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1.
Free Radic Biol Med ; 134: 76-86, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30605715

RESUMO

A radioprotective effect of exogenous recombinant peroxiredoxin 2 (Prx2) was revealed and characterized using an animal model of whole body X-ray irradiation at sublethal and lethal doses. Prx2 belongs to an evolutionarily ancient family of peroxidases that are involved in enzymatic degradation of a wide variety of organic and inorganic hydroperoxides. Apart from that, the oxidized form of Prx2 also exhibits chaperone activity, thereby preventing protein misfolding and aggregation under oxidative stress. Intravenous administration of Prx2 in animals at a concentration of 20 µg/g 15 min before exposure to ionizing radiation contributes to a significantly higher survival rate, suppresses the development of leucopenia and thrombocytopenia, as well as protects the bone marrow cells from genome DNA damage. Moreover, injection of Prx2 leads to suppression of apoptosis, stimulates cell proliferation and results in a more rapid recovery of the cell redox state. Exogenous Prx2 neutralizes the effect of the priming dose on the second irradiation of the cells. The radioprotective properties of exogenous Prx2 are stipulated by its broad substrate peroxidase activity, chaperone activity in the oxidized state, and are also due to the signal-regulatory function of Prx2 mediated by the regulation of the level of hydroperoxides as well as via interaction with redox-sensitive regulatory proteins.


Assuntos
Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/metabolismo , Leucopenia/prevenção & controle , Estresse Oxidativo/fisiologia , Radiação Ionizante , Protetores contra Radiação/administração & dosagem , Trombocitopenia/prevenção & controle , Animais , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Leucopenia/etiologia , Masculino , Camundongos , Oxirredução , Estresse Oxidativo/efeitos da radiação , Trombocitopenia/etiologia
2.
Elife ; 72018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29714688

RESUMO

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.


Assuntos
Diferenciação Celular , Reprogramação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Embrionárias/citologia , Magnetismo , Células-Tronco Neurais/citologia , RNA Mensageiro/administração & dosagem , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Sistemas de Liberação de Medicamentos , Células-Tronco Embrionárias/metabolismo , Fator 3-beta Nuclear de Hepatócito/administração & dosagem , Fator 3-beta Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM/administração & dosagem , Proteínas com Homeodomínio LIM/genética , Células-Tronco Neurais/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/administração & dosagem , Fatores de Transcrição/genética
3.
Int J Mol Med ; 41(2): 1055-1061, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29207034

RESUMO

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current available therapies. Inhibitor of growth 4 (ING4), a novel member of the ING family of proteins, has previously been shown to play a critical role in the development of multiple tumors by regulating apoptosis, proliferation, cell cycle progress, migration and invasion. However, the functional role of ING4 in human melanoma remains unclear. To fully understand its potential role in human melanoma, in the present study, lentivirus (LV)­ING4 and LV­ING4­short hairpin RNA were constructed and transfected into human melanoma A375 cells. First, the effect of overexpressing or downregulating ING4 on the apoptosis of the transfected melanoma cells and cluster of differentiation (CD)3+ T cells was investigated. In the present study, we found that the late apoptotic cells, and not the early apoptotic cells, were more in LV-ING4 group compared with LV-control, and both the early and late apoptosis of CD3+ T cells was significantly observed in A375 cells transfected with LV-ING4 compared with LV-control. Importantly, it was determined whether the overexpression of ING4 significantly induce apoptotic cell death via Fas/FasL (Fas death receptor/FasL) pathway activation and downregulation of poly(ADP­ribose) polymerase, caspase­3 and caspase­8 in the melanoma cells and CD3+ T cells. These results demonstrated that overexpression of ING4 can induce the apoptosis of melanoma cells and CD3+ T cells through signaling pathways such as the Fas/FasL pathway, and that ING4 gene therapy for melanoma treatment is a novel approach.


Assuntos
Proteínas de Ciclo Celular/genética , Proteína Ligante Fas/genética , Proteínas de Homeodomínio/genética , Melanoma/genética , Proteínas Supressoras de Tumor/genética , Receptor fas/genética , Apoptose/genética , Caspase 8/genética , Proteínas de Ciclo Celular/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Proteínas de Homeodomínio/administração & dosagem , Humanos , Lentivirus/genética , Melanoma/patologia , Transfecção , Proteínas Supressoras de Tumor/administração & dosagem
4.
Am J Med Genet A ; 170(12): 3157-3164, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27604636

RESUMO

Turner Syndrome (TS) is a developmental disorder caused by partial or complete loss of one sex chromosome. Bicuspid aortic valve and other left-sided congenital heart lesions (LSL), including thoracic aortic aneurysms and acute aortic dissections, are 30-50 times more frequent in TS than in the general population. In 454 TS subjects, we found that LSL are significantly associated with reduced dosage of Xp genes and increased dosage of Xq genes. We also showed that genome-wide copy number variation is increased in TS and identify a common copy number variant (CNV) in chromosome 12p13.31 that is associated with LSL with an odds ratio of 3.7. This CNV contains three protein-coding genes (SLC2A3, SLC2A14, and NANOGP1) and was previously implicated in congenital heart defects in the 22q11 deletion syndrome. In addition, we identified a subset of rare and recurrent CNVs that are also enriched in non-syndromic BAV cases. These observations support our hypothesis that X chromosome and autosomal variants affecting cardiac developmental genes may interact to cause the increased prevalence of LSL in TS. © 2016 Wiley Periodicals, Inc.


Assuntos
Cromossomos Humanos X/genética , Variações do Número de Cópias de DNA/genética , Cardiopatias Congênitas/genética , Síndrome de Turner/genética , Adulto , Feminino , Dosagem de Genes/genética , Genes Ligados ao Cromossomo X/genética , Genótipo , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportador de Glucose Tipo 3/genética , Cardiopatias Congênitas/fisiopatologia , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Humanos , Masculino , National Heart, Lung, and Blood Institute (U.S.) , Fatores de Transcrição/administração & dosagem , Fatores de Transcrição/genética , Síndrome de Turner/fisiopatologia , Estados Unidos
5.
Neurochem Res ; 41(6): 1375-80, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26846142

RESUMO

The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment.


Assuntos
Antidepressivos/administração & dosagem , Dependovirus , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Fatores de Crescimento Neural/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Administração Intranasal , Animais , Sequência de Bases , Dependovirus/genética , Depressão/tratamento farmacológico , Depressão/genética , Depressão/psicologia , Feminino , Vetores Genéticos/genética , Células HEK293 , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Resultado do Tratamento
6.
Biochem Biophys Res Commun ; 466(4): 656-63, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26403969

RESUMO

Type 1 diabetes is a T cell-mediated organ-specific autoimmune disease. Antigen-specific immune intervention allows the selective targeting of autoreactive T cell, while leaving the remainder of the immune system intact. However, immune intervention for type 1 diabetes has not yielded perfect results clinically. In our paper published previously, we asked whether pancreatic duodenal home box 1 (PDX1) is a target of anti-islet autoimmunity in type 1 diabetes. In this experiment, we assessed the therapeutic effect of oral administration of PDX1 on diabetes development of 4-week-old non-obese diabetic (NOD) mice. The results indicate that PDX1 immunization is an effective intervention strategy for delaying the onset of diabetes in NOD mice in association with: 1) reduced insulitis; 2) suppression of destructive autoreactive T cells; 3) augmentation of regulatory T cells; 4) a shift in cytokine production. The present observations suggest that immunization with PDX1 modulates immune cell responses in NOD mice, raising the possibility that it is beneficial in ameliorating autoimmune destruction of beta-cells and delaying type 1 diabetes development clinically.


Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/imunologia , Transativadores/administração & dosagem , Transativadores/imunologia , Administração Oral , Transferência Adotiva , Animais , Autoimunidade , Citocinas/biossíntese , Citocinas/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Imunização , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Transcriptoma
7.
Cell Rep ; 13(2): 242-50, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26411690

RESUMO

Engrailed homeoproteins are expressed in adult dopaminergic neurons of the substantia nigra. In Engrailed1 heterozygous mice, these neurons start dying at 6 weeks, are more sensitive to oxidative stress, and progressively develop traits similar to those observed following an acute and strong oxidative stress inflected to wild-type neurons. These changes include DNA strand breaks and the modification (intensity and distribution) of several nuclear and nucleolar heterochromatin marks. Engrailed1 and Engrailed2 are biochemically equivalent transducing proteins previously used to antagonize dopaminergic neuron death in Engrailed1 heterozygous mice and in mouse models of Parkinson disease. Accordingly, we show that, following an acute oxidative stress, a single Engrailed2 injection restores all nuclear and nucleolar heterochromatin marks, decreases the number of DNA strand breaks, and protects dopaminergic neurons against apoptosis.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteínas de Homeodomínio/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Substância Negra/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Dano ao DNA , Heterocromatina/genética , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Infusões Intraventriculares , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Substância Negra/citologia
8.
Pulm Pharmacol Ther ; 26(6): 672-6, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23380438

RESUMO

PRM-151, recombinant human Pentraxin-2 (PTX-2) also referred to as serum amyloid P (SAP), is under development for treatment of fibrosis. A First-in-Human (FIH) trial was performed to assess the safety, tolerability, and pharmacokinetics of single ascending intravenous doses of PRM-151 administered to healthy subjects, using a randomized, blinded, placebo controlled study design. Each cohort included three healthy subjects (PRM-151:placebo; 2:1). SAP levels were assessed using a validated ELISA method, non-discriminating between endogenous and exogenous SAP. At a dose level of 10 mg/kg, at which a physiologic plasma level of SAP was reached, two additional healthy volunteers and three pulmonary fibrosis (PF) patients were enrolled enabling comparison of the pharmacokinetic SAP profile between healthy volunteers and PF patients. In addition, the percentage of fibrocytes (CD45+/Procollagen-1+ cells) in whole blood samples was assessed to demonstrate biological activity of PRM-151 in the target population. PRM-151 administration was generally well tolerated. In two pulmonary fibrosis patients non-specific, transient skin reactions (urticaria and erythema) were observed. PRM-151 administration resulted in a 6-to 13-fold increase in mean baseline plasma SAP levels at dose levels of 5, 10, and 20 mg/kg. The estimated t1/2 of PRM-151 in healthy volunteers was 30 h. Pharmacokinetic profiles were comparable between healthy volunteers and PF patients. PRM-151 administration resulted in a 30-50% decrease in fibrocyte numbers 24 h post-dose. This suggests that administration of PRM-151 may be associated with a reduction of fibrocytes in PF patients, a population for which current pharmacotherapeutic options are limited. The pharmacological action of PRM-151 should be confirmed in future research.


Assuntos
Proteínas de Homeodomínio/administração & dosagem , Fibrose Pulmonar/tratamento farmacológico , Componente Amiloide P Sérico/administração & dosagem , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Relação Dose-Resposta a Droga , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Meia-Vida , Proteínas de Homeodomínio/efeitos adversos , Proteínas de Homeodomínio/farmacocinética , Humanos , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/fisiopatologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacocinética , Componente Amiloide P Sérico/efeitos adversos , Componente Amiloide P Sérico/farmacocinética , Adulto Jovem
9.
Exp Eye Res ; 93(5): 786-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21933674

RESUMO

This study investigated whether PRM-151 (Promedior, Inc., Malvern, PA), a recombinant form of human pentraxin-2 (PTX-2, also referred to as serum amyloid P, hSAP), that inhibits differentiation of circulating monocytes into fibrocytes and profibrotic macrophages, could modulate generation of myofibroblasts after opacity-producing corneal injury in rabbits, and, therefore, have potential to reduce or prevent haze after PRK. Nine diopter PRK for myopia was performed with the VISX S4 IR laser. Four groups of 6 animals were treated in masked fashion: Group 1: 30 µl of topical PRM-151 (20 mg/ml) 6 times a day for 5 days; Group 2: 30 µl topical vehicle 6 times a day for 5 days; Group 3: 200 µl sub-conjunctival PRM-151 (total injection of 4 mg) immediately after surgery and every other day until day 8; Group 4: 200 µl sub-conjunctival injections of vehicle according to the same schedule as group 3. At one month after PRK, the animals were euthanized and immunohistochemistry was performed for the myofibroblast marker α-smooth muscle actin (SMA). The density of SMA+ cells/400× field in the central stroma was determined in each cornea. Myofibroblast density at one month after surgery was significantly lower (p = 0.006) after sub-conjunctival PRM-151 treatment (5.8 ± 2.8 cells/400× stromal field) compared to sub-conjunctival vehicle treatment (15.3 ± 2.9 cells/400× stromal field). There was no significant (p = 0.27) decrease in stromal myofibroblasts triggered by topical PRM-151 treatment (11.8 ± 6.6 cells/400× stromal field) compared to the topical vehicle treatment (14.2.8 ± 6.2 cells/400× stromal field). PRM-151 inhibits myofibroblast generation when administered by sub-conjunctival injection, but not when administered topically, after opacity-producing corneal injury. This study provides additional confirmation that bone marrow-derived cells contribute to corneal myofibroblast generation.


Assuntos
Opacidade da Córnea/prevenção & controle , Substância Própria/efeitos dos fármacos , Proteínas de Homeodomínio/administração & dosagem , Monócitos/efeitos dos fármacos , Miofibroblastos/metabolismo , Componente Amiloide P Sérico/administração & dosagem , Actinas/metabolismo , Animais , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Lesões da Córnea , Opacidade da Córnea/metabolismo , Substância Própria/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Injeções Intraoculares , Lasers de Excimer , Miopia/cirurgia , Ceratectomia Fotorrefrativa , Coelhos , Proteínas Recombinantes/administração & dosagem
10.
Neuroscience ; 192: 37-53, 2011 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-21763404

RESUMO

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine ß-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.


Assuntos
Encéfalo/metabolismo , Dopamina beta-Hidroxilase/biossíntese , Proteínas de Homeodomínio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/biossíntese , Fatores de Transcrição/metabolismo , Regulação para Cima , Envelhecimento , Sequência de Aminoácidos , Animais , Linhagem Celular , Dopamina beta-Hidroxilase/genética , Vetores Genéticos , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Humanos , Masculino , Microinjeções , Dados de Sequência Molecular , Neurogênese/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/administração & dosagem , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos
11.
Acta Ophthalmol ; 89(2): e126-31, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21288307

RESUMO

PURPOSE: NAP is the smallest active element of activity-dependent neuroprotective protein (ADNP) in the non-myelinated neural tissue. This study evaluated the neuroprotective effect of NAP in reducing the spread of laser-induced retinal damage in rat. METHODS: Laser lesions were created in 72 DA pigmented rats. Two groups were treated by one intravenous or intravitreal injection of NAP immediately after exposure to laser. Two control groups were similarly administered saline injection. Histological and morphometrical evaluations of the lesions were preformed 3, 20 and 60 days after photocoagulation. RESULTS: After intravitreal treatment with NAP, a significant reduction in the diameter of the laser-induced lesions was found 3 days after photocoagulation (p < 0.001) but not after 20 and 60 days while the systemic treatment significantly reduced lesion diameter 20 and 60 days after photocoagulation (p = 0.001). Significant difference in photoreceptor cell loss was found in eyes treated intravitreally only 3 days after photocoagulation (p = 0.002). In the systemically treated animals such effect was found only after 20 and 60 days (p < 0.001). CONCLUSIONS: Treatment with NAP ameliorates laser-induced retinal lesions. Intravitreal treatment had an early short-term effect while the effect of systemic administration was delayed and prolonged. This treatment may be of clinical significance in reducing laser-induced retinal injuries in humans.


Assuntos
Traumatismos Oculares/prevenção & controle , Proteínas de Homeodomínio/administração & dosagem , Fotocoagulação a Laser/efeitos adversos , Proteínas do Tecido Nervoso/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Retina/lesões , Animais , Traumatismos Oculares/etiologia , Traumatismos Oculares/patologia , Injeções Intravenosas , Injeções Intravítreas , Lasers de Gás/efeitos adversos , Masculino , Ratos , Retina/patologia
12.
Blood ; 113(21): 5111-20, 2009 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-19270262

RESUMO

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.


Assuntos
Anemia de Fanconi/patologia , Células-Tronco Hematopoéticas/patologia , Proteínas de Homeodomínio/farmacologia , Fatores de Transcrição/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/administração & dosagem , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores do Fator de Necrose Tumoral/análise , Fatores de Transcrição/administração & dosagem
13.
Diabetes ; 57(3): 757-69, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18086901

RESUMO

OBJECTIVE: The key pancreatic transcription factor pancreatic duodenal homeobox-1 (Pdx1), known to control development and maintenance of pancreatic beta-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo-administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in mice with streptozotocin-induced diabetes. RESEARCH DESIGN AND METHODS: Cell entry and transcriptional regulatory properties of rPdx1 protein and its PTD-deletion mutant rPdx1Delta protein, as well as a PTD-green fluorescent protein, were evaluated in vitro. After intraperitoneal rPdx1 injection into mice with streptozotocin-induced diabetes, we assessed its action on blood glucose levels, insulin content, intraperitoneal glucose tolerance test (IPGTT), Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology. RESULTS: Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin, glucagon, and Ki67 immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated upregulation of INS and PDX1 genes and other genes relevant to pancreas regeneration. While the time course of beta-cell gene expression and serum/tissue insulin levels indicated that both liver- and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that beta-cell regeneration played the primary role in rPdx1-induced glucose homeostasis. CONCLUSIONS: rPdx1 treatment of mice with streptozotocin-induced diabetes promotes beta-cell regeneration and liver cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes while avoiding potential side effects associated with the use of viral vectors.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Transativadores/administração & dosagem , Transativadores/uso terapêutico , Animais , Glicemia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/sangue , Camundongos , Pâncreas/metabolismo , Estrutura Terciária de Proteína , Ratos , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo
14.
Blood Cells Mol Dis ; 40(1): 119-21, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17988908

RESUMO

Trib2 is a member of the Trib family of serine/threonine kinase-like proteins (Trib1, Trib2, Trib3). Mice reconstituted with hematopoietic stem cells (HSC) retrovirally expressing Trib2 uniformly developed fatal transplantable acute myelogenous leukemia (AML). Trib2-induced AML was clonal and we sought to identify cooperating genes in Trib2-induced AML. Using Splinkerette PCR techniques, we identified proviral insertion near HoxA9 in a Trib2 monoclonal tumor, which resulted in greatly elevated HoxA9 expression. Mice reconstituted with HSC cotransduced with HoxA9 and Trib2 had accelerated onset of AML compared to either gene alone. These data identify Trib2 and HoxA9 as cooperating genes in AML.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Leucemia Mieloide Aguda/etiologia , Proteínas Serina-Treonina Quinases/farmacologia , Animais , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Leucemia Mieloide Aguda/patologia , Camundongos , Proteínas Oncogênicas , Proteínas Serina-Treonina Quinases/administração & dosagem , Transdução Genética
15.
Blood ; 107(1): 63-72, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16174758

RESUMO

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation, which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time, attesting to a selective advantage of transduced progenitors. Notably, a high transgene copy number was detected in all lymphoid organs, and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether, these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases.


Assuntos
Terapia Genética/métodos , Proteínas de Homeodomínio/administração & dosagem , Sistema Imunitário/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Imunodeficiência Combinada Severa/terapia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Dosagem de Genes , Terapia Genética/efeitos adversos , Proteínas de Homeodomínio/efeitos adversos , Proteínas de Homeodomínio/genética , Sistema Imunitário/fisiologia , Transtornos Linfoproliferativos/etiologia , Camundongos , Camundongos Knockout , Retroviridae/genética , Imunodeficiência Combinada Severa/complicações , Linfócitos T/citologia , Linfócitos T/imunologia
16.
World J Surg ; 29(3): 339-43, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15706434

RESUMO

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells. Liposomes have been described as having better efficacy in gene delivery, and an advantage of using liposomes as gene carriers is that they can be used repeatedly in vivo. The objective of this study is to compare the effect of gene delivery routes and to determine whether systemic delivery of the rat insulin promoter (RIP)-directed suicide gene construct would permit cell-specific gene delivery in vivo. Severe combined immunodeficient (SCID) mice were injected with liposome-RIP-TK (thymidine kinase) complex by either the intraperitoneal or the intravenous route. Twenty-four hours post gene delivery, mice received ganciclovir (GCV) treatment twice daily for 14 days. Mice were sacrificed at various time points. Complete necropsy and serum chemistry analysis were performed. Islet morphology was determined using hematoxylin and eosin (H&E) staining. Serum glucose and insulin levels were also determined. To determine the toxic effect on pancreatic islet cells, immunostaining of insulin-producing and glucagon-producing cells was carried out at each time point. H&E staining indicated that both intravenous and intraperitoneal liposome-RIP-TK gene expression had no effect in normal endocrine islet cells. Both gene-delivery routes in mice resulted in normal glycemia and serum insulin levels. The endocrine islets were intact, with a normal distribution pattern of insulin-producing beta cells and glucagon-secreting alpha cells. However, serum chemistry analysis revealed significantly elevated levels of liver enzymes; suggesting that possible liver damage had occurred with the intraperitoneal gene delivery of liposome-pRIP-TK. Intravenous liposome-mediated gene delivery had no effect on liver enzyme levels. Liposome-mediated gene delivery via intravenous injection was less toxic than intraperitoneal delivery. This gene-delivery route requires fewer liposome-DNA complexes and maintains normal liver function. Thus, intravenous delivery of gene therapy would be superior to intraperitoneal administration of gene therapy in mice.


Assuntos
Terapia Genética/métodos , Proteínas de Homeodomínio/administração & dosagem , Ilhotas Pancreáticas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Timidina Quinase/genética , Transativadores/administração & dosagem , Animais , Sobrevivência Celular/genética , Feminino , Injeções Intraperitoneais , Injeções Intravenosas , Lipossomos , Fígado/enzimologia , Fígado/patologia , Camundongos , Camundongos SCID
17.
Am J Obstet Gynecol ; 189(3): 790-3, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14526315

RESUMO

OBJECTIVE: Fetal alcohol syndrome (FAS) is the most common nongenetic cause of mental retardation. Peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL), related to activity-dependent neuroprotective protein (ADNP), prevent alcohol-induced damage in a mouse model of FAS. Our objective was to characterize ADNP in this model to relate this protein to the mechanisms of damage and peptide neuroprotection. STUDY DESIGN: Timed, pregnant C57Bl6/J mice were treated on day 8. Groups were control, alcohol, peptide pretreatment, or peptide alone. Embryo and decidua were harvested at 6 and 24 hours and 10 days. To evaluate ADNP expression, real-time polymerase chain reaction was performed with results presented as the ratio of ADNP-to-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) concentration. Analysis of variance was performed for overall comparisons with P<.05 considered significant. RESULTS: At 6 hours, there was no difference in ADNP between alcohol-exposed embryos compared with control embryos. At 24 hours, there was an increase in ADNP in alcohol-exposed embryos compared with controls (P<.001); these findings persisted at 10 days (P<.001). In the decidua at 6 hours, there was no difference between alcohol and control. At 24 hours, there was greater ADNP in alcohol-exposed decidua compared with controls (P<.001), which did not persist at 10 days (P=.97). Peptide pretreatment did not prevent the alcohol-induced increase in ADNP in embryo or decidua. CONCLUSION: Alcohol increased embryonic and decidual ADNP expression at 24 hours and it persisted in the embryo for 10 days. Because ADNP is a known neuroprotectant, these findings suggest that it may be released as a protective mechanism in FAS. Changes in the embryo were persistent suggesting that the embryo is more vulnerable to alcohol-induced damage than the mother.


Assuntos
Modelos Animais de Doenças , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Decídua/química , Embrião de Mamíferos/química , Etanol/administração & dosagem , Feminino , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/genética , Gravidez , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Biosci Rep ; 22(2): 339-53, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12428909

RESUMO

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.


Assuntos
Células Dendríticas/imunologia , Epitopos/administração & dosagem , Lipossomos , Proteínas Nucleares , Fragmentos de Peptídeos/administração & dosagem , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição , Vacinação/métodos , Vacinas de Subunidades/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteína do Homeodomínio de Antennapedia , Apresentação do Antígeno , Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Archaea/química , Cátions , Sistemas de Liberação de Medicamentos , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/farmacocinética , Humanos , Fusão de Membrana , Lipídeos de Membrana/administração & dosagem , Lipídeos de Membrana/química , Lipídeos de Membrana/isolamento & purificação , Camundongos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Polietilenoglicóis/administração & dosagem , Vacinas de Subunidades/síntese química , Vacinas de Subunidades/imunologia
19.
Pharm Res ; 19(6): 744-54, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12134943

RESUMO

PURPOSE: The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate oligonucleotides. METHODS: The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. RESULTS: The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides. CONCLUSIONS: Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.


Assuntos
Produtos do Gene tat/farmacocinética , Proteínas de Homeodomínio/farmacocinética , Proteínas Nucleares , Oligonucleotídeos Antissenso/farmacocinética , Fatores de Transcrição , Sequência de Aminoácidos , Proteína do Homeodomínio de Antennapedia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Sistemas de Liberação de Medicamentos/métodos , Produtos do Gene tat/administração & dosagem , Produtos do Gene tat/genética , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/genética , Inibidores do Crescimento/farmacocinética , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Ligação Proteica/genética
20.
J Immunol Methods ; 254(1-2): 119-35, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11406158

RESUMO

Peptide carriers, such the homeodomain of Antennapedia molecule (AntpHD), which spontaneously cross cellular membranes, have been exploited to deliver antigenic peptide Cw3 to the major histocompatibility complex (MHC) class-I presentation pathway and to prime cytotoxic T cells (CTL). However, the in vivo use of AntpHD recombinant peptide has been limited because CTLs can only be primed in the presence of sodium dodecyl sulfate (SDS) as adjuvant. In this report, we have exploited liposomes to protect the AntpHD-Cw3 from serum degradation and to facilitate the delivery of the recombinant peptide into the MHC class-I pathway of antigen-presenting cells. We have demonstrated that AntpHD recombinant peptide spontaneously associates with liposomes and this association is stable in vitro. However, exchange studies assessing the transfer of the peptide to model membranes or cells in vitro indicates that approximately 50% of the liposome-associated peptide is readily exchangeable. This is consistent with trypsin-protection assays, which have shown that approximately 40% of the liposome-associated peptide is protected from hydrolysis. Importantly, macrophages and dendritic cells are able to internalize AntpHD recombinant peptide associated with liposomes resulting in efficient delivery of the CTL peptide into the cytosol. These studies have demonstrated that dendritic cells treated with AntpHD-Cw3 in liposomes sensitize CTL clones to lyse syngeneic target cells expressing Cw3 epitope. This strategy, which combines liposomes and a peptide vector, provides a new approach for introducing molecules into the MHC class-I antigen presentation pathway of dendritic cells.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-C/administração & dosagem , Antígenos HLA-C/imunologia , Proteínas de Homeodomínio/administração & dosagem , Proteínas Nucleares , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição , Animais , Proteína do Homeodomínio de Antennapedia , Apresentação do Antígeno/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos , Endocitose/imunologia , Vetores Genéticos , Antígenos HLA-C/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Lipossomos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/genética , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
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