RESUMO
The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.
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Testes de Toxicidade , Toxicologia , Animais , Toxicologia/métodos , Testes de Toxicidade/métodos , Humanos , Bases de Dados Factuais , Medição de RiscoRESUMO
The European Society of Toxicologic Pathology (ESTP) initiated a survey through its Pathology 2.0 workstream in partnership with sister professional societies in Europe and North America to generate a snapshot of artificial intelligence (AI) usage in the field of toxicologic pathology. In addition to demographic information, some general questions explored AI relative to (1) the current status of adoption across organizations; (2) technical and methodological aspects; (3) perceived business value and finally; and (4) roadblocks and perspectives. AI has become increasingly established in toxicologic pathology with most pathologists being supportive of its development despite some areas of uncertainty. A salient feature consisted of the variability of AI awareness and adoption among the responders, as the spectrum extended from pathologists having developed familiarity and technical skills in AI, to colleagues who had no interest in AI as a tool in toxicologic pathology. Despite a general enthusiasm for these techniques, the overall understanding and trust in AI algorithms as well as their added value in toxicologic pathology were generally low, suggesting room for the need for increased awareness and education. This survey will serve as a basis to evaluate the evolution of AI penetration and acceptance in this domain.
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Inteligência Artificial , Patologistas , Humanos , Algoritmos , Europa (Continente)RESUMO
In the last decade, numerous initiatives have emerged worldwide to reduce the use of animals in drug development, including more recently the introduction of Virtual Control Groups (VCGs) concept for nonclinical toxicity studies. Although replacement of concurrent controls (CCs) by virtual controls (VCs) represents an exciting opportunity, there are associated challenges that will be discussed in this paper with a more specific focus on anatomic pathology. Coordinated efforts will be needed from toxicologists, clinical and anatomic pathologists, and regulators to support approaches that will facilitate a staggered implementation of VCGs in nonclinical toxicity studies. Notably, the authors believe that a validated database for VC animals will need to include histopathology (digital) slides for microscopic assessment. Ultimately, the most important step lies in the validation of the concept by performing VCG and the full control group in parallel for studies of varying duration over a reasonable timespan to confirm there are no differences in outcomes (dual study design). The authors also discuss a hybrid approach, whereby control groups comprised both concurrent and VCs to demonstrate proof-of-concept. Once confidence is established by sponsors and regulators, VCs have the potential to replace some or all CC animals.
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Desenvolvimento de Medicamentos , Patologia , Animais , Grupos Controle , Projetos de PesquisaRESUMO
Nonclinical toxicology studies that are required to support human clinical trials of new drug candidates are generally conducted in a rodent and a non-rodent species. These studies typically contain a vehicle control group and low, intermediate, and high dose test article groups. In addition, a dosing-free recovery phase is sometimes included to determine reversibility of potential toxicities observed during the dosing phase and may include additional animals in the vehicle control and one or more dose groups. Typically, reversibility is determined by comparing the test article-related changes in the dosing phase animals to concurrent recovery phase animals at the same dose level. Therefore, for interpretation of reversibility, it is not always essential to euthanize the recovery vehicle control animals. In the absence of recovery vehicle control tissues, the pathologist's experience, historical control database, digital or glass slide repositories, or literature can be used to interpret the findings in the context of background pathology of the species/strain/age. Therefore, in most studies, the default approach could be not to euthanize recovery vehicle control animals. This article provides opinions on scenarios that may or may not necessitate euthanasia of recovery phase vehicle control animals in nonclinical toxicology studies involving dogs and nonhuman primates.
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Animais de Laboratório , Humanos , Animais , CãesRESUMO
Beagle dogs are a key nonrodent species in nonclinical safety evaluation of new biomedical products. The Society of Toxicologic Pathology (STP) has published "best practices" recommendations for nervous system sampling in nonrodents during general toxicity studies (Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the Beagle dog has not been defined specifically. Here we provide 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the dog brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen test articles with unknown or no anticipated neurotoxic potential; this plan using at least 7 coronal hemisections matches the STP "best practices" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses up to 14 coronal levels to investigate test articles where the brain is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves for dogs during nonclinical studies should follow published STP "best practices" recommendations for sampling the central (Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (Toxicol Pathol 46[4]: 372-402, 2018) nervous systems. This technical guide also demonstrates the locations and approaches to collecting uncommonly sampled peripheral nervous system sites.
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Síndromes Neurotóxicas , Testes de Toxicidade , Animais , Cães , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/veterinária , Sistema Nervoso Periférico , Manejo de Espécimes , Medula EspinalRESUMO
Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.
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Dependovirus , Vetores Genéticos , Animais , Dependovirus/genética , Vetores Genéticos/genética , Macaca fascicularis , Masculino , Neurônios Motores , Necrose , RNA Mensageiro , Ratos , Ratos Wistar , Suínos , Distribuição Tecidual , Transdução GenéticaRESUMO
Many pharmaceutical companies have recently elected to stop maintaining good laboratory practices (GLP) status of their R&D sites. Similar discussions have also been engaged in the (agro)chemical industry. This opinion paper examines the pros and cons of maintaining facility GLP status for the purposes of performing the pathology interpretation or peer reviews of GLP studies internally. The toxicologic pathologist provides gross and histomorphologic evaluation and interpretation of nonclinical exploratory and regulatory studies during drug and (agro)chemical development. This assessment significantly contributes to human risk assessment by characterizing the toxicological profile and discussing the human relevance of the findings. The toxicologic pathologist is a key contributor to compound development decisions (advancement or termination) and in the development of de-risking strategies for backup compounds, thus playing a critical role in helping to reduce the late attrition of drugs and chemicals. Maintaining GLP compliance is often perceived as a costly and cumbersome process; a common and short-term strategy to reduce the costs is to outsource regulatory toxicity studies. However, there are significant advantages in maintaining the GLP status for toxicologic pathology activities in-house including the sustainable retention of internal pathology expertise that has maintained the necessary training needed to manage GLP studies. [Box: see text].
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Patologia , Toxicologia , Humanos , Laboratórios , Patologistas , Revisão por Pares , Preparações Farmacêuticas , Projetos de PesquisaRESUMO
Lorlatinib is a potent small-molecule anaplastic lymphoma kinase inhibitor approved for the treatment of patients with nonsmall cell lung cancer. In a drug-drug interaction study in healthy human participants, liver enzyme elevations were observed when a single 100 mg dose of lorlatinib was administered after multiple doses of rifampin, a strong cytochrome P450 (CYP) 3A inducer and a pregnane X receptor (PXR) agonist. A series of in vitro and in vivo studies were conducted to evaluate potential mechanisms for the observed clinical toxicity. To investigate the involvement of CYP3A and/or PXR in the observed liver toxicity, studies were conducted in cynomolgus monkeys administered lorlatinib alone or with coadministration of multiple doses of known CYP3A inducers that are predominantly PXR agonists (rifampin, St. John's wort) or predominantly constitutive androstane receptor agonists (carbamazepine, phenytoin) and a net CYP3A inhibitory PXR agonist (ritonavir). Results from the investigative studies identified cynomolgus monkeys as a pharmacologically relevant nonclinical model, which recapitulated the elevated liver function test results observed in humans. Furthermore, liver toxicity was only observed in this model when lorlatinib was coadministered with strong CYP3A inducers, and the effects were not restricted to, or exclusively dependent upon, a PXR activation mechanism. These results generated mechanistic insights on the liver enzyme elevations observed in the clinical drug-drug interaction study and provided guidance on appropriate product safety label for lorlatinib.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Aminopiridinas , Animais , Citocromo P-450 CYP3A/genética , Indutores do Citocromo P-450 CYP3A/toxicidade , Interações Medicamentosas , Humanos , Lactamas , Lactamas Macrocíclicas , Fígado , Macaca fascicularis , PirazóisRESUMO
With advancements in whole slide imaging technology and improved understanding of the features of pathologist workstations required for digital slide evaluation, many institutions are investigating broad digital pathology adoption. The benefits of digital pathology evaluation include remote access to study or diagnostic case materials and integration of analysis and reporting tools. Diagnosis based on whole slide images is established in human medical pathology, and the use of digital pathology in toxicologic pathology is increasing. However, there has not been broad adoption in toxicologic pathology, particularly in the context of regulatory studies, due to lack of precedence. To address this topic, as well as practical aspects, the European Society of Toxicologic Pathology coordinated an expert international workshop to assess current applications and challenges and outline a set of minimal requirements needed to gain future regulatory acceptance for the use of digital toxicologic pathology workflows in research and development, so that toxicologic pathologists can benefit from digital slide technology.
RESUMO
The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.
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Células Epiteliais da Tireoide , Biomarcadores , Humanos , Hiperplasia , Hipertrofia , Medição de Risco , Estados UnidosRESUMO
Axonal dystrophy (AD) is a common age-related neurohistological finding in vertebrates that can be congenital or induced by xenobiotics, vitamin E deficiency, or trauma/compression. To understand the incidence and location of AD as a background finding in Beagle dogs used in routine toxicity studies, we examined central nervous system (CNS) and selected peripheral nervous system (PNS) tissues in twenty 18- to 24-month-old and ten 4- to 5-year-old control males and females. Both sexes were equally affected. The cuneate, gracile, and cochlear nuclei and the cerebellar white matter (rostral vermis) were the most common locations for AD. Incidence of AD increased with age in the cuneate nucleus, cerebellar white matter (rostral vermis), trigeminal nuclei/tracts, and lumbar spinal cord. Axonal dystrophy in the CNS was not accompanied by neuronal degeneration/necrosis, nerve fiber degeneration, and/or glial reaction. Axonal dystrophy was not observed in the PNS (sciatic nerve, vagus nerve branches, or gastrointestinal mural autonomic plexuses).
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Encéfalo/patologia , Doenças do Cão/patologia , Distrofias Neuroaxonais/patologia , Medula Espinal/patologia , Animais , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Cães , Feminino , Masculino , Bulbo/patologia , Degeneração Neural/patologiaRESUMO
The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Toxicologia/normas , Animais , Guias como Assunto , Humanos , Medição de Risco , Fenômenos ToxicológicosRESUMO
Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.
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Antibacterianos/toxicidade , Gentamicinas/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/urina , Acetilglucosaminidase/urina , Alanina Transaminase/sangue , alfa-Globulinas/urina , Animais , Antibacterianos/sangue , Antibacterianos/farmacocinética , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Biomarcadores/urina , Glicemia/análise , Nitrogênio da Ureia Sanguínea , Clusterina/urina , Creatina/sangue , Creatina/urina , Gentamicinas/sangue , Gentamicinas/farmacocinética , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/sangue , Nefropatias/patologia , Macaca fascicularis , Masculino , Necrose/induzido quimicamente , Osteopontina/urina , Albumina Sérica/análiseRESUMO
PURPOSE: To evaluate the histologic effects and clinical outcomes of high-intensity focused ultrasound (HIFU) delivered by miniaturized annular transducers for ciliary body coagulation in an animal study. METHODS: Eighteen eyes of 18 rabbits were insonified with a ring comprising a six-sector transducer, to produce a 12.8-mm-diameter circular lesion. Six sectors were activated in six rabbits (group 1), five sectors in six rabbits (group 2), and four sectors in six rabbits (group 3) at 2 W acoustic power. The rabbits were examined before treatment (day 0) and after treatment on days 1, 7, 15, 21, and 28. Detailed qualitative and semiquantitative histopathologic analyses of the enucleated eyes were performed. RESULTS: In the treated eyes, intraocular pressure changes ranged from -16.6 mm Hg (-55.3%) at day 28 to -8.9 mm Hg (-29.7%) at day 7 in group 1, from -4.7 mm Hg (-25.5%) at day 28 to -1.4 mm Hg (-7.6%) at day 21 in group 2 and from -7.9 mm Hg (-28.1%) at day 28 to +2.0 mm Hg (+7.1%) at day 7 in group 3. No macroscopic abnormalities were observed in the anterior segment or fundus. Histologic examination showed segmental-to-annular lesions in the ciliary processes, caused mainly by coagulation necrosis, whereas the sclera and lens appeared undamaged. Inflammation was very limited. CONCLUSIONS: Ultrasonic coagulation of the ciliary body with HIFU delivered via a circular miniaturized transducer seemed to be an effective and well-tolerated method of reducing intraocular pressure in an animal study.
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Corpo Ciliar/patologia , Pressão Intraocular , Terapia por Ultrassom/instrumentação , Animais , Artérias Ciliares , Corpo Ciliar/irrigação sanguínea , Desenho de Equipamento , Glaucoma/terapia , Masculino , Modelos Biológicos , Necrose , Coelhos , Tonometria Ocular , TransdutoresRESUMO
BACKGROUND: The purpose of this study was to evaluate the performance a newly developed nanocrystalline hydroxyapatite, OSTIM following functional implantation in femoral sites in thirty-eight sheep for 1, 2 or 3 months. Ostim 35 was compared to an established calcium phosphate, Alpha BSM. METHODS: Biomechanical testing, micro-CT analysis, histological and histomorphological analyses were conducted to compare the treatments including evaluation of bone regeneration level, material degradation, implant biomechanical characteristics. RESULTS: The micro-computed tomography (microCT) analysis and macroscopic observations showed that Ostim seemed to diffuse easily particularly when the defects were created in a cancellous bone area. Alpha BSM remained in the defect.The performance of Ostim was good in terms of mechanical properties that were similar to Alpha BSM and the histological analysis showed that the bone regeneration was better with Ostim than with Alpha BSM. The histomorphometric analysis confirmed the qualitative analysis and showed more bone ingrowth inside the implanted material with Ostim when compared to Alpha BSM at all time points. CONCLUSIONS: The successful bone healing with osseous consolidation verifies the importance of the nanocrystalline hydroxyapatite in the treatment of metaphyseal osseous volume defects in the metaphyseal spongiosa.
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Implantes Absorvíveis/tendências , Cimentos Ósseos/farmacologia , Doenças Ósseas/tratamento farmacológico , Regeneração Óssea/efeitos dos fármacos , Durapatita/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Cimentos Ósseos/uso terapêutico , Doenças Ósseas/diagnóstico por imagem , Doenças Ósseas/cirurgia , Regeneração Óssea/fisiologia , Modelos Animais de Doenças , Durapatita/uso terapêutico , Feminino , Nanopartículas/uso terapêutico , Avaliação de Resultados em Cuidados de Saúde , Carneiro Doméstico , Resultado do Tratamento , Cicatrização/fisiologia , Microtomografia por Raio-XRESUMO
Several transgenic mice models are accepted by regulatory agencies to determine the carcinogenic potential and predict the human response to exposure of chemicals, as an alternative to the conventional 2-year rodent bioassay. The rasH2 transgenic mouse model has been proposed to evaluate the carcinogenic potential of medical devices, but few data are currently available regarding study design--namely appropriate positive and negative controls to be used--as well as historical pathology data. BIOMATECH-NAMSA recently conducted a 26-week carcinogenicity study following subcutaneous implantation in the transgenic rasH2 mouse model. This paper describes the study design and the main results obtained in the positive and negative control groups. The survival rate statistical (Kaplan-Meier) analysis showed that the survival rate was significantly affected by the occurrence of tumors in the positive control group when compared to the negative control group, in both genders. Thymic malignant lymphomas and squamous cell papillomas were reported to occur at a higher incidence in rasH2 mice exposed to a known chemical carcinogen, for terminally sacrificed animals as well as for unscheduled and terminally sacrificed animals considered together. Background and age-related lesions were few. Taken together, these data confirmed the reliability and usefulness of the rasH2 transgenic model in the assessment of carcinogenic properties of medical devices. A major beneficial property of this animal model consisted in the ability to demonstrate chemical carcinogenesis response without the solid-state tumorigenesis response seen in traditional 2-year rodent bioassays.
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Camundongos Transgênicos , Testes de Mutagenicidade/métodos , Próteses e Implantes/efeitos adversos , Animais , Modelos Animais de Doenças , Feminino , Genes ras/genética , Humanos , Masculino , Camundongos , Proteínas ras/genéticaRESUMO
The main human forms of epidermolysis bullosa (EB), namely EB simplex, junctional EB and dystrophic EB, have also been described in domestic animals (small and large ruminants, and horses) and companion animals (cats and dogs). A recent description of dystrophic epidermolysis bullosa (DEB) in Golden Retriever dogs provided details of the principal clinical, morphological and genetic features. The disease is characterized by blisters and erosions in the oral and esophageal epithelia, together with milia, nails dystrophy and growth retardation. The cutaneous lesions regress spontaneously in adult dogs, whereas the epithelial lesions persist, aggravate and spread, notably to the cornea. Classical microscopic studies (light and electron microscopy, indirect immunofluorescence) have revealed anchoring fibril abnormalities and very low-level and heterogenous expression of collagen type VII. The culprit mutation (G1906S) in the canine gene COL7A1 (87.8% nucleotide sequence identity to the human counterpart) involves the replacement of guanine 5716 by adenine, leading to glycine substitution by serine at amino acid position 1906. Transmission in kennels occurs in recessive mode (RDEB). These features recall certain human forms of DEB, and particularly those with a phenotype intermediate between gravis (the so-called Hallopeau-Siemens form) and mitis. No curative treatment of human EB is currently available, and efforts are therefore being made to develop a gene therapy protocol in animals. The first steps have already been successfully achieved, namely the development of a recombinant virus vector able to insert the wild-type gene into the keratinocyte genome, and grafting of artificial skin containing transfected canine keratinocytes in nude mice. The recombinant vectors are Moloney-type retroviruses (MMLV-PCMV), and the Zeocin resistance gene is used to select transduced cells. The artificial skin reconstructed in vitro is of the full-thickness type. Despite the large size of the transduced (9 kb), 95% of cells are transduced and produce large amounts of wild-type collagen. Two key issues remain, however: the possible immunogenicity of the transgene product and the persistence of transgene expression in individuals with a functional immune system. Golden Retriever dogs will provide a suitable animal model for these studies.
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Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Terapia Genética , Animais , Modelos Animais de Doenças , Cães , Feminino , Glicina/genética , Masculino , Mutação , Serina/genéticaRESUMO
A 7-year-old, intact male Dachshund was presented to the Lyon veterinary school for lethargy and anorexia of several weeks duration. The main clinical signs were pale and icteric mucous membranes, hepatomegaly, splenomegaly, and lymphadenopathy. Results of a CBC and plasma biochemistry tests revealed severe nonregenerative anemia, thrombocytopenia, and increased alanine aminotransferase and alkaline phosphatase activities. Blood smear evaluation and cytologic examination of lymph node and bone marrow aspirate specimens revealed a large population of poorly differentiated blast cells with morphologic features suggesting megakaryocytic lineage. A low number of well-differentiated but dysplastic megakaryocytes also were observed in lymph node and bone marrow smears. A few blast cells were erythrophagocytic. Blast cells were positive for glycoprotein IIIa, factor VIII-related antigen, and factor XIII using immunocytochemistry. The dog was euthanized and necropsied. Histologic findings consisted of diffuse, massive infiltration of lymph nodes, liver, and spleen by megakaryoblasts and atypical megakaryocytes, with widespread thrombosis. This case confirms the usefulness of immunochemistry, including for factor XIII, in the diagnosis of megakaryoblastic leukemia, and demonstrates the unique features of tumor cell erythrophagocytosis and marked fibrinous thrombosis, which have not been reported previously in dogs.
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Doenças do Cão/patologia , Leucemia Megacarioblástica Aguda/veterinária , Trombose/veterinária , Animais , Doenças do Cão/sangue , Cães , Eritrócitos , Leucemia Megacarioblástica Aguda/sangue , Leucemia Megacarioblástica Aguda/patologia , Masculino , Fagocitose , Trombose/sangueRESUMO
A 28-day oral gavage toxicity study in the rat with 17alpha-methyltestosterone was conducted as part of the international validation exercise on the modified Enhanced OECD Test Guideline 407 (Organisation for Economic Co-operation and Development, Paris). Special emphasis was placed on the endocrine mediated effects exerted by 17alpha-methyltestosterone, a potent androgen agonist. The test compound was administered daily by oral gavage for at least 28 days to groups of 7-week-old-Wistar rats. Dose levels were 0, 10, 40 and 200 mg/kg body weight per day for males and 0, 10, 100 and 600 mg/kg body weight per day for females. In addition, and outside the remit of the enhanced protocol, testosterone levels in males, oestradiol levels in females and luteinizing hormone (LH) levels in both sexes were measured, to provide a broader profile on the hormonally mediated effects of 17alpha-methyltestosterone. Furthermore, stage-specific quantification of Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL)-labeled germ cells (apoptotic germ cells) in the seminiferous tubules was also performed, in an effort to demonstrate the precise stages in the spermatogenic cycle 17alpha-methyltestosterone exerts its effect. In this study, the most critical additional parameters contained in the Enhanced OECD Test Guideline 407 for the detection of endocrine disruption were considered to be the histopathological assessment and organ weight data of endocrine-related tissues. Beyond the scope of this validation exercise, an increase in apoptosis in specific germ cell types was detected using the TUNEL assay in male rats treated at 200 and 40 mg/kg.