RESUMO
BACKGROUND: Small round blue cell tumors or more commonly called small round cell tumors (SRCTs) are undifferentiated neoplasms, sharing an overlapping morphological pattern of small round blue cells. Diagnosing these tumors represents a complex challenge for cytopathologists and for general surgical pathologist alike. This stems from the fact that these tumors share not only similar morphological features, but also some immunophenotypic characteristics, thus requiring a broad panel of antibodies, which might not be included in the most basic immunohistochemistry panels, used in the routine work of most pathology laboratories. Furthermore, one should note that the diagnosis, prognosis, and/or therapeutic decision are often dependent on the knowledge of the existence of specific molecular alterations, which requires access to sophisticated molecular ancillary techniques. Cytological diagnosis of SRCT should be systematized. A thorough understanding of the morphological pattern of these tumors, the small details they entail, the background and cellular patterns, and the nuclear and cytoplasmic peculiarities, may hint to the most probable diagnosis. Minor clues, such as the presence of a fibrillar background, the presence of rosettes or a specific "salt and pepper" chromatin, are important clues toward a probable diagnosis of a neuroblastoma, or the presence of a tigroid background is a characteristic of rhabdomyosarcoma and the Ewing family tumors. However, in poorly differentiated tumors, morphology alone will not suffice, making it essential for the access to complementary diagnostic techniques in order to reach the final diagnosis. Summary and Key Messages: The cytological diagnosis and treatment of SRCTs require an experienced, well-articulated, proficient teamwork, and sophisticated complementary diagnostic techniques, only available in centers of reference.
Assuntos
Neuroblastoma , Sarcoma de Ewing , Núcleo Celular/patologia , Criança , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , Imunofenotipagem , Neuroblastoma/química , Neuroblastoma/diagnóstico , Neuroblastoma/patologia , Sarcoma de Ewing/patologiaRESUMO
In recent years, there has been an expansion in the use of flow cytometry (FC) immunophenotyping in the diagnosis and monitoring of childhood solid neoplasms. Neuroblastoma (NB), in turn, is the most common extracranial solid tumor in childhood. In the present study, we sought to compare FC and anatomopathological examination (PA) / immunohistochemistry (IHC) of children diagnosed or suspected with NB. The median age was 59 months (minimum 0; maximum 325 months), of these 12 were male (57.1%, 12/21). Forty-eight samples (27 bone marrow (BM), 10 peripheral blood (PB), 8 primary tumors (PT) and 2 liver nodules (HN) and 1 rib fragment (RF)) from 21 patients were evaluated. Twenty-nine samples were from patients with clinical suspicion while 19 samples were from patients with previously confirmed diagnosis. Thirteen samples (7 BM, 5 PT and 1 HN) presented NB when analyzed in FC while 8 (3 BM and 5 PT) samples were positive for NB in the PA/IHC. They were concordant in 88.9% of the cases. No NB cells were identified in any PB. Considering the PA as the gold standard, the FC obtained a sensitivity of 100%, a specificity of 86%, a positive predictive value of 67% and a negative predictive value of 100%. This study demonstrates that FC can be used as a methodology for diagnosis and assessment of NB involvement. In addition, FC has the advantage of allowing a quick diagnosis and accurate classification of the disease, and can also assist in monitoring the treatment.
Assuntos
Biomarcadores Tumorais/análise , Citometria de Fluxo , Imuno-Histoquímica , Neuroblastoma/diagnóstico , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Neuroblastoma/química , Neuroblastoma/genética , Neuroblastoma/imunologia , Ploidias , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Abnormal dopamine neurotransmission is associated with several neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, attention deficiency and hyperactivity disorder, and addiction. Developing highly sensitive, selective, and fast dopamine monitoring methods is of high importance especially for the early diagnosis of these diseases. Herein, we report a new ultrasensitive electrochemical sensing platform for in situ monitoring of cell-secreted dopamine using Au-coated arrays of micropyramid structures integrated directly into a Petri dish. This approach enables the monitoring of dopamine released from cells in real-time without the need for relocating cultured cells. According to the electrochemical analyses, our dopamine sensing platform exhibits excellent analytical characteristics with a detection limit of 0.50 ± 0.08 nM, a wide linear range of 0.01-500 µM, and a sensitivity of 0.18 ± 0.01 µA/µM. The sensor also has remarkable selectivity toward DA in the presence of different potentially interfering small molecules. The developed electrochemical sensor has great potential for in vitro analysis of neuronal cells as well as early diagnosis of different neurological diseases related to abnormal levels of dopamine.
Assuntos
Técnicas Biossensoriais , Dopamina/análise , Técnicas Eletroquímicas , Neuroblastoma/química , Técnicas Biossensoriais/instrumentação , Dopamina/metabolismo , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais CultivadasRESUMO
Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.
Assuntos
Membrana Celular/química , Lipídeos/química , Microdomínios da Membrana/química , Ácidos Siálicos/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Bicamadas Lipídicas , Lipossomos/química , Microscopia de Fluorescência , Neuroblastoma/química , Neurônios/citologia , Ligação Proteica , SolubilidadeRESUMO
Alternative radiolabeled, targeted agents are being investigated for children with relapsed neuroblastoma (NB) who do not respond to I-metaiodobenzylguanidine (MIBG) therapy. (DOTA-Tyr)-octreotate targets somatostatin receptors (SSTRs), particularly SSTR2, which are expressed on NB cells. We investigated SSTR2 expression in NB tumors (36 high-risk [HR]; 33 non-HR patients) and correlated SSTR2 levels with clinical features, norepinephrine transporter (NET) expression, and MIBG avidity. SSTR2 and NET immunohistochemistry scores (0 to 3) were calculated on biopsies using digital image analysis based on staining intensity and distribution. Clinical data were correlated with SSTR2 expression. Median SSTR2 score for 69 patients was 1.31 (0.26 to 2.55). Non-HR NB was associated with a higher SSTR2 score (P=0.032). The SSTR2 expression did not correlate with age, International Neuroblastoma Staging System (INSS) stage, MYCN amplification and histology. Higher SSTR2 scores were observed in MIBG-avid versus MIBG-nonavid NB. SSTR2 score was not significantly associated with NET score (r=-0.062, P=0.62). Twenty-six patients who relapsed or progressed had a median SSTR2 score of 1.33 (0.26 to 2.55). Patients with NB including relapsed or progressive disease showed SSTR2 expression at diagnosis, suggesting they could be candidates for radiolabeled-DOTA-conjugated peptide imaging or therapy.
Assuntos
Neuroblastoma/química , Receptores de Somatostatina/metabolismo , 3-Iodobenzilguanidina/análise , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Imagem Molecular/métodos , Terapia de Alvo Molecular/métodos , Neuroblastoma/diagnóstico por imagem , Neuroblastoma/radioterapia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/análise , Prevalência , Compostos Radiofarmacêuticos/uso terapêutico , Receptores de Somatostatina/análise , RecidivaRESUMO
The application of nanomaterials in the healthy system may induce some neurodegenerative diseases initiated by tau folding and neuronal cell death. Herein, aluminium oxide nanoparticles (Al2O3 NPs) were synthesized and characterized by XRD, TEM, DLS and zeta potential investigations. Afterwards, the interaction of Al2O3 NPs with tau protein was investigated by fluorescence and CD spectroscopic methods. The molecular docking and molecular dynamic were also run to explore the binding site and conformational changes of tau after interaction with Al2O3 cluster. Moreover, the MTT, LDH, caspase-9/-3 and flow cytometry assays were done to explore the Al2O3 NPs-induced cytotoxicity against SH-SY5Y cells. It was revealed that Al2O3 NPs bind to tau protein and form a static complex and fold the structure of tau toward a more packed structure. Molecular docking and molecular dynamic investigations revealed that NPs bind to the hydrophilic residues of the tau segments and promote some marginal structural folding of tau segment. The cellular assays displayed that Al2O3 NPs can elicit cell mortality through membrane leakage, caspase-9/-3 activations, and induction of both apoptosis and necrosis. This data may indicate that NPs can induce some adverse effects on the biological systems.
Assuntos
Óxido de Alumínio/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Neuroblastoma/tratamento farmacológico , Proteínas tau/química , Óxido de Alumínio/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neuroblastoma/química , Neuroblastoma/patologia , Conformação Proteica/efeitos dos fármacos , Proteínas tau/efeitos dos fármacosRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in children with contrasting outcomes. Precise risk assessment contributes to prognosis prediction, which is critical for treatment strategy decisions. In this study, we developed a 3-protein predictor model, including the neural stem cell marker Msi1, neural differentiation marker ID1, and proliferation marker proliferating cell nuclear antigen (PCNA), to improve clinical risk assessment of patients with NB. Kaplan-Meier analysis in the microarray data (GSE16476) revealed that low expression of ID1 and high expression of Msi1 and PCNA were associated with poor prognosis in NB patients. Combined application of these 3 markers to constitute a signature further stratified NB patients into different risk subgroups can help obtain more accurate prediction performance. Survival prognostic power of age and Msi1_ID1_PCNA signature by receiver operating characteristics analysis showed that this signature predicted more effectively and sensitively compared with classic risk stratification system, compensating for the deficiency of the prediction function of the age. Furthermore, we validated the expressions of these 3 proteins in neuroblastic tumor spectrum tissues by immunohistochemistry revealed that Msi1 and PCNA exhibited increased expression in NB compared with intermedial ganglioneuroblastoma and benign ganglioneuroma, whereas ID1 levels were reduced in NB. In conclusion, we established a robust risk assessment predictor model based on simple immunohistochemistry for therapeutic decisions of NB patients.
Assuntos
Imuno-Histoquímica , Proteína 1 Inibidora de Diferenciação/análise , Proteínas do Tecido Nervoso/análise , Neuroblastoma/química , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas de Ligação a RNA/análise , Adolescente , Criança , Pré-Escolar , Tomada de Decisão Clínica , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estadiamento de Neoplasias , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neuroblastoma/terapia , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco , Resultado do TratamentoRESUMO
BACKGROUND: Neuroblastoma is one of the most common pediatric solid tumors. Although the 5-year overall survival rate has increased over the past few decades, high-risk patients still have a poor prognosis due to a lack of biomonitoring therapy. This study was performed to investigate the role of Galectin-1 in neuroblastoma biomonitoring therapy. PROCEDURE: A tissue microarray containing 37 neuroblastoma tissue samples was used to evaluate the correlation between Galectin-1 expression and clinical features. Blood samples were examined to better understand whether serum Galectin-1 (sGalectin-1) could be used for biomonitoring therapy. Kaplan-Meier analysis and ROC analysis was conducted to distinguish the outcome associated with high or low expression of Galectin-1 in patients with neuroblastoma. RESULTS: Increased Galectin-1 expression was found in neuroblastoma and it was further demonstrated that elevated tissue Galectin-1 expression was related to INSS stage, histology, bone marrow metastasis, and poor survival. sGalectin-1 levels were higher in newly diagnosed patients with neuroblastoma than healthy subjects. Patients with elevated sGalectin-1 through treatment cycles correlated with the poor chemo-responses and tended to have worse outcomes, such as metastasis or stable tumor size, whereas gradually decreasing sGalectin-1 levels correlated with no observed progression in clinical symptoms. CONCLUSIONS: Tissue and serum Galectin-1 levels were associated with adverse clinical features in patients with neuroblastoma, and sGalectin-1 could be a potential biomarker for monitoring therapy.
Assuntos
Biomarcadores Tumorais/análise , Galectina 1/análise , Proteínas de Neoplasias/análise , Neuroblastoma/química , Neoplasias Retroperitoneais/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Neoplasias da Medula Óssea/secundário , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Galectina 1/biossíntese , Galectina 1/sangue , Humanos , Técnicas Imunoenzimáticas , Lactente , Estimativa de Kaplan-Meier , Masculino , Neoplasias do Mediastino/sangue , Neoplasias do Mediastino/química , Neoplasias do Mediastino/tratamento farmacológico , Neoplasias do Mediastino/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/sangue , Estadiamento de Neoplasias , Neuroblastoma/sangue , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Prognóstico , Intervalo Livre de Progressão , Neoplasias Retroperitoneais/sangue , Neoplasias Retroperitoneais/tratamento farmacológico , Neoplasias Retroperitoneais/patologia , Análise Serial de Tecidos , Carga TumoralRESUMO
Beta amyloid ( ) peptide, which is a common neuropathological hallmark deposit in the brain of patients with Alzheimer's disease, typically comprises 39-43 amino acid residues. peptides exist as isoforms of and with various lengths. In this research, atomic force microscopy (AFM) was applied to investigate aggregations in Hank's Balanced Salt Solution. Toxic effect of oligomer was investigated in live SH-SY5Y neuroblastoma cells by characterizing cell morphology and cell mechanics using high-resolution AFM scanning. oligomer-induced cytoskeleton reorganization was also observed under confocal microscopy, and it can account for reduction in Young's modulus of cells. Meanwhile, phosphorylation of tau increased after oligomer treatment, possibly resulting in microtubule disassembly. This paper demonstrates the linkage between cellular mechanical changes and neurodegeneration mediated by . The method used implies promising applications of real-time monitoring of cellular mechanical properties given the toxic effects of on living neuronal cells.
Assuntos
Peptídeos beta-Amiloides , Sobrevivência Celular/efeitos dos fármacos , Módulo de Elasticidade/efeitos dos fármacos , Neuroblastoma , Fragmentos de Peptídeos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Linhagem Celular Tumoral , Humanos , Microscopia de Força Atômica , Microscopia Confocal , Neuroblastoma/química , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologiaRESUMO
In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.
Assuntos
Álcoois/química , Anestésicos Gerais/química , Conexinas/química , Conformação Proteica/efeitos dos fármacos , Álcoois/farmacologia , Anestésicos Gerais/farmacologia , Animais , Conexinas/metabolismo , Junções Comunicantes/química , Junções Comunicantes/efeitos dos fármacos , Células HeLa , Hexanóis/química , Hexanóis/farmacologia , Humanos , Canais Iônicos/química , Isoflurano/química , Isoflurano/farmacologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Neuroblastoma/química , Técnicas de Patch-Clamp , Domínios Proteicos/efeitos dos fármacos , Proteína delta-2 de Junções ComunicantesRESUMO
BACKGROUND: Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers. METHODS: Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression. RESULTS: Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001). CONCLUSIONS: A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017;123:3807-3815. © 2017 American Cancer Society.
Assuntos
Antígeno B7-H1/análise , Linfócitos do Interstício Tumoral , Macrófagos , Proteínas de Neoplasias/análise , Neoplasias/química , Neoplasias Ósseas/química , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linfoma de Burkitt/química , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Criança , Glioblastoma/química , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias/patologia , Neuroblastoma/química , Neuroblastoma/imunologia , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Osteossarcoma/química , Osteossarcoma/imunologia , Osteossarcoma/patologia , Rabdomiossarcoma/química , Rabdomiossarcoma/imunologia , Rabdomiossarcoma/patologia , Sarcoma de Ewing/química , Sarcoma de Ewing/imunologia , Sarcoma de Ewing/patologia , Análise Serial de TecidosRESUMO
Small round blue cell tumors (SRBCTs) of children and adolescents are often diagnostically challenging lesions. With the increasing diagnostic approach based on small biopsies, there is the need of specific immunomarkers that can help in the differential diagnosis among the different tumor histotypes to assure the patient a correct diagnosis for proper treatment. Based on our recent studies showing cyclin D1 overexpression in both Ewing sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) and peripheral neuroblastic tumors (neuroblastoma and ganglioneuroblastoma), we immunohistochemically assessed cyclin D1 immunoreactivity in 128 cases of SRBCTs in children and adolescents to establish its potential utility in the differential diagnosis. All cases of EWS/pPNET and the undifferentiated/poorly differentiated neuroblastomatous component of all peripheral neuroblastic tumors exhibited strong and diffuse nuclear staining (>50% of neoplastic cells) for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of subtype) and lymphoblastic lymphoma (either B- or T-cell precursors), whereas it was only focally detected (<5% of neoplastic cells) in some cases of Wilms tumor (blastemal component) and desmoplastic small round cell tumor. Our findings suggest that cyclin D1 can be exploitable as a diagnostic adjunct to conventional markers in confirming the diagnosis of EWS/pPNET or neuroblastoma/ganglioneuroblastoma. Its use in routine practice may also be helpful for those cases of SRBCT with undifferentiated morphology that are difficult to diagnose after application of the conventional markers.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Ósseas/química , Ciclina D1/análise , Ganglioneuroblastoma/química , Neuroblastoma/química , Tumores Neuroectodérmicos Primitivos Periféricos/química , Sarcoma de Ewing/química , Adolescente , Biópsia , Neoplasias Ósseas/patologia , Diferenciação Celular , Criança , Pré-Escolar , Tumor Desmoplásico de Pequenas Células Redondas/química , Tumor Desmoplásico de Pequenas Células Redondas/patologia , Diagnóstico Diferencial , Feminino , Ganglioneuroblastoma/patologia , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Renais/química , Neoplasias Renais/patologia , Masculino , Neuroblastoma/patologia , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Rabdomiossarcoma/química , Rabdomiossarcoma/patologia , Sarcoma de Ewing/patologia , Tumor de Wilms/química , Tumor de Wilms/patologia , Adulto JovemRESUMO
INTRODUCTION: Dengue is a human disease caused by a virus with the same name, which is transmitted by the bite of Aedes mosquitoes. The infection has a wide range of clinical presentations ranging from asymptomatic to fatal cases, with the pediatric population being the most susceptible. According to the new classification of the disease, the neurological manifestations are considered a criterion for the diagnosis of severe dengue. OBJECTIVE: To evaluate the possible mechanisms involved in the onset of neurological signs in a cell line of human neurons as a model of infection with dengue virus type 2 (DENV-2). MATERIALS AND METHODS: Susceptibility and permissiveness of the SH-SY5Y line to infection by DENV-2 was analyzed, showing that the proportions of viral infection and production are similar to those of primate cells used as positive control for infection. RESULTS: Infection induced a cytopathic effect on the neuroblastoma line characterized by apoptotic cell death process, increasing the proportion of annexin V and TUNEL positive cells and an upregulation of TNF-α. Treatment with anti-TNF-α antibody increased slightly cell survival of infected cells. The addition of exogenous TNF-α to the infected cultures enhanced cell death. CONCLUSION: These results as a whole suggest that the upregulation of TNF-α could be part of the process that induces cell damage and death in cases of dengue encephalitis.
Assuntos
Apoptose , Vírus da Dengue/imunologia , Dengue/virologia , Encefalite/imunologia , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/imunologia , Animais , Linhagem Celular , Vírus da Dengue/química , Encefalite/metabolismo , Humanos , Neuroblastoma/química , Fator de Necrose Tumoral alfa/químicaRESUMO
Amyloid plaques and neurofibrillary tangles are the pathological hallmarks of Alzheimer's disease. However, there has been a long-standing discussion on the dynamic relations between Aß and tau proteins, partially due to the lack of a tool to track protein dynamics in individual live neurons at the early stage of Aß generation and tau phosphorylation. Here, we developed nanoplasmonic fiber tip probe (nFTP) technology to simultaneously monitor Aß42 generation and tau phosphorylation (at serine 262) in living, single neuroblastoma cells over 12 h. We observed that Aß42 generation, under clinically relevant anesthetic treatment, preceded tau phosphorylation, which then facilitated Aß42 generation. This observation is also supported by measuring proteins in cell lysates using the ultrasensitive label-free photonic crystal nanosensors. nFTP therefore provides an advanced method to investigate protein expression and post-translational modification in live cells and determine outcomes of intervention of Alzheimer's disease and other neurodegenerative disorders.
Assuntos
Peptídeos beta-Amiloides/química , Nanotecnologia , Neuroblastoma/química , Proteínas tau/química , Doença de Alzheimer , Linhagem Celular Tumoral , Humanos , FosforilaçãoRESUMO
MYCN and HDAC2 jointly repress the transcription of tumor suppressive miR-183 in neuroblastoma. Enforced miR-183 expression induces neuroblastoma cell death and inhibits xenograft growth in mice. Here we aimed to focus more closely on the miR-183 signaling network using a label-free mass spectrometric approach. Analysis of neuroblastoma cells transfected with either control or miR-183 expression vectors identified 85 differentially expressed proteins. All six members of the minichromosome maintenance (MCM) complex, which is indispensable for initiation and elongation during DNA replication and transcriptionally activated by MYCN in neuroblastoma, emerged to be down-regulated by miR-183. Subsequent annotation category enrichment analysis revealed a â¼14-fold enrichment in the "MCM" protein module category, which highlighted this complex as a critical node in the miR-183 signaling network. Down-regulation was confirmed by Western blotting. MCMs 2-5 were predicted by in silico methods as direct miR-183 targets. Dual-luciferase reporter gene assays with 3'-UTR constructs of the randomly selected MCMs 3 and 5 experimentally confirmed them as direct targets of miR-183. Our results reveal the MCM complex to be a critical and directly regulated node within the miR-183 signaling network in MYCN-amplified neuroblastoma cells.
Assuntos
MicroRNAs/farmacologia , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteína Proto-Oncogênica N-Myc/fisiologia , Neuroblastoma/patologia , Transdução de Sinais , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Espectrometria de Massas , MicroRNAs/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/efeitos dos fármacos , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 5 do Complexo de Manutenção de Minicromossomo/efeitos dos fármacos , Componente 5 do Complexo de Manutenção de Minicromossomo/metabolismo , Neuroblastoma/química , Neuroblastoma/metabolismo , Transfecção , Regulação para CimaRESUMO
In this study the effect of oxidative modification on micellar and drug delivery properties of copolymers of ethylene oxide (EO) and propylene oxide (PO) was investigated. Carboxylated trifunctional copolymers were synthesized in the reaction with chromium(VI) oxide. We found that carboxylation significantly improved the uniformity and stability of polymeric micelles by inhibiting the microphase transition. The cytotoxicity of copolymers was studied in relation to their aggregative state on two cell types (cancer line vs. primary fibroblasts). The accumulation of rhodamine 123 in neuroblastoma SH-SY5Y cells was dramatically increased in the presence of the oxidized block copolymer with the number of PO and EO units of 83.5 and 24.2, respectively. The copolymer was also tested as an enhancer for topical drug delivery to the spinal cord when applied subdurally. The oxidized copolymer facilitated the penetration of rhodamine 123 across spinal cord tissues and increased its intraspinal accumulation. These results show the potential of using oxidized EO/PO based polymers for non-invasive delivery of protective drugs after spinal cord injury.
Assuntos
Compostos de Epóxi/química , Óxido de Etileno/química , Rodamina 123/química , Medula Espinal/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromo/química , Compostos de Cromo/química , Sistemas de Liberação de Medicamentos/métodos , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Microscopia Confocal , Neuroblastoma/química , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ratos Wistar , Rodamina 123/administração & dosagem , Rodamina 123/farmacocinética , Espectroscopia de Infravermelho com Transformada de Fourier , Medula Espinal/químicaRESUMO
Stimulation with heavy metals is known to induce calcium (Ca(2+)) mobilization in many cell types. Interference with the measurement of intracellular Ca(2+) concentration by the heavy metals in cells loaded with Ca(2+) indicator fura-2 is an ongoing problem. In this study, we analyzed the effect of heavy metals on the fura-2 fluorescence ratio in human SH-SY5Y neuroblastoma cells by using TPEN, a specific cell-permeable heavy metal chelator. Manganese chloride (30-300 µM) did not cause significant changes in the fura-2 fluorescence ratio. A high concentration (300 µM) of lead acetate induced a slight elevation in the fura-2 fluorescence ratio. In contrast, stimulation with cadmium chloride, mercury chloride or MeHg (3-30 µM) elicited an apparent elevation of the fura-2 fluorescence ratio in a dose-dependent manner. In cells stimulated with 10 or 30 µM cadmium chloride, the addition of TPEN decreased the elevated fura-2 fluorescence ratio to basal levels. In cells stimulated with mercury or MeHg, the addition of TPEN significantly decreased the elevation of the fura-2 fluorescence ratio induced by lower concentrations (10 µM) of mercury or MeHg, but not by higher concentrations (30 µM). Pretreatment with Ca(2+) channel blockers, such as verapamil, 2-APB or lanthanum chloride, resulted in different effects on the fura-2 fluorescence ratio. Our study provides a characterization of the effects of several heavy metals on the mobilization of divalent cations and the toxicity of heavy metals to neuronal cells.
Assuntos
Intoxicação por Cádmio/tratamento farmacológico , Quelantes/farmacologia , Etilenodiaminas/farmacologia , Fura-2/metabolismo , Intoxicação por Mercúrio/tratamento farmacológico , Compostos de Metilmercúrio/intoxicação , Compostos de Boro/farmacologia , Cálcio/análise , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Lantânio/farmacologia , Neuroblastoma/química , Neuroblastoma/metabolismo , Verapamil/farmacologiaRESUMO
Aging and oxidative stress are two prominent pathological mechanisms for Parkinson's disease (PD) that are strongly associated with the degeneration of dopamine (DA) neurons in the midbrain. DA and other catechols readily oxidize into highly reactive o-quinone species that are precursors of neuromelanin (NM) pigment and under pathological conditions can modify and damage macromolecules. The role of DA oxidation in PD pathogenesis remains unclear in part due to the lack of appropriate disease models and the absence of a simple method for the quantification of DA-derived oxidants. Here, we describe a rapid, simple, and reproducible method for the quantification of o-quinones in cells and tissues that relies on the near-infrared fluorescent properties of these species. Importantly, we demonstrate that catechol-derived oxidants can be quantified in human neuroblastoma cells and midbrain dopamine neurons derived from induced pluripotent stem cells, providing a novel model to study the downstream actions of o-quinones. This method should facilitate further study of oxidative stress and DA oxidation in PD and related diseases that affect the dopaminergic system.
Assuntos
Neurônios Dopaminérgicos/química , Fluorescência , Raios Infravermelhos , Neuroblastoma/química , Quinonas/análise , Quinonas/química , Catecóis/química , Dopamina/química , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Neuroblastoma/patologia , Oxirredução , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
Multiple synchronous tumors presenting in infancy raise concern for inherited or sporadic cancer predisposition syndromes, which include Beckwith-Wiedemann syndrome, familial adenomatous polyposis syndrome, and Li-Fraumeni syndrome. We report a case of a 7-month-old previously healthy male born following an in vitro fertilization-assisted twin pregnancy who presented with new-onset refractory shock, severe acidosis, and rapid decline over several hours. An autopsy revealed a ruptured liver involved by hepatoblastoma, an adrenal gland involved by neuroblastoma, and multiple cutaneous capillary hemangiomas. Standard genetic testing demonstrated that both twins were Gaucher disease (GD) carriers without evidence of other known cancer predisposition syndromes. This report describes a unique association of multiple synchronous tumors, which underscores the utility and importance of the pediatric autopsy. Moreover, given that the reported child was a GD carrier, the possibility the tumors were the result of a GD-mediated cancer-associated phenotype or an unrecognized sporadic clinical syndrome remains an unanswered, but intriguing, question worthy of further investigation.
Assuntos
Hemangioma Capilar/patologia , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Neoplasias Primárias Múltiplas/patologia , Neuroblastoma/patologia , Neoplasias Cutâneas/patologia , Adulto , Autopsia , Biomarcadores Tumorais/análise , Causas de Morte , Evolução Fatal , Feminino , Fertilização in vitro , Doença de Gaucher/genética , Triagem de Portadores Genéticos , Hemangioma Capilar/química , Hepatoblastoma/química , Heterozigoto , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Hepáticas/química , Masculino , Neoplasias Primárias Múltiplas/química , Neuroblastoma/química , Gravidez , Gravidez de Gêmeos , Neoplasias Cutâneas/químicaRESUMO
OBJECTIVE: There is 25-35% mutation rate of p53 in cancerous neural cells and this rate reaches 70-76% in glioma cell line. Complement of wild-type p53 has become a potential strategy for protein therapy of cancerous neural cells. Here we investigated the feasibility of a novel RDP-p53 fusion protein for anti-proliferation of cancerous neural cell and the possible mechanism, which would provide an effective approach for targeted delivery of p53 protein to treat cancerous neural cells. METHODS: The RDP-p53 fusion proteins are expressed in Escherichia coli, and they are labeled with FITC and rhodamine B by chemical modification. The fluorescence-labeled proteins are added to human hepatocellular carcinoma cells (HepG-2) and human neuroblastoma cells (SH-SY5Y) in order to investigate the possibility of RDP enhancing the cell uptake efficiency into neural cells as a cell-permeable carrier. The inhibitory effect of RDP-p53 on SH-SY5Y and human glioma cells (U251) was evaluated by MTT assay. Moreover, the anti-proliferation mechanism of RDP-p53 was determined by Apoptosis and Necrosis Assay Kit and flow cytometric analysis. RESULTS: The results showed that RDP-p53 could enter SH-SY5Y cells with high efficiency and selectively inhibit the growth of cancerous neural cells, including SH-SY5Y and U251. Also, cell apoptosis pathway and cell-cycle arrest at the G2/M phase were associated with the inhibition mechanism of RDP-p53 according to the data of flow cytometric analysis. CONCLUSIONS: RDP-p53 could be a novel antitumor candidate for targeting treatment of cancerous neural cells.