A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

Management of cutaneous metastases using electrochemotherapy

Background. Cutaneous metastases may cause considerable discomfort as a consequence of ulceration, oozing, bleeding and pain. Electrochemotherapy has proven to be highly effective in the treatment of cutaneous metastases. Electrochemotherapy utilises pulses of electricity to increase the permeability of the cell membrane and thereby augment the effect of chemotherapy. For the drug bleomycin, the effect is enhanced several hundred-fold, enabling once-only treatment. The primary endpoint of this study is to evaluate the efficacy of electrochemotherapy as a palliative treatment. Methods. This phase II study is a collaboration between two centres, one in Denmark and the other in the UK. Patients with cutaneous metastases of any histology were included. Bleomycin was administered intratumourally or intravenously followed by application of electric pulses to the tumour site. Results. Fifty-two patients were included. Complete and partial response rate was 68% and 18%, respectively, for cutaneous metastases <3 cm and 8% and 23%, respectively, for cutaneous metastases >3 cm. Treatment was well-tolerated by patients, including the elderly, and no serious adverse events were observed. Conclusions. ECT is an efficient and safe treatment and clinicians should not hesitate to use it even in the elderly

Adaptive Strategy for the Statistical Analysis of Connectomes

We study an adaptive statistical approach to analyze brain networks represented by brain connection matrices of interregional connectivity (connectomes). Our approach is at a middle level between a global analysis and single connections analysis by considering subnetworks of the global brain network. These subnetworks represent either the inter-connectivity between two brain anatomical regions or by the intra-connectivity within the same brain anatomical region. An appropriate summary statistic, that characterizes a meaningful feature of the subnetwork, is evaluated. Based on this summary statistic, a statistical test is performed to derive the corresponding p-value. The reformulation of the problem in this way reduces the number of statistical tests in an orderly fashion based on our understanding of the problem. Considering the global testing problem, the p-values are corrected to control the rate of false discoveries. Finally, the procedure is followed by a local investigation within the significant subnetworks. We contrast this strategy with the one based on the individual measures in terms of power. We show that this strategy has a great potential, in particular in cases where the subnetworks are well defined and the summary statistics are properly chosen. As an application example, we compare structural brain connection matrices of two groups of subjects with a 22q11.2 deletion syndrome, distinguished by their IQ scores

Suicidal tendencies and attitude towards freedom to choose suicide among Lithuanian schoolchildren: results from three cross-sectional studies in 1994, 1998, and 2002

BACKGROUND: Suicidal behaviour is increasingly becoming a phenomenon associated with young people and an important public health issue in Lithuania. However, there are very few studies evaluating impact of young peoples' attitudes towards suicide to their suicidal behaviour. A better understanding of the relations among the variables associated with suicidal ideation and threats in the normal population of adolescents may eventually result in a better understanding of the more serious forms of adolescent suicidal behaviour. The aim of the present study was to evaluate prevalence of suicidal tendencies among Lithuanian schoolchildren and to estimate its association with an attitude towards suicide in 1994 – 2002. METHODS: Three country representative samples of schoolchildren, aged 11, 13 and 15, were surveyed in 1994 (n = 5428), 1998 (n = 4513), and 2002 (n = 5645) anonymously in conformity with the methodology of the World Health Organization Cross – National study on Health Behaviour in School-aged Children (HBSC). RESULTS: About one third of respondents reported about suicidal ideation, plans or attempts to commit suicide. In the study period of eight years, the percentage of adolescents who reported sometime suicidal ideation decreased but the percentage of adolescents who declared serious suicidal behaviour remained on the same high level (8.1%, 9.8% and 8.4% correspondingly in 1994, 1998 and 2002). Moreover, the number of suicidal attempts changed from 1.0% in 1994 to 1.8% in the year 1998 and to 1,7% in the year 2002. The schoolchildren's attitude towards suicide became more agreeable: 36.6%, 41.9% and 62.5% of respondents, correspondingly in 1994, 1998 and 2002, answered that they agree with a person's freedom to make a choice between life and suicide. A multiple logistic regression analysis with low level of suicidality and high level of suicidality versus non suicidal behaviour as dependent variables for gender, age, year of the survey and attitude towards freedom to choose suicide as independent variables approved a significant association between studied covariates over the entire study period. CONCLUSION: Suicidal tendencies are quite frequent among Lithuanian adolescents. An increasing number of schoolchildren are expressing an agreeable attitude towards suicide. The approving attitude towards suicide among adolescents correlates with suicidal ideation and behaviour

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder

<p>Abstract</p> <p>Background</p> <p>Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients.</p> <p>Methods</p> <p>We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA).</p> <p>Results</p> <p>No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients.</p> <p>Conclusions</p> <p>Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD.</p

Visuospatial working memory in children and adolescents with 22q11.2 deletion syndrome; an fMRI study

22q11.2 deletion syndrome (22q11DS) is a genetic disorder associated with a microdeletion of chromosome 22q11. In addition to high rates of neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder, children with 22q11DS have a specific neuropsychological profile with particular deficits in visuospatial and working memory. However, the neurobiological substrate underlying these deficits is poorly understood. We investigated brain function during a visuospatial working memory (SWM) task in eight children with 22q11DS and 13 healthy controls, using fMRI. Both groups showed task-related activation in dorsolateral prefrontal cortex (DLPFC) and bilateral parietal association cortices. Controls activated parietal and occipital regions significantly more than those with 22q11DS but there was no significant between-group difference in DLPFC. In addition, while controls had a significant age-related increase in the activation of posterior brain regions and an age-related decrease in anterior regions, the 22q11DS children showed the opposite pattern. Genetically determined differences in the development of specific brain systems may underpin the cognitive deficits in 22q11DS, and may contribute to the later development of neuropsychiatric disorders

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after geneelectrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest geneelectrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient

Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice

BACKGROUND: Bleomycin is poorly permeant but potent cytotoxic and radiosensitizing drug. The aim of the study was to evaluate whether a physical drug delivery system – electroporation can increase radiosensitising effect of bleomycin in vitro and in vivo. METHODS: LPB sarcoma cells and tumors were treated either with bleomycin, electroporation or ionizing radiation, and combination of these treatments. In vitro, response to different treatments was determined by colony forming assay, while in vivo, treatment effectiveness was determined by local tumor control (TCD(50)). Time dependence of partial oxygen pressure in LPB tumors after application of electric pulses was measured by electron paramagnetic oxyimetry. RESULTS: Electroporation of cells in vitro increased radiosensitising effect of bleomycin for 1.5 times, in vivo radiation response of tumors was enhanced by 1.9 fold compared to response of tumors that were irradiated only. Neither treatment of tumors with bleomycin nor application of electric pulses only, affected radiation response of tumors. Application of electric pulses to the tumors induced profound but transient reduction of tumor oxygenation. Although tumor oxygenation after electroporation partially restored at the time of irradiation, it was still reduced at the level of radiobiologically relevant hypoxia. CONCLUSION: Our study shows that application of electric pulses to cells and tumors increases radiosensitising effect of bleomycin. Furthermore, our results demonstrate that the radiobiologically relevant hypoxia induced by electroporation of tumors did not counteract the pronounced radiosensitising effect of electrochemotherapy with bleomycin