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1.
Anticancer Res ; 41(5): 2467-2471, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952472

RESUMO

BACKGROUND/AIM: Adaptive radiation therapy (ART) is a technique capable of reducing radiation dose to normal tissue without compromising local control. For potentially resectable thymoma, induction therapy is standard of care. Because large disease volume is common in this context, ART has been suggested to reduce toxicity from induction chemoradiation. This has not been previously illustrated in the literature. CASE REPORT: A 38-year-old man with initially unresectable thymoma was treated with induction chemoradiation including cisplatin and etoposide. He received 45 Gy in 25 fractions and ART was utilized to shrink the radiotherapy field for the final 10 fractions. RESULTS: Thymectomy showed Masaoka stage III disease with negative margins. He experienced no treatment-related toxicity and has no evidence of disease 8 years after diagnosis. CONCLUSION: Induction chemoradiotherapy with ART appears to be feasible, safe, and efficacious for locally advanced intact thymoma.


Assuntos
Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/radioterapia , Timoma/tratamento farmacológico , Timoma/radioterapia , Adulto , Cisplatino/uso terapêutico , Terapia Combinada , Etoposídeo/uso terapêutico , Humanos , Masculino , Recidiva Local de Neoplasia/patologia , Timoma/patologia
2.
Int J Radiat Oncol Biol Phys ; 104(1): 83-89, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30684664

RESUMO

PURPOSE: To determine the pain response and prevention of vertebral compression fractures (VCFs) after single-fraction stereotactic ablative radiation therapy (SABR) in conjunction with immediate vertebroplasty for spine metastases. METHODS AND MATERIALS: Patients with localized spine metastases free from VCF associated with loss of vertebral height with a pain score ≥4 using the visual analog scale were enrolled. Spine SABR was performed with 20 Gy delivered to the gross disease and 14 Gy to the contiguous bone marrow in a single fraction. Immediate, prophylactic vertebroplasty was performed within 1 month after spine SABR. The primary endpoint was pain response at 3 months compared to the historical control with external beam radiation therapy from Radiation Therapy Oncology Group study 9714. Secondary endpoints included pain response at 1 month, duration of pain response, vertebroplasty rate, VCF rate, local control, and overall survival. RESULTS: Thirty-five patients were enrolled, of whom 29 were deemed eligible and underwent single-fraction spine SABR. Twenty-three of these patients subsequently underwent prophylactic vertebroplasty. The 3-month pain response was significantly improved compared to Radiation Therapy Oncology Group study 9714: 95% versus 51% (P < .0001). The local control with a median follow-up of 9.6 months was 92%. The freedom from VCF was 90% at 1 year. Spine SABR was well tolerated with no grade 2 or higher toxicities. A single patient with disease extending from the vertebral body into the spinal canal developed vertebroplasty-related myelopathy, which was corrected with surgery. CONCLUSIONS: Single-fraction SABR immediately followed by prophylactic vertebroplasty improves pain response compared with conventional radiation therapy while providing long-term pain control and structural stability of the treated spine. Vertebroplasty is well tolerated as a prophylactic measure in patients without loss of vertebral height after spine SABR. Pain response and VCF rates are similar to patients undergoing SABR alone. Thus, patients who would benefit most from the addition of vertebroplasty need to be further identified.


Assuntos
Dor do Câncer/radioterapia , Fraturas por Compressão/prevenção & controle , Radiocirurgia/métodos , Fraturas da Coluna Vertebral/prevenção & controle , Neoplasias da Coluna Vertebral/radioterapia , Neoplasias da Coluna Vertebral/cirurgia , Vertebroplastia , Adulto , Idoso , Terapia Combinada/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Estudos Prospectivos , Dosagem Radioterapêutica , Neoplasias da Coluna Vertebral/secundário , Fatores de Tempo , Resultado do Tratamento
3.
J Investig Med ; 67(2): 295-302, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30530528

RESUMO

Survivors of childhood brain tumors may be at risk for early onset of metabolic syndrome, possibly secondary to surgery and/or radiation exposure. This study examines effects of radiation exposure to hypothalamus-pituitary-adrenal axis (HPA) on metabolic risk among survivors of childhood brain tumors. One hundred forty-two met inclusion criteria; 60 had tumor surgery plus radiation exposure (>1 Gray (Gy)) to HPA. The second subgroup of 82 subjects had surgery only and were not exposed to radiation. Both subgroups had survived for approximately 5 years at the time of study. All had clinical evaluation, vital signs, anthropometry, measurement of body composition by dual X-ray absorptiometry and fasting laboratory assays (metabolic panel, insulin, C-peptide, insulin-like growth factor-1, leptin and adiponectin). Body composition data for both subgroups was compared with the National Health and Nutrition Survey (NHANES) subgroup of similar age, gender and body mass index. Cranial surgery was associated with obesity of similar severity in both subgroups. However, survivors exposed to radiation to the HPA also had increased visceral fat mass and high prevalence of growth hormone deficiency and metabolic syndrome. Fat mass alone did not explain the prevalence of the metabolic syndrome in radiation exposure subgroup. Other factors such as growth hormone deficiency may have contributed to metabolic risk. We conclude that prevalence of metabolic syndrome among subjects exposed to hypothalamic radiation was higher than expected from hypothalamic obesity alone. Radiation exposure may exert untoward endocrinopathies due to HPA exposure that worsens metabolic risk. Early screening for metabolic syndrome in this population is indicated.


Assuntos
Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/radioterapia , Sobreviventes de Câncer , Hipotálamo/patologia , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/etiologia , Obesidade/complicações , Exposição à Radiação/efeitos adversos , Adolescente , Composição Corporal , Criança , Feminino , Hormônio do Crescimento/uso terapêutico , Humanos , Masculino , Fenótipo , Fatores de Risco
4.
Int J Radiat Oncol Biol Phys ; 102(1): 210-218, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29891202

RESUMO

PURPOSE: Radiation injury to the bronchial tree is an important yet poorly understood potential side effect in lung stereotactic ablative radiation therapy (SAbR). We investigate the integration of virtual bronchoscopy in radiation therapy planning to quantify dosage to individual airways. We develop a risk model of airway collapse and develop treatment plans that reduce the risk of radiation-induced airway injury. METHODS AND MATERIALS: Pre- and post-SAbR diagnostic-quality computerized tomography (CT) scans were retrospectively collected from 26 lung cancer patients. From each scan, the bronchial tree was segmented using a virtual bronchoscopy system and registered deformably to the planning CT. Univariate and stepwise multivariate Cox regressions were performed, examining factors such as age, comorbidities, smoking pack years, airway diameter, and maximum point dosage (Dmax). Logistic regression was utilized to formulate a risk function of segmental collapse based on Dmax and diameter. The risk function was incorporated into the objective function along with clinical dosage volume constraints for planning target volume (PTV) and organs at risk (OARs). RESULTS: Univariate analysis showed that segmental diameter (P = .014) and Dmax (P = .007) were significantly correlated with airway segment collapse. Multivariate stepwise Cox regression showed that diameter (P = .015), Dmax (P < .0001), and pack/years of smoking (P = .02) were significant independent factors associated with collapse. Risk management-based plans enabled significant dosage reduction to individual airway segments while fulfilling clinical dosimetric objectives. CONCLUSION: To our knowledge, this is the first systematic investigation of functional avoidance in lung SAbR based on mapping and minimizing doses to individual bronchial segments. Our early results show that it is possible to substantially lower airway dosage. Such dosage reduction may potentially reduce the risk of radiation-induced airway injury, while satisfying clinically prescribed dosimetric objectives.


Assuntos
Broncoscopia , Pulmão/efeitos da radiação , Radiocirurgia/efeitos adversos , Planejamento da Radioterapia Assistida por Computador/métodos , Fatores Etários , Relação Dose-Resposta à Radiação , Feminino , Humanos , Pulmão/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/radioterapia , Masculino , Modelos Estatísticos , Estudos Retrospectivos , Risco , Fatores Sexuais , Tomografia Computadorizada por Raios X
5.
PLoS One ; 13(3): e0194378, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29529069

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0192164.].

6.
PLoS One ; 13(2): e0192164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29420571

RESUMO

Culture-independent methods were used to study the microbiota of adult worms, third-stage larvae and eggs, both in faeces and laid in vitro, of Haemonchus contortus, a nematode parasite of the abomasa of ruminants which is a major cause of production losses and ill-health. Bacteria were identified in eggs, the female reproductive tract and the gut of adult and third-stage larvae (L3). PCR amplification of 16S rRNA sequences, denaturing gradient gel electrophoresis (DGGE) and clone libraries were used to compare the composition of the microbial communities of the different life-cycle stages of the parasites, as well as parasites and their natural environments. The microbiomes of adult worms and L3 were different from those in the abomasum or faeces respectively. The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria, Firmicutes and Bacteroidetes. Bacteria were localised in the gut, inside eggs and within the uterus of adult female worms using the universal FISH Eub338 probe, which targets most bacteria, and were also seen in these tissues by light and transmission electron microscopy. Streptococcus/Lactococcus sp. were identified within the distal uterus with the probe Strc493. Sequences from the genera Weissella and Leuconostoc were found in all life-cycle stages, except eggs collected from faeces, in which most sequences belonged to Clostridium sp. Bacteria affiliated with Weissella/Leuconostoc were identified in both PCR-DGGE short sequences and clone libraries of nearly full length 16S rRNA bacterial sequences in all life-cycle stages and subsequently visualised in eggs by fluorescent in situ hybridisation (FISH) with group-specific probes. This strongly suggests they are vertically transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, other field isolates will need to be examined to establish whether these bacteria are more widely dispersed and have potential as targets to control H. contortus infections.


Assuntos
Trato Gastrointestinal/parasitologia , Haemonchus/isolamento & purificação , Ovinos/parasitologia , Animais , Feminino , Trato Gastrointestinal/microbiologia , Enteropatias Parasitárias/microbiologia , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/veterinária , Lactococcus/genética , Lactococcus/isolamento & purificação , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Ovinos/microbiologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/parasitologia , Streptococcus/genética , Streptococcus/isolamento & purificação
7.
Sci Rep ; 5: 15115, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26463440

RESUMO

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.


Assuntos
Adenoviridae/genética , Glândulas Mamárias Humanas/fisiologia , Proteínas do Leite/biossíntese , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Transdução Genética/métodos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Feminino , Humanos , Camundongos , Proteínas do Leite/genética , Coelhos
9.
Comp Biochem Physiol B Biochem Mol Biol ; 161(3): 255-60, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22155552

RESUMO

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD(+)/H or NADP(+)/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD(+)/H than with NADP(+)/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.


Assuntos
Glutamato Desidrogenase/metabolismo , Haemonchus/enzimologia , Nucleotídeos/farmacologia , Trichostrongyloidea/enzimologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutamato Desidrogenase/genética , Guanosina Trifosfato/farmacologia , Haemonchus/efeitos dos fármacos , Haemonchus/genética , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Trichostrongyloidea/efeitos dos fármacos
10.
Radiat Oncol ; 6: 146, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-22035405

RESUMO

BACKGROUND: The retroperitoneal margin is a common site of positive surgical margins in patients with resectable pancreatic cancer. Preoperative margin-intensive therapy (MIT) involves delivery of a single high dose of ablative radiotherapy (30 Gy) focused on this surgically inaccessible margin, utilizing stereotactic techniques in an effort to reduce local failure following surgery. In this study, we investigated the motion of regional organs at risk (OAR) utilizing 4DCT, evaluated the dosimetric effects of abdominal compression (AC) to reduce regional motion, and compared various planning techniques to optimize MIT. METHODS: 10 patients were evaluated with 4DCT scans. All 10 patients had scans using AC and seven of the 10 patients had scans both with and without AC. The peak respiratory abdominal organ and major vessel centroid excursion was measured. A "sub-GTV" region was defined by a radiation oncologist and surgical oncologist encompassing the retroperitoneal margin typically lateral and posterior to the superior mesenteric artery (SMA), and a 3-5 mm margin was added to constitute the PTV. Identical 3D non-coplanar SABR (3DSABR) plans were designed for the average compression and non-compression scans. Compression scans were planned with 3DSABR, coplanar IMRT (IMRT), and Cyberknife (CK) planning techniques. Dose volume analysis was undertaken for various endpoints, comparing OAR doses with and without AC and for different planning methods. RESULTS: The mean PTV size was 20.2 cm3. Regional vessel motion of the SMA, celiac trunk, and renal vessels was small (< 5 mm) and not significantly impacted by AC. Mean pancreatic motion was > 5 mm, so AC has been used in all patients enrolled thus far. AC did not significantly increase OAR dose including the stomach and traverse colon. There were several statistically significant differences in the doses to OARs as a function of the type of planning modality used. CONCLUSIONS: AC does not significantly reduce the limited motion of structures in close proximity to the MIT target and does not significantly increase the dose to OARs that can be displaced by the compression plate. The treatment planning techniques evaluated in this study have different advantages with no clearly superior method in our analysis. Dose to adjacent vessels may be reduced with 3DSABR or IMRT techniques, while conformality is increased with IMRT or CK.


Assuntos
Carcinoma Ductal Pancreático/cirurgia , Diagnóstico por Imagem/métodos , Movimento , Neoplasias Pancreáticas/cirurgia , Radiocirurgia/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Humanos , Tomografia Computadorizada por Raios X
11.
J Gene Med ; 12(4): 385-98, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20352617

RESUMO

BACKGROUND: The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy, there are unique aspects that need to be addressed in biodistribution studies. Notably, the spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events; thus, it is critical to evaluate the risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. METHODS: In the present study, we conducted a comprehensive assessment of vector biodistribution throughout the brain, blood and major organs of rats that had been injected via the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. RESULTS: AAV genomes were not detected in blood or cerebrospinal fluid, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average of 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral subthalamic nucleus (STN). There were no discernable effects of AAV2-GAD on general health, and a behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. CONCLUSIONS: The present study met Food and Drug Administration requirements, in addition to efficacy and toxicity studies in rodents and nonhuman primates, to support and supplement a Phase II clinical trial invloving the gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson's disease.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/farmacocinética , Doença de Parkinson/terapia , Subtálamo/metabolismo , Animais , Crioultramicrotomia , Primers do DNA/genética , DNA Viral/sangue , DNA Viral/líquido cefalorraquidiano , Dependovirus/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Glutamato Descarboxilase/metabolismo , Imuno-Histoquímica , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subtálamo/virologia
12.
J Neurosci ; 29(40): 12497-505, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19812325

RESUMO

Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5 (cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by which cdk5 promotes death is unclear. Complicating the role of cdk5 are the observations that cdk5 can exist in multiple cellular regions and possess both prosurvival and prodeath characteristics. In particular, the critical role of cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we determined where cdk5 was activated in models of ischemia and how manipulation of cdk5 in differing compartments may affect neuronal death. Here, we show a critical function for cytoplasmic cdk5 in both focal and global models of stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5 localized to the cytoplasm is protective. Importantly, we also demonstrate the antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent. Following focal ischemia, nuclear cdk5 is activated and functionally relevant while there is no evidence for such activation following global ischemia. Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear target of cdk5 in vitro, is also phosphorylated by cdk5 following focal ischemia. In addition, MEF2D expression in this paradigm ameliorates death. Together, our results address the critical issue of cdk5 activity compartmentalization, as well as define critical substrates for both cytoplasmic and nuclear cdk5 activity in adult models of stroke.


Assuntos
Isquemia Encefálica/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Citoplasma/metabolismo , Peroxirredoxinas/metabolismo , Animais , Isquemia Encefálica/etiologia , Morte Celular/fisiologia , Células Cultivadas , Cerebelo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Ratos , Ratos Wistar , Acidente Vascular Cerebral/complicações
13.
Mol Ther ; 17(10): 1692-702, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19638961

RESUMO

Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1-9, with efficient gene transfer to neurons only-selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated-widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology.


Assuntos
Encéfalo/citologia , Vetores Genéticos/genética , Neuroglia/virologia , Primatas/virologia , Transdução Genética/métodos , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley
14.
J Neurochem ; 110(1): 23-33, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19490361

RESUMO

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.


Assuntos
Diferenciação Celular/genética , Neurônios/enzimologia , Proteínas Proto-Oncogênicas c-akt/genética , Substância Negra/enzimologia , Substância Negra/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Apoptose/genética , Proliferação de Células , Tamanho Celular , Dopamina/metabolismo , Vetores Genéticos/genética , Cones de Crescimento/enzimologia , Cones de Crescimento/ultraestrutura , Imuno-Histoquímica , Masculino , Vias Neurais/citologia , Vias Neurais/enzimologia , Vias Neurais/crescimento & desenvolvimento , Neurogênese/genética , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia , Transdução Genética/métodos
15.
Neurotherapeutics ; 6(2): 300-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19332323

RESUMO

Gene therapy may represent a promising alternative treatment of epileptic patients who are resistant to conventional anti-epileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant adeno-associated viral (AAV) vectors have been most widely used. Preclinical studies using a selection of "therapeutic" genes injected into the rodent brain to correct the compromised balance between inhibitory and excitatory transmission in epilepsy, showed significant reduction of seizures and inhibition of epileptogenesis. In particular, transduction of neuropeptide genes, such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. Recent findings showed a long-lasting NPY over-expression in the rat hippocampus by local application of recombinant AAV vectors associated with reduced generalization of seizures, delayed kindling epileptogenesis, and strong reduction of chronic spontaneous seizures. These results establish a proof-of-principle evidence of the efficacy of gene therapy as anticonvulsant treatment. Additional investigations are required to address safety concerns and possible side effects in more detail.


Assuntos
Adenoviridae/genética , Epilepsia/terapia , Terapia Genética/métodos , Vetores Genéticos , Neuropeptídeo Y/metabolismo , Animais , DNA Recombinante , Humanos , Neuropeptídeo Y/genética
16.
Brain ; 131(Pt 6): 1506-15, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18477594

RESUMO

Temporal lobe epilepsy remains amongst the most common and drug refractory of neurological disorders. Gene therapy may provide a realistic therapeutic approach alternative to surgery for intractable focal epilepsies. To test this hypothesis, we applied here a gene therapy approach, using a recombinant adeno-associated viral (rAAV) vector expressing the human neuropeptide Y (NPY) gene, to a progressive and spontaneous seizure model of temporal lobe epilepsy induced by electrical stimulation of the temporal pole of the hippocampus, which replicates many features of the human condition. rAAV-NPY or a control vector lacking the expression cassette (rAAV-Empty) was delivered into the epileptic rat hippocampi at an early progressive stage of the disease. Chronic epileptic rats were video-EEG monitored to establish pre-injection baseline recordings of spontaneous seizures and the effect of rAAV-NPY versus rAAV-Empty vector injection. Both non-injected stimulated controls and rAAV-empty injected rats showed a similar progressive increase of spontaneous seizure frequency consistent with epileptogenesis. The delivery of rAAV-NPY in epileptic rat brain leads to a remarkable decrease in the progression of seizures as compared to both control groups and this effect was correlated with the NPY over-expression in the hippocampus. Moreover, spontaneous seizure frequency was significantly reduced in 40% of treated animals as compared to their pre-injection baseline. Our data show that this gene therapy strategy decreases spontaneous seizures and suppresses their progression in chronic epileptic rats, thus representing a promising new therapeutic strategy.


Assuntos
Epilepsia do Lobo Temporal/terapia , Terapia Genética/métodos , Neuropeptídeo Y/genética , Animais , Doença Crônica , Dependovirus/genética , Eletroencefalografia , Epilepsia do Lobo Temporal/metabolismo , Expressão Gênica , Engenharia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Hipocampo/química , Hipocampo/metabolismo , Injeções , Masculino , Neurônios/química , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/uso terapêutico , Ratos , Ratos Sprague-Dawley , Transdução Genética/métodos , Gravação em Vídeo
17.
Mol Ther ; 16(6): 1048-55, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18398426

RESUMO

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição/metabolismo , Animais , Comportamento Animal , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Dependovirus/metabolismo , Dopamina/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Modelos Biológicos , Neurônios/metabolismo , Septinas
18.
J Neurosci ; 28(3): 672-80, 2008 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-18199767

RESUMO

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model.


Assuntos
Apoptose , Zíper de Leucina , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/fisiologia , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Análise de Variância , Animais , Carbazóis/uso terapêutico , Dependovirus/fisiologia , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indóis/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Oligopeptídeos , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/tratamento farmacológico , Peptídeos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
19.
Proc Natl Acad Sci U S A ; 104(47): 18748-53, 2007 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-18003894

RESUMO

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinson's disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinson's disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Biomarcadores , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Células Cultivadas , Cisteína/genética , Cisteína/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Oxirredução , Estresse Oxidativo , Doença de Parkinson/genética , Peroxirredoxinas , Proteína Desglicase DJ-1 , Ratos , Sensibilidade e Especificidade , Acidente Vascular Cerebral/genética
20.
Mol Neurodegener ; 2: 11, 2007 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-17559680

RESUMO

BACKGROUND: Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-beta peptide (Abeta) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Abeta42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Abeta40, the more prevalent Abeta peptide secreted by cells and a major component of cerebral Abeta deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Abeta42 and Abeta40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. RESULTS: Adeno-associated viral (AAV) vectors encoding BRI-Abeta cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Abeta peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Abeta40 and AAV-BRI-Abeta42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Abeta peptides. BRI-Abeta42 and the combination of BRI-Abeta40+42 overexpression resulted in elevated levels of detergent-insoluble Abeta. No significant increase in detergent-insoluble Abeta was seen in the rats expressing APPsw or BRI-Abeta40. No pathological features were noted in any rats, except the AAV-BRI-Abeta42 rats which showed focal, amorphous, Thioflavin-negative Abeta42 deposits. CONCLUSION: The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Abeta42 alone is sufficient to initiate Abeta deposition, both Abeta40 and Abeta42 may contribute to cognitive deficits.

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