In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

Differential patterns of multisensory interactions in core and belt areas of human auditory cortex

The auditory cortex is anatomically segregated into a central core and a peripheral belt region, which exhibit differences in preference to bandpassed noise and in temporal patterns of response to acoustic stimuli. While it has been shown that visual stimuli can modify response magnitude in auditory cortex, little is known about differential patterns of multisensory interactions in core and belt. Here, we used functional magnetic resonance imaging and examined the influence of a short visual stimulus presented prior to acoustic stimulation on the spatial pattern of blood oxygen level-dependent signal response in auditory cortex. Consistent with crossmodal inhibition, the light produced a suppression of signal response in a cortical region corresponding to the core. In the surrounding areas corresponding to the belt regions, however, we found an inverse modulation with an increasing signal in centrifugal direction. Our data suggest that crossmodal effects are differentially modulated according to the hierarchical core-belt organization of auditory cortex.

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

Role of the NFKB1 -94ins/delATTG promoter polymorphism in IBD and potential interactions with polymorphisms in the CARD15/NOD2, IKBL, and IL-1RN genes

BACKGROUND: Recently, an association of the NFKB1 polymorphism -94ins/delATTG with ulcerative colitis (UC) has been reported. This 4-bp insertion/deletion polymorphism is localized in the promoter region of the NFKB1 gene and appears to be functionally relevant. The aim of the present study was to confirm the association of the -94ins/delATTG (W/D) NFKB1 promoter polymorphism with UC in a population of German origin and to test for a potential association with Crohn's disease (CD). Furthermore, potential interactions of the -94ins/delATTG polymorphism with the IKBL and the IL-1RN genes should be determined. MATERIALS AND METHODS: The study population comprised 630 patients with CD, 365 patients with UC, and 974 healthy controls. Genotyping was performed using polymerase chain reaction and restriction fragment length polymorphism analysis. For statistical evaluation, the chi-square test and the Fisher exact test were used. RESULTS: No significant association of the W/D NFKB1 polymorphism with CD or UC was detected. In addition, no significant interactions between the -94ins/delATTG NFKB1 polymorphism and polymorphisms within the IKBL and the IL-1RN genes, respectively, were found in CD or UC. Also, no significant interactions of the NFKB1 polymorphism with mutations of the CARD15/NOD2 gene and with clinical phenotypes were detected in CD. Moreover, no associations of the NFKB1 polymorphism were found in UC depending on disease localization. CONCLUSIONS: The present study could not confirm the reported association of the -94ins/delATTG NFKB1 polymorphism with UC and also found no evidence for a role of this polymorphism in CD. The results do not give evidence for a role of this NFKB1 polymorphism in the pathogenesis of UC and CD.

Clinical evaluation of root filled teeth restored with or without post-and-core systems in a specialist practice setting

AIM: To assess survival rates and complications of root-filled teeth restored with or without post-and-core systems over a mean observation period of >or=4 years. METHODOLOGY: A total of 325 single- and multirooted teeth in 183 subjects treated in a private practice were root filled and restored with either a cast post-and-core or with a prefabricated titanium post and composite core. Root-filled teeth without post-retained restorations served as controls. The restored teeth served as abutments for single unit metal-ceramic or composite crowns or fixed bridges. Teeth supporting cantilever bridges, overdentures or telescopic crowns were excluded. RESULTS: Seventeen teeth in 17 subjects were lost to follow-up (17/325: 5.2%). The mean observation period was 5.2 +/- 1.8 (SD) years for restorations with titanium posts, 6.2 +/- 2.0 (SD) years for cast post-and-cores and 4.4 +/- 1.7 (SD) years for teeth without posts. Overall, 54% of build-ups included the incorporation of a titanium post and 26.5% the cementation of a cast post-and-core. The remaining 19.5% of the teeth were restored without intraradicular retention. The adjusted 5-year tooth survival rate amounted to 92.5% for teeth restored with titanium posts, to 97.1% for teeth restored with cast post-and-cores and to 94.3% for teeth without post restorations, respectively. The most frequent complications included root fracture (6.2%), recurrent caries (1.9%), post-treatment periradicular disease (1.6%) and loss of retention (1.3%). CONCLUSION: Provided that high-quality root canal treatment and restorative protocols are implemented, high survival and low complication rates of single- and multirooted root-filled teeth used as abutments for fixed restorations can be expected after a mean observation period of >or=4 years.

PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle

BACKGROUND: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE). Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin. RESULTS: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds. CONCLUSION: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.