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Pesquisa : D05.750.078.593.450.300.200 [Categoria DeCS]
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[PMID]:28400915
[Au] Autor:Cejka C; Kossl J; Hermankova B; Holan V; Cejkova J
[Ad] Endereço:Institute of Experimental Medicine, Czech Academy of Sciences, 14220 Prague 4, Czech Republic.
[Ti] Título:Molecular Hydrogen Effectively Heals Alkali-Injured Cornea via Suppression of Oxidative Stress.
[So] Source:Oxid Med Cell Longev;2017:8906027, 2017.
[Is] ISSN:1942-0994
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The aim of this study was to examine the effect of molecular hydrogen (H ) on the healing of alkali-injured cornea. The effects of the solution of H in phosphate buffered saline (PBS) or PBS alone topically applied on the alkali-injured rabbit cornea with 0.25 M NaOH were investigated using immunohistochemical and biochemical methods. Central corneal thickness taken as an index of corneal hydration was measured with an ultrasonic pachymeter. Results show that irrigation of the damaged eyes with H solution immediately after the injury and then within next five days renewed corneal transparency lost after the injury and reduced corneal hydration increased after the injury to physiological levels within ten days after the injury. In contrast, in injured corneas treated with PBS, the transparency of damaged corneas remained lost and corneal hydration elevated. Later results-on day 20 after the injury-showed that in alkali-injured corneas treated with H solution the expression of proinflammatory cytokines, peroxynitrite, detected by nitrotyrosine residues (NT), and malondialdehyde (MDA) expressions were very low or absent compared to PBS treated injured corneas, where NT and MDA expressions were present. In conclusion, H solution favorably influenced corneal healing after alkali injury via suppression of oxidative stress.
[Mh] Termos MeSH primário: Lesões da Córnea/etiologia
Hidrogênio/farmacologia
Estresse Oxidativo/efeitos dos fármacos
Hidróxido de Sódio/toxicidade
[Mh] Termos MeSH secundário: Actinas/metabolismo
Animais
Córnea/metabolismo
Córnea/patologia
Lesões da Córnea/metabolismo
Lesões da Córnea/patologia
Citocinas/metabolismo
Modelos Animais de Doenças
Feminino
Expressão Gênica/efeitos dos fármacos
Imuno-Histoquímica
Interleucina-1beta/genética
Interleucina-1beta/metabolismo
Queratina-12/metabolismo
Queratina-3/metabolismo
Malondialdeído/metabolismo
Ácido Peroxinitroso/metabolismo
Coelhos
Fator A de Crescimento do Endotélio Vascular/genética
Fator A de Crescimento do Endotélio Vascular/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Actins); 0 (Cytokines); 0 (Interleukin-1beta); 0 (Keratin-12); 0 (Keratin-3); 0 (Vascular Endothelial Growth Factor A); 14691-52-2 (Peroxynitrous Acid); 4Y8F71G49Q (Malondialdehyde); 55X04QC32I (Sodium Hydroxide); 7YNJ3PO35Z (Hydrogen)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170504
[Lr] Data última revisão:
170504
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170413
[St] Status:MEDLINE
[do] DOI:10.1155/2017/8906027


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[PMID]:26899008
[Au] Autor:Sasamoto Y; Hayashi R; Park SJ; Saito-Adachi M; Suzuki Y; Kawasaki S; Quantock AJ; Nakai K; Tsujikawa M; Nishida K
[Ad] Endereço:Department of Ophthalmology, Osaka University Medical School, Suita, Osaka 565-0871, Japan.
[Ti] Título:PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.
[So] Source:Sci Rep;6:20807, 2016 Feb 22.
[Is] ISSN:2045-2322
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.
[Mh] Termos MeSH primário: Olho/crescimento & desenvolvimento
Queratina-12/biossíntese
Queratina-3/biossíntese
Fatores de Transcrição Kruppel-Like/biossíntese
Fator 3 de Transcrição de Octâmero/biossíntese
Fator de Transcrição PAX6/genética
[Mh] Termos MeSH secundário: Linhagem Celular
Células Epiteliais/metabolismo
Epitélio Anterior/crescimento & desenvolvimento
Epitélio Anterior/metabolismo
Olho/metabolismo
Regulação da Expressão Gênica no Desenvolvimento
Seres Humanos
Queratina-12/genética
Queratina-3/genética
Fatores de Transcrição Kruppel-Like/genética
Fator 3 de Transcrição de Octâmero/genética
Fator de Transcrição PAX6/biossíntese
Isoformas de Proteínas/biossíntese
Isoformas de Proteínas/genética
Transdução Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (GKLF protein); 0 (KRT12 protein, human); 0 (KRT3 protein, human); 0 (Keratin-12); 0 (Keratin-3); 0 (Kruppel-Like Transcription Factors); 0 (Octamer Transcription Factor-3); 0 (PAX6 Transcription Factor); 0 (PAX6 protein, human); 0 (POU5F1 protein, human); 0 (Protein Isoforms)
[Em] Mês de entrada:1612
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160223
[St] Status:MEDLINE
[do] DOI:10.1038/srep20807


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[PMID]:26758872
[Au] Autor:Allen EH; Courtney DG; Atkinson SD; Moore JE; Mairs L; Poulsen ET; Schiroli D; Maurizi E; Cole C; Hickerson RP; James J; Murgatroyd H; Smith FJ; MacEwen C; Enghild JJ; Nesbit MA; Leslie Pedrioli DM; McLean WH; Moore CB
[Ad] Endereço:School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK, Centre for Dermatology and Genetic Medicine, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Scotland DD1 5EH, UK.
[Ti] Título:Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.
[So] Source:Hum Mol Genet;25(6):1176-91, 2016 Mar 15.
[Is] ISSN:1460-2083
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.
[Mh] Termos MeSH primário: Distrofia Corneana Epitelial Juvenil de Meesmann/genética
Queratina-12/genética
Queratina-3/genética
Mutação de Sentido Incorreto
[Mh] Termos MeSH secundário: Adulto
Animais
Apoptose/genética
Modelos Animais de Doenças
Éxons
Feminino
Heterozigoto
Seres Humanos
Camundongos
Camundongos Transgênicos
Mutação
Linhagem
Resposta a Proteínas não Dobradas
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (KRT12 protein, human); 0 (KRT3 protein, human); 0 (Keratin-12); 0 (Keratin-3)
[Em] Mês de entrada:1612
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160114
[St] Status:MEDLINE
[do] DOI:10.1093/hmg/ddw001


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[PMID]:26289666
[Au] Autor:Courtney DG; Moore JE; Atkinson SD; Maurizi E; Allen EH; Pedrioli DM; McLean WH; Nesbit MA; Moore CB
[Ad] Endereço:School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
[Ti] Título:CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.
[So] Source:Gene Ther;23(1):108-12, 2016 Jan.
[Is] ISSN:1476-5462
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas
Clivagem do DNA
Marcação de Genes
Queratina-12/genética
Polimorfismo de Nucleotídeo Único
[Mh] Termos MeSH secundário: Alelos
Animais
Sequência de Bases
Feminino
Terapia Genética
Heterozigoto
Queratina-12/metabolismo
Camundongos
Camundongos Endogâmicos C57BL
Modelos Animais
Dados de Sequência Molecular
Mutação de Sentido Incorreto
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (KRT12 protein, mouse); 0 (Keratin-12)
[Em] Mês de entrada:1610
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150821
[St] Status:MEDLINE
[do] DOI:10.1038/gt.2015.82


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[PMID]:25897888
[Au] Autor:Baradaran-Rafii A; Biazar E; Heidari-keshel S
[Ad] Endereço:a Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences , Tehran , Iran .
[Ti] Título:Cellular Response of Limbal Stem Cells on Polycaprolactone Nanofibrous Scaffolds for Ocular Epithelial Regeneration.
[So] Source:Curr Eye Res;41(3):326-33, 2016.
[Is] ISSN:1460-2202
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:PURPOSE: The aim of this study was to develop nanofibrous polycaprolactone (PCL) substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane (AM). MATERIALS AND METHODS: The human limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold and, human AM) based on their phenotypic profile, viability, proliferation and attachment ability. RESULTS: Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time-PCR results revealed no change in the expression profile of LECs grown on nanofibrous substrate when compared to those grown on human AM. CONCLUSION: In addition, electrospun nanofibrous PCL substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to AM.
[Mh] Termos MeSH primário: Epitélio Anterior/fisiologia
Limbo da Córnea/citologia
Poliésteres
Regeneração/fisiologia
Células-Tronco/citologia
Tecidos Suporte
[Mh] Termos MeSH secundário: Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo
Materiais Biocompatíveis
Biomarcadores/metabolismo
Adesão Celular/fisiologia
Proliferação Celular/fisiologia
Sobrevivência Celular/fisiologia
Citometria de Fluxo
Expressão Gênica
Seres Humanos
Queratina-12/genética
Queratina-12/metabolismo
Queratina-3/genética
Queratina-3/metabolismo
Limbo da Córnea/metabolismo
Microscopia Eletrônica de Varredura
Nanofibras
Proteínas de Neoplasias/genética
Proteínas de Neoplasias/metabolismo
Reação em Cadeia da Polimerase em Tempo Real
Células-Tronco/metabolismo
Fatores de Transcrição/genética
Fatores de Transcrição/metabolismo
Proteínas Supressoras de Tumor/genética
Proteínas Supressoras de Tumor/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (ABCG2 protein, human); 0 (ATP Binding Cassette Transporter, Sub-Family G, Member 2); 0 (Biocompatible Materials); 0 (Biomarkers); 0 (KRT12 protein, human); 0 (KRT3 protein, human); 0 (Keratin-12); 0 (Keratin-3); 0 (Neoplasm Proteins); 0 (Polyesters); 0 (TP63 protein, human); 0 (Transcription Factors); 0 (Tumor Suppressor Proteins); 24980-41-4 (polycaprolactone)
[Em] Mês de entrada:1612
[Cu] Atualização por classe:161230
[Lr] Data última revisão:
161230
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150422
[St] Status:MEDLINE
[do] DOI:10.3109/02713683.2015.1019004


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[PMID]:26788030
[Au] Autor:Chen JL; Lin BR; Gee KM; Gee JA; Chung DW; Frausto RF; Deng SX; Aldave AJ
[Ad] Endereço:Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA.
[Ti] Título:Identification of presumed pathogenic KRT3 and KRT12 gene mutations associated with Meesmann corneal dystrophy.
[So] Source:Mol Vis;21:1378-86, 2015.
[Is] ISSN:1090-0535
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:PURPOSE: To report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD). METHODS: Slit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity. RESULTS: Slit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo. CONCLUSIONS: We present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.
[Mh] Termos MeSH primário: Distrofia Corneana Epitelial Juvenil de Meesmann/genética
Queratina-12/genética
Queratina-3/genética
Mutação
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Substituição de Aminoácidos
Sequência de Bases
Criança
Distrofia Corneana Epitelial Juvenil de Meesmann/patologia
Análise Mutacional de DNA
Feminino
Heterozigoto
Seres Humanos
Mutação INDEL
Queratina-12/química
Queratina-3/química
Masculino
Meia-Idade
Mutação de Sentido Incorreto
Linhagem
Polimorfismo de Nucleotídeo Único
[Pt] Tipo de publicação:CASE REPORTS; JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (KRT12 protein, human); 0 (KRT3 protein, human); 0 (Keratin-12); 0 (Keratin-3)
[Em] Mês de entrada:1609
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160121
[St] Status:MEDLINE


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[PMID]:26673160
[Au] Autor:Sun CC; Chiu HT; Lin YF; Lee KY; Pang JH
[Ad] Endereço:Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan.
[Ti] Título:Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.
[So] Source:PLoS One;10(12):e0144571, 2015.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.
[Mh] Termos MeSH primário: Amidas/farmacologia
Células Epiteliais/patologia
Limbo da Córnea/patologia
Inibidores de Proteínas Quinases/farmacologia
Piridinas/farmacologia
Cicatrização/efeitos dos fármacos
Quinases Associadas a rho/antagonistas & inibidores
[Mh] Termos MeSH secundário: Animais
Apoptose/efeitos dos fármacos
Biomarcadores/metabolismo
Ciclo Celular/efeitos dos fármacos
Diferenciação Celular/efeitos dos fármacos
Proliferação Celular/efeitos dos fármacos
Ensaio de Unidades Formadoras de Colônias
Células Epiteliais/efeitos dos fármacos
Células Epiteliais/metabolismo
Seres Humanos
Queratina-12/metabolismo
Antígeno Ki-67/metabolismo
Ratos Sprague-Dawley
Proteína Supressora de Tumor p53/metabolismo
Quinases Associadas a rho/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Amides); 0 (Biomarkers); 0 (Keratin-12); 0 (Ki-67 Antigen); 0 (Protein Kinase Inhibitors); 0 (Pyridines); 0 (Tumor Suppressor Protein p53); 138381-45-0 (Y 27632); EC 2.7.11.1 (rho-Associated Kinases)
[Em] Mês de entrada:1606
[Cu] Atualização por classe:160101
[Lr] Data última revisão:
160101
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:151218
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0144571


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[PMID]:26050542
[Au] Autor:Pedrotti E; Passilongo M; Fasolo A; Nubile M; Parisi G; Mastropasqua R; Ficial S; Bertolin M; Di Iorio E; Ponzin D; Marchini G
[Ad] Endereço:Eye Clinic, Department of Neurological and Movement Sciences, University Hospital, Verona, Italy. Electronic address: emilio.pedrotti@univr.it.
[Ti] Título:In Vivo Confocal Microscopy 1 Year after Autologous Cultured Limbal Stem Cell Grafts.
[So] Source:Ophthalmology;122(8):1660-8, 2015 Aug.
[Is] ISSN:1549-4713
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:PURPOSE: To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation. DESIGN: Prospective, interventional, noncomparative, masked case series. PARTICIPANTS: Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye). METHODS: Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patient's injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohen's κ coefficient. MAIN OUTCOME MEASURES: Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface). RESULTS: We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea. CONCLUSIONS: Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft.
[Mh] Termos MeSH primário: Doenças da Córnea/patologia
Doenças da Córnea/terapia
Limbo da Córnea/citologia
Transplante de Células-Tronco
[Mh] Termos MeSH secundário: Células 3T3/citologia
Adulto
Idoso
Animais
Biomarcadores/metabolismo
Células Cultivadas
Técnicas de Cocultura
Doenças da Córnea/metabolismo
Epitélio Anterior/transplante
Feminino
Seres Humanos
Queratina-12/metabolismo
Limbo da Córnea/metabolismo
Masculino
Camundongos
Microscopia Confocal
Meia-Idade
Mucina-1/metabolismo
Estudos Prospectivos
Transplante Autólogo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Biomarkers); 0 (Keratin-12); 0 (MUC1 protein, human); 0 (Mucin-1)
[Em] Mês de entrada:1510
[Cu] Atualização por classe:151119
[Lr] Data última revisão:
151119
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150609
[St] Status:MEDLINE


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[PMID]:25970431
[Au] Autor:Poli M; Burillon C; Auxenfans C; Rovere MR; Damour O
[Ad] Endereço:*Clinique Ophtalmologique, Pavillon C, Hôpital Edouard Herriot, Lyon, France; and †Laboratoire des Greffes et Substituts Cutanés, Pavillon I, Hôpital Edouard Herriot, Lyon, France.
[Ti] Título:Immunocytochemical Diagnosis of Limbal Stem Cell Deficiency: Comparative Analysis of Current Corneal and Conjunctival Biomarkers.
[So] Source:Cornea;34(7):817-23, 2015 Jul.
[Is] ISSN:1536-4798
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:PURPOSE: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD). METHODS: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD. RESULTS: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed. CONCLUSIONS: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction.
[Mh] Termos MeSH primário: Biomarcadores/metabolismo
Túnica Conjuntiva/metabolismo
Doenças da Córnea/diagnóstico
Epitélio Anterior/metabolismo
Proteínas do Olho/metabolismo
Limbo da Córnea/patologia
Células-Tronco/patologia
[Mh] Termos MeSH secundário: Adulto
Idoso
Doenças da Córnea/metabolismo
Feminino
Técnica Indireta de Fluorescência para Anticorpo
Seres Humanos
Queratina-12/metabolismo
Queratina-13/metabolismo
Queratina-19/metabolismo
Queratina-7/metabolismo
Limbo da Córnea/metabolismo
Masculino
Meia-Idade
Mucina-5AC/metabolismo
Estudos Prospectivos
Células-Tronco/metabolismo
Adulto Jovem
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Biomarkers); 0 (Eye Proteins); 0 (KRT12 protein, human); 0 (KRT13 protein, human); 0 (KRT7 protein, human); 0 (Keratin-12); 0 (Keratin-13); 0 (Keratin-19); 0 (Keratin-7); 0 (MUC5AC protein, human); 0 (Mucin 5AC)
[Em] Mês de entrada:1601
[Cu] Atualização por classe:150608
[Lr] Data última revisão:
150608
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150514
[St] Status:MEDLINE
[do] DOI:10.1097/ICO.0000000000000457


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[PMID]:25651498
[Au] Autor:Takahashi H; Tajima K; Hattori T; Yamakawa N; Ito N; Goto H
[Ad] Endereço:*Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; †Department of Surgery, Keio University, Tokyo, Japan; and ‡Veterinary Medical Center, Faculty of Agriculture, Tottori University, Tottori, Japan.
[Ti] Título:Novel primary epithelial cell toxicity assay using porcine corneal explants.
[So] Source:Cornea;34(5):567-75, 2015 May.
[Is] ISSN:1536-4798
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:PURPOSE: The purpose of this study was to develop a novel primary epithelial cell toxicity assay using porcine corneal explant and evaluate the assay using benzalkonium chloride (BAK). METHODS: Circular corneal explants were trephined from the peripheral cornea of porcine eyes using 2-mm biopsy punches, and placed on 6-well culture dishes with a culture medium. After incubation for 12 hours, 50 µL of BAK at 0.00001%, 0.0001%, 0.001%, or 0.01% was applied to each well for 2 minutes. After washing, explants were cultured for another 24 hours, then epithelial outgrowth was photographed and measured. Corneal immunohistochemical characteristics were evaluated by cytokeratin (CK) 3, CK12, and ZO-1. Cell toxicity was evaluated by WST-8 assay, Ki-67 staining, and TUNEL assay. RESULTS: Epithelial cells migrated outward concentrically from the corneal explant as time elapsed and were positive for CK3, CK12, and ZO-1. The outgrowth rate decreased significantly with 0.0001%, 0.001%, and 0.01% BAK compared with the phosphate-buffered saline (PBS) control (P < 0.01), and the decrease was BAK concentration dependent. Numbers of viable cells and Ki-67-positive cells also decreased significantly with 0.01% BAK compared with the PBS control (P < 0.05). TUNEL-positive cells were present in the epithelial outgrowth. Moreover, TUNEL-positive cell density tended to increase with 0.01% BAK compared with the PBS control (P = 0.14) and 0.00001% BAK (P = 0.081). CONCLUSIONS: Our novel toxicity assay using porcine corneal explant is simple and reflects the effect of single and short-duration instillation of eye drops. The method is useful for evaluation of corneal toxicity at low concentrations of BAK.
[Mh] Termos MeSH primário: Compostos de Benzalcônio/toxicidade
Epitélio Anterior/efeitos dos fármacos
Conservantes Farmacêuticos/toxicidade
Testes de Toxicidade
[Mh] Termos MeSH secundário: Animais
Biomarcadores/metabolismo
Contagem de Células
Movimento Celular/efeitos dos fármacos
Células Cultivadas
Epitélio Anterior/metabolismo
Marcação In Situ das Extremidades Cortadas
Queratina-12/metabolismo
Queratina-3/metabolismo
Antígeno Ki-67/metabolismo
Técnicas de Cultura de Órgãos
Suínos
Sais de Tetrazólio/metabolismo
Proteína da Zônula de Oclusão-1/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt); 0 (Benzalkonium Compounds); 0 (Biomarkers); 0 (Keratin-12); 0 (Keratin-3); 0 (Ki-67 Antigen); 0 (Preservatives, Pharmaceutical); 0 (Tetrazolium Salts); 0 (Zonula Occludens-1 Protein)
[Em] Mês de entrada:1509
[Cu] Atualização por classe:151119
[Lr] Data última revisão:
151119
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150205
[St] Status:MEDLINE
[do] DOI:10.1097/ICO.0000000000000377



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