RESUMO
3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a genetic disorder biochemically characterized by predominant accumulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA) acids in tissues and biological fluids of affected individuals. Clinically, the patients present neurological symptoms and basal ganglia injury, whose pathomechanisms are partially understood. In the present study, we investigated the ex vivo effects of intrastriatal administration of HMG and MGA on important parameters of oxidative stress in striatum of developing rats. Our results demonstrate that HMG and MGA induce lipid and protein oxidative damage. HMG and MGA also increased 2',7'-dichlorofluorescein oxidation, whereas only HMG elicited nitric oxide production, indicating a role for reactive oxygen (HMG and MGA) and nitrogen (HMG) species in these effects. Regarding the enzymatic antioxidant defenses, both organic acids decreased reduced glutathione concentrations and the activities of superoxide dismutase and glutathione reductase and increased glutathione peroxidase activity. HMG also provoked an increase of catalase activity and a diminution of glucose-6-phosphate dehydrogenase activity. We finally observed that antioxidants fully prevented or attenuated HMG-induced alterations of the oxidative stress parameters, further indicating the participation of reactive species in these effects. We also observed that MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, prevented some of these effects, indicating the involvement of the NMDA receptor in HMG effects. The present data provide solid evidence that oxidative stress is induced in vivo by HMG and MGA in rat striatum and it is presumed that this pathomechanism may explain, at least in part, the cerebral alterations observed in HL deficiency.
Assuntos
Acetil-CoA C-Acetiltransferase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Gânglios da Base/metabolismo , Meglutol/análogos & derivados , Meglutol/metabolismo , Estresse Oxidativo , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Antioxidantes/farmacologia , Gânglios da Base/crescimento & desenvolvimento , Gânglios da Base/patologia , Catalase/metabolismo , Maleato de Dizocilpina/farmacologia , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Masculino , Malondialdeído/metabolismo , Carbonilação Proteica , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Superóxido Dismutase/metabolismo , Vitamina E/farmacologiaRESUMO
Cell encapsulation, although a promising strategy to deliver therapeutic products, is hampered by immune response against biomaterials. The aim of this article is to assess the effect of prednisolone on enzyme release by microencapsulated cells implanted in vivo. Recombinant cells encapsulated were implanted in the peritoneum of wild-type mice and mucopolysaccharidosis (MPS) I mice, with or without prednisolone. Later, microcapsules were recovered for histological and enzyme analysis. Blood was collected from MPS I mice. All animals receiving prednisolone had a smaller inflammatory infiltrate. In vitro, prednisolone increased the amount of enzyme released from the recovered capsules, but this was not accompanied by an increase in the amount of circulating enzyme in vivo after 15 days. However, in 7 days, prednisolone significantly increased the amount of enzyme detected in the serum. Although prednisolone improved enzyme release in vitro and in vivo after 7 days, it was unable to maintain this effect for a longer period.
Assuntos
Anti-Inflamatórios/farmacologia , Terapia de Reposição de Enzimas/métodos , Iduronidase , Mucopolissacaridose I/terapia , Prednisolona/farmacologia , Animais , Anti-Inflamatórios/farmacocinética , Linhagem Celular , Células Imobilizadas/transplante , Cricetinae , Feminino , Humanos , Iduronidase/biossíntese , Iduronidase/genética , Iduronidase/farmacocinética , Iduronidase/uso terapêutico , Camundongos , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Prednisolona/farmacocinéticaRESUMO
Lysosomal storage disorders (LSD) are monogenic diseases caused by the deficiency of different lysosomal enzymes that degrade complex substrates such as glycosaminoglycans, sphingolipids, and others. As a consequence there is multisystemic storage of these substrates. Most treatments for these disorders are based in the fact that most of these enzymes are soluble and can be internalized by adjacent cells via mannose-6-phosphate receptor. In that sense, these disorders are good candidates to be treated by somatic gene therapy based on cell microencapsulation. Here, we review the existing data about this approach focused on the LSD treatments, the advantages and limitations faced by these studies.
Assuntos
Transplante de Células/métodos , Composição de Medicamentos/estatística & dados numéricos , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/terapia , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/uso terapêutico , Transplante de Células/instrumentação , Composição de Medicamentos/métodos , Técnicas de Transferência de Genes , Terapia Genética/instrumentação , Terapia Genética/métodos , Humanos , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/genética , Modelos BiológicosRESUMO
Metachromatic leukodystrophy (MLD) is a lysosomal disorder caused by arylsulfatase A (ARSA) deficiency. It is classified into three forms according to the age of onset of symptoms (late infantile, juvenile, and adult). We carried out a cross-sectional and retrospective study, which aimed to determine the epidemiological, clinical, and biochemical profile of MLD patients from a national reference center for Inborn Errors of Metabolism in Brazil. Twenty-nine patients (male, 17) agreed to participate in the study (late infantile form: 22; juvenile form: 4; adult form: 1; asymptomatic: 2). Mean ages at onset of symptoms and at biochemical diagnosis were, respectively, 19 and 39 months for late infantile form and 84.7 and 161.2 months for juvenile form. The most frequently reported first clinical symptom/sign of the disease was gait disturbance and other motor abnormalities (72.7%) for late infantile form and behavioral and cognitive alterations (50%) for juvenile form. Leukocyte ARSA activity level did not present significant correlation with the age of onset of symptoms (r = -0.09, p = 0.67). Occipital white matter and basal nuclei abnormalities were not found in patients with the late infantile MLD. Our results suggest that there is a considerable delay between the age of onset of signs and symptoms and the diagnosis of MLD in Brazil. Correlation between ARSA activity and MLD clinical form was not found. Further studies on the epidemiology and natural history of this disease with larger samples are needed, especially now when specific treatments should be available in the near future.
Assuntos
Cerebrosídeo Sulfatase/deficiência , Leucócitos/enzimologia , Leucodistrofia Metacromática/diagnóstico , Adolescente , Idade de Início , Biomarcadores/sangue , Biomarcadores/urina , Brasil/epidemiologia , Cerebrosídeo Sulfatase/sangue , Criança , Pré-Escolar , Estudos Transversais , Técnicas de Diagnóstico Oftalmológico , Progressão da Doença , Eletroencefalografia , Oftalmopatias/diagnóstico , Oftalmopatias/enzimologia , Oftalmopatias/epidemiologia , Feminino , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/enzimologia , Transtornos Neurológicos da Marcha/epidemiologia , Humanos , Lactente , Leucodistrofia Metacromática/tratamento farmacológico , Leucodistrofia Metacromática/enzimologia , Leucodistrofia Metacromática/epidemiologia , Leucoencefalopatias/diagnóstico , Leucoencefalopatias/enzimologia , Leucoencefalopatias/epidemiologia , Imageamento por Ressonância Magnética , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/enzimologia , Transtornos Mentais/epidemiologia , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Sulfoglicoesfingolipídeos/urina , Fatores de Tempo , Adulto JovemRESUMO
Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.
Assuntos
Criopreservação , Iduronidase/administração & dosagem , Iduronidase/metabolismo , Rim/citologia , Alginatos/química , Animais , Linhagem Celular , Sobrevivência Celular , Células Imobilizadas/enzimologia , Temperatura Baixa , Cricetinae , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Iduronidase/genética , Mucopolissacaridose I/enzimologia , Regulação para CimaRESUMO
Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by deficiency of α-l-iduronidase (IDUA), which results in the lysosomal accumulation of glycosaminoglycans (GAG) leading to widespread clinical manifestations. The microencapsulation of IDUA overexpressing recombinant cells has been considered as a promising strategy for the treatment of MPS I. This study aimed at the optimization of alginate microcapsules containing recombinant BHK (Baby Hamster Kidney) cells (rBHK) overexpressing IDUA produced by electrostatic extrusion technique. The alginate microcapsule (MC-A) optimization study was carried out by means of an experimental Box-Behnken Design that allowed the simultaneous evaluation of the influence of voltage (kV), alginate/cell suspension flow (mL/h), and alginate concentration (%) on size and IDUA activity. The optimal conditions of voltage (10kV), flow (25mL/h), and alginate concentration (1.3%) made possible to obtain the smallest microcapsules showing the highest IDUA activity. After optimization, the microcapsules were sequentially coated with PLL and alginate (MC-APA) to increase their stability. MC-A and MC-APA presented monodisperse populations (span<1.22) with an average diameter of less than 350µm. The coating increased the mechanical stability of MC-APA by about 6-fold and modulated the permeability to the enzyme. Surface analyzes of MC-APA showed the presence of PLL bands, suggesting that the last alginate layer appears to have only partially coated the PLL. After 30days of subcutaneous implantation of the MC-APA microcapsules containing rBHK cells in a MPS I murine model, a significant increase in IDUA activity was observed in the skin near the implant. Histological analysis revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the conditions evaluated. Taken together, the overall results demonstrate the feasibility of MC-APA as a potential alternative for local treatment of MPS I.
Assuntos
Alginatos/química , Células Imobilizadas/enzimologia , Portadores de Fármacos/química , Iduronidase/administração & dosagem , Mucopolissacaridose I/tratamento farmacológico , Animais , Cápsulas , Linhagem Celular , Sobrevivência Celular , Cricetinae , Modelos Animais de Doenças , Composição de Medicamentos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Iduronidase/genética , Iduronidase/metabolismo , Rim/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Propriedades de Superfície , TransfecçãoRESUMO
Bioenergetics dysfunction has been postulated as an important pathomechanism of brain damage in glutaric aciduria type I, but this is still under debate. We investigated activities of citric acid cycle (CAC) enzymes, lactate release, respiration and membrane potential (ΔΨm) in mitochondrial preparations from cerebral cortex and striatum of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh-/-) and wild type mice fed a baseline or a high lysine (Lys, 4.7%) chow for 60 or 96h. Brain histological analyses were performed in these animals, as well as in 90-day-old animals fed a baseline or a high Lys chow during 30 days starting at 60-day-old. A moderate reduction of citrate synthase and isocitrate dehydrogenase activities was observed only in the striatum from 30-day-old Gcdh-/- animals submitted to a high Lys chow. In contrast, the other CAC enzyme activities, lactate release, the respiratory parameters state 3, state 4, the respiratory control ratio and CCCP-stimulated (uncoupled) state, as well as ΔΨm were not altered in the striatum. Similarly, none of the evaluated parameters were changed in the cerebral cortex from these animals under baseline or Lys overload. On the other hand, histological analyses revealed the presence of intense vacuolation in the cerebral cortex of 60 and 90-day-old Gcdh-/- mice fed a baseline chow and in the striatum of 90-day-old Gcdh-/- mice submitted to Lys overload for 30 days. Taken together, the present data demonstrate mild impairment of bioenergetics homeostasis and marked histological alterations in striatum from Gcdh-/- mice under a high Lys chow, suggesting that disruption of energy metabolism is not mainly involved in the brain injury of these animals.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias Metabólicas/metabolismo , Encefalopatias/metabolismo , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Metabolismo Energético , Glutaril-CoA Desidrogenase/deficiência , Lisina/administração & dosagem , Animais , Encefalopatias/patologia , Córtex Cerebral/patologia , Corpo Estriado/patologia , Dieta , Modelos Animais de Doenças , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Isocitrato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Camundongos da Linhagem 129 , Camundongos Knockout , Consumo de Oxigênio/fisiologiaRESUMO
We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Encefalopatias Metabólicas/patologia , Córtex Cerebral/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Neostriado/metabolismo , Receptores de Glutamato/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Encefalopatias Metabólicas/enzimologia , Córtex Cerebral/patologia , Dieta , Feminino , Regulação da Expressão Gênica , Glutaril-CoA Desidrogenase/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Neostriado/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glutamato/genéticaRESUMO
Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by mutations on α-L-iduronidase (IDUA) gene, leading to low or null enzyme activity. As nonsense mutations are present in about two thirds of the patients, stop codon read through (SCRT) is a potential alternative to achieve enhanced enzyme activity. This mechanism suppresses premature stop codon mutations allowing the protein to be fully translated. Chloramphenicol is a peptidyl transferase inhibitor able to induce readthrough and is efficient in cross the blood brain barrier. In this work, fibroblasts from MPS I patients (p.W402X/p.W402X; p.R89W/p.W402X and p.Q70X/c.1739-1g > t) were treated with chloramphenicol, which resulted in 100-fold increase on IDUA activity on compound heterozygous fibroblasts. cDNA sequencing showed that only the alleles without the nonsense mutation were being amplified, even after treatment, leading us to suggest that the nonsense alleles were targeted to nonsense-mediated mRNA decay and that chloramphenicol acts through a mechanism other than SCRT.
Assuntos
Cloranfenicol/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Iduronidase/metabolismo , Mucopolissacaridose I/metabolismo , Peptidil Transferases/antagonistas & inibidores , Células Cultivadas , Códon sem Sentido , Códon de Terminação , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Iduronidase/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Cationic nanoemulsions have been recently considered as potential delivery systems for nucleic acids. This study reports the influence of phospholipids on the properties of cationic nanoemulsions/DNA plasmid complexes. METHODS: Nanoemulsions composed of medium-chain triglycerides, stearylamine, egg lecithin or isolated phospholipids, ie, DSPC, DOPC, DSPE, or DOPE, glycerol, and water were prepared by spontaneous emulsification. Gene transfer to Hep G2 cells was analyzed using real-time polymerase chain reaction. RESULTS: The procedure resulted in monodispersed nanoemulsions with a droplet size and zeta potential of approximately 250 nm and +50 mV, respectively. The complexation of cationic nanoemulsions with DNA plasmid, analyzed by agarose gel retardation assay, was complete when the complex was obtained at a charge ratio of ≥ 1.0. In these conditions, the complexes were protected from enzymatic degradation by DNase I. The cytotoxicity of the complexes in Hep G2 cells, evaluated by MTT assay, showed that an increasing number of complexes led to progressive toxicity. Higher amounts of reporter DNA were detected for the formulation obtained with the DSPC phospholipid. Complexes containing DSPC and DSPE phospholipids, which have high phase transition temperatures, were less toxic in comparison with the formulations obtained with lecithin, DOPC, and DOPE. CONCLUSION: The results show the effect of the DNA/nanoemulsion complexes composition on the toxicity and transfection results.
Assuntos
DNA/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Aminas , Cátions/química , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Estabilidade de Medicamentos , Emulsões/química , Emulsões/farmacologia , Células Hep G2 , Humanos , Nanoestruturas/química , Plasmídeos/química , Transfecção/métodosRESUMO
The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Formação de Anticorpos , Vacina BCG/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Vacinação , Adesinas Bacterianas/administração & dosagem , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antibacterianos/sangue , Vacina BCG/administração & dosagem , Vacina BCG/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Clonagem Molecular , Enterotoxinas/administração & dosagem , Enterotoxinas/genética , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/imunologia , Tuberculose/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder due to arylsulfatase A (ARSA) deficiency that affects primarily the central nervous system. Ongoing treatments include enzyme replacement therapy and bone marrow transplantation, both limited in their effects due to the blood-brain barrier. An alternative approach would be the in situ implantation of encapsulated cells over expressing ARSA. Based on that, we tested the ability of encapsulated BHK cells over expressing ARSA to correct the enzyme deficiency in MLD patients' fibroblasts. Three groups were analyzed: fibroblasts treated with ARSA-over expressing BHK cells (rBHK) trapped in alginate capsules (capsules group), fibroblasts treated with supernatant of non-encapsulated rBHK (uptake control) and fibroblasts treated with empty capsules (empty group). Untreated and normal fibroblasts were used as controls. rBHK obtained by clone selection after non-viral transfection with pTARSA-CMV2. ARSA activity was measured after 1, 2, 3 and 4 weeks of treatment and beta-gal was used as reference enzyme. Statistical analysis was performed using ANOVA and Tukey's test. Normal fibroblasts showed ARSA activity of 23.9 + /- 2.01 nmol/h/mg of protein, whereas untreated MLD fibroblasts had the low ARSA activity (2.22 + /- 0.17). In the empty group, ARSA activity was equal to that of untreated fibroblasts (2.71 + /- 0.34). Capsules and uptake control groups showed higher enzymatic activity levels, compared to MLD untreated, 23.42 + /- 6.39 and 42.35 + /- 5.20, respectively (p < 0.01 for all groups). Encapsulated rBHK clones show potential as a new therapeutic strategy for the treatment of MLD, reaching normal enzyme levels in human MLD fibroblasts.
Assuntos
Transplante de Células/métodos , Cerebrosídeo Sulfatase/genética , Fibroblastos/enzimologia , Vetores Genéticos/uso terapêutico , Leucodistrofia Metacromática/terapia , Análise de Variância , Cápsulas , Células Cultivadas , Cerebrosídeo Sulfatase/deficiência , Engenharia Genética , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Implantes Experimentais , Rim/citologia , Leucodistrofia Metacromática/enzimologia , Leucodistrofia Metacromática/genética , Estatísticas não Paramétricas , Transgenes/genéticaRESUMO
Tendons are part of the connective tissue that joins muscle to bone. Tendon injuries are a problem, since they have a poor ability to regenerate spontaneously. Alternative treatments involving the injection of local growth factors and gene transfer has been evaluated. Thus, we compared two methods for non-viral gene transfer tendons, using the GFP gene as reporter gene. Methods: Wistar rats had the medial quadriceps tendon exposed and the plasmid was transferred by direct injection or complexed with liposomes. Quantification of GFP in the tendom and in the spleen was evaluated by histological analysis with a fluorescence microscope. Results: gene transfer to the tendon was successfully obtained in both treatments. Lipoplex, as expected, showed the highest efficiency in transducing tenocytes, however we have found GFP expression also in the spleen. Naked DNA also showed fluorescence values above the control group and the signal was limited to the tendom. Discussion: the use of GFP as a reporter gene is a classical approach to evaluate gene transfer efficiency. Non-viral gene transfer methods are safe but show low levels of transduction and transient expression. For tendon repair, however, these characteristics may prove beneficial because a transient expression may be desirable to avoid the risk of adverse effects. GFP distribution in the spleen was probably a result of lipoplexes uptake by cells from the reticular endothelial system. Conclusion: taking into account the distribution of GFP in another tissue when using lipoplex, we believe that naked DNA is a more appropriate way to perform gene transfer to the tendon, ensuring safety, low cost and easy handling
Injúrias no tendão representam um problema, uma vez que estes têm pobre capacidade de regeneração espontânea. Tratamentos envolvendo injeção local de fatores de crescimento e transferência gênica tem sido avaliados. Assim, comparamos dois métodos de transferência gênica não viral para tendões, usando o gene GFP como gene repórter. Métodos: ratos Wistar tiveram a porção medial do tendão quadriciptal exposto e o plasmídeo foi transferido através de injeção direta ou complexado com lipossoma. A quantificação de GFP no tendão e no baço foi avaliada por análise histológica. Resultados: a transferência gênica para o tendão foi obtida com sucesso nos dois tratamentos. Lipoplexo demonstrou maior eficiência na transfecção, porém a presença de GFP foi detectada também no baço. A transfecção com DNA nu demonstrou valores de fluorescência superiores ao grupo controle e o sinal foi limitado ao tendão. Discussão: o uso de GFP como gene repórter é uma abordagem clássica para avaliar a eficiência da transferência de genes. A transferência não-viral é segura embora apresente expressão transiente. Para o reparo do tendão, no entanto, essas características podem ser benéficas, pois uma expressão transiente pode ser desejável para evitar o risco de efeitos adversos. A distribuição de GFP no baço foi provavelmente resultado da absorção dos lipoplexos por células do sistema retículo endotelial. Conclusão: Tendo em conta a distribuição de GFP em outro tecido quando utilizamos lipoplexo, pensamos que o DNA nu é uma forma mais adequada para realizar a transferência de genes para o tendão, garantindo segurança, baixo custo e fácil manuseio
Assuntos
MedicinaRESUMO
A diarréia neonatal em suínos causada por Escherichia coli produtora de enterotoxinas (ETEC) é responsável por alta mortalidade e baixa taxa de crescimento de leitões. A habilidade de tais cepas causar doença é dependente principalmente da capacidade de E. coli aderir-se a mucosa do intestino delgado, que é mediada por fímbrias. Neste estudo o gene faeC, que codifica a subunidade menor da fímbria de E. coli K88ab, foi clonado no vetor de expressão em eucariotos pcDNA3, associado ou não à seqüência de KozaK. DNA plasmidial das duas versões da vacina foi inoculado em camundongos via intra-muscular, em duas doses, nos dias 0 e 21. Os animais que receberam a vacina de DNA contendo o faeC associado a seqüência de Kozak apresentaram soroconversões significativamente maiores (p<0,05) que os vacinados com pcDNA3/faeC sem a seqüência de Kozak.
RESUMO
The neonatal diarrhea in swine caused by enterotoxigenic Escherichia coli (ETEC) is responsible for high mortality and low growth rate in pigs and it is mainly dependent on the capacity of E. coli to attach to the surface of the small intestine, a property mediated by fimbria. In this study the faeC gene, which codes for the minor fimbrial subunit of E. coli K88ab, was cloned in the eukaryotic expression vector pcDNA3, associated or not to the Kozak sequence. Plasmid DNA of the two versions of the vaccine candidate was inoculated in mice by the intramuscular route, in two doses, at 0 and 21 days. The animals that received the DNA vaccine containing faeC associated to the Kozak sequence presented seroconversion significantly higher (P 0.05) than the one vaccinated with pcDNA3/faeC without the Kozak sequence.
A diarréia neonatal em suínos causada por Escherichia coli produtora de enterotoxinas (ETEC) é responsável por alta mortalidade e baixa taxa de crescimento de leitões. A habilidade de tais cepas causar doença é dependente principalmente da capacidade de E. coli aderir-se a mucosa do intestino delgado, que é mediada por fímbrias. Neste estudo o gene faeC, que codifica a subunidade menor da fímbria de E. coli K88ab, foi clonado no vetor de expressão em eucariotos pcDNA3, associado ou não à seqüência de KozaK. DNA plasmidial das duas versões da vacina foi inoculado em camundongos via intra-muscular, em duas doses, nos dias 0 e 21. Os animais que receberam a vacina de DNA contendo o faeC associado a seqüência de Kozak apresentaram soroconversões significativamente maiores (p 0,05) que os vacinados com pcDNA3/faeC sem a seqüência de Kozak.