RESUMO
Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.
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Bilateral cataracts were diagnosed in two rescued juvenile, immature loggerhead sea turtles (Caretta caretta), weighing 1.65 and 1.7 kg. Both animals showed vision impairment and difficulty in feeding without assistance. In fact, they did not notice the presence of the food in the tank unless it was brought close to touching the mouth. Ocular ultrasonography and electroretinography showed no lesions of the vitreal body and retinal layer, therefore, both animals were candidates for bilateral cataract surgery. Topical administration of tropicamide + phenylephrine alternating with rocuronium resulted in only minimal mydriasis. Administration of intracameral rocuronium did not improve mydriasis. Phacoemulsification using a one-handed technique was performed bilaterally with a phacoemulsification device (Sovereign, AMO (Abbott Medical Optics®). After surgery, the systemic anti-inflammatory drug (dexamethasone 0.2 mg/kg, IM daily for one week) and antibiotics (enrofloxacin 10 mg/kg IM q 72 h, for 4 weeks; ceftazidime 20 mg/kg IM q 72 h for 3 weeks) were administered. Topical ofloxacin, flurbiprofen and tobramycin/dexamethasone were instilled TID for 4 weeks. Both turtles regained vision in both eyes. Results at a 10-month follow-up were satisfactory. This is the first report of cataracts in turtles rescued in the Mediterranean Sea and the first description of surgical treatment of cataracts in loggerhead turtles so young.
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Adhesions involving the bulbar and the palpebral conjunctiva (Symblepharon) may interfere with tear drainage, cause chronic conjunctivitis, and reduce ocular motility. This condition may be associated with adhesion of the edges of the upper and lower eyelids (ankyloblepharon). The present case describes bilateral symblepharon, ankyloblepharon and salt gland dysfunction in a juvenile Caretta caretta. The loggerhead presented both eyelids swollen, ulcerated, and not separable when rescued. Eye examination was not possible, but ultrasonography showed right bulbar integrity, while the left eye was smaller, with a thicker cornea that had lost its normal doubled lined structure. Surgical dissection of the fibrous adhesions between the palpebral and bulbar conjunctiva, cornea, and third eyelid was performed, and large dacryoliths were removed. The microscopic findings were consistent with chronic keratoconjunctivitis. Ultrastructurally, no virus-like particles were observed. In addition, tissue samples were negative for herpesvirus by qualitative PCR. The eyelids of both eyes and the corneal epithelium of the right eye healed; moreover, the vision was restored in the right eye. There were no recurrences after 12 months of follow-up, and the turtle was released 16 months after the end of treatments on the southern Tyrrhenian coast in the western Mediterranean Sea. To the authors' knowledge, this is the first report of symblepharon with ankyloblepharon and salt gland dysfunction in Caretta caretta turtle. Ocular ultrasonography was helpful in the preliminary diagnostic work-up.
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This study aimed to evaluate intraocular pressure (IOP) estimates in healthy eyes of Caretta caretta using rebound tonometry in comparison with applanation tonometry. Twenty-three healthy C. caretta (housed at the Marine Turtle Research Center) without preexisting ophthalmic disease were enrolled in the study. IOP measurements were obtained by the same ophthalmologist, with the turtle in ventral recumbency between 2:30 p.m. and 4:30 p.m., using a rebound tonometer (RT; TonoVet) in dog calibration mode, and after topical anesthesia, an applanation tonometer (AT; Tono-Pen) in both eyes. The average of three readings per instrument was used for analysis. The agreement between the two tonometers was assessed by Bland-Altman analysis and intraclass correlation coefficient (ICC). Moreover, differences in IOP between the two tonometers were analyzed using the Mann-Whitney test. Moderate agreement was found between the two tonometers (ICC, 0.663; 95% confidence interval, 0.206-0.857). The median, Q1, and Q3 IOP obtained with AT (6.2, 4.7, and 9.1 mm Hg) were significantly lower (P = 0.001) than that obtained with RT (9.7, 8.3, and 11.6 mm Hg). It was not possible to obtain an instrument automatically generated mean of four values with AT because of retraction of the globe by the animals, and IOP measurement was unsuccessful in 7 eyes. In conclusion, IOP readings from the RT were statistically higher than those from the AT. RT proved to be more feasible because of the light, short-lasting contact with the cornea.
Assuntos
Pressão Intraocular/fisiologia , Tonometria Ocular/veterinária , Tartarugas/fisiologia , Animais , Tonometria Ocular/instrumentação , Tonometria Ocular/métodosRESUMO
BACKGROUND: Parasites of the family Spirorchiidae cause disease and mortality in marine and freshwater turtles; two species, Hapalotrema mistroides and Neospirorchis sp., are reported in the resident population of loggerhead turtles of the Mediterranean Sea, with the first being the most widespread. In vivo diagnosis of spirorchidiasis can represent a challenge in guaranteeing prompt control and treatment of the disease and is currently limited to copromicroscopy. The aim of this study was the development of a real time PCR assay with TaqMan probe for the detection of H. mistroides infection in the blood of live loggerhead turtles, Caretta caretta, hospitalized in rehabilitation centres. Its potential use for in vivo diagnosis is explored. RESULTS: The developed real time PCR successfully detected H. mistroides DNA from both positive controls and experimental blood samples of live loggerhead sea turtles, showing good specificity, sensitivity and good reaction efficiency. Two out of three turtles which had demonstrated positivity at copromicroscopy also tested positive to this blood assay; DNA of H. mistroides was detected within the blood of one sea turtle, which tested negative for copromicroscopy. CONCLUSIONS: This study describes a specific and rapid molecular assay to detect H. mistroides infection from live sea turtles and highlights for the first time the presence of DNA of this species in turtle blood samples. Since this assay is able to detect low amounts of the parasitic free DNA in blood samples, its application could be helpful for in vivo diagnosis of H. mistroides infection as well as for epidemiological purposes.
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Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções por Trematódeos/veterinária , Tartarugas/parasitologia , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Mar Mediterrâneo , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Trematódeos/genética , Trematódeos/isolamento & purificação , Infecções por Trematódeos/diagnóstico , Tartarugas/sangueRESUMO
Bacterial bone infections have been occasionally reported in wild sea turtles. This study reports on a sub-adult Caretta caretta affected by Aeromonas hydrophila osteomyelitis, with extensive involvement of cranial and caudal flippers. The turtle was severely debilitated, had limited flipper mobility and showed signs of pain in reaction to manipulation. Radiographs and computed tomography revealed multiple lytic bone lesions. Since an infectious polyostotic osteomyelitis was suspected, the turtle was subjected to echo-assisted fine needle aspiration to characterize the etiology of the disease. Bacterial cultures and antibiotic susceptibility testing led to the isolation of Aeromonas hydrophila responsive to amikacin and doxycycline. Therefore, the turtle was treated with these antibiotics and monitored through repeat bacterial cultures and diagnostic imaging. The turtle was released 17 mo after admission, upon resolution of clinical signs. The documentation of this case provides a treatment approach that may improve the outcome of Aeromonas-associated osteomyelitis, especially in endangered wildlife species.
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Aeromonas , Tartarugas , Envelhecimento , Animais , Espécies em Perigo de ExtinçãoRESUMO
This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of â¼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.
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Comitês de Cuidado Animal/organização & administração , Criação de Animais Domésticos/métodos , Bem-Estar do Animal/normas , Projetos de Pesquisa/legislação & jurisprudência , Médicos Veterinários/legislação & jurisprudência , Animais , Cefalópodes , Consenso , União Europeia , Estados UnidosRESUMO
BACKGROUND: Comparison of toxicogenomic data facilitates the identification of deregulated gene patterns and maximizes health risk prediction in human. RESULTS: Here, we performed phenotypic anchoring on the effects of acute exposure to low-grade polluted groundwater using mouse and zebrafish. Also, we evaluated two windows of chronic exposure in mouse, starting in utero and at the end of lactation. Bioinformatic analysis of livers microarray data showed that the number of deregulated biofunctions and pathways is higher after acute exposure, compared to the chronic one. It also revealed specific profiles of altered gene expression in all treatments, pointing to stress response/mitochondrial pathways as major players of environmental toxicity. Of note, dysfunction of steroid hormones was also predicted by bioinformatic analysis and verified in both models by traditional approaches, serum estrogens measurement and vitellogenin mRNA determination in mice and zebrafish, respectively. CONCLUSIONS: In our report, phenotypic anchoring in two vertebrate model organisms highlights the toxicity of low-grade pollution, with varying susceptibility based on exposure window. The overlay of zebrafish and mice deregulated pathways, more than single genes, is useful in risk identification from chemicals implicated in the observed effects.
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Água Subterrânea/química , Fenótipo , Toxicogenética , Poluição da Água/efeitos adversos , Animais , Biomarcadores , Exposição Ambiental/efeitos adversos , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Água Subterrânea/análise , Fígado/efeitos dos fármacos , Fígado/metabolismo , Testes de Função Hepática , Masculino , Camundongos , Reprodutibilidade dos Testes , Especificidade da Espécie , Fatores de Tempo , Testes de Toxicidade Aguda , Testes de Toxicidade Crônica , Peixe-ZebraRESUMO
NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.
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Replicação do DNA , Coativadores de Receptor Nuclear/fisiologia , Origem de Replicação , Animais , Células Cultivadas , Senescência Celular , Células HeLa , Humanos , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevisRESUMO
Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.
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Experimentação Animal/normas , Bem-Estar do Animal/normas , Cefalópodes , Neurociências/normas , Animais , União Europeia , Guias como AssuntoRESUMO
Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.
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Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Glioblastoma/patologia , Animais , Neoplasias Encefálicas/genética , Glioblastoma/genética , Humanos , Cariotipagem , CamundongosRESUMO
Axl is a tyrosine kinase receptor that was first identified as a transforming gene in human myeloid leukemia. Recent converging evidence suggests its implication in cancer progression and invasion for several solid tumors, including lung, breast, brain, thyroid, and pancreas. In the last decade, Axl has thus become an attractive target for therapeutic development of more aggressive cancers. An emerging class of therapeutic inhibitors is now represented by short nucleic acid aptamers. These molecules act as high affinity ligands with several advantages over conventional antibodies for their use in vivo, including their small size and negligible immunogenicity. Furthermore, these molecules can easily form conjugates able to drive the specific delivery of interfering RNAs, nanoparticles, or chemotherapeutics. We have thus generated and characterized a selective RNA-based aptamer, GL21.T that binds the extracellular domain of Axl at high affinity (12 nmol/l) and inhibits its catalytic activity. GL21.T blocked Axl-dependent transducing events in vitro, including Erk and Akt phosphorylation, cell migration and invasion, as well as in vivo lung tumor formation in mice xenografts. In this respect, the GL21.T aptamer represents a promising therapeutic molecule for Axl-dependent cancers whose importance is highlighted by the paucity of available Axl-specific inhibitory molecules.
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Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias/genética , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Experimental procedures on animals often require anesthesia and/or analgesia to obtain adequate immobilization and to reduce stress or pain. Mice anesthesia is challenging for several reasons including the animals' size, metabolic rate, and the high risk of hypothermia and hypoglycemia. Moreover, anesthetic agents influence physiological parameters, further interfering with experimental results. Small animal imaging procedures are increasingly used in biomedical research both because the animals allow in vivo monitoring and because they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. Anesthesia must adapt to the imaging technique, the procedure length, and the aim of the study. The purpose of this article is to review the existing literature on anesthetic protocols adopted in mice for molecular imaging studies and to report our experience.
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Analgesia/efeitos adversos , Anestesia/efeitos adversos , Imagem Molecular/métodos , Anestésicos/efeitos adversos , Animais , CamundongosRESUMO
Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Mice imaging procedures are increasingly used in preclinical research because they allow in vivo monitoring and they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. New imaging techniques and sophisticated laboratory animal imaging tools are currently producing a large body of evidence about the possible interference of anesthesia with different imaging methods that have the potential to compromise the results of in vivo studies. The purpose of this article is to review the existing literature on molecular imaging studies in mice, to describe the effects of different anesthetic protocols on their outcome, and to report our own experience with such studies.
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Analgesia/efeitos adversos , Anestesia/efeitos adversos , Imagem Molecular/métodos , Anestésicos/efeitos adversos , Animais , CamundongosRESUMO
Emerging evidence suggests that tumors contain and are driven by a cellular component that displays stem cell properties, the so-called cancer stem cells (CSCs). CSCs have been identified in several solid human cancers; however, there are no data about CSCs in primary human gastric cancer (GC). By using CD133 and CD44 cell surface markers we investigated whether primary human GCs contain a cell subset expressing stem-like properties and whether this subpopulation has tumor-initiating properties in xenograft transplantation experiments. We examined tissues from 44 patients who underwent gastrectomy for primary GC. The tumorigenicity of the cells separated by flow cytometry using CD133 and CD44 surface markers was tested by subcutaneous or intraperitoneum injection in NOD/SCID and nude mice. GCs included in the study were intestinal in 34 cases and diffuse in 10 cases. All samples contained surface marker-positive cells: CD133(+) mean percentage 10.6% and CD133(+)/CD44(+) mean percentage 27.7%, irrespective of cancer phenotype or grade of differentiation. Purified CD133(+) and CD133(+)/CD44(+) cells, obtained in sufficient number only in 12 intestinal type GC cases, failed to reproduce cancer in two mice models. However, the unseparated cells produced glandular-like structures in 70% of the mice inoculated. In conclusion, although CD133(+) and CD133(+)/CD44(+) were detectable in human primary GCs, they neither expressed stem-like properties nor exhibited tumor-initiating properties in xenograft transplantation experiments.
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Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Glicoproteínas/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/imunologia , Peptídeos/metabolismo , Neoplasias Gástricas/imunologia , Antígeno AC133 , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Separação Celular/métodos , Feminino , Citometria de Fluxo , Gastrectomia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.
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Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Immunoblotting , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos NusRESUMO
Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.
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Adenocarcinoma/genética , Adenocarcinoma/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adenocarcinoma de Pulmão , Idoso , Idoso de 80 Anos ou mais , Aldeído Desidrogenase/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Adesão Celular , Separação Celular , Biologia Computacional , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The objective of the study was to explore high-frequency ultrasound (HFUS) for noninvasive microimaging of thyroid in living mice. Thyroid examination was performed by HFUS in 10 normal C57BL/6 mice, eight mice treated by propylthiouracil, and 22 Tg-TRK-T1 transgenic mice. The dimension of the gland and the presence of nodules were evaluated. Nodules were classified as malignant (hypoechogenicity, poorly defined margins, internal microcalcification, irregular shapes, and extra glandular extension) or not, and the findings were compared with histological data. Thyroid images were successfully obtained in all the animals analyzed. Normal thyroid reached a volume of 4.92 microl (range 2.11-4.92 microl). Mice with propylthiouracil-induced goiter showed diffuse thyroid enlargement (median volume 6.67 microl, range 4.09-8.82 microl). In 19 of 22 Tg-TRK-T1 mice (86%), HFUS identified a nodular process (the smallest detected nodule had a diameter of 0.46 mm). Eleven nodules were classified as malignant and eight as benign. Compared with histological analysis, HFUS showed a sensitivity of 100% in the detection of thyroid nodules and a specificity of 60% (two of the nodules identified by HFUS were not confirmed at the histology). The specificity and sensitivity of HFUS in predicting the malignancy of the thyroid nodules were 83 and 91%, respectively. Thus, HFUS is an accurate imaging modality that can potentially replace more invasive techniques, and, therefore, it represents a significant advancement in phenotypic assessment of mouse models of thyroid cancer.
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Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/diagnóstico por imagem , Animais , Feminino , Hiperplasia/diagnóstico por imagem , Hiperplasia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/patologia , UltrassonografiaRESUMO
In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.
Assuntos
Ácido Aspártico/metabolismo , Cognição/fisiologia , Hipocampo/metabolismo , Potenciação de Longa Duração/genética , Animais , Ácido Aspártico/farmacologia , Cognição/efeitos dos fármacos , D-Aspartato Oxidase/genética , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Memória/fisiologia , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.