Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

Background

Intrathecal (IT) gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks) impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF) modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV), one of the most promising vector types for clinical use.

Results

Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV) vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP) for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots.

Conclusion

sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2. Experiments presented here will provide a rational basis for utilizing IT rAAV gene transfer in basic and translational studies on chronic pain.     

High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction  

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.     

Novel micropropagation and weaning methods for the integrated conservation of a critically endangered tree species, Medusagyne oppositifolia

Medusagyne oppositifolia is a critically endangered monotypic species with only a few trees left in the wild. Due to invasion and habitat clearance, genetic diversity of this species has been reduced to an alarmingly low level. Long-term in vitro cultures of M. oppositifolia were slow-growing and eventually lost the ability to multiply and root. Because of the lack of new seed and vegetative materials, efforts were made to rejuvenate the existing cultures. This was attempted by a series of steps involving serial pruning of the shoot tips and growing on a modified MS medium containing silver nitrate and thidiazuron. Rapid cycling clonal propagules were produced this way from cultured nodes. Isolated shoots were rooted on Florialite?, and rooted plants were weaned in a transition stage using a Nutrient Film Technique (NFT) hydroponic system before being completely weaned under glasshouse conditions. The importance of screening cultures for multiplication and rooting efficiency during long-term maintenance is discussed in the context of stress metabolite production and its influence on growth in culture. Factors contributing to rejuvenation of old cultures, application of unconventional supporting systems for rooting and application of the hydroponic system as a weaning tool are also discussed.     

Aphid parasitoid responses to semiochemicals — Genetic, conditioned or learnt?

Parasitoid foraging behaviour is known to be influenced by interactions of genetic, physiological, environmental and experiential factors. Although the role of genetics, learning and conditioning in determining responses to foraging cues has been studied in lepidopteran parasitoids, aphid parasitoids have been less intensively researched. Using the tritrophic system,Vicia faba — Acyrthosiphon pisum — Aphidius ervi, evidence for the role of genetics and learning in parasitoid foraging is presented, and the difficulty of differentiating between genetic responses and those conditioned during parasitoid development is discussed. Aphidius ervi responds to aphid sex pheromones both in the field and in the laboratory. Since laboratory reared individuals have never experienced sexual aphids, the response must be genetic as it cannot have been conditioned during development. An example of a response conditioned during development is the variable response ofA. rhopalosiphi to different wheat cultivars depending upon host feeding. Aphid parasitoids also are adept at learning as shown by their responses to plant-derived cues which are learnt as Conditioned Stimuli (CS). Host products such as honeydew, as well as the host itself, can act as the Unconditioned Stimulus (US) in the learning process. Aphidius ervi offers a good model for investigating the role of these factors in parasitoid foraging behaviour. Finally, the value of such research for biological control programmes involving aphid parasitoids is discussed.     

Multi-dimensional modulations of α and γ cortical dynamics following mindfulness-based cognitive therapy in Major Depressive Disorder

To illuminate candidate neural working mechanisms of Mindfulness-Based Cognitive Therapy (MBCT) in the treatment of recurrent depressive disorder, parallel to the potential interplays between modulations in electro-cortical dynamics and depressive symptom severity and self-compassionate experience. Linear and nonlinear α and γ EEG oscillatory dynamics were examined concomitant to an affective Go/NoGo paradigm, pre-to-post MBCT or natural wait-list, in 51 recurrent depressive patients. Specific EEG variables investigated were; (1) induced event-related (de-) synchronisation (ERD/ERS), (2) evoked power, and (3) inter-/intra-hemispheric coherence. Secondary clinical measures included depressive severity and experiences of self-compassion. MBCT significantly downregulated α and γ power, reflecting increased cortical excitability. Enhanced α-desynchronisation/ERD was observed for negative material opposed to attenuated α-ERD towards positively valenced stimuli, suggesting activation of neural networks usually hypoactive in depression, related to positive emotion regulation. MBCT-related increase in left-intra-hemispheric α-coherence of the fronto-parietal circuit aligned with these synchronisation dynamics. Ameliorated depressive severity and increased self-compassionate experience pre-to-post MBCT correlated with α-ERD change. The multi-dimensional neural mechanisms of MBCT pertain to task-specific linear and non-linear neural synchronisation and connectivity network dynamics. We propose MBCT-related modulations in differing cortical oscillatory bands have discrete excitatory (enacting positive emotionality) and inhibitory (disengaging from negative material) effects, where mediation in the α and γ bands relates to the former.     

Potential side effects of insect-resistant transgenic plants on arthropod natural enemies.

Engineering genes encoding insecticidal proteins into crop plants offers numerous benefits to agriculture. However, like many conventional insecticides, this new technology has the potential to disrupt natural biological control through both direct and indirect side effects of the plants on the fitness or behaviour of arthropod predators and parasitoids. Interactions between transgenic plants and these beneficial insects are being assessed to avoid incompatibility.