Evaluation of bone texture imaging parameters on panoramic radiographs of patients with Sheehan's syndrome: a STROBE-compliant case-control study.

Sheehan's syndrome (SHS) is a rare condition related to the risk of osteoporosis and evaluation of bone texture imaging features on panoramic radiographs would be suitable for this condition, which was the aim of the present study. Fractal dimension, lacunarity, and trabecular morphologic aspects were significantly altered in these patients. INTRODUCTION: SHS is an important public health problem particularly in developing countries. It is characterized as postpartum hypopituitarism secondary to obstetric complications-related ischemic pituitary necrosis that shows significant systemic metabolic repercussions. Thus, this study aimed to evaluate bone texture parameters in digital panoramic radiographs of patients with SHS. METHODS: A case-control study was conducted with 30 SHS patients from an Endocrinology and Diabetology Service of reference in Brazil, and 30 age- and sex-matched healthy controls. A custom computer program measured fractal dimension, lacunarity, and some morphologic features in the following mandibular regions of interest (50 × 50 pixels): below the mental foramen (F1), between the first and second molars (M1), and at the center of the mandibular ramus (R1). RESULTS: The fractal analysis showed a statistically significant difference between the studied groups in all regions of interest. The fractal dimension in F1 (p = 0.016), M1 (p = 0.043), and R1 (p = 0.028) was significantly lower in SHS group, as well as lacunarity in R1 (p = 0.008). Additionally, several morphologic features were statistically significant in the SHS group (p < 0.05). CONCLUSION: Therefore, individuals with SHS showed altered imaging texture parameters on panoramic radiographs, which reflect a smaller spatial organization of the bone trabeculae and, possibly, a state of reduced mineral bone density.

The Long and Winding Road: From the High-Affinity Choline Uptake Site to Clinical Trials for Malignant Brain Tumors.

Malignant brain tumors are one of the most lethal cancers. They originate from glial cells which infiltrate throughout the brain. Current standard of care involves surgical resection, radiotherapy, and chemotherapy; median survival is currently ~14-20 months postdiagnosis. Given that the brain immune system is deficient in priming systemic immune responses to glioma antigens, we proposed to reconstitute the brain immune system to achieve immunological priming from within the brain. Two adenoviral vectors are injected into the resection cavity or remaining tumor. One adenoviral vector expresses the HSV-1-derived thymidine kinase which converts ganciclovir into a compound only cytotoxic to dividing glioma cells. The second adenovirus expresses the cytokine fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L differentiates precursors into dendritic cells and acts as a chemokine that attracts dendritic cells to the brain. HSV-1/ganciclovir killing of tumor cells releases tumor antigens that are taken up by dendritic cells within the brain tumor microenvironment. Tumor killing also releases HMGB1, an endogenous TLR2 agonist that activates dendritic cells. HMGB1-activated dendritic cells, loaded with glioma antigens, migrate to cervical lymph nodes to stimulate a systemic CD8+ T cells cytotoxic immune response against glioma. This immune response is specific to glioma tumors, induces immunological memory, and does neither cause brain toxicity nor autoimmune responses. An IND was granted by the FDA on 4/7/2011. A Phase I, first in person trial, to test whether reengineering the brain immune system is potentially therapeutic is ongoing.

Ecocardiografia de cães da raça Yorkshire Terrier clinicamente normais / Echocardiography of clinically normal Yorkshire Terrier dogs

Determinaram-se os parâmetros ecocardiográficos em modo-M normais de cães da raça Yorkshire Terrier. Foram utilizados 30 cães clinicamente sadios, com peso médio de 2,42±0,64kg e idades entre um e seis anos. O diâmetro do átrio esquerdo e da aorta, a dimensão interna do ventrículo esquerdo na diástole e na sístole, a espessura do septo interventricular na diástole e na sístole, e a espessura da parede posterior do ventrículo esquerdo na diástole e na sístole correlacionaram-se com o peso corporal. As demais variáveis do modo-M não se correlacionaram com peso corporal, sexo ou idade. Os valores ecocardiográficos obtidos neste estudo podem ser utilizados como referência para cães dessa raça.

The aim of this study was to establish the normal echocardiographic parameters in M-mode for Yorkshire Terrier dogs. Thirty clinically normal dogs with mean weight of 2.42±0.64kg and ages varying from one to six years old were studied. The left atrial diameter, the aortic diameter, the left ventricular internal dimension at end - diastole and end - systole, end - diastolic and end - systolic interventricular septal thickness, and end - diastolic and end - systolic left ventricular posterior wall thickness had correlation with body weight. The other M - mode variables showed no linear correlations with body weight, sex or age. The echocardiographic values found in this study can be used as reference for this breed.

Avaliação radiográfica da silhueta cardíaca pelo método vertebral heart size em cães da raça Yorkshire Terrier clinicamente normais / Radiographic evaluation of the cardiac silhouette in clinically normal Yorkshire Terrier dogs through the vertebral heart size method

Determinou-se valor médio do vertebral heart size (VHS) em cães da raça Yorkshire Terrier. Foram selecionados 30 cães clinicamente normais, com média de peso de 2,42±0,64kg e idades entre um e seis anos. Os animais foram submetidos ao exame radiográfico do tórax nas projeções lateral direita, ventrodorsal e dorsoventral. Foram feitas mensurações para avaliação cardíaca e da profundidade e largura torácicas. O valor médio de VHS foi de 9,9±0,6 vértebras na projeção lateral, 10,1±0,6 vértebras na projeção ventrodorsal e 10,0±0,6 vértebras na projeção dorsoventral. Não houve diferença entre esses valores. Cinco animais (16,7 por cento) apresentaram VHS acima de 10,5 vértebras, valor sugerido como limite superior para a maioria das raças, em projeção lateral. Os valores de VHS correlacionaram-se com peso corporal nas projeções lateral e ventrodorsal. A razão profundidade:largura torácica apresentou valor médio de 0,75±0,06. Não foi observada correlação entre a qualidade do tórax e o VHS.

The mean vertebral heart size (VHS) was established for Yorkshire Terrier dogs. Thirty clinically normal dogs with mean weight of 2.42±0.64kg and ages varying from one to six years of age, were studied. The animals were submitted to right lateral, ventrodorsal and dorsoventral thoracic radiographs. The Buchanan e Bücheler method was applied to the cardiac silhouette and thoracic depth and width. The VHS was 9.9±0.6 vertebrae on lateral, 10.1±0.6 on ventrodorsal and 10.0±0.6 on dorsoventral radiographs. There was no difference among these values. Five animals (16.7 percent) presented VHS values exceeding 10.5, the value suggested as upper limit for most breeds in lateral view. However, 95 percent of the animals in this study provide VHS values below the upper limit, and this should be equal to 11 vertebrae. The VHS values had significant correlation with body weight on lateral and VD radiographs. The mean depth:width ratio was 0.75±0.06. There was no correlation among VHS and depth:width ratios.

ECG as a part of the preparticipation screening programme: an old and still present international dilemma.

INTRODUCTION: Long-term Italian experience has provided evidence that preparticipation screening in competitive athletes with 12-lead ECG, history and physical examination is effective in identifying potentially lethal cardiovascular diseases. However, it is not being routinely practised in other countries. OBJECTIVES: To evaluate the usefulness of a preparticipation screening programme in a sample of players belonging to different disciplines. MATERIAL AND METHODS: From September 2006 to June 2008, 1220 young athletes from different sports disciplines underwent a cardiovascular examination that included personal and family history, physical examination and a resting 12-lead ECG. Those with abnormal findings were referred for additional tests. RESULTS: 1220 Athletes were screened: 96% males; mean age 23 (4) years. 90 (7.4%) players were referred for additional tests because of abnormal findings on baseline examination: 11 (0.9%) personal or family history, 4 (0.08%) physical examination and 75 (6.14%) 12-lead ECG. Echocardiographic assessment fulfilled left ventricular hypertrophy criteria in 8 of the 90 players. Of those, one case was considered an athlete's heart and one case was diagnosed with hypertrophic cardiomyopathy (septal thickness 23 mm). Further tests were needed in the remaining six, included in the "grey area", with one additional case of hypertrophic cardiomyopathy (apical variant) suggested by cardiac MRI. CONCLUSION: Given the ability of 12-lead ECG to detect individuals with structural heart disease, we suggest its inclusion as a part of preparticipation screening programmes.

Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors.

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.

Kupfer-type immunological synapse characteristics do not predict anti-brain tumor cytolytic T-cell function in vivo.

To analyze the in vivo structure of antigen-specific immunological synapses during an effective immune response, we established brain tumors expressing the surrogate tumor antigen ovalbumin and labeled antigen-specific anti-glioma T cells using specific tetramers. Using these techniques, we determined that a significant number of antigen-specific T cells were localized to the brain tumor and surrounding brain tissue and a large percentage could be induced to express IFNgamma when exposed to the specific ovalbumin-derived peptide epitope SIINFEKL. Detailed morphological analysis of T cells immunoreactive for tetramers in direct physical contact with tumor cells expressing ovalbumin indicated that the interface between T cells and target tumor cells displayed various morphologies, including Kupfer-type immunological synapses. Quantitative analysis of adjacent confocal optical sections was performed to determine if the higher frequency of antigen-specific antiglioma T cells present in animals that developed an effective antitumor immune response could be correlated with a specific immunological synaptic morphology. Detailed in vivo quantitative analysis failed to detect an increased proportion of immunological synapses displaying the characteristic Kupfer-type morphology in animals mounting a strong and effective antitumor immune response as compared with those experiencing a clinically ineffective response. We conclude that an effective cytolytic immune response is not dependent on an increased frequency of Kupfer-type immunological synapses between T cells and tumor cells.

Study of the efficacy, biodistribution, and safety profile of therapeutic gutless adenovirus vectors as a prelude to a phase I clinical trial for glioblastoma.

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in approximately 70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials.

Estudo retrospectivo ecodopplercardiográfico das principais cardiopatias diagnosticadas em cães / Echodopplercardiographic retrospective study of the main cardiopathies diagnosed in dogs

A retrospective study was carried out to evaluate echodopplercardiographic examinations of 854 dogs referred to the service of echocardiography. This work presents the frequency of the main cardiopathies according to breed, gender, and presence of echocardiographic alterations. The valvular acquired disease was the most common cardiopathy (76.7%), mainly occurring in male dogs of small breeds. The dilated cardiomyopathy was the second more diagnosed cardiovascular pathology (9.8%), and most frequently noticed in male dogs of large or giant breeds. However, Cocker Spaniel breed also presented a high prevalence for this cardiopathy.

Herpes simplex virus type 1 thymidine kinase sequence fused to the lacz gene increases levels of {beta}-galactosidase activity per genome of high-capacity but not first-generation adenoviral vectors in vitro and in vivo.

Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3' end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.

PURPOSE: Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. METHODS: rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. RESULTS: By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. CONCLUSIONS: rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.

Screening of the endothelin1 gene (EDN1) in a cohort of patients with essential left ventricular hypertrophy.

Our objective was to analyse the role of endothelin1 gene (EDN1) variation in essential left ventricular hypertrophy (LVH). We searched for EDN1 variants in 145 Spanish patients with an essential form of LVH (not secondary to hypertension, aortic stenosis, or any other disease that could explain the hypertrophy). The five EDN1 coding exons and 1.5 kilobases of the promoter region were analysed through single strand conformation analysis and direct sequencing. We found four nucleotide changes: -1224 C/A (promoter), -131 ins/del A (exon 1, 5'-non-translated sequence), A/G in codon 106 (exon 3, silent), and G/T in codon 198 (exon 5, lys198asn). To determine the association between these polymorphisms and cardiac hypertrophy, we compared the genotype frequencies from these 145 patients with 250 healthy controls. We found a higher frequency of patients homozygous for 198 lys (198 KK) (65% vs. 52%; p = 0.01; OR = 1.76) and for -1224 AA (73% vs. 66%; p = 0.19). Homozygotes for -1224 A + 198 K (AA+KK) were significantly more frequent in patients (62% vs. 45%; p = 0.0007; OR = 2.10; 95% CI = 1.35-3.25). The expression of the -1224 C/A and exon 5 K198N variants was analysed with cells in culture. These in vitro studies showed that these variations did not differ in their expression levels. In conclusion, our work has shown that EDN1 variation, and in particular homozygosity for the -1224A/198K haplotype, is associated with the risk of developing cardiac hypertrophy. However, these EDN1 variants do not affect in vitro gene expression.

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.

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.

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.

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.

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.

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.

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.

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.