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.

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.

Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.

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.

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.

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.

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.

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.

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.

Adenovirus-mediated gene transfer of a secreted transforming growth factor-beta type II receptor inhibits luminal loss and constrictive remodeling after coronary angioplasty and enhances adventitial collagen deposition.

BACKGROUND: Extracellular matrix (ECM) remodeling is central to the development of restenosis after coronary angioplasty (PTCA). As a regulator of ECM deposition by vascular cells, substantial evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of restenosis. We investigated the effects of intracoronary expression of a transgenic antagonist of TGF-beta1 on luminal loss after PTCA. METHODS AND RESULTS: Porcine coronary arteries were randomized to receive a recombinant adenovirus expressing a secreted form of TGF-beta type II receptor (Ad5-RIIs), an adenovirus expressing beta-galactosidase (Ad5-lacZ), or vehicle only by intramural injection at the site of PTCA. Computerized morphometry 28 days after angioplasty revealed a greater minimum luminal area in Ad5-RIIs-injected arteries (1.71+/-0.12 mm(2)) than in the Ad5-lacZ (1.33+/-0.13 mm(2)) or vehicle-only (1.08+/-0.17 mm(2); P=0.010 by ANOVA) groups. This was accompanied by greater areas within the internal (P=0.013) and external (P=0.031) elastic laminae in Ad5-RIIs-treated vessels. Adventitial collagen content at the site of injury was increased in the Ad5-RIIs group, in contrast to decreases in the Ad5-lacZ and vehicle-only groups (P=0.004). CONCLUSIONS: Adenovirus-mediated antagonism of TGF-beta1 at the site of PTCA reduces luminal loss after PTCA by inhibiting constrictive remodeling. Antagonism of TGF-beta1 stimulates the formation of a dense collagenous adventitia, which prevents constrictive remodeling by acting as an external scaffold. These findings demonstrate the potential of gene therapy-mediated antagonism of TGF-beta1 as prophylactic therapy for restenosis.

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.

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.

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.

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.

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.

Molecular therapy in a model neuroendocrine disease: developing clinical gene therapy for pituitary tumours.

The main objectives of pituitary tumour treatment are to restore normal function of the pituitary gland and prevent tumour recurrences. In spite of the success of current therapies in the treatment of relatively small tumours, new therapeutic alternatives need to be explored for large invasive tumours, tumour recurrences postsurgery, and when intolerance to drug treatment develops. Gene therapy, which uses nucleic acids as drugs, is a very attractive alternative to classic therapeutic modalities. With the development of efficient gene delivery vectors, which allow widespread distribution and long-term transgene expression with limited side effects, the clinical implementation of gene therapy for the treatment of pituitary tumours will become a reality within the next five to ten years.

Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.

The utility of first-generation adenovirus vectors for long-term gene transfer in humans is limited by preexisting antiadenoviral immunity. We demonstrate here that new-generation high-capacity adenovirus vectors (HC-Ads) can efficiently transduce the brain and mediate stable transgene expression for at least 2 months, even in the presence of a preexisting antiadenoviral immune response. First-generation vector-mediated transduction was almost completely abolished in preimmunized animals within 60 days of the vector injection. Levels of HC-Ad-mediated transduction by 3 days postinjection were not significantly affected by preimmunization, were reduced within 14 days to 56% of those levels seen in nonimmunized animals, and remained stable until day 60 postinjection. Acute brain inflammation elicited by the HC-Ad vector injection was more transient, and was reduced in intensity compared with brain inflammation elicited by the first-generation vector injection in immunized animals. Inflammation was significantly higher in all immunized animals than in nonimmunized animals. Our results show that preexisting antiadenoviral immunity does not significantly reduce initial HC-Ad-mediated infection of the brain and is not a barrier to stable HC-Ad vector-mediated transduction of the CNS. Although input HC-Ad capsid proteins injected into the brain may contain transient targets for a brain-infiltrating cellular adenovirus-specific immune response, this fails to eliminate transgene expression. Thus HC-Ads show promise for gene therapy of chronic brain disease.

Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand.

Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.