demarcation at 274 μm in rabbits and 173 μm in horses. In non-CXL-treated controls, we observed a median actin cytoskeleton loss with a demarcation at 134 μm in rabbits and 149 μm in horses. No effect was detected in the epithelium-on procedure. Conclusions: CXL penetration depth, as determined by a novel ex vivo fluorescent assay, shows clear differences between species. A distinct effect was observed following epithelium-off CXL treatment in the anterior stroma of rabbits, but no different effect was observed in horses in comparison with nontreated controls. Different protocols need to be established to effectively treat equine patients with infectious corneal disease.

Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15-60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

A major obstacle for preclinical testing of Alzheimer’s disease (AD) therapies is the availability of translationally relevant AD models. Critical for the validation of such models is the application of the same approaches and techniques used for the neuropathological characterization of AD. Deposition of amyloid-β 42 (Aβ42) plaques and neurofibrillary tangles containing phospho-Tau (pTau) are the pathognomonic features of AD. In the neuropathologic evaluation of AD, immunohistochemistry (IHC) is the current standard method for detection of Aβ42 and pTau. Although IHC is indispensable for determining the distribution of AD pathology, it is of rather limited use for assessment of the quantity of AD pathology. We have recently developed Luminex-based assays for the quantitative assessment of Aβ42 and pTau in AD brains. These assays are based on the same antibodies that are used for the IHC-based diagnosis of AD neuropathologic change. Here we report the application and extension of such quantitative AD neuropathology assays to commonly used genetically engineered AD models and to animals that develop AD neuropathologic change as they age naturally. We believe that identifying AD models that have Aβ42 or pTau levels comparable to those observed in AD will greatly improve the ability to develop AD therapies. Abbreviations: Alzheimer’s disease (AD); amyloid β 42 (Aβ42); phospho-Tau (pTau); immunohistochemistry (IHC)

This satellite meeting to the 12th International Conference on Malignant Lymphoma was conceived to bring together European researchers focused on canine lymphoma to explore several facets of this promising model of human disease. A series of invited lectures showed striking similarities between the two diseases namely in topics related to pathogenesis, diagnosis and classification and therapy. In particular, the potential value of the model was shown at the level of the NF-kB/p65 pathway, the Bcl-2 family of proteins, Ki67 and the S-phase fraction, as well as the MMPs, VEGF and PDGF. The utility of the growing body of well-characterized canine cell lines was stressed. The value of cytology and flow cytometry as tools for diagnosis, disease progression monitoring and prognosis were emphasized, whereas the failure so far of the standard immunohistochemical panel to differentiate between germinal centre and non-germinal centre diffuse large B-cell lymphomas subtypes in dogs was discussed. Further contributions included the report of encouraging results from a chemo-immunotherapy trial administered to dogs with diffuse large B-cell lymphoma, an overview on the use of radiation therapy for canine lymphoma and the role of surgery in splenic lymphoma. Altogether, the success of this meeting, attended by more than 160 participants, documents the rising interest for the spontaneous canine lymphoma model. Copyright © 2013 John Wiley & Sons, Ltd.

Aelurostrongylus abstrusus infection is common in endemic areas and may cause severe respiratory clinical signs. Computed tomography (CT) is an important tool to diagnose pulmonary disease, because it allows detection of small lesions and discrimination of superimposed structures. The purpose of this study was to characterize by CT and angiographic CT the pulmonary lesions in six cats before, and 48 and 81 days after inoculation with 100 or 800 A. abstrusus infective larvae. Histological examination of the accessory lung lobe was performed to determine the microscopic, pathomorphologic correlate of the CT findings. The predominant CT lesion consisted of multiple nodules of varying size distributed throughout the lungs, severity depending on infectious dose. The histological correlate of the nodular lesions was multifocal dense granulomatous to mixed inflammatory cell infiltrates, including eosinophils distributed in the parenchyma and obliterating the alveoli. Marked, multifocal, dose-dependent thickening of the bronchi and adjacent interstitial changes blurred the margins of the outer serosal surface of the bronchi and vessels. Histologically, this was due to peribronchial mixed cell inflammation. During the course of infection some of the nodular and peribronchial changes were replaced by areas of ground-glass opacity. In addition to providing detailed depiction of pulmonary lesions resulting from an infectious cause and clearly defining lesions with respect to time and severity of infection, CT allowed quantitative assessment of bronchial thickness and lymph node size during the course of disease. Findings indicated that CT characteristics of this disease are consistent with pathologic findings.

Schweizer Archiv für Tierheilkunde, 155 (4), ISSN:0036-7281, ISSN:1664-2848

The BH3-only protein Bad is a proapoptotic Bcl-2 family member that acts as a sensitizer in intrinsic apoptosis by inactivating antiapoptotic members through heterodimer formation. Bad has been shown to contribute to tumorigenesis, including lymphoma formation in humans and mice, through alteration in expression or functional status. Here, its immunohistochemical expression was analyzed in canine nonneoplastic and lymphoma tissues using tissue microarrays. Bad was expressed in the cytoplasm of a wide range of nonneoplastic tissues, especially epithelial cells. Nonneoplastic lymph nodes displayed weak immunostaining in the follicular germinal centers only. Immunoblotting supported these observations but also revealed presence of nonspecific labeling in some organs. Of 81 lymphomas, 29 (35.8%) displayed moderate to strong immunohistochemical Bad labeling, and a significant expression increase was found in lymphomas (especially B cell and double negative) compared to nonneoplastic lymph nodes. These findings warrant further investigations of the functional status, the involvement of partner proteins, and a possible impact of Bad on prognosis in canine lymphoma.

SummaryCancer registries are valuable sources for epidemiological research investigating risk factors underlying different types of cancer incidence. The present study is based on the Swiss Feline Cancer Registry that comprises 51,322 feline patient records, compiled between 1965 and 2008. In these records, 18,375 tumours were reported. The study analyses the influence of sex, neutering status, breed, time and age on the development of the most common tumour types and on their locations, using a multiple logistic regression model. The largest differences between breeds were found in the development of fibrosarcomas and squamous cell carcinomas, as well as in the development of tumours in the skin/subcutis and mammary gland. Differences, although often small, in sex and neutering status were observed in most analyses. Tumours were more frequent in middle-aged and older cats. The sample size allowed detailed analyses of the influence of sex, neutering status, breed and age. Results of the study are mainly consistent with previous analyses; however, some results cannot be compared with the existing literature. Further investigations are necessary, since feline tumours have not been investigated in depth to date. More accurate comparisons would require the definition of international standards for animal cancer registries.

This study is based on the Swiss Canine Cancer Registry, comprising 121,963 diagnostic records of dogs compiled between 1955 and 2008, in which 63,214 (51.83%) animals were diagnosed with tumour lesions through microscopical investigation. Adenoma/adenocarcinoma (n = 12,293, 18.09%) was the most frequent tumour diagnosis. Other common tumour diagnoses were: mast cell tumour (n = 4,415, 6.50%), lymphoma (n = 2,955, 4.35%), melanocytic tumours (n = 2,466, 3.63%), fibroma/fibrosarcoma (n = 2,309, 3.40%), haemangioma/haemangiosarcoma (n = 1,904, 2.80%), squamous cell carcinoma (n = 1,324, 1.95%) and osteoma/osteosarcoma (n = 842, 1.24%). The relative occurrence over time and the most common body locations of those tumour diagnoses are presented.Analyses of the influence of age, breed, body size, sex and neutering status on tumour development were carried out using multiple logistic regression. In certain breeds/breed categories the odds ratios (ORs) for particular tumours were outstandingly high: the boxer had higher ORs for mast cell tumour and haemangioma/haemangiosarcoma, as did the shepherd group for haemangioma/haemangiosarcoma, the schnauzer for squamous cell carcinoma and the rottweiler for osteoma/osteosarcoma. In small dogs, the risk of developing mammary tumours was three times higher than in large dogs. However, small dogs were less likely to be affected by many other tumour types (e.g. tumours of the skeletal system).Examination of the influence of sex and neutering status on tumour prevalence showed that the results depend on the examination method. In all sampling groups the risk for female dogs of developing adenoma/adenocarcinoma was higher than for male dogs. Females had a lower risk of developing haemangioma/haemangiosarcoma and squamous cell carcinoma than males. Neutered animals were at higher risk of developing specific tumours outside the genital organs than intact animals.The sample size allows detailed insight into the influences of age, breed, body size, sex and neutering status on canine tumour development. In many cases, the analysis confirms the findings of other authors. In some cases, the results are unique or contradict other studies, implying that further investigations are necessary.

Pancreatitis has been described in cats with diabetes mellitus, although the number of studies currently available is very limited. In addition, ketoacidosis has been hypothesized to be associated with pancreatitis in diabetic cats. The aims of the present study were to investigate whether diabetic cats have pancreatitis and to determine if pancreatitis is more frequent with ketoacidosis. Samples of pancreas were collected postmortem from 37 diabetic cats, including 15 with ketoacidosis, and 20 control cats matched for age, sex, breed, and body weight. Sections were stained with hematoxylin and eosin, double-labeled for insulin/CD3, insulin/CD20, insulin/myeloperoxidase, insulin/PCNA, and glucagon/Ki67, and single-labeled for Iba1. A previously proposed semiquantitative score was used to characterize pancreatitis, along with counts of inflammatory cells. Scores of pancreatitis and the number of neutrophils, macrophages, and lymphocytes in the exocrine pancreas did not differ between diabetic and control cats or between diabetic cats with and without ketoacidosis. Of note, PCNA-positive acinar cells were increased ( P = .002) in diabetic cats, particularly near islets ( P < .001). Ki67-positive acinar cells were increased only near islets ( P = .038). Ketoacidosis was not linked to proliferation. The results suggest that histopathologic evidence of pancreatitis may not be more frequent in diabetic cats and that ketoacidosis may not be associated with it at the time of death. Augmented PCNA-positive acinar cells might indicate increased proliferation due to chronic pancreatitis. The reason behind the prevalent proliferation of acinar cells surrounding pancreatic islets deserves further investigation.

For various types of tumor therapy, it is suggested that co-targeting of tumor microenvironment, mainly tumor vasculature, mediates tumor response mechanisms. Immunohistochemistry for glucose transporter-1 (GLUT-1), carbonic anhydrase-IX (CAIX), Ki-67, and von Willebrand factor VIII for microvessel density (MVD) were performed on formalin-fixed paraffin-embedded samples of canine oral malignant neoplasms. Polarographic oxygen measurements (median pO2) and perfusion data via contrast-enhanced power Doppler ultrasound (median vascularity, median blood volume) provided additional information. Ninety-two samples were analyzed: sarcomas (n = 32), carcinomas (n = 30), and malignant melanomas (n = 30). Polarographic oxygen and perfusion data was available in 22.8% (sarcomas n = 9, carcinomas n = 7, melanomas n = 5), and 27.1% (sarcomas n = 10, carcinomas n = 8, melanomas n = 7) of cases, respectively. GLUT-1 expression was detected in 46.7% of all samples, and was generally weak. CAIX expression was found in 34.8% of all samples. Median Ki-67 score and MVD count was 19% and 17, respectively. The evaluation of the GLUT-1 score and continuous data showed significantly lower GLUT-1 levels in sarcomas (mean 5.1%, SD 6.2) versus carcinomas and melanomas (mean 16.5%/ 19.0%, SD 17.3/ 20.9, p = 0.001). The expression of CAIX correlated mildly positively with GLUT-1 (p = 0.018, rho = 0.250) as well as with Ki-67 (p = 0.014, rho = 0.295). MVD showed a significantly lower level in melanomas (mean 12.6, SD 7.7) versus sarcomas and carcinomas (mean 21.8/ 26.9, SD 13.0/20.4, p = 0.001). Median vascularity and blood volume were significantly lower in sarcomas (mean 10.4%, SD 11.0, and mean 6.3%, SD 6.5, respectively) versus carcinomas (mean 39.2%, SD 16.4 and mean 33.0%, SD 25.6, respectively) and melanomas (mean 36.0%, SD 18.3, and 31.5%, SD 24.5). Between the 3 histological groups, there was neither a significant difference in the GLUT-1 and CAIX score and continuous data, nor the Ki67 score, or polarographic oxygen measurements. GLUT-1 continuous data and Ki-67 (p

Background: Progesterone receptor (PR) antagonist aglepristone (RU534) has been used successfully for pregnancy termination and therapy of pyometra, vaginal tumors, and mammary hyperplasia in bitches and queens. All of these conditions share with canine mammary carcinomas the expression of PR. Objectives: To study the effect of RU534 on proliferation and apoptosis in canine mammary carcinomas in relation to PR expression. Animals: Twenty-seven nonspayed bitches with mammary carcinomas were treated with either 2 doses of 20 mg/kg RU534 (n = 22, RU534-treated group) or oil placebo (n = 5, control group) on days 1 and 8. Methods: Tumor samples were collected before (day 1) and after (day 15) treatment for immunohistochemistry. PR expression, proliferation index (PI), and apoptotic index (AI) were determined using antibodies against PR, Ki67, and cleaved lamin A/C antigens, respectively. The effect of treatment on these parameters was analyzed. Results: Differential expression of PR between day 1 (59.1% PR-positive tumors) and day 15 (36.4% PR-positive tumors) was observed in RU534-treated tumors exclusively. After RU534 treatment, mean PI was significantly decreased in PR-positive but unchanged in PR-negative RU534-treated tumors. A reduction of ≥20% in PI was found in 61.5% of RU534-treated tumors with PR expression. Conversely, no effect on AI was observed after RU534 treatment. Conclusions and Clinical Importance: Neoadjuvant RU534 treatment had PR expression-related inhibiting effects on proliferation of canine mammary carcinoma cells.

Adrenal necrosis has been reported as a complication of trilostane application in dogs with hyperadrenocorticism. One suspicion was that necrosis results from the increase of adrenocorticotropic hormone (ACTH) during trilostane therapy. The aim of the current study was to assess the effects of ACTH and trilostane on adrenal glands of rats. For experiment 1, 36 rats were divided into 6 groups. Groups 1.1 to 1.4 received ACTH in different doses (60, 40, 20, and 10 μg/d) infused subcutaneously with osmotic minipumps for 16 wk. Group 1.5 received saline, and group 1.6 received no therapy. For experiment 2, 24 rats were divided into 3 groups. Group 2.1 and 2.2 received 5 and 50 mg/kg trilostane/d orally mixed into chocolate pudding for 16 wk. Eight control rats received pudding alone. At the end of the experiments, adrenal glands were assessed for necrosis by histology and immunohistochemistry; levels of endogenous ACTH and nucleosomes were assessed in the blood. Rats treated with 60 μg ACTH/d showed more hemorrhage and vacuolization and increased numbers of apoptotic cells in the adrenal glands than rats treated with 20 or 10 μg ACTH/d, trilostane, or control rats. Rats treated with 60 μg ACTH/d had a higher amount of nucleosomes in the blood compared with rats treated with 10 μg ACTH/d, trilostane, or saline. We conclude that in healthy rats ACTH, but not trilostane, causes adrenal degeneration in a dose-dependent manner. Results of this study support the hypothesis that adrenal gland lesions seen in trilostane-treated dogs are caused by ACTH and not by trilostane.

The hair follicle has a lifelong capacity to cycle through recurrent phases of controlled growth (anagen), regression (catagen) and quiescence (telogen), each associated with specific morphological changes. A comprehensive classification scheme is available for mice to distinguish the cycle stages anagen I-VI, catagen I-VIII and telogen. For dogs, such a classification system does not exist, although alopecia associated with hair cycle arrest is common. We applied analogous morphological criteria and various staining techniques to subdivide the canine hair cycle stages to the same extent as has been done in mice. Of all the staining techniques applied, haematoxylin and eosin stain, Sacpic, Masson Fontana and immunohistochemistry for vimentin and laminin proved to be most useful. To evaluate the applicability of our criteria, we investigated skin biopsies from healthy beagle dogs (n=20; biopsies from shoulder and thigh) kept in controlled conditions. From each biopsy, at least 50 hair follicles were assessed. Statistical analysis revealed that 30% of the follicles were in anagen (12% early and 18% late), 8% in catagen (2% early, 5% late and 1% not determinable) and 27% in telogen. Thirty-five per cent of hair follicles could not be assigned to a specific cycle stage because not all follicles within one biopsy were oriented perfectly. In conclusion, this guide will not only be helpful for the investigation of alopecic disorders and possibly their pathogenesis, but may also serve as a basis for research projects in which the comparison of hair cycle stages is essential, e.g. comparative analysis of gene expression patterns.

Aims/hypothesis: In vitro studies point to a toxic effect of high glucose and non-esterified fatty acids on beta cells. Whether elevated levels of glucose and lipids induce beta cell loss in vivo is less clear. The domestic cat has recently been proposed as a valuable animal model for human type 2 diabetes because feline diabetes shows several similarities with diabetes in humans, including obesity-induced insulin resistance, impaired beta cell function, decreased number of beta cells and pancreatic amyloid deposition. Methods: We infused healthy cats with glucose or lipids for 10days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: 25-30mmol/l; triacylglycerols: 3-7mmol/l). Results: Glucose and lipid levels were adequately targeted. Plasma non-esterified fatty acids were increased by lipid infusion 1.7-fold. A dramatic and progressive decline of plasma insulin levels was observed in glucose-infused cats beginning after 2days of hyperglycaemic clamp. In contrast, plasma insulin concentration and glucose tolerance test were not affected by hyperlipidaemia. Compared with controls, glucose-infused cats had a 50% decrease in beta cells per pancreatic area. Apoptotic islet cells and cleaved caspase-3-positive beta cells were observed in glucose-infused cats only. Conclusions/interpretation: Sustained hyperglycaemia but not hyperlipidaemia induces early and severe beta cell dysfunction in cats, and excess glucose causes beta cell loss via apoptosis in vivo. Hyperglycaemic clamps in cats may provide a good model to study the pathogenesis of glucose toxicity in beta cells

In most validation studies of tissue microarrays (TMAs), a fixed number of cores with a given diameter are analyzed to determine the degree of accuracy by which the TMA represents the whole section. The statistical model described in the present study predicts this property for various combinations of 2 core sizes (0.6 mm and 1.2 mm) and different core numbers. The model was based on artificial TMA core biopsies generated from Ki-67 and active caspase-3 immunostains of 40 canine lymphoma samples. Positivity was scored on a continuous scale, and a large number of cells were analyzed with the help of semiautomated cell counting. Despite considerable differences in range and distribution of Ki-67 and active caspase-3 positivity values, the model predictions showed a high degree of agreement for both markers. Comparison of 0.6 mm and 1.2 mm cores indicated that the use of small cores necessitates inclusion of a larger number of samples but requires counting a markedly smaller number of cells. Suitability of TMAs to determine the immunophenotype of the whole section was assessed using 2 different combinations of core sizes and numbers. Both displayed a high degree of concordance with the whole section (κ0.6 = 0.79; κ1.2 = 0.91). The present study provides a basis for the use of TMAs in future high-throughput immunohistochemical investigations of selected markers in canine lymphomas. The statistical model presented can be used to determine an optimal TMA design depending on a desired accuracy.

PTK787/ZK 222584 is an inhibitor of the vascular endothelial growth factor (VEGF) receptor tyrosine kinases. In this study, the effectiveness of PTK787/ZK 222584 and radiation on canine oral melanoma xenografts was assessed. Xenografts were treated with radiotherapy (4 · 6 Gy), or with PTK787/ZK 222584, or with a combination of both. Treatment efficacy was assessed using a tumour growth delay assay, serial power Doppler and pO2 measurements during and after therapy. There was a significant growth delay for the group treated with radiation alone and for the combined treatment group. However, tumour growth delay was similar in both groups. Tumours were hypoxic before irradiation and no significant re-oxygenation occurred during therapy. In all tumours, vascularity and perfusion were significantly lower at the end of the study but no significant differences in perfusion, vascularity and oxygenation status were observed between and within treatment groups. The combination of PTK787/ZK 222584 and radiotherapy did not perform better than radiotherapy alone in this model.

Equine and feline dysautonomias are characterized histopathologically by degenerating neurons with chromatolysis, pyknotic and sometimes eccentric nuclei, and loss of Nissl substance in the peripheral autonomic ganglia. Because it may be difficult to distinguish pathological from post-mortem changes in affected ganglia by histopathological examination, synaptophysin was evaluated as an immunohistochemical marker. Degenerating neurons showed strong intracytoplasmic labelling indicating abnormal accumulation of synaptophysin. It was concluded that synaptophysin immunohistochemistry is a helpful tool for detecting degenerating neurons in equine (grass sickness) and feline (Key-Gaskell syndrome) dysautonomias.

The presence of a complete (BH1-3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells. It is unclear, however, what distinct roles the members of the large family of BH3-only proapoptotic molecules play in apoptosis. Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict. We have, therefore, established three functional categories of BH3-only human proapoptotic proteins based on their toxicity after expression in budding yeast: directly killing (tBid), sensitizing in Bax/Bcl-2 expressing cells (Bad or Puma), and non-toxic (BNip3, BNip3L, and Noxa). The mechanism of killing by the proapoptotic molecules in yeast, however, is not due to activation of the recently described yeast metacaspase MCA1.

Objective—To determine responses of canine and feline lenses to incubation in a medium with a high glucose concentration. Sample Population—Lenses from 35 dogs and 26 cats. Procedure—Glucose concentrations were measured in paired lenses from 25 dogs and 17 cats after incubation for 14 days in high-glucose (30 mmol of glucose/ L) or control (6 mmol of glucose/L) medium. Aldose reductase activity was measured spectrophotometrically in the incubated lenses and in freshly frozen lenses from 10 dogs and 9 cats. Two lenses of each group were studied histologically. Results—Canine and feline lenses in high-glucose medium developed glucose-specific opacities of variable localization and extent. Canine lenses developed equatorial vacuoles, but severity of the lesions was not associated with the age of the dog. Lenses from young cats (≤ 4 years old) developed extensive posterior cortical opacities, whereas those from older cats (> 4 years old) did not. Glucose concentrations were similar in all lenses incubated in high-glucose medium; however aldose reductase activity was significantly lower in lenses from older cats, compared with lenses from young cats and from dogs. Conclusions and Clinical Relevance—High aldose reductase activity and glucose-related opacities suggest a central role for this enzyme in the pathogenesis of diabetic cataracts in dogs and cats. Because onset of diabetes mellitus usually occurs in cats > 7 years of age, low activity of aldose reductase in lenses of older cats may explain why diabetic cataracts are rare in this species despite hyperglycemia. (Am J Vet Res 2002;63:1591–1597)

Dogs present with spontaneous neoplasms biologically similar to human cancers. Apoptotic pathways are deregulated during cancer genesis and progression and are important for therapy. We have assessed the degree of conservation of a set of canine Bcl-2 family members with the human and murine orthologs. To this end, seven complete canine open reading frames were cloned in this family, four of which are novel for the dog, their sequences were analyzed, and their functional interactions were studied in yeasts. We found a high degree of overall and domain sequence homology between canine and human proteins. It was slightly higher than between murine and human proteins. Functional interactions between canine pro-apoptotic Bax and Bak and anti-apoptotic Bcl-xL, Bcl-w, and Mcl-1 were recapitulated in yeasts. Our data provide support for the notion that systems based on canine-derived proteins might faithfully reproduce Bcl-2 family member interactions known from other species and establish the yeast as a useful tool for functional studies with canine proteins.