Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Pulse oximetry in pediatric intensive care: comparison with measured saturations and transcutaneous oxygen tension.

We evaluated a new pulse oximeter designed to monitor beat-to-beat arterial oxygen saturation (SaO2) and compared the monitored SaO2 with arterial samples measured by co-oximetry. In 40 critically ill children (112 data sets) with a mean age of 3.9 years (range 1 day to 19 years), SaO2 ranged from 57% to 100%, and PaO2 from 27 to 128 mm Hg, heart rates from 85 to 210 beats per minute, hematocrit from 20% to 67%, and fetal hemoglobin levels from 1.3% to 60%; peripheral temperatures varied between 26.5 degrees and 36.5 degrees C. Linear correlation analysis revealed a good agreement between simultaneous pulse oximeter values and both directly measured SaO2 (r = 0.95) and that calculated from measured arterial PaO2 (r = 0.95). The device detected several otherwise unrecognized drops in SaO2 but failed to function in four patients with poor peripheral perfusion secondary to low cardiac output. Simultaneous measurements with a tcPO2 electrode showed a similarly good correlation with PaO22 (r = 0.91), but the differences between the two measurements were much wider (mean 7.1 +/- 10.3 mm Hg, range -14 to +49 mm Hg) than the differences between pulse oximeter SaO2 and measured SaO2 (1.5% +/- 3.5%, range -7.5% to -9%) and were not predictable. We conclude that pulse oximetry is a reliable and accurate noninvasive device for measuring saturation, which because of its rapid response time may be an important advance in monitoring changes in oxygenation and guiding oxygen therapy.

Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Oxidative potential of a panel of carbonaceous and metallic nanoparticles

Characterisation of nanoparticles (NP) based on size distribution, surface area, reactivity, and aggregation status of nanoparticles (NP) are of prime importance because they are usually closely related to toxicity. To date, most of the toxicity studies are quite time and money consuming. In the present study we report the oxidative properties of a panel of various NP (four Carbonaceous, nine Metal oxides, and one Metal as showed in Table 1) assessed with an acellular reactivity test measuring dithiothreitol (DTT) consumption (Sauvain et al. 2008). Such a test allows determining the ability of NP to catalyse the transfer of electrons from DTT to oxygen. DTT is used as a reductant species. NP were diluted and sonicated in Tween 80® to a final concentration of 50 g/mL. Printex 90 was diluted 5 times before doing the DTT assay because of its expected higher activity. Suspensions were characterised for NP size distribution by Nanoparticle Tracking Analysis (Nanosight©). Fresh solutions were incubated with DTT (100 μM). Aliquots were taken every 5 min and the remaining DTT was determined by reacting it with DTNB. The reaction rate was determined for NP suspensions and blank in parallel. The mean Brownian size distribution of NP agglomerates in suspension is presented in Table 1. D values correspond to 10th, and 50th percentiles of the particle diameters. All the NP agglomerated in Tween 80 with a D50 size corresponding to at least twice their primary size, except for Al2O3 (300 nm). The DTT test showed Printex 90 sample to be the most reactive one, followed by Diesel EPA and Nanotubes. Most of the metallic NP was nonresponding toward this test, except for NiO and Ag which reacted positively and ZnO which presented the most negative reactivity (see Figure 1). This last observation suggests that electron transfer between DTT and oxygen is hindered in presence of ZnO compared with the blank. Such "stabilization" could be attributable to ZnO dissolution and complexation between Zn2+ ions and DTT.

CD4+CD25-mTGFbeta+ T cells induced by nasal application of ovalbumin transfer tolerance in a therapeutic model of asthma.

Background: Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. Objectives: To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. Methods: After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in the induction of tolerance was analysed. Results: Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T(h)1 and T(h)2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4(+)CD25(-) T cells, which strongly expressed membrane-bound transforming growth factor beta (mTGF beta), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4(+)CD25(+)(Foxp3(+)) T cells during the process of tolerization was indispensable to CD4(+)CD25(-) T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4(+)CD25(-)mTGF beta(+) T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Conclusion: Nasal application of OVA in established asthma led to the induction of CD4(+)CD25(-)mTGF beta(+) T cells with regulatory properties, able to confer protection upon allergen re-exposure.

Physiological differences between cycling and running: lessons from triathletes

The purpose of this review was to provide a synopsis of the literature concerning the physiological differences between cycling and running. By comparing physiological variables such as maximal oxygen consumption (V O(2max)), anaerobic threshold (AT), heart rate, economy or delta efficiency measured in cycling and running in triathletes, runners or cyclists, this review aims to identify the effects of exercise modality on the underlying mechanisms (ventilatory responses, blood flow, muscle oxidative capacity, peripheral innervation and neuromuscular fatigue) of adaptation. The majority of studies indicate that runners achieve a higher V O(2max) on treadmill whereas cyclists can achieve a V O(2max) value in cycle ergometry similar to that in treadmill running. Hence, V O(2max) is specific to the exercise modality. In addition, the muscles adapt specifically to a given exercise task over a period of time, resulting in an improvement in submaximal physiological variables such as the ventilatory threshold, in some cases without a change in V O(2max). However, this effect is probably larger in cycling than in running. At the same time, skill influencing motor unit recruitment patterns is an important influence on the anaerobic threshold in cycling. Furthermore, it is likely that there is more physiological training transfer from running to cycling than vice versa. In triathletes, there is generally no difference in V O(2max) measured in cycle ergometry and treadmill running. The data concerning the anaerobic threshold in cycling and running in triathletes are conflicting. This is likely to be due to a combination of actual training load and prior training history in each discipline. The mechanisms surrounding the differences in the AT together with V O(2max) in cycling and running are not largely understood but are probably due to the relative adaptation of cardiac output influencing V O(2max) and also the recruitment of muscle mass in combination with the oxidative capacity of this mass influencing the AT. Several other physiological differences between cycling and running are addressed: heart rate is different between the two activities both for maximal and submaximal intensities. The delta efficiency is higher in running. Ventilation is more impaired in cycling than in running. It has also been shown that pedalling cadence affects the metabolic responses during cycling but also during a subsequent running bout. However, the optimal cadence is still debated. Central fatigue and decrease in maximal strength are more important after prolonged exercise in running than in cycling.

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat.

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.

Initial clinical experience with the admiral oxygenator combined with separated suction.

The Admiral, a new microporous membrane oxygenator with a low surface area, decreased priming volume and two separate reservoirs, was tested in 30 adult patients. This study was undertaken to evaluate blood path resistance, gas exchange capabilities and blood trauma in clinical use, with and without shed blood separation. Patients were divided into 3 groups. Group 1 had valve surgery without separation of suction, Group 2 had coronary artery bypass grafting (CABG) with direct blood aspiration and Group 3 had coronary artery bypass grafting with shed blood separation. The suctioned, separated, cardiotomy blood in Group 3 was treated with an autotransfusion device at the end of bypass before being returned to the patient. Theoretical blood flow could be achieved in all cases without problem. The pressure drop through the oxygenator averaged 88 +/- 13 mmHg at 4 l/min and 109 +/- 12 mmHg at 5 l/min. O(2) transfer was 163 +/- 27 ml/min. Free plasma haemoglobin rose in all groups, but significantly less in group 3. Lactate dehydrogenase (LDH) rose significantly in Groups 1 and 2. Platelets decreased in all groups without significant differences. Clinical experience with this new oxygenator was safe, the reduced membrane surface did not impair gas exchange and blood trauma could be minimized easily by separating shed blood, using the second cardiotomy reservoir.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Study of Staphylococcus aureus pathogenic genes by transfer and expression in the less virulent organism Streptococcus gordonii.

Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P &lt; 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

Are initial radiographic and clinical scales associated with subsequent intracranial pressure and brain oxygen levels after severe traumatic brain injury?

BACKGROUND: Prediction of clinical course and outcome after severe traumatic brain injury (TBI) is important. OBJECTIVE: To examine whether clinical scales (Glasgow Coma Scale [GCS], Injury Severity Score [ISS], and Acute Physiology and Chronic Health Evaluation II [APACHE II]) or radiographic scales based on admission computed tomography (Marshall and Rotterdam) were associated with intensive care unit (ICU) physiology (intracranial pressure [ICP], brain tissue oxygen tension [PbtO2]), and clinical outcome after severe TBI. METHODS: One hundred one patients (median age, 41.0 years; interquartile range [26-55]) with severe TBI who had ICP and PbtO2 monitoring were identified. The relationship between admission GCS, ISS, APACHE II, Marshall and Rotterdam scores and ICP, PbtO2, and outcome was examined by using mixed-effects models and logistic regression. RESULTS: Median (25%-75% interquartile range) admission GCS and APACHE II without GCS scores were 3.0 (3-7) and 11.0 (8-13), respectively. Marshall and Rotterdam scores were 3.0 (3-5) and 4.0 (4-5). Mean ICP and PbtO2 during the patients' ICU course were 15.5 ± 10.7 mm Hg and 29.9 ± 10.8 mm Hg, respectively. Three-month mortality was 37.6%. Admission GCS was not associated with mortality. APACHE II (P = .003), APACHE-non-GCS (P = .004), Marshall (P < .001), and Rotterdam scores (P < .001) were associated with mortality. No relationship between GCS, ISS, Marshall, or Rotterdam scores and subsequent ICP or PbtO2 was observed. The APACHE II score was inversely associated with median PbtO2 (P = .03) and minimum PbtO2 (P = .008) and had a stronger correlation with amount of time of reduced PbtO2. CONCLUSION: Following severe TBI, factors associated with outcome may not always predict a patient's ICU course and, in particular, intracranial physiology.

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Oxygen consumption in offspring tawny owls Strix aluco is associated with colour morph of foster mother

In several colour polymorphic species, morphs differ in thermoregulation either because dark and pale surfaces absorb solar radiation to a different extent and/or because morphs differ in key metabolic processes. Morph-specific thermoregulation may potentially account for the observation that differently coloured individuals are frequently not randomly distributed among habitats, and differ in many respects, including behaviour, morphology, survival and reproductive success. In a wild population of the colour polymorphic tawny owl Strix aluco, a recent cross-fostering experiment showed that offspring raised and born from red mothers were heavier than those from grey mothers. In the present study, we tested in the same individuals whether these morph-specific offspring growth patterns were associated with a difference in metabolic rate between offspring of red and grey mothers. For this purpose, we measured nestling oxygen consumption under two different temperatures (laboratory measurements: 4 and 20 degrees C), and examined the relationships between these data sets and the colour morph of foster and biological mothers. After controlling for nestling body mass, oxygen consumption at 20 degrees C was greater in foster offspring raised by grey foster mothers. No relationship was found between nestling oxygen consumption and coloration of their biological mother. Therefore, our study indicates that in our experiment offspring raised by grey foster mothers showed not only a lower body mass than offspring raised by red foster mothers, but also consumed more oxygen under warm temperature. This further indicates that rearing conditions in nests of grey mothers were more stressful than in nests of red mothers.

Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.Gene Therapy advance online publication, 27 June 2013; doi:10.1038/gt.2013.36.