PubMed Health⌕ Search

Biomedical subjects

M G Castro

Publications and source records attributed to M G Castro.

At least 19 recordsLinked to original sources
PubMed2006

Quantification of high-capacity helper-dependent adenoviral vector genomes in vitro and in vivo, using quantitative TaqMan real-time polymerase chain reaction.

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.

Adenoviridae↗
PubMed2006

Adenoviral vectors encoding tumor necrosis factor-alpha and FasL induce apoptosis of normal and tumoral anterior pituitary cells.

Our previous work showed that tumor necrosis factor (TNF)-alpha and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-alpha, FasL or, as a control, beta-galactosidase (beta-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF-alpha and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF-alpha or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-alpha, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF-alpha or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF-alpha or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF-alpha or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of beta-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas.

Adenoviridae↗
PubMed2005

Delivery of sonic hedgehog or glial derived neurotrophic factor to dopamine-rich grafts in a rat model of Parkinson's disease using adenoviral vectors Increased yield of dopamine cells is dependent on embryonic donor age.

The poor survival of dopamine grafts in Parkinson's disease is one of the main obstacles to the widespread application of this therapy. One hypothesis is that implanted neurons, once removed from the embryonic environment, lack the differentiation factors needed to develop the dopaminergic phenotype. In an effort to improve the numbers of dopamine neurons surviving in the grafts, we have investigated the potential of adenoviral vectors to deliver the differentiation factor sonic hedgehog or the glial cell line-derived neurotrophic factor GDNF to dopamine-rich grafts in a rat model of Parkinson's disease. Adenoviral vectors containing sonic hedgehog, GDNF, or the marker gene LacZ were injected into the dopamine depleted striatum of hemiparkinsonian rats. Two weeks later, ventral mesencephalic cell suspensions were prepared from embryos of donor ages E12, E13, E14 or E15 and implanted into the vector-transduced striatum. Pre-treatment with the sonic hedgehog vector produced a three-fold increase in the numbers of tyrosine hydroxylase-positive (presumed dopaminergic) cells in grafts derived from E12 donors, but had no effect on E13-E15 grafts. By contrast, pre-treatment with the GDNF vector increased yields of dopamine cells in grafts derived from E14 and E15 donors but had no effect on grafts from younger donors. The results indicate that provision of both trophic and differentiation factors can enhance the yields of dopamine neurons in ventral mesencephalic grafts, but that the two factors differ in the age and stage of embryonic development at which they have maximal effects.

Adenoviridae↗
PubMed2005

Estrogens up-regulate the Fas/FasL apoptotic pathway in lactotropes.

The Fas/FasL system provides the major apoptotic mechanism for many cell types, participating in cell turnover in hormone-dependent tissues. In the present study, we localized both Fas and FasL in anterior pituitary cells, mainly in lactotropes and somatotropes. The percentage of anterior pituitary cells showing immunoreactivity for Fas or FasL was higher in cells from rats killed in proestrus than in diestrus. Also, the proportion of pituitary cells from ovariectomized (OVX) rats expressing Fas or FasL increased in the presence of 17beta-estradiol (10(-9) M). This steroid increased the percentage of lactotropes with immunoreactivity for Fas or FasL and the percentage of somatotropes expressing Fas. Activation of Fas by an agonist anti-Fas antibody (Mab-Fas) decreased the vi-ability-3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay)-of anterior pituitary cells from OVX rats cultured in the presence of 17beta-estradiol. Also, membrane-bound FasL decreased cell viability-[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (MTS assay)-only when anterior pituitary cells from OVX rats were incubated with 17beta-estradiol. Moreover, FasL increased the percentage of hypodiploid anterior pituitary cells (flow cytometry). Mab-Fas increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive pituitary cells and lactotropes from OVX rats only when cells were incubated in the presence of 17beta-estradiol. Also, Mab-Fas triggered apoptosis of anterior pituitary cells from rats killed at proestrus but not at diestrus. Our results show that 17beta-estradiol up-regulates the expression of the Fas/FasL system in anterior pituitary cells and increases Fas-induced apoptosis in lactotropes, suggesting that Fas-induced apoptosis could be involved in the pituitary cell renewal during the estrous cycle.

Animals↗
PubMed2005

In vivo transgene expression from an adenoviral vector is altered following a 6-OHDA lesion of the dopamine system.

We have investigated the in vivo dynamics of an adenovirus-based, LacZ expressing vector, RAd36, at different doses, when injected unilaterally into the corpus striatum of normal rats. We have further investigated the characteristics of this vector in the presence of a 6-OHDA lesion of the nigrostriatal pathway. The dopamine-depleting lesion had an effect on both the number and the distribution of cells transduced by the adenoviral vector. The lesioned side of the brain contained significantly greater numbers of beta-galactosidase positive cells than the unlesioned side at 3 days, 1 week and 4 weeks post-injection and the distribution of transduced cells was altered by the presence of a dopamine lesion. We conclude that the increased levels of transgene expression seen in the lesioned hemisphere are due to a change in the diffusion characteristics of the injected vector in the lesioned hemisphere. These results indicate that, when investigating the use of virus-based vectors, ultimately for use in gene therapies in the CNS, the in vivo dynamics of the vector need to be assessed not only in the normal brain, but also in the pathological brain state such as animal models of target diseases.

Adenoviridae↗
PubMed2005

Regulatable gene expression systems for gene therapy applications: progress and future challenges.

Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life of a few hours (as opposed to minutes or days) so that when withdrawn or added (depending on the regulatable system used) gene expression can be turned "on" or "off" quickly and effectively. Also, the genetic switches employed should ideally be nonimmunogenic in the host. The ability to switch transgenes on and off would be of paramount importance not only when the therapy is no longer needed, but also in the case of the development of adverse side effects to the therapy. Many regulatable systems are currently under development and some, i.e., the tetracycline-dependent transcriptional switch, have been used successfully for in vivo preclinical applications. Despite this, there are no examples of switches that have been employed in a human clinical trial. In this review, we aim to highlight the main regulatable systems currently under development, the gene transfer systems employed for their expression, and also the preclinical models in which they have been used successfully. We also discuss the substantial challenges that still remain before these regulatable switches can be employed in the clinical setting.

Gene Expression Regulation↗
PubMed2004

Progesterone antagonizes the permissive action of estradiol on tumor necrosis factor-alpha-induced apoptosis of anterior pituitary cells.

We previously reported that TNF-alpha-induced apoptosis of lactotropes is estrogen dependent and predominant at proestrus. Here we observed that TNF-alpha (50 ng/ml) failed to induce apoptosis of anterior pituitary cells from ovariectomized rats cultured in the presence of progesterone (10(-6) m). However, progesterone blocked the apoptotic effect of TNF-alpha in anterior pituitary cells and lactotropes cultured with 17beta-estradiol (10(-9) m). In addition, 17beta-estradiol induced apoptosis of somatotropes and triggered the proapoptotic action of TNF-alpha in these cells, effects completely blocked by ICI 182 780 (10(-6) m), an estrogen receptor antagonist. Progesterone reverted the permissive effect of 17beta-estradiol on TNF-alpha-induced apoptosis of somatotropes. TNF-alpha induced apoptosis of somatotropes from rats killed at proestrus but not at diestrus. The antiprogestine ZK 98,299 (10(-6) m) completely inhibited the protective action of progesterone on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Although progesterone can interact with glucocorticoid receptors, dexamethasone (10(-6) m) had no effect on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Our results show that progesterone, by interacting with progesterone receptors, antagonizes the permissive action of estrogens on TNF-alpha-induced apoptosis of lactotropes and somatotropes. These observations suggest that the steroid milieu may modulate the apoptotic response of anterior pituitary cells during the estrous cycle.

Animals↗
PubMed2004

Estrogens sensitize anterior pituitary gland to apoptosis.

Tissue homeostasis results from a balance between cell proliferation and cell death by apoptosis. Estradiol affects proliferation as well as apoptosis in hormone-dependent tissues. In the present study, we investigated the apoptotic response of the anterior pituitary gland to lipopolysaccharide (LPS) in cycling female rats, and the influence of estradiol in this response in ovariectomized (OVX) rats. The OVX rats were chronically estrogenized with implanted Silastic capsules containing 1 mg of 17beta-estradiol (E2). Cycling or OVX and E2-treated rats were injected with LPS (250 microg/rat ip). Apoptosis was determined by the terminal deoxynucleotidyl-mediated dUTP nick-end labeling (TUNEL) method in sections of the anterior pituitary gland and spleen. Chronic estrogenization induced apoptosis in the anterior pituitary gland. Acute endotoxemia triggered apoptosis of cells in the anterior pituitary gland of E2-treated rats but not of OVX rats. No differences were observed in the apoptotic response to LPS in spleen between OVX and E2-treated rats. The apoptotic response of the anterior pituitary to LPS was variable along the estrous cycle, being higher at proestrus than at estrus or diestrus I. Approximately 75% of the apoptotic cells were identified as lactotropes by immunofluorescence. In conclusion, our results indicate that estradiol induces apoptosis and enables the proapoptotic action of LPS in the anterior pituitary gland. Also, our study suggests that estrogens may be involved in anterior pituitary cell renewal during the estrous cycle, sensitizing lactotropes to proapoptotic stimuli.

Animals↗
PubMed2004

Differentiation and transcription factor gene therapy in experimental parkinson's disease: sonic hedgehog and Gli-1, but not Nurr-1, protect nigrostriatal cell bodies from 6-OHDA-induced neurodegeneration.

We tested the activity of the dopaminergic neuron differentiation factor sonic hedgehog, its downstream transcription factor target Gli-1, and an orphan nuclear receptor, Nurr-1, necessary for the induction of the dopaminergic phenotype of nigrostriatal neurons, in an in vivo model of nigrostriatal neurodegeneration. Our preliminary experiments demonstrated that all three constructs expressed the proper molecules and that these had the predicted biological activities in vitro. We expressed the N-terminal of sonic hedgehog (ShhN) and the Gli-1 and Nurr-1 entire coding regions from highly purified, and quality controlled, replication-defective adenoviral vectors injected into the brains of rats and used the dopaminergic growth factor GDNF as a positive control. The neurotoxin 6-hydroxydopamine was used to lesion the nigrostriatal dopaminergic innervation; RAd-ShhN and RAd-Gli-1 protected dopaminergic neuronal cell bodies in the substantia nigra, but not axonal terminals in the striatum, from 6-OHDA-induced cell death, while RAd-Nurr-1 was ineffective in protecting either cell bodies or axons. RAd-GDNF was able to protect both the dopaminergic cell bodies and the striatal axon terminals. Our results establish for the first time, to the best of our knowledge, that gene transfer of ShhN and one of its target transcription factors can selectively protect dopaminergic nigrostriatal neuronal cell bodies from a specific neurotoxic insult. Selective protection of nigrostriatal dopaminergic cell bodies by the differentiation factor ShhN and the transcription factor Gli-1 was achieved in a neurotoxic model that eliminates more than 70% of the nigral neurons under consideration. Differentiation and transcription factors can thus be used for the treatment of neurodegeneration by gene therapy.

Animals↗
PubMed2004

Neuronal expression of the transcription factor Gli1 using the Talpha1 alpha-tubulin promoter is neuroprotective in an experimental model of Parkinson's disease.

Nigrostriatal neurons degenerate during Parkinson's disease. Experimentally, neurotoxins such as 6-hydroxydopamine (6-OHDA) in rodents, and MPTP in mice and non-human primates, are used to model the disease-induced degeneration of midbrain dopaminergic neurons. Glial-cell-derived neurotrophic factor (GDNF) is a very powerful neuroprotector of dopaminergic neurons in all species examined. However, recent reports have indicated the possibility that GDNF may, in the long term and if expressed in an unregulated manner, exert untoward effects on midbrain dopaminergic neuronal structure and function. Although GDNF remains a powerful neurotrophin, the search for alternative therapies based on alternative and complementary mechanisms of action to GDNF is warranted. Recently, recombinant adenovirus-derived vectors encoding the differentiation factor Sonic Hedgehog (Shh) and its downstream transcriptional activator (Gli1) were shown to protect dopaminergic neurons in the substantia nigra pars compacta from 6-OHDA-induced neurotoxicity in rats in vivo. A pancellular human CMV (hCMV) promoter was used to drive the expression of both Shh and Gli1. Since Gli1 is a transcription factor and therefore exerts its actions intracellularly, we decided to test whether expression of Gli1 within neurons would be effective for neuroprotection. We demonstrate that neuronal-specific expression of Gli1 using the neuron-specific Talpha1 alpha-tubulin (Talpha1) promoter was neuroprotective, and its efficiency was comparable to the pancellular strong viral hCMV promoter. These results suggest that expression of the transcription factor Gli1 solely within neurons is neuroprotective for dopaminergic neurons in vivo and, furthermore, that neuronal-specific promoters are effective within the context of adenovirus-mediated gene therapy-induced neuroprotection of dopaminergic midbrain neurons. Since cell-type specific promoters are known to be weaker than the viral hCMV promoter, our data demonstrate that neuronal-specific expression of transcription factors is an effective, specific, and sufficient targeted approach for neurological gene therapy applications, potentially minimizing side effects due to unrestricted promiscuous gene expression within target tissues.

Animals↗
PubMed2003

Current and future strategies for the treatment of malignant brain tumors.

Glioblastoma (GB) is the most common subtype of primary brain tumor in adults. These tumors are highly invasive, very aggressive, and often infiltrate critical neurological areas within the brain. The mean survival time after diagnosis of GB has remained unchanged during the last few decades, in spite of advances in surgical techniques, radiotherapy, and also chemotherapy; patients' survival ranges from 9 to 12 months after initial diagnosis. In the same time frame, with our increasing understanding and knowledge of the physiopathology of several cancers, meaningful advances have been made in the treatment and control of several cancers, such as breast, prostate, and hematopoietic malignancies. Although a number of the genetic lesions present in GB have been elucidated and our understanding of the progressions of this cancer has increased dramatically over the last few years, it has not yet been possible to harness this information towards developing effective cures. In this review, we will focus on the classical ways in which GB is currently being treated, and will introduce a novel therapeutic modality, i.e., gene therapy, which we believe will be used in combination with classical treatment strategies to prolong the life-span of patients and to ultimately be able to control and/or cure these brain tumors. We will discuss the use of several vector systems that are needed to introduce the therapeutic genes within either the tumor mass, if these are not resectable, or the tumor bed, after successful tumor resection. We also discuss different therapeutic modalities that could be exploited using gene therapy, i.e., conditional cytotoxic approach, direct cytotoxicity, immunotherapy, inhibition of angiogenesis, and the use of pro-apoptotic genes. The advantages and disadvantages of each of the current vector systems available to transfer genes into the CNS are also discussed. With the advances in molecular techniques, both towards the elucidation of the physiopathology of GB and the development of novel, more efficient and less toxic vectors to deliver putative therapeutic genes into the CNS, it should be possible to develop new rationale and effective therapeutic approaches to treat this devastating cancer.

Animals↗
PubMed2003

Environmental assessment of agriculture at a regional scale in the Pampas of Argentina.

Governments need good information to design policies. However, in the Argentine Pampas there are neither sufficient knowledge on environmental issues, nor clear perception of environmental alterations across space and time. The general objective of this work was to provide decision makers with a scientifically sound set of indicators aiming at the assessment of current status and future trends in the rural environment of this sensitive region. As driving criteria to select indicators, we assumed that they had to be sound, simple to calculate, easy to understand, and easily applicable by decision makers. They are related closely to significant ecological structures and functions. Twelve basic indicators were identified: (1) land use, (2) fossil energy use, (3) fossil energy use efficiency, (4) nitrogen (N) balance, (5) phosphorus (P) balance, (6) nitrogen contamination risk, (7) phosphorus contamination risk, (8) pesticide contamination, (9) soil erosion risk, (10) habitat intervention, (11) changes in soil carbon stock, and (12) balance of greenhouse gases. Indicators were geographically referenced using a geographic information system (GIS). The strength of this study is not in the absolute value of environmental indicators, but rather in the conceptualization of indicator and the identification of changing patterns, gradients and trends in space and time. According to our results, we can not definitely say that agriculture in the Pampas, as a whole, tends to be sustainable or not. While some indicators tend to improve, others keep stable, and the rest worsen. The relative importance among indicators must also be considered. The indicators that showed a negative net change are key to the identification of critical problems that will require special attention in the close future.

Agriculture↗
PubMed2003

A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer.

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of beta-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Animals↗
PubMed2003

Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells.

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

Adenoviridae↗
PubMed2002

Molecular indetermination in the transition to error catastrophe: systematic elimination of lymphocytic choriomeningitis virus through mutagenesis does not correlate linearly with large increases in mutant spectrum complexity.

Studies with several RNA viruses have shown that enhanced mutagenesis resulted in decreases of infectivity or virus extinction, as predicted from virus entry into error catastrophe. Here we report that lymphocytic choriomeningitis virus, the prototype arenavirus, is extremely susceptible to extinction mutagenesis by the base analog 5-fluorouracil. Virus elimination was preceded by increases in complexity of the mutant spectra of treated populations. However, careful molecular comparison of the mutant spectra of several genomic segments suggests that the largest increases in mutation frequency do not predict virus extinction. Highly mutated viral genomes have escaped detection presumably because lymphocytic choriomeningitis virus replicates at or near the error threshold, and genomes in the transition toward error catastrophe may have an extremely short half-life and escape detection with state-of-the-art cloning and sequencing technologies.

Animals↗
PubMed2002

Expression of prolactin receptors and regulation of cell proliferation by prolactin, corticotropin-releasing factor, and corticosterone in a neuroblastoma cell line.

The aetiology of neuroblastoma remains obscure, although a number of neuropeptides have been implicated in its pathogenesis. Using the mouse neuroblastoma cell line Neuro2a as a model, we have investigated the mitogenic actions of prolactin (PRL) and two hypothalamo-pituitary-adrenal stress axis hormones, corticotropin-releasing factor (CRF) and corticosterone. Using established polyclonal PRL receptor antisera with immunofluorescence cytochemistry, we show that the Neuro2a cells possess immunoreactive forms of both the long and short forms of the receptor. PRL and CRF were effective as mitogens in Neuro2a cell cultures, where a 10(-7) M concentration of PRL or CRF elicited a two-fold increase in the numbers of cells after 72 h (p < 0.0001). Corticosterone, however, attenuated their proliferation. These data suggest that prolactin may act to increase the proliferation and regulation of neuroblastomas and that the effects of PRL may be modified by hypothalamo-pituitary-adrenal hormones.

Animals↗