Genetic Basis of Dilated Cardiomyopathy in Dogs and Its Potential as a Bidirectional Model

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment

De ontwikkeling van ex vivo/ in vitro immuunfunctie-testen voor de bot, Platichthys flesus

De stand van zaken betreffende de ontwikkeling van immuunfunctie testen voor de bot, Platichthys flesus is beschreven. Epidemiologische veldstudies hebben aangetoond dat deze platvis species een hoge prevalentie aan infectieuze ziekten heeft, hetgeen gecorreleerd kan worden met de aanwezigheid van contaminanten in het aquatische milieu. Om een mogelijk causaal verband vast te kunnen stellen en om de bot als indicator species te kunnen gebruiken, zijn immuunfunctie-testen ontwikkeld. Het rapport beschrijft de ontwikkeling en toepassing van assays voor het bepalen van niet-specifieke cytotoxie, lymfocytenstimulatie en de immunohistochemische identificatie van leukocyt populaties in de bot. Deze assays, tezamen met andere testen die momenteel in ontwikkeling zijn, zullen gebruikt worden om de immuunstatus van de bot te 'peilen' in immunotoxicologische experimenten.The state of the art concerning the development of immune function tests in flounder, Platichthys flesus has been described. It is known from epidemiological field surveys that this flatfish species has a high prevalence of infectious diseases which is correlated with aquatic pollution. In order to provide more evidence for a possible causal link and to use flounder as an indicator species, immune function tests were developed. This report describes the development and application of assays to measure non-specific cytotoxicity, lymphocyte stimulation and immunohistochemical identification of leukocyte populations of flounder. These assays, as well as others which are developed at the moment, will be applied to monitor the immune status of flounder in immunotoxicological experiments.DGM/DW

Short term toxicity of polycyclic aromatic hydrocarbons (PAC) in flounder (Platichthys flesus): preliminary experiments with benzo[a]pyrene en 7,12-dimethylbenz[a]anthracene

Dit rapport beschrijft een aantal pilot studies waarin de bot (Platichthys flesus) onder gecontroleerde laboratoriumomstandigheden kortdurend werd blootgesteld aan twee PAK's, te weten benzo[a]pyreen (BaP) en 7,12-dimethylbenz[a]anthraceen (DMBA). Blootstelling vond plaats met hoge concentraties in de waterfase of via injectie in de lichaamsholte. Effecten op uiterlijk en gedrag werden geregistreerd. Met behulp van histopathologisch onderzoek werd een inventarisatie gemaakt van de effecten op diverse organen waaronder kieuwen, thymus, hepatopancreas, maagdarmkanaal, pro- en mesonephros, milt, ovaria of testikels. Hierbij werd tevens gekeken naar inductie van het enzym cytochroom P450 1A1 (CYP 1A1) als maat voor de biotransformatie van de PAKþs en de vorming van biologisch actieve metabolieten. Blootstelling van de botten aan de PAK's resulteerde ondanks de hoge concentraties in de waterfase niet in sterke effecten. Slechts een geringe toename van immunoreactiviteit voor CYP 1A1 in levercellen van geinjecteerde botten werd geconstateerd. De bot blijkt dus bij deze experimentele opzet weinig gevoelig voor PAK's te zijn. Daarom spelen de PAK's mogelijk een weinig belangrijke rol bij het ontstaan van ziektes bij botten in het veld.In this report 5 pilot studies on short term exposure of flounder (Platichthys flesus) to benzo[a]pyrene (BaP) and 7,12-dimehtylbenz[a]anthracene (DMBA) under controlled laboratory conditions are described. Exposure took place with high concentrations through the water-phase and by intraperitoneal injection. Effects on behaviour and exterior were checked, and the histopathological effects on gills, thymus, hepatopancreas, digestive tract, pro- and mesonephros, spleen, and ovary or testicles were investigated. We also looked for the induction of the enzyme cytochrome P450 1A1 (CYP 1A1) as a measure for the biotransformation of PAH's and the formation of biologically active metabolites. Exposure of flounder to the PAH's did not result in striking effects in spite of the high concentrations. Only a slight increase in immunoreactivity for CYP 1A1 in hepatocytes of the injected animals was noted. Flounder appears to be not very sensitive to PAHs under the used experimental conditions. Therefore PAHs may not play a very important role in the induction of diseases in flounder as seen in the field.DGM/DW

Laboratoriumcondities, anatomie en histologie van de bot, Platichthys flesus

Diverse veldonderzoeken suggereren een relatie tussen watervervuiling en enkele ziekten (b.v. huidzweren, vin-rot, lymphocystis infectie en levertumoren) bij vissen. Onderzoeken met de bot (Platichthys flesus), gehouden onder semi-veld (mesocosmos) condities, wijzen op een mogelijk causaal verband tussen watervervuiling, de inductie van lever tumoren en lymphocystis virus infectie. Onderzoek onder gecontroleerde laboratorium omstandigheden blijft echter noodzakelijk om een causaal verband tussen vervuiling met specifieke xenobiotische stoffen en de inductie van deze ziekten aan te tonen. Voor gebruik in laboratorium onderzoek is de bot gedurende anderhalf jaar zonder grote problemen gehouden en opgekweekt. Er wordt een korte beschijving gegeven van de huisvesting en verzorging van de botten onder laboratoriumomstandigheden. De botten worden in een multi-stress project (chemische- en microbiologische stress) gebruikt om de carcinogene- en immunotoxische effecten van polyclisch aromatische koolwaterstoffen te evalueren. In dit rapport wordt de normale histologie van de bot beschreven om pathologische veranderingen te kunnen interpreteren waarbij de nadruk wordt gelegd op de organen die betrokken zijn bij de immunologische afweer (thymus, nier, milt, melanomacrofagen-centra en het bloed), de lever, huid en kieuwen. Verschillen in anatomie en histologie tussen de bot, andere teleosta en zoogdieren worden beschreven aan de hand van literatuurgegevens en eigen waarnemingen.Several field survey's suggest a relation between marine/estuarine pollution and some diseases (e.g. skin ulcer, fin rot, lymphocystis disease and liver tumours in fish. Studies with flounder (Platichthys flesus) kept under semi-field (mesocosm) conditions indicate a causal link between aquatic contamination, hepatic tumour induction and lymphocystis virus infection. Research under controlled laboratory conditions is necessary in order to establish causal links between pollution with specific xenobiotics and disease induction. For laboratory investigations flounder has been kept and raised successfully for 1.5 years without major problems. A short description is given of the maintenance of flounder under laboratory conditions. They are used in a multi-stress project (chemical- and microbiological stress) evaluating the carcinogenic- and immunotoxic effects of polycyclic aromatic hydrocarbons. In order to be able to interpret pathological lesions, the normal anatomy and histology of flounder is described in this report. The emphasis is put on the organs involved in the immunological response (thymus, kidney, spleen, melanomacrophage centres and the blood), the liver, skin and gills. Differences in anatomy and histology between flounder, other teleost fish and mammals are discussed, using data from literature and our own findings.DGM/DW

Sequential Treatment of a Large Pituitary Corticotroph Neoplasm and Associated Neurological Signs in a Dog

No standardized treatment guidelines are reported in veterinary medicine for dogs with large pituitary corticotroph neoplasms causing neurological signs, and such dogs usually have a short overall survival. When these dogs undergo pituitary surgery and the tumor regrows there are few reports of subsequent treatments. A 7 yr old male Maltese diagnosed with pituitary-dependent hypercortisolism developed seizures in conjunction with a large pituitary corticotroph adenoma and underwent transsphenoidal hypophysectomy. After 3 yr of clinical remission, hypercortisolism recurred, and trilostane therapy was initiated. One year later, the dog developed new neurological signs and computed tomography revealed regrowth of a large pituitary mass that was then treated with radiation therapy. The dog lived disease-free for 3 more yr. At postmortem examination, a more aggressive pituitary neoplasm than the one examined at the time of surgery was found, which is suggestive of malignant transformation into a carcinoma despite the absence of convincing metastasis

Korte termijn toxiciteit van bis (tri-n-butyltin) oxide in de bot (Platichthys flesus) ; pathologie en immuunfunctie

Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relation between pollution and the increase in prevalence of tumours and infectious diseases in fish. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders (Platichthys flesus) were exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The possible histopathological effects on several organs (gill, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non specific and specific resistance was studied using ex vivo/ in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which are in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 mug TBT ; field: 7.2 mug TBT), caused mortality after 7-12 days, decreased the condition factor, resulted in gill lesions, and induced significant reduction of the non specific resistance. No marked effects on the relative thymus volume, or the specific immune system were noted after exposure to TBTO.Veldonderzoeken uitgevoerd in diverse verontreinigde kustwateren in zowel Europa als de Verenigde Staten laten een duidelijke relatie tussen vervuiling en een toegenomen prevalentie van tumoren en infectieziekten bij vissen zien. Een van de chemische verbindingen in de veelheid van xenobiotica die gevonden kunnen worden in vervuilde wateren en sedimenten is de organotinverbinding tributyltin (TBT), hoofdzakelijk afkomstig van verf waarmee scheepshuiden worden behandeld om ongewenste aangroei van onder andere algen en schelpdieren tegen te gaan. Dit rapport beschrijft een onderzoek waarin botten (Platichthys flesus) via het water werden blootgesteld aan bis(tri-n-butyltin)oxide (TBTO) onder gecontroleerde laboratoriumomstandigheden. De mogelijke histopathologische effecten aan diverse organen (kieuw, huid, ogen, lever, buiknier, ovarium/testikel, milt en maagdarmkanaal) werden onderzocht en tevens werd er een morfometrisch onderzoek van de thymus (zwezerik) uitgevoerd om de doelorganen van TBTO bij de bot vast te stellen. Daarnaast werd het functioneren van zowel de specifieke- als aspecifieke afweer onderzocht door gebruik te maken van ex vivo / in vitro immuun functietesten. Blootstelling van botten aan TBTO gehaltes in dezelfde orde van grootte als de maximaal gemeten TBT gehaltes in de veldsituatie (experiment: 17.3 mug TBT ; veld: 7.2 mug TBT) veroorzaakte sterfte na 7-12 dagen, een vermindering van de conditiefactor, kieuwlaesies en een significante onderdrukking van de aspecifieke weerstand. Er werden geen duidelijke effecten waargenomen op het relatieve volume van de thymus en op het specifieke immuunsysteem na blootstelling aan TBTO

Korte termijn toxiciteit van bis (tri-n-butyltin) oxide in de bot (Platichthys flesus) ; pathologie en immuunfunctie

Veldonderzoeken uitgevoerd in diverse verontreinigde kustwateren in zowel Europa als de Verenigde Staten laten een duidelijke relatie tussen vervuiling en een toegenomen prevalentie van tumoren en infectieziekten bij vissen zien. Een van de chemische verbindingen in de veelheid van xenobiotica die gevonden kunnen worden in vervuilde wateren en sedimenten is de organotinverbinding tributyltin (TBT), hoofdzakelijk afkomstig van verf waarmee scheepshuiden worden behandeld om ongewenste aangroei van onder andere algen en schelpdieren tegen te gaan. Dit rapport beschrijft een onderzoek waarin botten (Platichthys flesus) via het water werden blootgesteld aan bis(tri-n-butyltin)oxide (TBTO) onder gecontroleerde laboratoriumomstandigheden. De mogelijke histopathologische effecten aan diverse organen (kieuw, huid, ogen, lever, buiknier, ovarium/testikel, milt en maagdarmkanaal) werden onderzocht en tevens werd er een morfometrisch onderzoek van de thymus (zwezerik) uitgevoerd om de doelorganen van TBTO bij de bot vast te stellen. Daarnaast werd het functioneren van zowel de specifieke- als aspecifieke afweer onderzocht door gebruik te maken van ex vivo / in vitro immuun functietesten. Blootstelling van botten aan TBTO gehaltes in dezelfde orde van grootte als de maximaal gemeten TBT gehaltes in de veldsituatie (experiment: 17.3 mug TBT ; veld: 7.2 mug TBT) veroorzaakte sterfte na 7-12 dagen, een vermindering van de conditiefactor, kieuwlaesies en een significante onderdrukking van de aspecifieke weerstand. Er werden geen duidelijke effecten waargenomen op het relatieve volume van de thymus en op het specifieke immuunsysteem na blootstelling aan TBTO.Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relation between pollution and the increase in prevalence of tumours and infectious diseases in fish. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders (Platichthys flesus) were exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The possible histopathological effects on several organs (gill, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non specific and specific resistance was studied using ex vivo/ in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which are in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 mug TBT ; field: 7.2 mug TBT), caused mortality after 7-12 days, decreased the condition factor, resulted in gill lesions, and induced significant reduction of the non specific resistance. No marked effects on the relative thymus volume, or the specific immune system were noted after exposure to TBTO.DGM/DW

Gene Expression Profiling of Histiocytic Sarcomas in a Canine Model: The Predisposed Flatcoated Retriever Dog

Background: The determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed. Methods: Microarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses. Results: QPCR analyses identified the significantly altered expression of nine genes; PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process. Conclusions: This study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human

A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects

Biological-effects monitoring in estuarine environments is complex as a result of strong gradients and fluctuations in salinity and other environmental conditions, which may influence contaminant bioavailability and the physiology and metabolism of the organisms. To select the most robust and reliable biological-effect methods for monitoring and assessment programmes, a large-scale field study was conducted in two estuarine transects in the Netherlands. The locations ranged from heavily polluted harbour areas (the ports of Rotterdam and Amsterdam) to cleaner coastal and freshwater sites. Assessment methods used included a variety of biomarkers in flounder (Platichthys flesus) and a range of in vitro (sediment extracts) and in vivo bioassays. Multivariate statistical analysis was applied to investigate correlations and relationships between various biological effects and contaminant levels in flounder liver or sediments. Several biological methods seemed to be too much affected by salinity differences for routine use in estuaries. The most discriminative biomarkers in the study were hepatic metallothionein content and biliary 1-OH pyrene in fish. Mechanism-based in vitro assays DR-CALUX and ER-CALUX applied to sediment extracts for screening of potential toxicity were much more responsive than in vivo bioassays with macro-invertebrates using survival as an endpoin