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1.
Gene Ther ; 31(1-2): 12-18, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37985879

RESUMO

Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH deficiency leads to increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. These metabolites are the main triggers of brain damage. Mechanistic studies supporting neurotoxicity in mouse models have been conducted. However, the different vulnerability to some stressors between mouse and human brain cells reveals the need to have a reliable human neuronal model to study GA1 pathogenesis. In the present work we generated a GCDH knockout (KO) in the human neuroblastoma cell line SH-SY5Y by CRISPR/Cas9 technology. SH-SY5Y-GCDH KO cells accumulate GA, 3-OHGA, and glutarylcarnitine when exposed to lysine overload. GA or lysine treatment triggered neuronal damage in GCDH deficient cells. SH-SY5Y-GCDH KO cells also displayed features of GA1 pathogenesis such as increased oxidative stress vulnerability. Restoration of the GCDH activity by gene replacement rescued neuronal alterations. Thus, our findings provide a human neuronal cellular model of GA1 to study this disease and show the potential of gene therapy to rescue GCDH deficiency.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Encefalopatias Metabólicas , Lisina , Neuroblastoma , Humanos , Animais , Camundongos , Lisina/genética , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Camundongos Knockout , Terapia Genética
2.
HPB (Oxford) ; 25(1): 73-80, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36123222

RESUMO

BACKGROUND: Clinically relevant postoperative pancreatic fistula (CR-POPF) is the most feared complication following pancreaticoduodenectomy (PD). There is increasing evidence that very early postoperative factors can be helpful to identify high-risk patients. The aim of this study is to analyze whether postoperative day one (POD1) systemic inflammatory response can be used as an early biomarker of CR-POPF development. METHODS: All patients undergoing PD from 2014 to 2020 were considered. Variables were extracted from a prospectively held database. Clinical and perioperative variables, including POD1 systemic inflammatory response syndrome (SIRS) and C-reactive protein level were collected. To elucidate the independent role of early CR-POPF biomarkers, multivariate hierarchical logistic regression analyses were planned. RESULTS: Out of 243, 213 patients were included in this analysis. CR-POPF occurred in 49 (23.0%) patients and 90-day mortality was 1.4%. POD1 SIRS was reported in 65 (30.5%) patients. Following hierarchical logistic regression analyses, CR-POPF was independently associated with body mass index (OR = 2.787, p = 0.003), soft pancreatic texture (OR = 4.258, p = 0.002) and POD1 SIRS (OR = 50.067, p = 0.001). CONCLUSION: POD1 SIRS is powerfully associated with CR-POPF and therefore it could be used as a tool to optimize postoperative care of PD patients. Further prospective studies are needed to validate these findings.


Assuntos
Pâncreas , Fístula Pancreática , Humanos , Fístula Pancreática/diagnóstico , Fístula Pancreática/etiologia , Fatores de Risco , Pâncreas/cirurgia , Pancreaticoduodenectomia/efeitos adversos , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Biomarcadores , Estudos Retrospectivos
3.
Surgery ; 172(6): 1651-1655, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36272774

RESUMO

BACKGROUND: The Coronavirus pandemic outbreak in 2019 and the saturation of healthcare system led to an increased use of digital tools for surveillance. In this study we described our experience using telemedicine to follow-up on patients with intraductal papillary mucinous neoplasms during the COVID-19 era and analyze those factors associated to patients' satisfaction. METHODS: This 1-year retrospective observational study enrolled patients with intraductal papillary mucinous neoplasms followed-up by telemedicine during COVID-19 outbreak. Patients with high-risk features needing on-site physical examination or declining remote follow-up were excluded. A 13-question survey was conducted; demographic, geographic, and employment information was collected. Univariate and multivariate analyses were performed to evaluate those factors associated to patients' satisfaction. RESULTS: Out of 287, a total of 177 patients with intraductal papillary mucinous neoplasms were included: the mean age was 69 (44-87) years and the male/female ratio was 0.78. A total of 80 (45.2%) patients had previously experienced abdominal pain. Most patients (85.3%) were satisfied with telemedicine: at univariate analysis, age ≥70 years (P = .007), retirement (P = .001), and absence of previous abdominal pain (P = .05) were significantly associated with patient satisfaction. At multivariate analysis, the absence of previous abdominal pain was the only factor independently associated with patient satisfaction (odds ratio 5.964, 95% confidence interval 2.21-16.11, P < .001). CONCLUSION: Telemedicine allows a new follow-up strategy that can be used in selected patients with intraductal papillary mucinous neoplasms. The absence of previous abdominal pain is associated with patient satisfaction during follow-up. Further studies are needed to evaluate safety of remote follow-up in patients with intraductal papillary mucinous neoplasms.


Assuntos
Adenocarcinoma Mucinoso , COVID-19 , Carcinoma Ductal Pancreático , Carcinoma Papilar , Neoplasias Pancreáticas , Telemedicina , Humanos , Feminino , Masculino , Idoso , Seguimentos , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/diagnóstico , Surtos de Doenças , Dor Abdominal
4.
SD, Rev. med. int. Síndr. Down (Ed. castell.) ; 18(2): 21-28, mayo-ago. 2014.
Artigo em Espanhol | IBECS | ID: ibc-126439

RESUMO

La terapia génica nos ofrece la posibilidad de manipular los virus para convertir un agente infeccioso en un vehículo que transporta en su genoma secuencias de DNA con potencial terapéutico. En este trabajo hemos aprovechado la metodología que nos proporciona la terapia génica para desarrollar vectores virales derivados de virus adenoasociados y lentivirus con el objetivo de identificar genes clave (proteínas o microRNA [miRNA]) implicados en las alteraciones cognitivas presentes en el síndrome de Down (SD). Hemos demostrado que en un contexto de trisomía, como es el modelo de ratón Ts65Dn, la normalización de la expresión de Dyrk1A a través de la administración de los virus adenoasociados AAV2/1-shDyrk1A contribuye a restablecer la regulación de moléculas clave en los procesos de memoria y aprendizaje. Ello permite una atenuación de los defectos en plasticidad sináptica y facilita el desarrollo de una estrategia de aprendizaje visuoespacial más eficiente. Estos estudios refuerzan el papel destacado de Dyrk1A en los procesos cognitivos. Por otro lado, la estrategia de control de la expresión de miRNA desarrollada mediante los lentivirus Lv-anti-miR155-802 nos permite proponer a MeCP2 como un gen cuya desregulación en el síndrome de Down puede tener un papel clave en el deterioro cognitivo (AU)


Viruses have evolved ways of encapsulating and delivering their genes into human cells. Gene therapy takes advantage of this capability to manipulate the viral genome and convert an infectious agent into an efficient vector that delivers therapeutic genes. In the current work we have applied gene therapy approaches based on adeno-associated virus and lentivirus delivery to identify candidate genes (protein-coding or miRNAs) involved in the cognitive deficits in Down Syndrome. We show that the hippocampal injection of the adeno-associated virus AAV2/1-shDyrk1A normalized Dyrk1A expression in the trisomic Ts65Dn mice. As a consequence the regulation of key molecular players in memory and learning processes was rescued and mice showed an attenuation of synaptic plasticity defects and improved efficacy in learning strategies. All together these results reinforce the role of Dyrk1A in cognition. On the other hand, with the lentiviral strategydeveloped to specifically inhibit miR-155 and miR-802 (Lv-anti-miR155/802), we were able to show a tight control of the miRNAs target Mecp2 suggesting that the downregulation of MeCP2 in Down syndromecould be a contributing factor to the cognitive defects (AU)


Assuntos
Animais , Masculino , Feminino , Camundongos , Síndrome de Down/genética , Terapia Genética/métodos , Terapia Genética/tendências , Terapia Genética , Expressão Gênica , Terapia Genética/instrumentação , Terapia Genética/psicologia , Terapia Genética/normas
5.
Gene Ther ; 21(8): 767-74, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24942629

RESUMO

Endovenously administered oncolytic viruses extravasate and penetrate poorly into tumors. iRGD is a cyclic peptide that enhances tumor penetration when conjugated or coadministered with different types of molecules such as drugs, nanoparticles or phages. iRGD-mediated tumor penetration occurs in three steps: binding to αv-integrins on tumor vasculature or tumor cells, exposure by proteolysis of a C-terminal motif that binds to neuropilin-1 (NRP-1) and cell internalization. We have genetically inserted the iRGD peptide in the fiber C terminus of ICOVIR15K, an oncolytic tumor-retargeted adenovirus to increase its tumor penetration. In vitro, NRP-1 interaction improved binding and internalization of the virus in different cancer cells overexpressing integrins and NRP-1. However, such NRP-1-mediated internalization did not affect transduction or cytotoxicity. In vivo, iRGD did not change the normal organ transduction pattern, with liver and spleen as main targeted organs. In tumors, however, iRGD enhanced transduction and early adenovirus dissemination through the tumor mass leading to an improved antitumor efficacy.


Assuntos
Terapia Genética/métodos , Oligopeptídeos/uso terapêutico , Terapia Viral Oncolítica/métodos , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Neuropilina-1/metabolismo , Internalização do Vírus , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cereb Cortex ; 22(12): 2867-77, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22215728

RESUMO

Down syndrome (DS) is the most frequent genetic cause of mental retardation. Cognitive dysfunction in these patients is correlated with reduced dendritic branching and complexity, along with fewer spines of abnormal shape that characterize the cortical neuronal profile of DS. DS phenotypes are caused by the disruptive effect of specific trisomic genes. Here, we report that overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A, DYRK1A, is sufficient to produce the dendritic alterations observed in DS patients. Engineered changes in Dyrk1A gene dosage in vivo strongly alter the postnatal dendritic arborization processes with a similar progression than in humans. In cultured mammalian cortical neurons, we determined a reduction of neurite outgrowth and synaptogenesis. The mechanism underlying neurite dysgenesia involves changes in the dynamic reorganization of the cytoskeleton.


Assuntos
Córtex Cerebral/metabolismo , Citoesqueleto/metabolismo , Síndrome de Down/metabolismo , Neurogênese , Neurônios/metabolismo , Neurônios/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Córtex Cerebral/patologia , Citoesqueleto/patologia , Síndrome de Down/patologia , Camundongos , Camundongos Transgênicos , Quinases Dyrk
7.
Gene Ther ; 18(1): 73-81, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20720574

RESUMO

The thymidine kinase/ganciclovir (TK/GCV) cancer gene therapy approach is based on inducing GCV metabolite cytotoxicity in tumor cells expressing the herpes simplex virus TK gene and exposed to GCV. A bystander effect, mediated by gap junctions, accounts for the transfer of toxic metabolites from TK-expressing cells to neighboring cells. It has been proposed that E-cadherin participates in the formation and function of such gap junctions. In this study we investigate the influence of E-cadherin on TK/GCV suicide therapy with a panel of cellular and in vivo models of pancreatic ductal adenocarcinoma. We observed a strong correlation of E-cadherin expression and the TK/GCV bystander effect, associated with the modulation of gap junction communication and connexin expression or localization. Importantly, the co-expression of TK and E-cadherin genes in the adenoviral vector AdTat8TKIE improved TK/GCV cytotoxicity and triggered a potent antitumoral effect, superior to standard AdTat8TK/GCV in MIAPaCa-2 xenografts. The increased expression of E-cadherin resulted in the reduction of the bcl-2 content. Interestingly, the knockdown of bcl-2 sensitized cells to TK/GCV. Thus, we propose that by restoring E-cadherin in pancreatic tumor cells we will improve TK/GCV therapy, both by enhancing the bystander effect and by facilitating the induction of apoptosis.


Assuntos
Antineoplásicos/administração & dosagem , Caderinas/genética , Ganciclovir/administração & dosagem , Neoplasias Pancreáticas/terapia , Timidina Quinase/genética , Antineoplásicos/farmacologia , Efeito Espectador/genética , Caderinas/metabolismo , Ganciclovir/farmacologia , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias Pancreáticas/genética , Retroviridae/genética , Retroviridae/metabolismo , Simplexvirus/genética , Simplexvirus/metabolismo , Timidina Quinase/administração & dosagem , Timidina Quinase/metabolismo , Transfecção , Células Tumorais Cultivadas
8.
Genes Brain Behav ; 9(2): 160-72, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19840121

RESUMO

BACE2 is homologous to BACE1, a beta-secretase that is involved in the amyloidogenic pathway of amyloid precursor protein (APP), and maps to the Down syndrome critical region of chromosome 21. Alzheimer disease neuropathology is common in Down syndrome patients at relatively early ages, and it has thus been speculated that BACE2 co-overexpression with APP would promote the early neurodegenerative phenotype. However, the in vivo function of BACE2 has not yet been elucidated. The aim of the present work has been to analyse the impact of in vivo BACE2 overexpression using a transgenic mouse model. Our results suggest that BACE2 is not involved in the amyloidogenic pathway, cognitive dysfunction or cholinergic degeneration. However, TgBACE2 animals showed increased anxiety-like behaviour along with increased numbers of noradrenergic neurones in locus coeruleus, thus suggesting an unexpected role of BACE2 overexpression.


Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Animais , Ansiedade/genética , Escuridão , Síndrome de Down/enzimologia , Síndrome de Down/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Locus Cerúleo/patologia , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Modelos Animais , Atividade Motora/genética , Neurônios/patologia , Transtornos de Fotossensibilidade/genética , Prosencéfalo/enzimologia , RNA/genética , Transcrição Gênica
9.
Neurobiol Dis ; 32(3): 377-84, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18773961

RESUMO

Transgenic mice overexpressing Dyrk1A (TgDyrk1A), a Down syndrome (DS) candidate gene, exhibit motor and cognitive alterations similar to those observed in DS individuals. To gain new insights into the molecular consequences of Dyrk1A overexpression underlying TgDyrk1A and possibly DS motor phenotypes, microarray studies were performed. Transcriptome analysis showed an upregulation of the NR2A subunit of the NMDA type of glutamate receptors in TgDyrk1A cerebellum. NR2A protein overexpression was also detected in TgDyrk1A cerebellar homogenates, in the synaptosome-enriched fraction and in TgDyrk1A primary cerebellar granular neuronal cultures (CGNs). In TgDyrk1A synaptosomes, calcium-imaging experiments showed a higher calcium uptake after NMDA stimulation. Similarly, NMDA administration promoted longer calcium transients in TgDyrk1A CGNs. Taken together, these results show that NMDA-induced calcium rise is altered in TgDyrk1A cerebellar neurons and indicate that calcium signaling is dysregulated in TgDyrk1A mice cerebella. These findings suggest that DYRK1A overexpression might contribute to the dysbalance in the excitatory transmission found in the cerebellum of DS individuals and DS mouse models.


Assuntos
Cálcio/metabolismo , Cerebelo/metabolismo , Síndrome de Down/genética , N-Metilaspartato/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Regulação para Cima , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinaptossomos/metabolismo , Quinases Dyrk
10.
Ther Drug Monit ; 29(5): 612-8, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17898652

RESUMO

Gene therapy is anticipated as being an important medical development. Essential to its effectiveness is the appropriate activity (protein expression) in the expected target cells. A noninvasive diagnostic procedure of successful gene expression will be of paramount importance to validate its use or its misuse (eg, sports gene doping). Externally detectable labeled oligonucleotide hybridizing with the messenger RNA generated by the transferred gene has been proposed as a possibility to monitor successful gene therapy. The authors selected the erythropoietin gene (Epo) for a pilot study on erythropoietin protein expression in mouse muscle. Oligonucleotides of peptide nucleic acid (PNA) type capable of antisense binding to unique murine Epo-mRNA sequences were synthesized by solid phase methods, and elongated at the N-terminus with the HIV Tat (48-60) cell penetrating peptide. They were labeled with fluorescence and radioactive tags to verify penetration and longer half-life properties in Epo gene transfected C2C12 mouse muscle cells as compared with corresponding wild-type cells. Downregulation of newly expressed erythropoietin protein in such cells additionally confirmed the penetration and hybridizing properties of the selected labeled oligonucleotide. I-labeled Tat-PNAs were intravenously injected into mice that had previously received the Epo gene into the right tibialis muscle by DNA electrotransfer. Preferential accumulation of radioactivity in the transferred limb as compared with the contralateral limb was ascertained, especially for I-Tat-CTA CGT AGA CCA CT (labeled Tat-PNA 1). This study provides experimental data to support the potential use of external noninvasive image detection to monitor gene therapy. The extension of the approach to more sensitive methods for whole-body external detection such as positron emission tomography appears feasible.


Assuntos
Eritropoetina/genética , Músculo Esquelético/química , Animais , DNA Antissenso , Monitoramento de Medicamentos/métodos , Terapia Genética , Camundongos , Ácidos Nucleicos Peptídicos , Projetos Piloto , RNA Mensageiro/análise
11.
Gene Ther ; 14(20): 1471-80, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17713568

RESUMO

Replication-competent adenoviruses carrying the herpes simplex thymidine kinase (TK) gene have shown contradictory evidence with regard to their antitumoural efficacy in combination with ganciclovir (GCV) treatment. We generated a replication-competent adenovirus carrying Tat8-TK, a modified form of the TK gene, under the control of the adenoviral major late promoter (AdRGDTat8-TK-L). Pancreatic cancer cell lines with different sensitivity to the TK/GCV system were infected with AdRGDTat8-TK-L, both in the presence and absence of GCV, and tested for treatment efficacy. We observed that, although the presence of GCV reduced viral replication in all infected cell lines, in three out of four GCV significantly enhanced the efficacy of the virotherapy. Interestingly, the cytotoxicity of the AdRGD-Tat8-TK-L/GCV was found more potent than that of a first generation AdTK/GCV system. In tumour xenografts from BxPC-3 and NP-18 pancreatic cells, both AdRGDTat8-TK-L and AdRGDTat8-TK-L/GCV treatment showed antitumoural activity. In BxPC-3 tumours scheduling of virus and prodrug was a key factor to determine the outcome of the therapy. Importantly, the addition of GCV enhanced the antitumoural effect of AdRGDTat8-TK-L only when applied in two rounds of virus+GCV. Interestingly, in spite of interfering with viral replication in vitro, GCV treatment of NP-18 tumours did not compromise the antitumoural efficacy of the AdRGDTat8-TK-L adenovirus. Thus, our results show that the combination therapy of a replicative adenovirus and the Tat8-TK/GCV suicide system can prove beneficial, when the appropriate regimen of virus and GCV is applied.


Assuntos
Adenoviridae/genética , Antivirais/uso terapêutico , Ganciclovir/uso terapêutico , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Pancreáticas/terapia , Animais , Efeito Citopatogênico Viral/efeitos dos fármacos , Expressão Gênica , Genes tat , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Nus , Modelos Animais , Transplante de Neoplasias , Neoplasias Pancreáticas/virologia , Distribuição Aleatória , Timidina Quinase/genética , Transdução Genética/métodos , Resultado do Tratamento , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacos
12.
Kidney Int ; 70(2): 265-74, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16710352

RESUMO

Inflammatory processes and tissue scarring are characteristic features of chronic allograft nephropathy. Hepatocyte growth factor (HGF) has beneficial effects on renal fibrosis and it also ameliorates renal interstitial inflammation as it has been recently described. Contrarily to protein administration, intramuscular gene electrotransfer allows sustained release of HGF. So, here we hypothesized that gene therapy with human HGF would diminish the characteristic scarring of chronic allograft nephropathy either by antagonizing tissue fibrosis mechanisms or by reducing inflammation. Lewis rats transplanted with cold preserved Fischer kidneys received vehicle (NoHGF) or intramuscular plasmid DNA encoding HGF plus electroporation either before transplantation (IniHGF, early post-transplant cytoprotection of tubular cells) or 8/10 weeks after transplantation (DelHGF, delayed prevention of chronic mechanisms). Serum creatinine and proteinuria were measured every 4 weeks for 24 weeks. Grafts at 12 or 24 weeks were evaluated for glomerulosclerosis, fibrosis inflammatory cells and mediators, cell regeneration and tubulo-interstitial damage. Nontreated animals developed renal insufficiency, progressive proteinuria and fibrosis among other characteristic histological features of chronic allograft nephropathy. Treatment with human HGF, especially when delayed until the onset of fibrogenic mechanisms, reduced renal failure and mortality, diminished tubule-interstitial damage, induced cell regeneration, decreased inflammation, NF-kappaB activation, and profibrotic markers at 12 weeks and prevented late interstitial fibrosis and glomerulosclerosis. The effectiveness of HGF-gene therapy in the prevention of renal allograft scarring is related with the halt of profibrotic inflammatory-induced mechanisms.


Assuntos
Terapia Genética/métodos , Fator de Crescimento de Hepatócito/genética , Falência Renal Crônica/terapia , Transplante de Rim , Nefrite/prevenção & controle , Animais , Biomarcadores , Cicatriz/patologia , Cicatriz/prevenção & controle , Cicatriz/terapia , Fibrose , Interferon gama/imunologia , Interleucina-12/imunologia , Rim/imunologia , Rim/patologia , Falência Renal Crônica/patologia , Falência Renal Crônica/cirurgia , Masculino , Nefrite/patologia , Nefrite/terapia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/prevenção & controle , Complicações Pós-Operatórias/terapia , Proteinúria/patologia , Proteinúria/cirurgia , Proteinúria/terapia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Fator de Transcrição RelA/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/imunologia
13.
Gene Ther ; 12(21): 1551-8, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15973441

RESUMO

In the early phase of kidney transplantation, the transplanted kidney is exposed to insults like ischemia/reperfusion, which is a leading cause of acute renal failure (ARF). ARF in the context of renal transplantation predisposes the graft to developing chronic damage and to long-term graft loss. Hepatocyte growth factor (HGF) has been suggested to support the intrinsic ability of the kidney to regenerate in response to injury by its morphogenic, mitogenic, motogenic and antiapoptotic activities. In the present paper, we examine whether human HGF (hHGF) gene electrotransfer helps in the recovery from ARF in a model of rat renal warm ischemia. We also assess the advantages of this form of gene therapy by direct electroporation of the kidney, given that transplantation offers the possibility of manipulating the organ in vivo. We have compared the therapeutic efficiency of two electroporation methodologies in a rat ARF model. Although they both targeted the same organ, the two methods were applied to different parts of the animal: muscle and kidney. Kidney direct electrotransfer was shown to be more efficient not only in pharmacokinetic but also in therapeutic terms, so it may become a clinically practical alternative in renal transplantation.


Assuntos
Injúria Renal Aguda/prevenção & controle , Eletroporação/métodos , Terapia Genética/métodos , Fator de Crescimento de Hepatócito/genética , Transplante de Rim , Injúria Renal Aguda/patologia , Injúria Renal Aguda/terapia , Animais , Apoptose , Morte Celular , Proliferação de Células , Expressão Gênica , Sobrevivência de Enxerto , Fator de Crescimento de Hepatócito/administração & dosagem , Fator de Crescimento de Hepatócito/metabolismo , Imuno-Histoquímica , Isquemia/metabolismo , Isquemia/terapia , Rim/irrigação sanguínea , Rim/metabolismo , Rim/patologia , Músculo Esquelético/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
14.
Prog Neurobiol ; 74(2): 111-26, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15518956

RESUMO

Since the discovery in the 1970s that dendritic abnormalities in cortical pyramidal neurons are the most consistent pathologic correlate of mental retardation, research has focused on how dendritic alterations are related to reduced intellectual ability. Due in part to obvious ethical problems and in part to the lack of fruitful methods to study neuronal circuitry in the human cortex, there is little data about the microanatomical contribution to mental retardation. The recent identification of the genetic bases of some mental retardation associated alterations, coupled with the technology to create transgenic animal models and the introduction of powerful sophisticated tools in the field of microanatomy, has led to a growth in the studies of the alterations of pyramidal cell morphology in these disorders. Studies of individuals with Down syndrome, the most frequent genetic disorder leading to mental retardation, allow the analysis of the relationships between cognition, genotype and brain microanatomy. In Down syndrome the crucial question is to define the mechanisms by which an excess of normal gene products, in interaction with the environment, directs and constrains neural maturation, and how this abnormal development translates into cognition and behaviour. In the present article we discuss mainly Down syndrome-associated dendritic abnormalities and plasticity and the role of animal models in these studies. We believe that through the further development of such approaches, the study of the microanatomical substrates of mental retardation will contribute significantly to our understanding of the mechanisms underlying human brain disorders associated with mental retardation.


Assuntos
Transtornos Cognitivos/patologia , Transtornos Cognitivos/fisiopatologia , Dendritos/efeitos dos fármacos , Dendritos/patologia , Síndrome de Down/patologia , Síndrome de Down/fisiopatologia , Plasticidade Neuronal/efeitos dos fármacos , Animais , Transtornos Cognitivos/tratamento farmacológico , Modelos Animais de Doenças , Síndrome de Down/tratamento farmacológico , Síndrome de Down/genética , Terapia Genética/métodos , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia
15.
Neurobiol Dis ; 15(1): 132-42, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14751778

RESUMO

Motor deficits are among the most frequent impairments in Down syndrome (DS), but their neuropathological and molecular bases remain elusive. Here we investigate the motor profile of transgenic mice overexpressing Dyrk1a, Tg(Dyrk1a)1Cff (hereafter TgDyrk1a), a candidate gene hypothesized to cause some of the neurological defects associated with DS. We have previously shown DYRK1A expression in the cerebellum and functionally related structures, most brainstem motor nuclei and spinal cord, supporting a role for Dyrk1a in controlling motor function. Here we demonstrate that TgDyrk1a mice present DYRK1A overexpression in these areas along with specific motor dysfunction. The main finding that emerged was impairment of motor learning and alteration of the organization of locomotor behavior, which agrees with reported clinical observations in subjects with DS. These results confirm and extend previous data and provide further insight to the functional domains that might be altered in TgDyrk1a mice and underlying molecular mechanisms of DS motor dysfunction.


Assuntos
Encéfalo/metabolismo , Síndrome de Down/complicações , Transtornos dos Movimentos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Coxeadura Animal/genética , Coxeadura Animal/metabolismo , Coxeadura Animal/fisiopatologia , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/metabolismo , Deficiências da Aprendizagem/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/fisiopatologia , Debilidade Muscular/genética , Debilidade Muscular/metabolismo , Debilidade Muscular/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Quinases Dyrk
16.
Curr Gene Ther ; 3(1): 13-26, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12553532

RESUMO

Gene-directed enzyme prodrug therapy (GDEPT) is a two step therapeutic approach for cancer gene therapy. In the first step, the transgene is delivered into the tumor and expressed. In the second step a prodrug is administered and is selectively activated by the expressed enzyme. The first GDEPT system described was the thymidine kinase gene of the Herpes Simplex virus (HSVtk) in combination with the prodrug Ganciclovir (GCV). A large number of experiments have been performed with this system, in different types of tumors and initial studies in animal models were very promising. This encouraged investigators to move into clinical trials although poor results have been obtained so far. A large effort has been made with numerous different strategies to enhance HSVtk/GCV efficacy in cellular and in vivo models and very strong cytotoxic effects have been obtained. The present review describes the current state of preclinical research and summarizes the results of the clinical trials undertaken.


Assuntos
Ganciclovir/metabolismo , Terapia Genética , Vetores Genéticos , Simplexvirus , Timidina Quinase/genética , Morte Celular/genética , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia
17.
Pharmacol Biochem Behav ; 73(1): 19-25, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12076721

RESUMO

The present study was aimed at establishing the differences in the neurodevelopmental profile between two F2 lines derived from two F1 hybrid mouse strains (129 x C57BL/6 and C57BL/6 x SJL). The choice of the given strains was based on the frequent use of these mice in transgenic research. For the neurodevelopment phenotyping, we employed a test battery consisting of 23 somatometric, sensorial and motor tests. Significant variations between the strains were established in different functional domains. Some specific delays in the appearance of developmental landmarks were observed in F2 mice derived from crosses of F1 C57BL/6 x 129, whereas they acquired early developmental functions, such as the righting reflex, sooner than C57BL/6 x SJL-derived mice. C57BL/6 x 129 F2 offspring were spontaneously hypoactive, and their poorer motor performance was confirmed by low performance in the negative geotaxis test. However, there were no differences in the general psychomotor development as shown by the good performance in the homing test in both F2 lines. Both strains were susceptible to the handling procedures used, presenting a similar alteration in the response observed in the homing test as compared to nonhandled control mice. In conclusion, our work highlights the importance of the genetic background for transgenesis experiments and also the need for well-established testing protocols to obtain sufficient information at the first stage of behavioral screening of genetically modified mice.


Assuntos
Animais Geneticamente Modificados/genética , Atividade Motora/genética , Destreza Motora/fisiologia , Animais , Animais Recém-Nascidos , Cruzamento/métodos , Feminino , Manobra Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Especificidade da Espécie
18.
Int J Cancer ; 94(1): 81-8, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11668482

RESUMO

Currently, there is no effective treatment for pancreatic cancer and prodrug-activating gene therapy with the herpes simplex virus thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) has been suggested as a candidate approach against this disease. In the present study, we have evaluated the efficacy of the HSV-tk/GCV treatment in a panel of pancreatic tumor cells (NP-9, NP-18, NP-31) and the potentiation of the cytotoxic effect in combination with the overexpression of the connexin 26 gene (Cx26). Pancreatic cells transduced with a retrovirus containing the HSV-tk gene showed different sensitivities to GCV that seemed to be independent of HSV-tk expression levels. The extent of the bystander effect also varied among the pancreatic tumor cells and correlated with the level of gap junction intercellular communication (GJIC). Transduction of the pancreatic tumor cells with a retrovirus carrying the connexin 26 gene resulted in high levels of connexin 26 expression and in an increase in the GJIC that correlated to an extent in the bystander effect in both NP-9Cx26 and NP-18Cx26 cells. Neither an increment in GJIC nor an increase in the bystander killing was detected in NP-31Cx26. The bystander effect in NP-18 Cx26 cells was also prevented by the long term inhibitor of GJIC, 18-alpha-glycyrrhetinic acid (AGA). Together, these results demonstrate that pancreatic tumor cells are highly different as regards the susceptibility to HSV-tk/GCV treatment. Moreover, they indicate that overexpression of the Cx26 gene does not always correspond to an increase in GJIC although they clearly suggest the role of GJIC in mediating the bystander effect.


Assuntos
Conexinas/genética , Terapia Genética , Neoplasias Pancreáticas/terapia , Retroviridae/genética , Simplexvirus/enzimologia , Timidina Quinase/genética , Comunicação Celular , Conexina 26 , Conexinas/fisiologia , Ganciclovir/uso terapêutico , Junções Comunicantes/fisiologia , Humanos , Neoplasias Pancreáticas/metabolismo , Transfecção , Células Tumorais Cultivadas
19.
Hum Mol Genet ; 10(18): 1915-23, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11555628

RESUMO

Down's syndrome (DS) is a major cause of mental retardation, hypotonia and delayed development. Murine models of DS carrying large murine or human genomic fragments show motor alterations and memory deficits. The specific genes responsible for these phenotypic alterations have not yet been defined. DYRK1A, the human homolog of the Drosophila minibrain gene, maps to the DS critical region of human chromosome 21 and is overexpressed in DS fetal brain. DYRK1A encodes a serine-threonine kinase, probably involved in neuroblast proliferation. Mutant Drosophila minibrain flies have a reduction in both optic lobes and central brain, showing learning deficits and hypoactivity. We have generated transgenic mice (TgDyrk1A) overexpressing the full-length cDNA of Dyrk1A. TgDyrk1A mice exhibit delayed cranio-caudal maturation with functional consequences in neuromotor development. TgDyrk1A mice also show altered motor skill acquisition and hyperactivity, which is maintained to adulthood. In the Morris water maze, TgDyrk1A mice show a significant impairment in spatial learning and cognitive flexibility, indicative of hippocampal and prefrontal cortex dysfunction. In the more complex repeated reversal learning paradigm, this defect turned out to be specifically related to reference memory, whereas working memory was almost unimpaired. These alterations are comparable with those found in the partial trisomy chromosome 16 murine models of DS and suggest a causative role of DYRK1A in mental retardation and in motor anomalies of DS.


Assuntos
Transtornos Cognitivos/genética , Síndrome de Down/genética , Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transtornos Psicomotores/genética , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Síndrome de Down/patologia , Síndrome de Down/fisiopatologia , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Quinases Dyrk
20.
Physiol Behav ; 73(5): 859-71, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11566219

RESUMO

The availability of the recently published DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS). Trisomy 21 or DS is the only autosomal aneuploidy that is not lethal in the fetal or early postnatal period. DS phenotypes show variable penetrance, affecting many different organs, including brain (mental retardation, early onset of Alzheimer's disease, AD), muscle (hypotonia), skeleton, and blood. DS phenotypes may stem directly from the cumulative effect of overexpression of specific HSA21 gene products or indirectly through the interaction of these gene products with the whole genome, transcriptome, or proteome. Mouse genetic models have played an important role in the elucidation of the contribution of specific genes to the DS phenotype. To date, the strategies used for modeling DS in mice have been three: (1) to assess single-gene contributions to DS phenotype, using transgenic techniques to create models overexpressing single or combinations of genes, (2) to assess the effects of overexpressing large foreign DNA pieces, introduced on yeast artificial chromosomes (YACs) or bacterial artificial chromosomes (BACs) into transgenic mice, and (3) mouse trisomies that carry all or part of MMU16, which has regions of conserved homology with HSA21. Here we review the existing murine models and the relevance of their contribution to DS research.


Assuntos
Modelos Animais de Doenças , Síndrome de Down/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/patologia , Cromossomos Humanos Par 21 , Expressão Gênica/fisiologia , Genética Comportamental , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos
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