RESUMO
Side population (SP) cells were isolated by FACS from a human amnion mesenchymal cell (AMC) layer soon after enzyme treatment. The yield of SP cells from AMC layer (AMC-SP cells) was about 0.1-0.2%. AMC-SP cells grew well with cell doublings of 40-80 days of culture. FACS profiles and immunocytostaining showed that AMC-SP cells were composed of two different cells immunologically: HLA I(-)/II(-) and HLA P/II(-). Oct-3/4 was detected in the nucleus of AMC-SP cells, when the culture was examined at the third, sixth, and 10th passages. RT-PCR showed that AMC-SP cells expressed the Oct-4, Sox-2, and Rex-1 genes. Immunocytochemistry revealed that all AMC-SP cells were vimentin+, CK19+, and nestin+. In addition, flow cytometry analysis showed that SP cells had high expression of CD13, CD29, CD44, CD46, CD49b, CD49c, CD49e, CD59, CD140a, and CD166 but low expression of CD 49d, and CD51. No evidence of expression was obtained for CD34, CD45, CD49a, CD56, CD90, CD105, CD106, CD117, CD133, CD271, or Flk-1. Upon appropriate differentiation protocols, AMC-SP cells differentiated to several cell lineages such as neuroectodermal, osteogenic, chondrogenic, and adipogenic cells. These results indicate that AMC-SP cells have multilineage potential to several cell lineages with unique immunological characteristics such as HLA I(-)/II(-) or HLA I+/II(-). AMC-SP cells should be of considerable value for regenerative medicine because they do not induce acute rejection after allotransplantation, they do not cause ethical issues, and there is no limit of supply.
Assuntos
Âmnio/citologia , Linhagem da Célula , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Âmnio/metabolismo , Antígenos CD13/análise , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Proteínas HMGB/genética , Humanos , Imuno-Histoquímica , Integrina beta1/análise , Proteínas de Filamentos Intermediários/análise , Queratina-19/análise , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/análise , Nestina , Fator 3 de Transcrição de Octâmero/genética , Osteócitos/citologia , Osteócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genética , Vimentina/análiseRESUMO
In this study we tested the ability of monkey amniotic epithelial cells (MAEC) to take up and decarboxylate l-3,4-dihydroxyphenylalanine (l-DOPA) by incubating the cells in buffer containing l-DOPA under different experimental conditions followed by assaying cellular dopamine (DA) content using high performance liquid chromatography with electrochemical detection. Cellular contents of DA were significantly increased in a time- and l-DOPA-concentration-dependent manner, suggesting the uptake of l-DOPA by MAEC and indicating the presence of aromatic l-amino acid decarboxylase (AADC). This was confirmed by the decreased DA content in the presence of benserazide, an AADC inhibitor. Neither d-DOPA nor DA uptake blockers such as mazindol and GBR 12935 significantly affected l-DOPA uptake and hence DA levels. Further, synthesis of DA from l-DOPA was decreased in the presence of the amino acids tyrosine, phenylalanine and tryptophan, whereas the amino acids glycine and proline were without any significant effect. These findings suggest that MAEC have the capacity to selectively take up and decarboxylate l-DOPA with subsequent production of DA.
Assuntos
Âmnio/metabolismo , Dopa Descarboxilase/metabolismo , Células Epiteliais/metabolismo , Levodopa/metabolismo , Gravidez/metabolismo , Animais , Células Cultivadas , Descarboxilação , Dopamina/biossíntese , Feminino , Macaca fascicularis , Placenta/metabolismoRESUMO
This study was to investigate the presence of dopamine (DA) D(2)receptors mRNA and binding sites in human amniotic epithelial cells (HAEC). RT-PCR revealed that HAEC express DA D(2)receptor mRNA that is having 100 per cent homology with human DA D(2)receptors. Radioligand saturation binding studies showed a [3H]YM-09151-2 high affinity binding site with a K(D)and B(max)values of 0.53+/-0.09 nM and 119.6+/-8.5 fmol/mg protein, respectively. Competition experiments demonstrated that selective D(2)antagonists such as spiroperidol, domperidone and eticlopride potently competed with [3H]YM-09151-2 binding, whereas selective D(1)antagonists like SCH 23390 displayed weaker competition for the binding sites. The rank order of potency of these compounds in competing with [3H]YM-09151-2 for the binding sites was consistent with the pharmacology of the DA D(2)receptors. All competition curves were better fitted to a one-site model with a Hill coefficient around unity, indicating that [3H]YM-09151-2 is labelling a single population of receptors. These results provide evidence that HAEC natively express DA D(2)receptor mRNA and binding sites. Although the physiological function of D2 receptors in HAEC is currently unclear, the present results suggest that these cells could represent a source of human DA D(2)receptors without transformation or cloning procedures.
Assuntos
Âmnio/metabolismo , Células Epiteliais/metabolismo , Receptores de Dopamina D2/metabolismo , Adulto , Âmnio/efeitos dos fármacos , Benzamidas/metabolismo , Benzamidas/farmacologia , Ligação Competitiva , Células Cultivadas , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Modelos Biológicos , Gravidez , RNA Mensageiro/metabolismo , Receptores de Dopamina D2/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fabry disease is an X-linked recessive disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-gal). The deficiency of this enzyme leads to the systemic deposition of ceramide trihexoside (CTH) in various tissues and organs. Enzyme replacement using IV doses of recombinant human alpha-gal produced in CHO cells or in human fibroblasts is currently being evaluated in clinical trials as a potential therapy for this disease. However, it requires lifelong therapy involving a large amount of purified alpha-gal. As a novel approach for treatment of Fabry disease we used polymer encapsulated Chinese hamster ovary (CHO) cells genetically modified to express alpha-gal. The secreted high levels of alpha-gal passed through the semipermeable polymeric membrane. Using coculture system with Fabry fibroblasts, the secreted enzyme was taken up in cells, resulting in reduced accumulation of CTH in Fabry fibroblasts. This in vitro study demonstrated that an encapsulated alpha-gal-secreting cell line can be used to treat Fabry mice by transplantation in vivo. Judging from the protection against immune rejection by a semipermeable synthetic membrane, this novel approach may be applied to treat patients with Fabry disease and other lysosomal storage diseases.
Assuntos
Doença de Fabry/genética , Doença de Fabry/terapia , Terapia Genética/métodos , alfa-Galactosidase/genética , Animais , Células CHO , Técnicas de Cocultura , Cricetinae , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Vetores Genéticos , Glicoesfingolipídeos/farmacologia , Humanos , Immunoblotting , Imuno-Histoquímica , Doenças por Armazenamento dos Lisossomos/terapia , Mutação , alfa-Galactosidase/metabolismoRESUMO
We investigated the potential use of rat amniotic epithelial (RAE) cells as donor cells for transplantation-based therapy in brain ischemia. In vitro, RAE cells were positive for both neuronal and neural stem cell markers, neurofilament microtubule-associated protein 2 and nestin. RT-PCR revealed that these cells express nestin mRNA. The RAE cells were transplanted into the hippocampus of adult gerbils that were subjected to temporal occlusion of bilateral carotid arteries. Five weeks after transplantation, grafted cells migrated into the CA1 pyramidal layer that showed selective neuronal death, and survived in a manner similar to CA1 pyramidal neurons. These results suggest that intracerebral transplantation of amniotic epithelial cells may have therapeutic potential for the treatment of ischemic damage in neuronal disorders.
Assuntos
Âmnio/citologia , Isquemia Encefálica/terapia , Células Epiteliais/transplante , Proteínas do Tecido Nervoso , Neurônios/citologia , Animais , Movimento Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Feminino , Expressão Gênica , Gerbillinae , Hipocampo/citologia , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/genética , Proteínas Associadas aos Microtúbulos/análise , Nestina , Neurônios/química , Gravidez , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 10(6) cells per pregnant female (10(5) cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells. Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.
Assuntos
Âmnio/citologia , Transplante de Células , Células Epiteliais/transplante , Fígado/cirurgia , Albuminas/metabolismo , Animais , Divisão Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Gravidez , Ratos , Ratos Endogâmicos , Telomerase/metabolismo , Transplante IsogênicoRESUMO
Cell-mediated therapy for mucopolysaccharidosis type VII (MPSVII) was studied using monkey amniotic epithelial cells (mAEC). The cells were transduced with a recombinant adenovirus expressing human beta-glucuronidase (GUSB), and cells overexpressing GUSB were generated. The cells expressed 2000-fold higher activities than the endogenous GUSB activities of nontransduced mAEC, demonstrating that mAEC were successfully transduced with adenoviral vectors. These cells also secreted high levels of GUSB. To clarify the cross-correction of GUSB secreted from mAEC, the conditioned medium containing high levels of GUSB was added into the medium for culturing human or murine fibroblasts established from an MPSVII patient or a mouse model of the disease. Dramatic increases in GUSB activities were observed in both fibroblasts. We then transplanted the cells transduced with an adenovirus expressing LacZ into the caudate-putamen of monkey brain. Survival and distribution of the transplanted cells 1 month after the treatment were evaluated. Histochemical analysis showed that LacZ-positive cells were widely distributed in the brain, suggesting that the transplanted cells had migrated and were distributed even at regions far from the implantation site. These findings suggest that local intracerebral engraftment of genetically engineered amniotic epithelial cells is favorable for the treatment of lysosome storage disorders, whose pathological abnormalities are not restricted to specific regions of the brain.
Assuntos
Adenoviridae/genética , Âmnio/citologia , Encéfalo/citologia , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Adenoviridae/metabolismo , Animais , Encéfalo/metabolismo , Transplante de Células , Células Cultivadas , Ventrículos Cerebrais/citologia , Meios de Cultivo Condicionados , Células Epiteliais/virologia , Fibroblastos/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Glucuronidase/genética , Glucuronidase/metabolismo , Humanos , Óperon Lac/genética , Macaca fascicularis , Camundongos , Mucopolissacaridose VII/terapia , Receptor IGF Tipo 2/metabolismo , Transdução Genética , Transplante HomólogoRESUMO
To determine the effect of flunarizine therapy on patients with alternating hemiplegia of childhood (AHC), we sent a questionnaire by mail to council members of the Japanese Society of Child Neurology. We collected 28 AHC patients, and studied their clinical courses and the effects of drug therapy. All of the patients had received flunarizine. In 18 of the 28 patients, flunarizine reduced the severity, duration, or frequency of the hemiplegic attacks. No other drug was more effective than flunarizine. Some flunarizine non-effective patients were severely deteriorated, for example, they had dementia or were ventilator-assisted. Flunarizine had not only a short-term effect, i.e. it reduced the hemiplegic attacks, but also a long-term effect on the motor and intellectual development in some patients with AHC. Flunarizine is still an essential drug for treating AHC.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Sistema Nervoso Central/efeitos dos fármacos , Flunarizina/uso terapêutico , Hemiplegia/tratamento farmacológico , Adolescente , Adulto , Benzodiazepinas/uso terapêutico , Bloqueadores dos Canais de Cálcio/efeitos adversos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Criança , Pré-Escolar , Hidrato de Cloral/uso terapêutico , Esquema de Medicação , Feminino , Flunarizina/efeitos adversos , Hemiplegia/metabolismo , Hemiplegia/fisiopatologia , Humanos , Japão , Masculino , Estudos Retrospectivos , Inquéritos e Questionários , Resultado do TratamentoRESUMO
This study has demonstrated the potential of human amniotic epithelial cells (HAEC) as a transgene carrier to treat patients with familial hypercholesterolemia (FH). One approach to liver-directed gene therapy is represented by transplantation of autologous hepatocytes that have been genetically modified in vitro. However, the hepatocytes must be isolated from surgically resected tissue and it is difficult to expand the hepatocytes in culture. In contrast, the advantages for using HAEC are the higher availability and the nonimmunogenicity after allotransplantation. Our strategy involved isolating HAEC from an amnion, transducing a human low-density lipoprotein receptor (LDLR) gene into these cells with a recombinant adenovirus, and transplanting the genetically modified cells into the liver of an animal model of FH. Each animal, treated with the LDLR-transduced HAEC, exhibited a substantial decrease in serum cholesterol with an eventual return to pretreatment level. Moreover, the transplanted HAEC migrated out of the sinusoids into the hepatic parenchyma and expressed the LDLRs until at least 20 days after transplantation. However, the transplanted HAEC markedly decreased in number after 10 days post-transplant with an increase of inflammatory cells. The temporary nature of the metabolic improvement may be associated with xenograft rejection and transient function of the adenoviral vector.
Assuntos
Âmnio/citologia , Transplante de Células , Terapia Genética/métodos , Hiperlipoproteinemia Tipo II/terapia , Receptores de LDL/genética , Adenovírus Humanos/genética , Animais , Movimento Celular , Células Cultivadas/transplante , Colesterol/sangue , Vírus Defeituosos/genética , Modelos Animais de Doenças , Células Epiteliais/transplante , Vetores Genéticos/genética , Rejeição de Enxerto/imunologia , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/genética , Fígado/patologia , Coelhos , Receptores de LDL/deficiência , Deleção de Sequência , Transplante Heterólogo/imunologia , Transplante HeterotópicoRESUMO
Human amniotic epithelial cells (HAEC) are formed from epiblasts on the 8th day after fertilization. Because they lack major histocompatibility complex (MHC) antigen, human amniotic tissue transplantation has been used for allotranplantation to treat patients with lysosomal diseases. We have provided evidence that HAEC have multiple functions such as synthesis and release of acetylcholine (ACh) and catecholamine (CA) as well as expressing mRNA coding for dopamine receptors and dopamine (DA) transporter (DAT). On the other hand, we showed that monkey amniotic epithelial cells (MAEC) synthesize and release CA and posses DA receptors and DAT. Detection of muscarinic actylcholine receptors indicates the presence of an autocrine mechanism in HAEC. Recently, we found that HAEC have neurotrophic function in conditioned medium from HAEC, indicating the presence of a novel neurotrohpic factor that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors as well as neurotransmitters to the amniotic fluid, suggesting an important function in the early stages of neural development of the embryo. This review will focus on the neuropharmacological aspects of HAEC and MAEC in relation to the physiology of amniotic membrane.
Assuntos
Acetilcolina/metabolismo , Âmnio/metabolismo , Catecolaminas/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Dopamina/metabolismo , Células Epiteliais/metabolismo , Haplorrinos , Humanos , Receptores Dopaminérgicos/metabolismoRESUMO
Cell-mediated gene therapy for visceral lesions of lysosomal storage diseases is promising; however, the treatment of central nervous system (CNS) lesions remains a challenge. In this study, we generated rat amniotic epithelial cells (AEC) that overexpress and secrete human beta-glucuronidase (GUSB) following transduction with an adenoviral vector encoding human GUSB. The AEC were used as donor cells for cell-mediated gene therapy of CNS lesions in mice with mucopolysaccharidosis type VII (MPSVII), a lysosomal storage disorder caused by an inherited deficiency of GUSB activity. After confirmation that the secreted GUSB was taken up mainly via mannose 6-phosphate receptors in primary cultured neurons, the AEC were transplanted into the brains of adult MPSVII mice. Histochemical analysis showed extensive GUSB activity throughout the ipsilateral hemisphere of the recipient brains, and pathological improvement of the lysosomal storage was observed even in regions far from the site of injection. These results suggest that intracerebral transplantation of genetically engineered AEC has therapeutic potential for the treatment of CNS lesions in lysosomal storage disorders.
Assuntos
Líquido Amniótico/citologia , Encéfalo/metabolismo , Células Epiteliais/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Lisossomos/metabolismo , Mucopolissacaridose VII/genética , Mucopolissacaridose VII/terapia , Adenoviridae/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Vetores Genéticos/metabolismo , Glucuronidase/genética , Humanos , Camundongos , Microscopia de Fluorescência , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptor IGF Tipo 2/metabolismo , Fatores de Tempo , Transdução Genética , TransplanteRESUMO
This study correlates changes in neutrophilic activity and endothelial injury with markers of hemostatic activity following the infusion of increasing concentrations of E. coli organisms. It focuses on the hemostatic response as a marker of microvascular injury and uses the response to increasing concentrations of E. coli to refine our definition of disseminated intravascular coagulation (DIC) and distinguish between a compensated (non-overt DIC) and uncompensated (overt DIC) response. We observed that the global coagulation tests reflected activation of the hemostatic system in a dose dependent manner (overt DIC) in the early phases (T+2 to 6 h) of the response to increasing concentrations of E. coli, but that they failed to do so in the late phases (T+ 24 to 48 h). We observed that molecular markers, soluble thrombomodulin and elastase, unlike thrombin/antithrombin and plasmin/antiplasmin complexes, remained elevated out to T+24 to 48 h indicating endothelial injury that persists beyond the initial inflammatory insult in compensated as well as uncompensated DIC.
Assuntos
Testes de Coagulação Sanguínea , Coagulação Intravascular Disseminada/sangue , Endotélio Vascular/fisiopatologia , Infecções por Escherichia coli/sangue , Elastase de Leucócito/sangue , Neutrófilos/fisiologia , Sepse/sangue , Trombomodulina/análise , Animais , Antifibrinolíticos/sangue , Antígenos/análise , Antitrombina III , Biomarcadores , Coagulação Intravascular Disseminada/etiologia , Infecções por Escherichia coli/complicações , Fator VII/análise , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinogênio/análise , Fibrinolisina , Hemostasia , Isquemia/sangue , Contagem de Leucócitos , Masculino , Modelos Animais , Papio , Peptídeo Hidrolases/sangue , Contagem de Plaquetas , Valor Preditivo dos Testes , Reperfusão , Sepse/complicações , alfa 2-AntiplasminaRESUMO
Eight different sulfated polysaccharides were isolated from Chlorophyta. All exhibited thrombin inhibition through a heparin cofactor II (HCII)-dependent pathway, and their effects on the inhibition of thrombin were more potent than those of heparin or dermatan sulfate. In particular, remarkably potent thrombin inhibition was found for the sulfated polysaccharides isolated from the Codiales. In the presence of these sulfated polysaccharides, both the recombinant HCII (rHCII) variants Lys(173)-->Leu and Arg(189)-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHCII. This result indicates that the binding site of HCII for each of these eight sulfated polysaccharides is different from the heparin- or dermatan sulfate-binding site. All the sulfated polysaccharides but RS-2 significantly stimulated the inhibition of thrombin by an N-terminal deletion mutant of HCII (rHCII-Delta74). Furthermore, hirudin(54-65) decreased only 2-5-fold the rate of thrombin inhibition by HCII stimulated by the sulfated polysaccharides, while HD22, a single-stranded DNA aptamer that binds exosite II of thrombin, produced an approximately 10-fold reduction in this rate. These results suggest that, unlike heparin and dermatan sulfate, the sulfated polysaccharides isolated from Chlorophyta activate HCII primarily by an allosteric mechanism different from displacement and template mechanisms.
Assuntos
Clorófitas/metabolismo , Cofator II da Heparina/farmacologia , Polissacarídeos/farmacologia , Trombina/antagonistas & inibidores , Antitrombinas/farmacologia , Sítios de Ligação , Clorófitas/química , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Glicosaminoglicanos/farmacologia , Hirudinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Polissacarídeos/química , Polissacarídeos/isolamento & purificaçãoRESUMO
Human amniotic epithelial cells (HAEC) may have pluripotent function because they are formed from the epiblast cells at the 8th day of fertilization. Previously, we reported that HAEC have the capacity to synthesize and release acetylcholine and catecholamine associated with the binding sites of catecholamine receptors. We show the neurotrophic function of a conditioned medium from HAEC using cultured cortical neurons of E18 rats. Extensive analyses with various techniques demonstrated that HAEC and immortalized HAEC synthesize and release brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF). Other neurotrophic factors were not detected in a cultured medium of HAEC by enzyme immunoassay. Various neurotrophic factors or growth factors did not show neurotrophic effects on E18 rat neuron except for EGF. Because EGF was not detected in the conditioned medium of HAEC, these data indicate an unidentified neurotrophic factor presently that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors to the amniotic fluid as well as neurotransmitters, suggesting an important function to the early stages of neural development in the embryo.
Assuntos
Líquido Amniótico/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/metabolismo , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Líquido Amniótico/citologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Meios de Cultivo Condicionados/metabolismo , Células Epiteliais/citologia , Feminino , Feto , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/biossíntese , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
We screened 22 Japanese patients with acid maltase deficiency (seven with the infantile type, eight with the juvenile type and seven with the adult type) for three previously described mutations, D645E, S529V and R672Q, and a novel mutation, R600C. Although D645E has been reported to be common in Chinese patients with the infantile type, only three of 44 alleles (two of 14 infantile type alleles) from Japanese patients harbored the D645E mutation. The S529V mutation was identified in six of 14 alleles from adult-onset patients. None of the infantile or juvenile patients harbored the S529V mutation. Therefore, S529V apparently results in the adult type disease and is common in Japanese adult-onset patients. R672Q was identified in two pairs of siblings with the juvenile type. A novel mutation, R600C, was identified in eight of 22 patients (nine of 44 alleles). Therefore, R600C is another common Japanese mutation occurring at a CpG dinucleotide "hot spot". Homozygosity for this mutation apparently results in the infantile phenotype. Genetic diagnosis by detecting these four mutations might be feasible for most Japanese patients with acid maltase deficiency.
Assuntos
Doença de Depósito de Glicogênio Tipo II/genética , Mutação/genética , Adolescente , Adulto , Alelos , Análise Mutacional de DNA , Testes Genéticos , Humanos , Lactente , JapãoRESUMO
We have recently found that human amniotic epithelial (HAE) cells synthesize catecholamines including dopamine (DA). The present study was designed to explore the possibility of HAE cells to serve as a donor for transplantation therapy of Parkinson's disease (PD). Thus, we investigated their ability to produce DA in vitro and the survival and function of HAE cells grafted into a rat model of PD. RT-PCR and Western blotting revealed that HAE cells express tyrosine hydroxylase (TH) mRNA and protein, respectively. TH-immunohistochemistry on cultured HAE cells demonstrated that around 10% of the total cells are immunopositive for this protein. The production of DA by HAE cells was increased with time in the presence of L-tyrosine and BH(4), and was abolished with a specific TH inhibitor, alpha-methyl-rho-tyrosine. Dissociated HAE cells transduced with the Escherichia coli LacZ marker gene (beta-gal) were implanted into the previously DA-depleted striatum of immunosuppressed rats. Two weeks postgrafting HAE grafts were demonstrated to survive without overgrowth, as evidenced by the presence of beta-gal-positive cells and TH-immunoreactive cells within the grafts. The grafts also provided partial amelioration of apomorphine-induced rotational asymmetry. The results clearly indicate that HAE cells capable of producing DA can survive and function in the brain of a rat model of PD. Although DA replacement therapy of PD could possibly be achieved with implantation of HAE cells, further studies are needed to develop strategies to enhance the ability of HAE cells to produce DA as well as the graft survival.
Assuntos
Líquido Amniótico/citologia , Líquido Amniótico/metabolismo , Transplante de Células , Doença de Parkinson/cirurgia , Animais , Apomorfina/farmacologia , Comportamento Animal/efeitos dos fármacos , Catecolaminas/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Humanos , Doença de Parkinson/psicologia , Ratos , Ratos Sprague-Dawley , Comportamento Estereotipado , Doadores de Tecidos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
As human amniotic epithelial tissue is formed on about the eighth day after fertilization, human amniotic epithelial cells (hAEC) may have multipotency to differentiate into various organs, such as brain, heart, or liver. In this study, we showed evidence of the synthesis and excretion of albumin by hAEC, by immunostaining and enzyme-linked immunoassay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses revealed the expression of albumin mRNA and protein, respectively. In addition, hAEC also demonstrated immunoreactivity to genetic markers of liver lineage, such as human serum albumin and alpha-fetoprotein. Transplanted hAEC to Scid mouse liver showed positive immunoreactivity to albumin and alpha-fetoprotein. Genetically modified cells containing the beta-galactosidase (LacZ) gene (AxCALacZ) were integrated in liver parenchyma. Human polymorphic gene analysis in Scid mouse liver after the implantation of hAEC showed that these Scid mouse livers obviously contained this human-specific gene until day 7 after the cell transplantation. As hAEC do not cause any acute rejection by allotransplantation, we conclude that hAEC may be useful as a transgene carrier to treat patients with inherited liver diseases.
Assuntos
Líquido Amniótico/citologia , Transplante de Células , Células Epiteliais/transplante , Fígado/cirurgia , Transgenes , Albuminas/biossíntese , Líquido Amniótico/metabolismo , Animais , Western Blotting , Técnicas de Transferência de Genes , Engenharia Genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HomólogoRESUMO
In the present study, the positive rate of thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex (PPIC), soluble fibrin monomer (sFM), and D-dimer for the diagnosis of disseminated intravascular coagulation (DIC) was evaluated. The study comprised 307 patients with DIC, 123 with pre-DIC, and 121 with non-DIC. Plasma levels of TAT, PPIC, sFM, and D-dimer were significantly higher in DIC and pre-DIC patients than in non-DIC patients. In DIC patients, the positive rate of sFM was high and that of D-dimer was low; the positive rate of PPIC was higher in patients with hematopoietic malignancy than in those without this disease. In pre-DIC patients, the positive rate of all markers was low (<0.16), and the positive rate of PPIC was relatively high. In non-DIC patients, the positive rate of all hemostatic markers was low (<0.16), that of sFM being the lowest. Scoring the positive rate of TAT, PPIC, and sFM disclosed the following results: 72% of DIC patients had three or more points, 17.6% of pre-DIC patients had three or more points, and almost all (96.6%) non-DIC patients had two or less points. Scoring the positive rate of TAT, PPIC, and D-dimer disclosed the following results: 52.9% of DIC patients and 27.4% of pre-DIC patients had three or more points and almost all (96.7%) non-DIC patients had 2 or less points. These data suggest that the combination of TAT, PPIC, and sFM is useful for making the diagnosis of DIC.