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1.
Exp Cell Res ; 359(1): 101-111, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28800962

RESUMO

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing.


Assuntos
Técnicas de Cocultura/métodos , Células-Tronco Embrionárias Humanas/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Microvasos/citologia , Epitélio Pigmentado da Retina/citologia , Forma Celular , Matriz Extracelular/metabolismo , Células-Tronco Embrionárias Humanas/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microvilosidades/ultraestrutura , Epitélio Pigmentado da Retina/ultraestrutura
2.
Exp Eye Res ; 108: 129-39, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23352832

RESUMO

Ca(2+) signaling is vitally important in cellular physiological processes and various drugs also affect Ca(2+) signaling. Thus, knowledge of Ca(2+) dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca(2+) communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca(2+) wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) in the monolayer were tracked with fluorescence microscopy imaging using Ca(2+)-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca(2+), after depletion of intracellular Ca(2+) stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca(2+)](i) increase, and percentage of responsive cells were calculated to quantitatively characterize Ca(2+) wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca(2+)](i), which propagated to neighboring cells in a wave-like manner. Ca(2+) wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca(2+) reduced [Ca(2+)](i) increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca(2+) stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca(2+)](i) increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca(2+) wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca(2+) wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca(2+) wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca(2+) wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca(2+) dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca(2+) wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca(2+) wave propagated mainly through gap junctions and required Ca(2+) from both intracellular Ca(2+) stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca(2+). The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.


Assuntos
Sinalização do Cálcio , Células Epiteliais/metabolismo , Mecanotransdução Celular , Epitélio Pigmentado da Retina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Forma Celular , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Corantes Fluorescentes , Fura-2 , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Ácido Glicirretínico/farmacologia , Humanos , Cinética , Mecanotransdução Celular/efeitos dos fármacos , Microscopia de Fluorescência , Estimulação Física , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suramina/farmacologia , Tapsigargina/farmacologia
3.
Ann Biomed Eng ; 39(12): 3055-69, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21904797

RESUMO

The development and differentiation of stem cell-derived impermeable retinal pigment epithelium (RPE) with tight junctions (TJs) is a gradual process that is, at confluence, controlled by cell-to-cell contact. The objective of this study was to evaluate the use of electric impedance spectroscopy (EIS) to follow the maturation and development of barrier function in human embryonic stem cell-derived RPE (hESC-RPE). Barrier function was assessed using EIS, permeability measurements, and microscopic inspection in intact cells and following calcium sequestration with ethylene glycol tetraacetic acid (EGTA). The results showed that the cultures with the most mature morphology had the highest impedance and the lowest permeability values. The EIS of samples of high integrity fitted well to the equivalent model of a single RC circuit, whereas the semicircular shape of the Nyquist plots was distorted for samples of lower integrity. EGTA treatment resulted in lower impedance values and changes in the shapes of plots. Our results show that EIS-as a measure of overall maturity and integrity of the epithelium-is useful when evaluating the maturity of cell cultures. It is highly warranted in future transplantation therapies and in in vitro cell culture models in drug development.


Assuntos
Diferenciação Celular , Espectroscopia Dielétrica , Células-Tronco Embrionárias/citologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Linhagem Celular , Humanos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/fisiologia , Junções Íntimas/fisiologia
4.
Clin Exp Immunol ; 152(3): 552-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18422736

RESUMO

Wheat gliadin induces severe intestinal symptoms and small-bowel mucosal damage in coeliac disease patients. At present, the only effective treatment for the disease is a strict life-long gluten-free diet. In this study we investigated whether probiotics Lactobacillus fermentum or Bifidobacterium lactis can inhibit the toxic effects of gliadin in intestinal cell culture conditions. The ability of live probiotics to inhibit peptic-tryptic digested gliadin-induced damage to human colon cells Caco-2 was evaluated by measuring epithelial permeability by transepithelial resistance, actin cytoskeleton arrangements by the extent of membrane ruffling and expression of tight junctional protein ZO-1. B. lactis inhibited the gliadin-induced increase dose-dependently in epithelial permeability, higher concentrations completely abolishing the gliadin-induced decrease in transepithelial resistance. The same bacterial strain also inhibited the formation of membrane ruffles in Caco-2 cells induced by gliadin administration. Furthermore, it also protected the tight junctions of Caco-2 cells against the effects of gliadin, as evinced by the pattern of ZO-1 expression. We conclude thus that live B. lactis bacteria can counteract directly the harmful effects exerted by coeliac-toxic gliadin and would clearly warrant further studies of its potential as a novel dietary supplement in the treatment of coeliac disease.


Assuntos
Bifidobacterium , Gliadina/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Probióticos/farmacologia , Células CACO-2 , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gliadina/toxicidade , Humanos , Mucosa Intestinal/metabolismo , Limosilactobacillus fermentum , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/efeitos dos fármacos , Triticum/química , Proteína da Zônula de Oclusão-1
5.
J Med Genet ; 45(4): 222-7, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18077767

RESUMO

BACKGROUND: Coeliac disease is caused by dietary gluten, which triggers chronic inflammation of the small intestine in genetically predisposed individuals. In one quarter of the patients the disease manifests in the skin as dermatitis herpetiformis. Recently, a novel candidate gene, myosin IXB on chromosome 19p13, was shown to be associated with coeliac disease in the Dutch and Spanish populations. The same gene has previously been associated with inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis risk, making myosin IXB a potential shared risk factor in these inflammatory disorders. METHODS: In this study, previously reported myosin IXB variants were tested for genetic linkage and association with coeliac disease in 495 Hungarian and Finnish families and in an additional 270 patients and controls. RESULTS AND CONCLUSION: The results show significant linkage (logarithm of odds (LOD) 3.76, p = 0.00002) to 19p13 which supports the presence of a genuine risk factor for coeliac disease in this locus. Myosin IXB variants were not associated with coeliac disease in this study; however, weak evidence of association with dermatitis herpetiformis was found. The association could not explain the strong linkage seen in both phenotypes, indicating that the role of other neighbouring genes in the region cannot be excluded. Therefore, more detailed genetic and functional studies are required to characterise the role of the myosin IXB gene in both coeliac disease and dermatitis herpetiformis.


Assuntos
Doença Celíaca/genética , Dermatite Herpetiforme/genética , Miosinas/genética , Alelos , Estudos de Casos e Controles , Doença Celíaca/complicações , Cromossomos Humanos Par 19/genética , Dermatite Herpetiforme/complicações , Feminino , Finlândia , Predisposição Genética para Doença , Variação Genética , Glutens/efeitos adversos , Haplótipos , Homozigoto , Humanos , Hungria , Doenças Inflamatórias Intestinais/genética , Desequilíbrio de Ligação , Masculino , Fatores de Risco
6.
Clin Exp Immunol ; 150(2): 294-305, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17888028

RESUMO

In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.


Assuntos
Doença Celíaca/genética , Duodeno/imunologia , Regulação da Expressão Gênica/imunologia , Glutens/imunologia , Mucosa Intestinal/patologia , Adulto , Idoso , Biópsia , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Doença Celíaca/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Duodeno/metabolismo , Duodeno/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
7.
J Autoimmun ; 22(3): 249-65, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15041046

RESUMO

In coeliac disease in the jejunum of a genetically susceptible person wheat gliadin and related prolamins from rye and barley trigger an immunological reaction, which induces small-bowel mucosal transformations, villous atrophy and crypt hyperplasia. Though CD4+ specific T cells, intraepithelial lymphocytes, infiltrating plasma cells and autoantibodies are known to have an effect on the coeliac disease, the pathogenic mechanisms leading to the tissue injury remain to be elucidated. Our aim was to find novel gene transcripts, which might have a role in coeliac disease pathogenesis. The gene expression in duodenal biopsy samples from untreated coeliac patients (n=4), patients on gluten-free diet (n=4) and healthy controls (n=4) was studied by cDNA microarray analysis. The method allows monitoring of the expression of thousands of genes simultaneously. Compared to healthy controls, the expression of 156 and 60 genes was changed in untreated and treated coeliac disease, respectively. Between treated and untreated coeliac disease, 98 genes had altered expression. Of the 5184 genes or expressed sequence tags, altogether 263 were affected. Many of these genes are directly or indirectly connected to T-cell activation, B-cell maturation or epithelial cell differentiation. By the microarray method, numerous genes were found to evince altered mRNA expression in coeliac disease. The method holds promise in exploring the pathogenetic mechanisms in the small bowel and may reveal new target genes for the therapy of coeliac disease.


Assuntos
Doença Celíaca/genética , Expressão Gênica , Jejuno/patologia , Adulto , Idoso , Biópsia , Doença Celíaca/etiologia , Doença Celíaca/patologia , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Eur J Cell Biol ; 79(7): 458-68, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10961445

RESUMO

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.


Assuntos
Avidina/metabolismo , Endossomos/metabolismo , Receptor IGF Tipo 2/metabolismo , Motivos de Aminoácidos , Animais , Avidina/química , Avidina/genética , Transporte Biológico , Biotinilação , Brefeldina A/farmacologia , Cátions , Bovinos , Membrana Celular/metabolismo , Galinhas , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , Citoplasma/metabolismo , Dimerização , Endocitose/fisiologia , Endossomos/efeitos dos fármacos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Povidona/farmacologia , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Floresta de Semliki/genética , Dióxido de Silício/farmacologia , Fatores de Tempo
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