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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33446504

RESUMO

Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.


Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Receptores Imunológicos/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proliferação de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/classificação , Microglia/efeitos dos fármacos , Microglia/patologia , Mutação , Ligação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Fatores Sexuais
2.
Anal Chem ; 95(42): 15477-15485, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37812809

RESUMO

The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (∼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.


Assuntos
COVID-19 , Humanos , Glicosilação , Cromatografia Líquida , Enzima de Conversão de Angiotensina 2/metabolismo , Espectrometria de Massas em Tandem , Anticorpos Antivirais , Ligação Proteica , Cromatografia em Gel
3.
Hum Mutat ; 40(5): 619-630, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30740813

RESUMO

The lipid phosphatase gene FIG4 is responsible for Yunis-Varón syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.


Assuntos
Alelos , Doenças Desmielinizantes/diagnóstico , Doenças Desmielinizantes/genética , Flavoproteínas/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Monoéster Fosfórico Hidrolases/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Doenças Desmielinizantes/metabolismo , Fibroblastos/metabolismo , Genótipo , Humanos , Padrões de Herança , Imageamento por Ressonância Magnética , Masculino , Neuroimagem , Linhagem , Fenótipo
4.
Dev Med Child Neurol ; 61(4): 490-496, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30146710

RESUMO

This case series describes three children with chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), an inflammatory condition characterized by a relapsing-remitting disease course responsive to steroids. The patients (two males, age 3y and 13y; one female, age 14y) presented with ataxia, dysarthria, and multiple cranial neuropathies. All patients demonstrated bilateral nodular lesions with contrast enhancement in the brainstem and cerebellum on magnetic resonance imaging, and perivascular lymphocytes and macrophages infiltrates on brain biopsies. Despite an initially good response to corticosteroids, all patients eventually became steroid-dependent or -resistant, with frequent relapses on maintenance immunosuppressive therapy. Natalizumab and intravenous immunoglobulin stopped neurological disease progression in Patient 1 but he died at 17 years from respiratory complications. Patient 2 went into remission on infliximab and intravenous methylprednisolone for several months but was then diagnosed with Epstein-Barr virus driven B-cell lymphoma 3 years after symptom onset. Patient 3 failed to respond to treatment and died 4 years after diagnosis. CLIPPERS disease in children is aggressive, with poor response to immunotherapy. Earlier use of newer immunotherapeutic agents such as natalizumab may be beneficial. Potential side effects need to be considered carefully. WHAT THIS PAPER ADDS: Paediatric chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) appears a more severe condition than previously reported in adults. Aggressive treatment before neuroaxonal loss may be required for a better outcome.


¿ES LA INFLAMACIÓN LINFOCÍTICA CRÓNICA CON REALCE PERIVASCULAR PONTINO SENSIBLE A LOS ESTEROIDES (CLIPPERS) EN LOS NIÑOS CON LA MISMA CONDICIÓN QUE EN LOS ADULTOS?: Esta serie de casos describe a tres niños con inflamación linfocítica crónica con realce pontinal perivascular sensible a esteroides (CLIPPERS), una enfermedad inflamatoria caracterizada por un curso de enfermedad recurrente-remitente sensible a los esteroides. Los pacientes (dos varones, edad 3 y 13 años, una mujer, edad 14 años) presentaron ataxia, disartria y neuropatías craneales múltiples. Todos los pacientes demostraron lesiones nodulares bilaterales con realce de contraste en el tallo cerebral y el cerebelo en imágenes de resonancia magnética y linfocitos perivasculares y infiltrados de macrófagos en biopsias cerebrales. A pesar de una respuesta inicialmente buena a los corticosteroides, todos los pacientes finalmente se volvieron dependientes de esteroides o resistentes, con recaídas frecuentes en la terapia inmunosupresora de mantenimiento. El natalizumab y la inmunoglobulina intravenosa suspendieron la progresión de la enfermedad neurológica en el paciente 1, pero falleció a los 17 años por complicaciones respiratorias. El paciente 2 entró en remisión con infliximab y metilprednisolona por vía intravenosa durante varios meses, pero luego se le diagnosticó linfoma de células B dirigido por el virus de Epstein-Barr, 3 años después del inicio de los síntomas. El paciente 3 no respondió al tratamiento y murió 4 años después del diagnóstico. La enfermedad de CLIPPERS en los niños es agresiva, con una respuesta deficiente a la inmunoterapia. El uso previo de agentes inmunoterápicos más nuevos como natalizumab puede ser beneficioso. Los posibles efectos secundarios deben considerarse cuidadosamente.


A INFLAMAÇÃO LINFOCÍTICA CRÔNICA COM REALCE PERIVASCULAR PONTINO RESPONSIVA A ESTERÓIDES (CLIPPERS) É A MESMA CONDIÇÃO EM CRIANÇAS E ADULTOS?: Esta série de casos descreve três crianças com inflamação linfocítica crônica com realce perivascular pontino responsiva a esteróides (CLIPPERS), uma condição inflamatória caracterizada por uma doença com curso remissivo-recidivante responsive a esteróides. Os pacientes (dois meninos, idades 3 e 13 anos; uma menina, idade 14 anos) apresentaram ataxia, disartria, e múltiplas neuropatias craniais. Todos os pacientes demonstraram lesões nodulares bilaterais com realce no tronco cerebral e cerebelo ao exame ne ressonância magnética, e infiltrados perivasculares de linfócitos e macrófagos nas biópsias cerebrais. Apesar de uma resposta inicialmente boa aos corticoesteróides, todos os pacientes eventualmente se tornaram esteróide-dependentes ou resistentes, com frequentes recidivas com manutenção de imunoterapia supressora. Natalizumab e imunoglobulina intravenosa interromperam a progressão neurológica da doença no Paciente 1, mas ele veio a óbito na idade de 17 anos devido a complicações respiratórias. O Paciente 2 entrou em remissão com infliximab e metilprednosolona intravenosa por vários meses, mas foi então diagnosticado com linfoma de células B causado por virus Epstein-Barr 3 anos após o início dos sintomas. O Paciente 3 não respondeu ao tratamento e veio a óbito 4 anos após o diagnóstico. Patient 2 went into remission on infliximab and intravenous methylprednisolone for several months but was then diagnosed with Epstein-Barr virus driven B-cell lymphoma 3 years after symptom onset. Patient 3 failed to respond to treatment and died 4 years after diagnosis. INTERPRETAÇÃO: A doença CLIPPERS em crianças é agressiva, com pouca resposta à imunoterapia. O uso precoce de agentes imunoterapêuticos mais novos como natalizumab pode ser benéfico. Potenciais efeitos colaterais devem ser considerados com cautela.


Assuntos
Encefalite/diagnóstico , Encefalite/terapia , Glucocorticoides/uso terapêutico , Metilprednisolona/uso terapêutico , Ponte , Adolescente , Fatores Etários , Pré-Escolar , Doença Crônica , Encefalite/etiologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino
5.
Neuropediatrics ; 49(2): 118-122, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29253910

RESUMO

Alexander disease (AD) is a leukodystrophy caused by heterozygous mutations in the gene encoding the glial fibrillary acidic protein (GFAP). Currently, de novo heterozygous missense mutations in the GFAP gene are identified in over 95% of patients with AD. However, patients with biopsy-proven AD have been reported in whom no GFAP mutation has been identified. We report identical twin boys presenting in infancy with seizures and developmental delay in whom MR appearances were suggestive of AD with the exception of an unusual, bilateral, arc of calcification at the frontal white-gray junction. Initial mutation screening of the GFAP gene did not identify a mutation. Whole exome sequencing in both brothers revealed a de novo heterozygous in-frame deletion of the whole of exon 5 of the GFAP gene. Mutations in the GFAP gene are thought to result in a toxic effect of mutant GFAP disrupting the formation of the normal intermediate filament network and resulting in Rosenthal fiber formation, which has hitherto not been linked to exonic scale copy number variants in GFAP. Further studies on mutation negative AD patients are warranted to determine whether a similar mechanism underlies their disease.


Assuntos
Doença de Alexander/genética , Éxons/genética , Deleção de Genes , Proteína Glial Fibrilar Ácida/genética , Doença de Alexander/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Análise Mutacional de DNA , Seguimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Tomógrafos Computadorizados
6.
Arch Dis Child Educ Pract Ed ; 103(5): 267-273, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29150423

RESUMO

The ability to interact with children and young people (CYP), appropriately examine and competently interpret signs is an essential skill for many medical practitioners and allied healthcare professionals; yet, how do we ensure competence in our students and trainees? One method is to include CYP in both formative and summative assessments; this provides an invaluable opportunity for examiners not only to evaluate the clinical interaction but also to gain an understanding of the CYP experience and what characteristics they value in a 'good children's doctor'. This paper explores the benefits and challenges of involving CYP in assessments and provides practical advice for course organisers, assessors and students when encountering CYP in assessment.


Assuntos
Participação do Paciente , Exame Físico , Relações Médico-Paciente , Criança , Humanos , Consentimento Livre e Esclarecido , Consentimento dos Pais , Autonomia Pessoal
7.
Proc Natl Acad Sci U S A ; 111(26): E2721-30, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24979794

RESUMO

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.


Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Quinazolinonas/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Sinergismo Farmacológico , Linfoma , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismo
8.
J Cell Sci ; 125(Pt 6): 1591-604, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22344259

RESUMO

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.


Assuntos
Adenosina Trifosfatases/fisiologia , Cromossomos/genética , Proteínas de Ligação a DNA/fisiologia , Mitose/genética , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Linhagem Celular Tumoral , Galinhas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes/métodos , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética
9.
Eur J Hum Genet ; 30(7): 860-864, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35217805

RESUMO

Leukodystrophies are a heterogenous group of genetic disorders, characterised by abnormal development of cerebral white matter. Pelizaeus-Merzbacher disease is caused by mutations in PLP1, encoding major myelin-resident protein required for myelin sheath assembly. We report a missense variant p.(Ala109Asp) in MAL as causative for a rare, hypomyelinating leukodystrophy similar to Pelizaeus-Merzbacher disease. MAL encodes a membrane proteolipid that directly interacts with PLP1, ensuring correct distribution during myelin assembly. In contrast to wild-type MAL, mutant MAL was retained in the endoplasmic reticulum but was released following treatment with 4-phenylbutyrate. Proximity-dependent identification of wild-type MAL interactants implicated post-Golgi vesicle-mediated protein transport and protein localisation to membranes, whereas mutant MAL interactants suggested unfolded protein responses. Our results suggest that mislocalisation of MAL affects PLP1 distribution, consistent with known pathomechanisms for hypomyelinating leukodystrophies.


Assuntos
Doenças Neurodegenerativas , Doença de Pelizaeus-Merzbacher , Humanos , Mutação , Mutação de Sentido Incorreto , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Doença de Pelizaeus-Merzbacher/genética , Transporte Proteico
10.
Sci Transl Med ; 14(646): eabn1252, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35412328

RESUMO

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Humanos , Camundongos , Glicoproteína da Espícula de Coronavírus
11.
bioRxiv ; 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34268509

RESUMO

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC 50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. ONE-SENTENCE SUMMARY: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

12.
Ann Clin Transl Neurol ; 7(1): 83-93, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31814314

RESUMO

OBJECTIVE: Biallelic variants in RARS1, encoding the cytoplasmic tRNA synthetase for arginine (ArgRS), cause a hypomyelinating leukodystrophy. This study aimed to investigate clinical, neuroradiological and genetic features of patients with RARS1-related disease, and to identify possible genotype-phenotype relationships. METHODS: We performed a multinational cross-sectional survey among 20 patients with biallelic RARS1 variants identified by next-generation sequencing techniques. Clinical data, brain MRI findings and genetic results were analyzed. Additionally, ArgRS activity was measured in fibroblasts of four patients, and translation of long and short ArgRS isoforms was quantified by western blot. RESULTS: Clinical presentation ranged from severe (onset in the first 3 months, usually with refractory epilepsy and early brain atrophy), to intermediate (onset in the first year with nystagmus and spasticity), and mild (onset around or after 12 months with minimal cognitive impairment and preserved independent walking). The most frequent RARS1 variant, c.5A>G, led to mild or intermediate phenotypes, whereas truncating variants and variants affecting amino acids close to the ArgRS active centre led to severe phenotypes. ArgRS activity was significantly reduced in three patients with intermediate and severe phenotypes; in a fourth patient with intermediate to severe presentation, we measured normal ArgRS activity, but found translation mainly of the short instead of the long ArgRS isoform. INTERPRETATION: Variants in RARS1 impair ArgRS activity and do not only lead to a classic hypomyelination presentation with nystagmus and spasticity, but to a wide spectrum, ranging from severe, early-onset epileptic encephalopathy with brain atrophy to mild disease with relatively preserved myelination.


Assuntos
Arginina-tRNA Ligase/genética , Estudos de Associação Genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Estudos Transversais , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico por imagem , Humanos , Lactente , Imageamento por Ressonância Magnética , Índice de Gravidade de Doença , Adulto Jovem
13.
Wound Repair Regen ; 17(5): 639-48, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19769717

RESUMO

Differences in cellular competence offer an explanation for the differences in the healing capacity of tissues of various ages and conditions. The homeobox family of genes plays key roles in governing cellular competence. Of these, we hypothesize that Msx2 is a strong candidate regulator of competence in skin wound healing because it is expressed in the skin during fetal development in the stage of scarless healing, affects postnatal digit regeneration, and is reexpressed transiently during postnatal skin wound repair. To address whether Msx2 affects cellular competence in injury repair, 3 mm full-thickness excisional wounds were created on the back of C.Cg-Msx2(tm1Rilm)/Mmcd (Msx2 null) mice and the healing pattern was compared with that of the wild type mice. The results show that Msx2 null mice exhibited faster wound closure with accelerated reepithelialization plus earlier appearance of keratin markers for differentiation and an increased level of smooth muscle actin and tenascin in the granulation tissue. In vitro, keratinocytes of Msx2 null mice exhibit increased cell migration and the fibroblasts show stronger collagen gel contraction. Thus, our results suggest that Msx2 regulates the cellular competence of keratinocytes and fibroblasts in skin injury repair.


Assuntos
Fibroblastos/metabolismo , Proteínas de Homeodomínio/genética , Queratinócitos/metabolismo , Pele/metabolismo , Cicatrização/genética , Animais , Modelos Animais de Doenças , Proteínas de Homeodomínio/biossíntese , Camundongos , Camundongos Transgênicos , Fatores de Tempo
14.
Cell Signal ; 30: 104-117, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27890558

RESUMO

Elevated expression of TLX (also called as NR2E1) in neuroblastoma (NB) correlates with unfavorable prognosis, and TLX is required for self-renewal of NB cells. Knockdown of TLX has been shown to reduce the NB sphere-forming ability. ASK1 (MAP3K5) and TLX expression are both enhanced in SP (side population) NB and patient-derived primary NB sphere cell lines, but the majority of non-SP NB lines express lower ASK1 expression. We found that ASK1 phosphorylated and stabilized TLX, which led induction of HIF-1α, and its downstream VEGF-A in an Akt dependent manner. In depleting ASK1 upon hypoxia, TLX decreased and the apoptosis ratio of NB cells was enhanced, while low-ASK1-expressing NB cell lines were refractory in TUNEL assay by using flow cytometry. Interestingly, primary NB spheres cell lines express only high levels of active pASK1Thr-838 but the established cell lines expressed inhibitory pASK1Ser-966, and both could be targeted by ASK1 depletion. We report a novel pro-survival role of ASK1 in the tumorigenic NB cell populations, which may be applied as a therapeutic target, inducing apoptosis specifically in cancer stem cells.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzimidazóis/metabolismo , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase Quinase 5/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos SCID , Receptores Nucleares Órfãos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Res ; 76(8): 2376-83, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-26941288

RESUMO

Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376-83. ©2016 AACR.


Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
16.
F1000Res ; 5: 2764, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27990272

RESUMO

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.

17.
Oncotarget ; 7(45): 73200-73215, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27689322

RESUMO

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Estilbenos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Variações do Número de Cópias de DNA , Sinergismo Farmacológico , Feminino , Gefitinibe , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estilbenos/uso terapêutico , Transcriptoma
18.
Patient Educ Couns ; 98(8): 1025-34, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25913245

RESUMO

OBJECTIVE: To adapt marketing approaches in a health services environment. METHODS: Researchers and advertising professionals partnered in developing advertising-style messages designed to activate patients pre-identified as having chronic kidney disease to ask providers about recommended medications. We assessed feasibility of the development process by evaluating partnership structure, costs, and timeframe. We tested messages with patients and providers using preliminary surveys to refine initial messages and subsequent focus groups to identify the most persuasive ones. RESULTS: The partnership achieved an efficient structure, $14,550 total costs, and 4-month timeframe. The advertising team developed 11 initial messages. The research team conducted surveys and focus groups with a total of 13 patients and 8 providers to identify three messages as most activating. Focus group themes suggested the general approach of using advertising-style messages was acceptable if it supported patient-provider relationships and had a credible evidence base. Individual messages were more motivating if they elicited personal identification with imagery, particular emotions, active patient role, and message clarity. CONCLUSION: We demonstrated feasibility of a research-advertising partnership and acceptability and likely impact of advertising-style messages on patient medication-seeking behavior. PRACTICE IMPLICATIONS: Healthcare systems may want to replicate our adaptation of marketing approaches to patients with chronic conditions.


Assuntos
Publicidade/métodos , Comunicação em Saúde , Relações Interprofissionais , Marketing de Serviços de Saúde , Comunicação Persuasiva , Adulto , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Doença Crônica , Rotulagem de Medicamentos , Estudos de Viabilidade , Feminino , Grupos Focais , Humanos , Aprendizagem , Masculino , Motivação , Insuficiência Renal Crônica/tratamento farmacológico , Inquéritos e Questionários
19.
J Cell Biol ; 199(5): 755-70, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23166350

RESUMO

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic "X" shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the "intrinsic structure" of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.


Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Cromossomos/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/genética , Animais , Galinhas , Cromátides/metabolismo , Proteínas de Ligação a DNA/genética , Cinesinas/genética , Complexos Multiproteicos/genética , Mutação , Proteínas Nucleares/genética , Células Tumorais Cultivadas
20.
Mult Scler ; 10(2): 170-5, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15124763

RESUMO

CONTEXT: Osteoporosis and the increased fracture risk associated with osteoporosis become apparent in men approximately 10 years later than women. However, in recent studies, approximately 20% of healthy men in the age range 55-64 years were found to be osteopenic. Emerging data suggest a significantly increased prevalence of osteoporosis in men and women with multiple sclerosis (MS) compared to age-matched controls, but no specific clinical testing recommendations are available for men. OBJECTIVE: To determine the proportion of male MS patients with osteoporosis and to identify the factors associated with the reduction in bone mass. DESIGN: Consecutive male MS patients seen at our MS clinic were screened with dual-X-ray absorptiometry (DEXA) scan for determining the bone mineral density (BMD). All patients had neurological Expanded Disability Status Scale (EDSS) evaluation. The results were compared to healthy age-matched male reference population using the Z score and to a cohort of women MS patients and women controls. Calcium, total testosterone, sex-hormone binding globulin (SHBG), 25-hydroxy-vitamin-D, and parathyroid hormone (PTH) were evaluated in male patients with decreased BMD. Relevant data on body mass index (BMI), medication, alcohol consumption, smoking, and sexual dysfunction were recorded. SETTING: Academic MS Centre. PATIENTS AND OTHER PARTICIPANTS: Forty consecutive male MS patients, age mean 51.2 +/- 8.7 years, and mean EDSS of 5.8 +/- 1.9 were evaluated with DEXA scan. Of these, 17.5% patients were relapsing-remitting (RR) MS, 57.5% were secondary progressive (SP) MS and 25% were primary progressive (PP) MS. MAIN OUTCOME MEASURE: Proportion of male MS patients with reduced BMD at the lumbar spine and femoral neck. RESULTS: Thirty-two (80%) of our patients had a reduced bone mass of either lumbar spine or the femoral neck; of these 17 patients (42.5%) had osteopenia and 15 patients (37.5%) had osteoporosis. Twenty-one per cent (eight out of 38 patients) had vertebral, rib or extremities fractures. Multivariate linear regression analysis indicated that the EDSS (P < 0.0001) and BMI (P = 0.0004) were the important factors associated with low BMD at the femoral neck and the EDSS was the important factor (P = 0.0017) associated with low BMD at the lumbar spine. The same factors emerged as significantly associated with the corresponding Z scores, which are corrected for age and sex. No clear association between intravenous steroid therapy and BMD was evident in the multivariate analysis. Low levels of 25-hydroxy-vitamin-D were seen in 37.5% of patients. CONCLUSIONS: The proportion of male MS patients with reduced bone mass is high and disproportionate to their age and ambulation, consistent with an association between the MS disease process and pathological bone loss. Increased awareness and bone density screening of male and female MS patients over 40 years of age is warranted.


Assuntos
Doenças Ósseas Metabólicas/epidemiologia , Esclerose Múltipla/epidemiologia , Absorciometria de Fóton , Adulto , Densidade Óssea , Doenças Ósseas Metabólicas/sangue , Cálcio/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Hormônio Paratireóideo/sangue , Prevalência , Fatores de Risco , Globulina de Ligação a Hormônio Sexual/metabolismo , Esteroides/uso terapêutico , Testosterona/sangue
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