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BACKGROUND: Endovascular selective intra-arterial (ESIA) infusion of cellular oncotherapeutics is a rapidly evolving strategy for treating glioblastoma. Evaluation of ESIA infusion requires a unique animal model. Our goal was to create a rabbit human GBM model to test IA infusions of cellular therapies and to test its usefulness by employing clinical-grade microcatheters and infusion methods to deliver mesenchymal stem cells loaded with an oncolytic adenovirus, Delta-24-RGD (MSC-D24). METHODS: Rabbits were immunosuppressed with mycophenolate mofetil, dexamethasone, and tacrolimus. They underwent stereotactic xenoimplantation of human GBM cell lines (U87, MDA-GSC-17, and MDA-GSC-8-11) into the right frontal lobe. Tumor formation was confirmed on magnetic resonance imaging, histologic, and immunohistochemistry analysis. Selective microcatheter infusion of MSC-D24 was performed via the ipsilateral internal carotid artery to assess model utility and the efficacy and safety of this approach. RESULTS: Twenty-five rabbits were implanted (18 with U87, 2 MDA-GSC-17, and 5 MDA-GSC-8-11). Tumors formed in 68% of rabbits (77.8% for U87, 50.0% for MDA-GSC-17, and 40.0% for MDA-GSC-8-11). On MRI, the tumors were hyperintense on T2-weighted image with variable enhancement (evidence of blood brain barrier breakdown). Histologically, tumors showed phenotypic traits of human GBM including varying levels of vascularity. ESIA infusion into the distal internal carotid artery of 2 ml of MSCs-D24 (107 cells) was safe in the model. Examination of post infusion specimens documented that MSCs-D24 homed to the implanted tumor at 24 hours. CONCLUSIONS: The intracranial immunosuppressed rabbit human GBM model allows testing of ESIA infusion of novel therapeutics (eg, MSC-D24) in a clinically relevant fashion.
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Neoplasias Encefálicas , Glioblastoma , Animais , Humanos , Coelhos , Glioblastoma/patologia , Infusões Intra-Arteriais , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Células-Tronco/patologiaRESUMO
Half a million patients in the USA alone require treatment for burns annually. Following an extensive burn, it may not be possible to provide sufficient autografts in a single setting. Genetic manipulations (GM) of pigs offer the possibility of reducing primate humoral and cellular rejection of pig skin xenografts and thus extending graft survival. We compared the survival of skin grafts from pigs with 9-GM with that of autografts and allografts in squirrel monkeys. Monitoring for rejection was by (1) macroscopic examination, (2) histopathological examination of skin biopsies, and (3) measurement of anti-monkey and anti-pig IgM and IgG antibodies. Autografts (n = 5) survived throughout the 28 days of follow-up without histopathological features of rejection. Median survival of allografts (n = 6) was 14 days and of pig xenografts (n = 12) 21 days. Allotransplantation was associated with an increase in anti-monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti-pig IgM and IgG. In all cases, histopathologic features of rejection were similar. 9-GM pig skin xenografts survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti-pig IgM and IgG antibodies, these did not cross-react with monkey antigens, indicating that a primary 9-GM pig skin graft would not be detrimental to a subsequent monkey skin allograft.
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Queimaduras , Transplante de Pele , Animais , Queimaduras/terapia , Rejeição de Enxerto , Sobrevivência de Enxerto , Imunoglobulina G , Imunoglobulina M , Saimiri , Suínos , Transplante HeterólogoRESUMO
With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculated were similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.
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Transplante de Rim , Animais , Suínos , Humanos , Papio , Inulina , Cefoxitina , Transplante Heterólogo , RimRESUMO
Ferret systemic coronavirus (FRSCV) causes a highly fatal disease of ferrets (Mustela putorius furo). It is believed to be a mutated variant of ferret enteric coronavirus (FRECV) and has a clinical presentation similar to that of feline infectious peritonitis virus (FIPV) in cats. The interplay of infectious diseases and host genetics will become a greater issue in the research environment as genetically modified species other than rodents become available due to advances in gene editing technology. In this case series, we present the clinical and histopathologic features of a FRSCV outbreak that affected 5 out of 10 ferrets with α-1 antitrypsin knockout (AAT KO) over an approximately 1-y period. Clinical features varied, with the affected ferrets presenting with some combination of wasting, hind limb paralysis, incontinence or sudden death. Multiple ferrets had gross pathologic lesions consistent with FRSCV, but the lesions were typically mild. Microscopic pyogranulomatous inflammation was present in 4 ferrets. Immunohistochemistry using an anti-feline coronavirus antibody that cross reacts with ferret coronavirus confirmed infection of intralesional macrophages in 4 out of 5 animals with suspected FRSCV infection. PCR testing of formalin fixed tissue was negative for all ferrets. PCR testing of feces from healthy wild-type ferrets indicated that the endemic presence of FRECV genotype 2, while PCR surveillance testing of other in-house AAT KO ferrets revealed both enteric coronavirus genotypes 1 and 2. This case series highlights the potential for greater disease incidence in the future as genetically modified ferrets are used more often, and may support exclusion of FRECV and similar viruses from highly susceptible ferret genotypes.
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Infecções por Coronavirus , Coronavirus , Animais , Gatos , Furões , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/patologia , Inflamação , Reação em Cadeia da Polimerase , Coronavirus/genéticaRESUMO
Congestion, granular platelet debris both within macrophage and extracellularly, and neutrophil infiltration in the spleen of a baboon that was euthanized with profound thrombocytopenia.
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Trombocitopenia , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/uso terapêutico , Papio , Trombocitopenia/tratamento farmacológico , Transplante HeterólogoRESUMO
BACKGROUND: Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE: To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS: ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS: ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 106 cells/20 mL to 1 × 108 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION: This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 108 cells/10 mL (107 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.
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Vacinas Anticâncer/administração & dosagem , Transplante de Células-Tronco Mesenquimais/métodos , Terapia Viral Oncolítica/métodos , Animais , Cães , Xenoenxertos , Humanos , Infusões Intra-Arteriais , Masculino , Modelos AnimaisRESUMO
BACKGROUND: Polyglactin 910 is a synthetic braided, absorbable suture commonly used in surgery. Though polyglactin 910 suture-related pseudoinfection is well documented in the human literature, it has not been previously reported in the veterinary literature. CASE DESCRIPTION: A 3-year-old female, ovariectomized but otherwise experimentally naïve Yucatan pig was evaluated for a several week history of bilateral multifocal abscesses in the area of the paralumbar fossa, which continued to worsen despite oral antibiotics and non-steroidal anti-inflammatory medications. The multi-focal abscesses continued to worsen and additional diagnostics were pursued including cytology, culture (aerobic, anaerobic and fungal), and bloodwork. All supported a non-infectious etiology. Biopsy results indicated a suture-related pseudoinfection. Despite treatment including parenteral antibiotics, pain medications and superficial surgical debridement, the dermatologic lesions worsened. Euthanasia was elected. Post-mortem necropsy demonstrated a suture-related pseudoinfection with extrusion of suture material from the ovarian pedicle ligatures through the body wall and skin leading to numerous sterile abscesses in the bilateral paralumbar fossa. CONCLUSIONS: This is the first published report of a significantly delayed polyglactin 910 suture-related pseudoinfection in a Yucatan pig. While likely an isolated incident, it supports further research into this area. Additionally for critical research studies using Yucatan pigs, pre-surgical assessment with hypersensitivity patch testing may be appropriate.
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Abscesso/veterinária , Ovariectomia/veterinária , Poliglactina 910/efeitos adversos , Suturas/efeitos adversos , Abscesso/tratamento farmacológico , Abscesso/cirurgia , Animais , Antibacterianos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Feminino , Ovariectomia/efeitos adversos , SuínosRESUMO
PURPOSE: With advancing endovascular technology and increasing interest in minimally invasive intra-arterial therapies such as stem cell and chemotherapy for cerebral disease, the establishment of a translational model with cerebral circulation accessible to microcatheters is needed. We report our experience catheterizing canine cerebral circulation with microcatheters, present high-resolution angiographic images of the canine vascular anatomy, describe arterial branch flow patterns and provide measurements of canine arterial conduits. MATERIALS AND METHODS: Angiograms were performed on 10 intact purpose-bred hounds. Angiography, measurements of arterial conduits and catheterization information for intracranial arterial branches were obtained. RESULTS: Selective and superselective cerebral angiography was successful in all subjects. Relevant arterial mean diameters include the femoral (4.64 mm), aorta (9.38 mm), external carotid (3.65 mm), internal carotid arteries (1.6 mm), vertebrobasilar system and Circle of Willis branches. Catheterization of the Circle of Willis was achieved via the posterior circulation in all subjects tested (n=3) and the use of flow directed microcatheters resulted in reduced arterial tree deformation and improved superselection of intracranial vessels. Catheterization of the intracranial circulation was attempted but not achieved via the internal carotid artery (n=7) due to its tortuosity and subsequent catheter related vasospasm. CONCLUSION: The canine cerebral vasculature is posterior circulation dominant. Anterior circulation angiography is achievable via the internal carotid artery, but direct cerebral arterial access is best achieved via the posterior circulation using flow-directed microcatheters. It is feasible to deliver intra-arterial therapies to selective vascular territories within the canine cerebral circulation, thus making it a viable animal model for testing novel intra-arterial cerebral treatments.
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BACKGROUND: Endovascular technological advances have revolutionized the field of neurovascular surgery and have become the mainstay of treatment for many cerebrovascular pathologies. Digital subtraction angiography (DSA) is the 'gold standard' for visualization of the vasculature and deployment of endovascular devices. Nonetheless, with recent technological advances in optics, angioscopy has emerged as a potentially important adjunct to DSA. Angioscopy can offer direct visualization of the intracranial vasculature, and direct observation and inspection of device deployment. However, previous iterations of this technology have not been sufficiently miniaturized or practical for modern neurointerventional practice. OBJECTIVE: To describe the evolution, development, and design of a microangioscope that offers both high-quality direct visualization and the miniaturization necessary to navigate in the small intracranial vessels and provide examples of its potential applications in the diagnosis and treatment of cerebrovascular pathologies using an in vivo porcine model. METHODS: In this proof-of-concept study we introduce a novel microangioscope, designed from coherent fiber bundle technology. The microangioscope is smaller than any previously described angioscope, at 1.7 F, while maintaining high-resolution images. A porcine model is used to demonstrate the resolution of the images in vivo. RESULTS: Video recordings of the microangioscope show the versatility of the camera mounted on different microcatheters and its ability to navigate external carotid artery branches. The microangioscope is also shown to be able to resolve the subtle differences between red and white thrombi in a porcine model. CONCLUSION: A new microangioscope, based on miniaturized fiber optic technology, offers a potentially revolutionary way to visualize the intracranial vascular space.
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Angioscopia/tendências , Desenho de Equipamento/tendências , Neuronavegação/tendências , Estudo de Prova de Conceito , Angiografia Digital/instrumentação , Angiografia Digital/métodos , Angiografia Digital/tendências , Angioscopia/instrumentação , Angioscopia/métodos , Animais , Desenho de Equipamento/métodos , Feminino , Tecnologia de Fibra Óptica/instrumentação , Tecnologia de Fibra Óptica/métodos , Tecnologia de Fibra Óptica/tendências , Humanos , Neuronavegação/instrumentação , Neuronavegação/métodos , SuínosRESUMO
Pigeons (Columba livia) are used in biomedical research for studies of vision, cognition, neuronal pathways, and spatial orientation. Because there are few commercial laboratory sources, research pigeons are typically acquired from local fancier breeders or bred onsite. For acquired pigeons, the health and vaccine status is often unknown. A juvenile pigeon, born onsite and living in an enclosed outdoor loft, presented with small, bleeding, wart-like lesions on the medial aspects of digits 1 and 4. Topical treatment was initiated. Within a week, 4 fledglings were reported for small, dark papular lesions on the face, head, neck, and beak, and shortly thereafter, 2 additional juvenile pigeons developed similar lesions. The fledglings were euthanized, and histologic examination revealed numerous intralesional eosinophilic cytoplasmic viral inclusions (Bollinger bodies) confirming a diagnosis of poxvirus infection, likely pigeon pox. Although usually self-limiting, pigeon pox can cause moderate to severe lesions in fledgling and juvenile birds. Vaccination with a modified live poxvirus labeled for chickens was used to create herd immunity to pigeon poxvirus. Since vaccination of our entire flock and implementation of more stringent health protocols, all lesions have resolved, and no new lesions have been noted.
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Avipoxvirus , Doenças das Aves/virologia , Columbidae/virologia , Infecções por Poxviridae/veterinária , Animais , Animais de Laboratório/virologia , Avipoxvirus/imunologia , Avipoxvirus/patogenicidade , Doenças das Aves/patologia , Doenças das Aves/prevenção & controle , Galinhas , Columbidae/imunologia , Surtos de Doenças/prevenção & controle , Surtos de Doenças/veterinária , Infecções por Poxviridae/patologia , Infecções por Poxviridae/prevenção & controle , Pele/patologia , Vacinação/veterinária , Vacinas Virais/administração & dosagemRESUMO
Fibrocytes are unique cells with innate and adaptive immune functions, but these mechanisms have not been fully explored. The aim of this study was to explain the mechanism by which adoptive transfer of exogenous fibrocytes improved bacterial clearance and increased sepsis survival. Initial flow cytometry-based, in vitro assays demonstrated phagocytosis by fibrocytes and intracellular bacterial killing was confirmed by direct plating of cell lysates after exposure to live bacteria. Intravenous adoptive transfer of fibrocytes at the time of cecal ligation and puncture (CLP) or 2 h after CLP in mice increased survivability. Decreased intraperitoneal bacterial burden was also observed. Quantification of peritoneal cell populations using flow cytometry demonstrated transferred and endogenous fibrocytes were significantly increased after CLP, while macrophage and neutrophil numbers were unchanged. To determine the impact in vivo, fluorescently labeled, killed bacteria were injected i.p. into mice 10 h after CLP or sham surgeryâ±âadoptive transfer. Two hours later, flow cytometry of peritoneal cell populations after CLP alone revealed increased phagocytosis by macrophages, neutrophils, and endogenous fibrocytes. Transferred fibrocytes had significantly increased phagocytic activity in the septic peritoneum compared with sham and greater activity than any other cell type. Therefore, adoptive transfer may enhance bacterial clearance in early sepsis through the cumulative effects of endogenous and transferred fibrocytes rather than modulating the function of other endogenous phagocytes. Direct phagocytic activity coupled with previously described influences on T cell responses may explain the benefits of fibrocyte transfer in sepsis.
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Fagocitose/fisiologia , Sepse/microbiologia , Sepse/patologia , Animais , Ceco/lesões , Células Cultivadas , Escherichia coli/patogenicidade , Ligadura/efeitos adversos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/metabolismo , Peritonite/microbiologia , Peritonite/patologia , Punções/efeitos adversosRESUMO
Viral vector research presents unique occupational health and safety challenges to institutions due to the rapid development of both in vivo and in vitro gene-editing technologies. Risks to human and animal health make it incumbent on institutions to appropriately evaluate viral vector usage in research on the basis of available information and governmental regulations and guidelines. Here we review the factors related to risk assessment regarding viral vector usage in animals and the relevant regulatory documents associated with this research, and we highlight the most commonly used viral vectors in research today. This review is particularly focused on the background, use in research and associated health and environmental risks related to adenoviral, adeno-associated viral, lentiviral, and herpesviral vectors.
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Experimentação Animal , Vetores Genéticos , Exposição Ocupacional/prevenção & controle , Saúde Ocupacional , Vírus/genética , Humanos , Medição de Risco , Controle Social FormalRESUMO
Estrogen supplementation is a key component of numerous mouse research models but can adversely affect the urinary system. The goal of this study was to develop a clinical scoring system and identify biomarkers of occult urinary tract lesions prior to the development of systemic illness in mice. Ovariectomized or sham-surgery SCID mice were implanted subcutaneously with a placebo pellet or one containing sustained-release estradiol (0.18 mg 60-d release 17ß-estradiol). Mice were assessed twice weekly for 4 to 6 wk by using a clinical scoring system that included body condition, general activity, posture, hair coat, hydration, abdominal distension, urine staining of coat and skin, and ability to urinate. Samples were collected weekly for urinalysis, BUN, creatinine, and serum estradiol levels. Terminal samples were analyzed for histopathologic lesions. Compared with placebo controls, estradiolsupplemented mice had higher serum estradiol levels at weeks 2 and 3; significant differences in total clinical scores by the 3-wk time point; and in body condition, general activity, posture, hair coat, and urine staining scores by the 6-wk terminal time point. Urinary tract lesions included hydronephrosis, pyelonephritis, cystitis, and urolithiasis. All mice with urolithiasis had crystalluria, and 5 of the 6 mice with pyelonephritis or hydroureter had dilute urine (that is, specific gravity less than 1.030). However, these findings were not specific to mice with lesions. A total clinical score of 3.5 (maximum, 24) identified estradiol-supplemented mice with 83% specificity and 50% sensitivity, but no single clinical parameter, biomarker, or the total clinical score accurately predicted occult urinary tract lesions. Considering the lesions we observed, prudence is warranted when using pelleted sustained-release estradiol in mice, and important parameters to monitor for animal health include urine staining, body condition score, urine sediment, and urine specific gravity.
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Estradiol/efeitos adversos , Estrogênios/efeitos adversos , Sistema Urinário/efeitos dos fármacos , Animais , Biomarcadores/sangue , Biomarcadores/urina , Cistite/induzido quimicamente , Preparações de Ação Retardada , Modelos Animais de Doenças , Estradiol/sangue , Estrogênios/sangue , Feminino , Hidronefrose/induzido quimicamente , Camundongos , Camundongos SCID , Pielonefrite/induzido quimicamente , Urolitíase/induzido quimicamenteRESUMO
A 6-d-old Indian-origin female rhesus macaque (Macaca mulatta) presented with bradycardia shortly after sedation with ketamine. No other cardiac abnormalities were apparent. Approximately 2 wk after the initial presentation, the macaque was again bradycardic and exhibited a regularly irregular arrhythmia on a prestudy examination. ECG, echocardiography, blood pressure measurement, SpO2 assessment, and a CBC analysis were performed. The echocardiogram and bloodwork were normal, but the infant was hypotensive at the time of echocardiogram. The ECG revealed ventricular parasystole. Ventricular parasystole is considered a benign arrhythmia caused by an ectopic pacemaker that is insulated from impulses from the sinus node. Given this abnormality, the macaque was transferred to a short-term study protocol, according to veterinary recommendation. On the final veterinary exam, a grade 3 systolic murmur and a decrease in arrhythmia frequency were noted. Gross cardiac lesions were not identified at necropsy the following day. Cardiac tissue sections were essentially normal on microscopic examination. This infant did not display signs of cardiovascular insufficiency, and a review of the medical record indicated normal growth, feed intake and activity levels. This case demonstrates the importance of appropriate screening of potential neonatal and juvenile research candidates for occult cardiovascular abnormalities. Whether the arrhythmia diagnosed in this case was truly innocuous is unclear, given the documented hypotension and the development of a systolic heart murmur.
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Ventrículos do Coração/patologia , Macaca mulatta , Parassístole/veterinária , Animais , Animais de Laboratório , Bradicardia/veterinária , Ecocardiografia/veterinária , Feminino , Ventrículos do Coração/anormalidades , Ventrículos do Coração/diagnóstico por imagem , Humanos , Doenças dos Macacos/patologia , Parassístole/patologiaRESUMO
An experimentally naïve 37.7-kg Yorkshire-crossbred gilt died unexpectedly 2 d after arrival. Necropsy revealed severe dilated cardiomyopathy characterized grossly by markedly dilated ventricles and thinned ventricular walls and interventricular septum. Histologically there was multifocal myofiber attenuation and patchy loss of myofiber cross striations. The liver contained submassive to massive, diffuse hepatic centrilobular hemorrhage and degeneration. These lesions supported a diagnosis of dilated cardiomyopathy with right heart failure and secondary hepatic degeneration due to marked acute passive congestion. To our knowledge, this case is the first report of spontaneous dilated cardiomyopathy in swine and represents a potential diagnostic challenge regarding the differentiation of the cardiac-associated liver lesion from hepatosis dietetica. The diagnosis of dilated cardiomyopathy and right-sided heart failure was supported by the character of the hepatic lesion, absence of typical gross or histologic lesions of mulberry heart disease, and normal selenium levels.
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Cardiomiopatia Dilatada/veterinária , Insuficiência Cardíaca/veterinária , Hepatopatias/veterinária , Doenças dos Suínos/diagnóstico , Animais , Autopsia/veterinária , Biópsia/veterinária , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Diagnóstico Diferencial , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/patologia , Fígado/patologia , Hepatopatias/diagnóstico , Hepatopatias/patologia , Miocárdio/patologia , Índice de Gravidade de Doença , Sus scrofa , Suínos , Doenças dos Suínos/patologiaRESUMO
Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.