Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Multi-color magnetic nanoparticle imaging using magnetorelaxometry.

Magnetorelaxometry (MRX) is a well-known measurement technique which allows the retrieval of magnetic nanoparticle (MNP) characteristics such as size distribution and clustering behavior. This technique also enables the non-invasive reconstruction of the spatial MNP distribution by solving an inverse problem, referred to as MRX imaging. Although MRX allows the imaging of a broad range of MNP types, little research has been done on imaging different MNP types simultaneously. Biomedical applications can benefit significantly from a measurement technique that allows the separation of the resulting measurement signal into its components originating from different MNP types. In this paper, we present a theoretical procedure and experimental validation to show the feasibility of MRX imaging in reconstructing multiple MNP types simultaneously. Because each particle type has its own characteristic MRX signal, it is possible to take this a priori information into account while solving the inverse problem. This way each particle type's signal can be separated and its spatial distribution reconstructed. By assigning a unique color code and intensity to each particle type's signal, an image can be obtained in which each spatial distribution is depicted in the resulting color and with the intensity measuring the amount of particles of that type, hence the name multi-color MNP imaging. The theoretical procedure is validated by reconstructing six phantoms, with different spatial arrangements of multiple MNP types, using MRX imaging. It is observed that MRX imaging easily allows up to four particle types to be separated simultaneously, meaning their quantitative spatial distributions can be obtained.

How to include the variability of TMS responses in simulations: a speech mapping case study.

When delivered over a specific cortical site, TMS can temporarily disrupt the ongoing process in that area. This allows mapping of speech-related areas for preoperative evaluation purposes. We numerically explore the observed variability of TMS responses during a speech mapping experiment performed with a neuronavigation system. We selected four cases with very small perturbations in coil position and orientation. In one case (E) a naming error occurred, while in the other cases (NEA, B, C) the subject appointed the images as smoothly as without TMS. A realistic anisotropic head model was constructed of the subject from T1-weighted and diffusion-weighted MRI. The induced electric field distributions were computed, associated to the coil parameters retrieved from the neuronavigation system. Finally, the membrane potentials along relevant white matter fibre tracts, extracted from DTI-based tractography, were computed using a compartmental cable equation. While only minor differences could be noticed between the induced electric field distributions of the four cases, computing the corresponding membrane potentials revealed different subsets of tracts were activated. A single tract was activated for all coil positions. Another tract was only triggered for case E. NEA induced action potentials in 13 tracts, while NEB stimulated 11 tracts and NEC one. The calculated results are certainly sensitive to the coil specifications, demonstrating the observed variability in this study. However, even though a tract connecting Broca's with Wernicke's area is only triggered for the error case, further research is needed on other study cases and on refining the neural model with synapses and network connections. Case- and subject-specific modelling that includes both electromagnetic fields and neuronal activity enables demonstration of the variability in TMS experiments and can capture the interaction with complex neural networks.

Toward 2D and 3D imaging of magnetic nanoparticles using EPR measurements.

PURPOSE: Magnetic nanoparticles (MNPs) are an important asset in many biomedical applications. An effective working of these applications requires an accurate knowledge of the spatial MNP distribution. A promising, noninvasive, and sensitive technique to visualize MNP distributions in vivo is electron paramagnetic resonance (EPR). Currently only 1D MNP distributions can be reconstructed. In this paper, the authors propose extending 1D EPR toward 2D and 3D using computer simulations to allow accurate imaging of MNP distributions. METHODS: To find the MNP distribution belonging to EPR measurements, an inverse problem needs to be solved. The solution of this inverse problem highly depends on the stability of the inverse problem. The authors adapt 1D EPR imaging to realize the imaging of multidimensional MNP distributions. Furthermore, the authors introduce partial volume excitation in which only parts of the volume are imaged to increase stability of the inverse solution and to speed up the measurements. The authors simulate EPR measurements of different 2D and 3D MNP distributions and solve the inverse problem. The stability is evaluated by calculating the condition measure and by comparing the actual MNP distribution to the reconstructed MNP distribution. Based on these simulations, the authors define requirements for the EPR system to cope with the added dimensions. Moreover, the authors investigate how EPR measurements should be conducted to improve the stability of the associated inverse problem and to increase reconstruction quality. RESULTS: The approach used in 1D EPR can only be employed for the reconstruction of small volumes in 2D and 3D EPRs due to numerical instability of the inverse solution. The authors performed EPR measurements of increasing cylindrical volumes and evaluated the condition measure. This showed that a reduction of the inherent symmetry in the EPR methodology is necessary. By reducing the symmetry of the EPR setup, quantitative images of larger volumes can be obtained. The authors found that, by selectively exciting parts of the volume, the authors could increase the reconstruction quality even further while reducing the amount of measurements. Additionally, the inverse solution of this activation method degrades slower for increasing volumes. Finally, the methodology was applied to noisy EPR measurements: using the reduced EPR setup's symmetry and the partial activation method, an increase in reconstruction quality of ≈ 80% can be seen with a speedup of the measurements with 10%. CONCLUSIONS: Applying the aforementioned requirements to the EPR setup and stabilizing the EPR measurements showed a tremendous increase in noise robustness, thereby making EPR a valuable method for quantitative imaging of multidimensional MNP distributions.

Effective electric fields along realistic DTI-based neural trajectories for modelling the stimulation mechanisms of TMS.

In transcranial magnetic stimulation (TMS), an applied alternating magnetic field induces an electric field in the brain that can interact with the neural system. It is generally assumed that this induced electric field is the crucial effect exciting a certain region of the brain. More specifically, it is the component of this field parallel to the neuron's local orientation, the so-called effective electric field, that can initiate neuronal stimulation. Deeper insights on the stimulation mechanisms can be acquired through extensive TMS modelling. Most models study simple representations of neurons with assumed geometries, whereas we embed realistic neural trajectories computed using tractography based on diffusion tensor images. This way of modelling ensures a more accurate spatial distribution of the effective electric field that is in addition patient and case specific. The case study of this paper focuses on the single pulse stimulation of the left primary motor cortex with a standard figure-of-eight coil. Including realistic neural geometry in the model demonstrates the strong and localized variations of the effective electric field between the tracts themselves and along them due to the interplay of factors such as the tract's position and orientation in relation to the TMS coil, the neural trajectory and its course along the white and grey matter interface. Furthermore, the influence of changes in the coil orientation is studied. Investigating the impact of tissue anisotropy confirms that its contribution is not negligible. Moreover, assuming isotropic tissues lead to errors of the same size as rotating or tilting the coil with 10 degrees. In contrast, the model proves to be less sensitive towards the not well-known tissue conductivity values.

Actin cytoskeleton control of the comings and goings of T lymphocytes.

T lymphocytes are key players of adaptive immune responses. Upon recognition of specific peptides presented by human leukocyte antigen (HLA) molecules on antigen presenting cells (APC), these cells execute subset-related functions such as killing, help and regulation. The ontogeny, the activation and the effector functions of T lymphocytes are all steps of T-lymphocyte life cycle that rely on high motility properties. These cells travel through the organism in a succession of steps, including entry into tissues, interstitial migration, APC scanning, synapse formation and tissue exit. Such ability is possible because of a plastic motility behavior, which is highly controlled in time and space. The molecular basis for the adaptable motility behavior of T lymphocytes is only starting to be unraveled. The scope of this review is to discuss recent data pointing to the key role of regulators of actin cytoskeleton remodeling in tuning distinct aspects of T-lymphocyte motility during their entry, residency and exit from tissues.

Bayesian phylogeographic inferences reveal contrasting colonization dynamics among European groundwater isopods.

The potentially important role of northern microrefugia during postglacial dispersal is challenging the view of southern Europe as a refuge and source area of European biota. In groundwaters, large geographic ranges of presumably good dispersers are increasingly suspected to consist of assemblages of cryptic species with narrow ranges. Moreover, a large species range, even when confirmed by molecular evidence, tells us little about the spatiotemporal dynamics of dispersal. Here, we used phylogenetic inferences, species delineation methods and Bayesian phylogeographic diffusion models to test for the likelihood of postglacial colonization from distant refugia among five morphospecies of Proasellus (Isopoda, Asellidae). All morphospecies except one were monophyletic, but they comprised a total of 15-17 cryptic species. Three cryptic species retained ranges that spanned a distance >650 km, similar to that of the nominal morphospecies. Bayesian diffusion models based on mitochondrial markers revealed considerable spatiotemporal heterogeneity in dispersal rates, suggesting that short-time dispersal windows were instrumental in shaping species ranges. Only one species was found to experience a recent, presumably postglacial, range expansion. The Jura and Alpine foothills probably played a major role in maintaining diversity within Proasellus in northern regions by acting both as diversification hotspots and Pleistocene refugia. Gaining insight into the spatiotemporal heterogeneity of dispersal rates revealed contrasting colonization dynamics among species that were not consistent with a global postglacial colonization of Europe from distant refugia.

Reduction of the impact of multiple uncertain conductivity values on EEG dipole source analysis.

It is well known that the uncertain knowledge of the conductivity values of the head tissues has an important impact upon the accuracy of the electroencephalogram source reconstruction. Assuming a certain value of the conductivity often leads to high reconstruction error values when solving the inverse problem. It is possible to quantify the impact of multiple uncertain conductivity values on the localization accuracy. We propose an approach that reduces the impact of these multiple uncertainties on the reconstruction accuracy of the dipole parameters. This paper elaborates the numerical method and shows results of localization accuracy in a five-shell spherical head model. Sensitivity analysis, when considering multiple layers in the head model, shows the different scales of the influence of the various uncertain conductivity values on the potential values. We propose a cost function that reduces the impact of multiple uncertainties of the conductivity value on the electroencephalogram dipole reconstruction and two strategies for selecting potential values on the basis of the sensitivity analysis. Numerical simulations, when considering multiple uncertainties in the model, provide results with higher reconstruction accuracy compared with the case where only a single uncertainty is taken into account.

A DTI-based model for TMS using the independent impedance method with frequency-dependent tissue parameters.

Accurate simulations on detailed realistic head models are necessary to gain a better understanding of the response to transcranial magnetic stimulation (TMS). Hitherto, head models with simplified geometries and constant isotropic material properties are often used, whereas some biological tissues have anisotropic characteristics which vary naturally with frequency. Moreover, most computational methods do not take the tissue permittivity into account. Therefore, we calculate the electromagnetic behaviour due to TMS in a head model with realistic geometry and where realistic dispersive anisotropic tissue properties are incorporated, based on T1-weighted and diffusion-weighted magnetic resonance images. This paper studies the impact of tissue anisotropy, permittivity and frequency dependence, using the anisotropic independent impedance method. The results show that anisotropy yields differences up to 32% and 19% of the maximum induced currents and electric field, respectively. Neglecting the permittivity values leads to a decrease of about 72% and 24% of the maximum currents and field, respectively. Implementing the dispersive effects of biological tissues results in a difference of 6% of the maximum currents. The cerebral voxels show limited sensitivity of the induced electric field to changes in conductivity and permittivity, whereas the field varies approximately linearly with frequency. These findings illustrate the importance of including each of the above parameters in the model and confirm the need for accuracy in the applied patient-specific method, which can be used in computer-assisted TMS.

Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients.

Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.

An objective assessment of nerve stimulators used for peripheral nerve blockade*.

Nerve stimulation is considered by many to be the current 'gold standard' for locating peripheral nerves. Thirteen nerve stimulators were subjected to a battery of tests performed by two independent observers under standardised conditions using a digital oscilloscope, a calibrated resistance and a novel scoring system. Individual scores were assigned for the signal waveform, current intensity, impulse duration, maximum load output and functionality of each nerve stimulator; the maximum score achievable was 20. A score of > or = 15/20 was achieved by six nerve stimulators: Stimuplex HNS 12 (B Braun); MultiStim Vario (Pajunk); Plexival (Medival, Vygon); MultiStim Sensor (Pajunk); Plexygon (Aryon, Vygon); Stimuplex HNS 11 (B Braun). Seven nerve stimulators scored < 15/20. Anaesthetists should be aware of the limitations of the nerve stimulator being used. Standardisation of features is desirable when manufacturing nerve stimulators.

Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization.

DNA vaccination induces antigen-specific immune responses with characteristics distinct from other vaccination modes. In the present study, the contribution of the plasmid backbone adjuvant effect to the quality of the DNA-raised antibody response was investigated. For this purpose, three intradermal primings were compared in mice using: (1) the recombinant Schistosoma haematobium glutathione S-transferase antigen (rSh28GST): (2) rSh28GST supplemented with a non-coding plasmid; and (3) a Sh28GST-encoding plasmid. In contrast to immunization with the protein, DNA immunization elicited a very stable antibody (Ab) response over a prolonged period of time. This feature was attributed to the plasmid backbone, because co-administration of the non-coding plasmid with rSh28GST allowed the maintenance of the specific Ab response. A strong anamnestic Ab response was induced after intradermal boost with rSh28GST only in the mice primed with pMSh. This indicated that the selective ability of DNA vaccination to induce memory humoral response was independent of the plasmid backbone. In contrast the plasmid backbone was found to strongly participate in the preferential IgG2a Ab production observed. These results suggest that, following DNA immunization, the Th1-biased profile and the maintenance of the long-lived Ab response could be attributed to an adjuvant effect of the plasmid backbone during priming, whereas the strength of B-cell memory was independent of this effect.

Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection.

In vivo delivery of DNA encoding antigens is a simple tool to induce immune responses against pathogens. This approach to vaccination also offers the possibility to codeliver plasmids encoding immunomodulatory molecules in order to drive immune responses towards optimal protective effects. In the murine model of Schistosoma mansoni infection, vaccination inducing a Th1 profile has been shown to be protective. In this study, we used a plasmid encoding the Th1-promoting cytokine IL-18, since we observed that percutaneous infection of Balb/c mice strongly induced the production of IL-18 mRNA in the skin. Intradermal injection of the IL-18-encoding plasmid prior to infection did not interfere with parasite migration through the skin although it led to a local and transient cellular infiltration. When the IL-18-encoding plasmid was codelivered with a S. mansoni glutathione S-transferase (Sm28GST)-encoding plasmid, a 30-fold increase of antigen-specific IFN-gamma secretion by spleen cells was observed in comparison to spleen cells from mice that had received only the Sm28GST-encoding plasmid. This immunostimulatory effect was related to a significant protective effect (28% reduction in egg laying and 23% reduction in worm burden) which was attributed to a cooperative effect between both plasmids. Therefore, this study shows that codelivery of an IL-18-encoding plasmid with an antigen-encoding plasmid can stimulate specific cellular responses and induce protective effects against S. mansoni infection.

Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine.

Many different HIV-1 vaccine strategies have been developed, but as yet none has been completely successful. Promising results from combined DNA prime/protein boost vaccines have been reported. Specific immune responses generated by DNA vaccines can be modulated by the co-delivery of genes coding for cytokines. In this study, we have used the intradermal route by needle injection of a plasmid coding for the HIV-1 Nef accessory protein. We show that DNA prime/protein boost vaccine combinations increase the humoral and cellular immune responses against HIV-1 Nef and that the co-injection of DNA encoding Interleukin-18 (IL-18) modulates the specific immune response towards a Th1 type.

CpG motifs induce Langerhans cell migration in vivo.

Cytosine-guanosine (CpG) oligonucleotide (CpG-oligo) sequences are immunostimulatory motifs that are present in bacterial DNA and their presence in plasmids might contribute to the immune response generated by DNA vaccination. The cell targets of CpG motifs in vivo have not been characterized yet. In this report we assessed the in vivo effects of CpG motifs on Langerhans cells (LC) migration. We showed that intradermal injection of 10 microg of CpG-containing oligonucleotides in mouse ear induced the local depletion of LC within 2 h of exposure as shown by CD11c and Ia immunohistological staining. To demonstrate that LC depletion was due to LC migration, CpG oligonucleotides were injected into the explants ex vivo, and the CD11c(+) cells emigrating from the cultured isolated skin within medium were evaluated by immunostaining and FACS analysis. Our findings demonstrate that CpG motifs induce LC/dendritic cell (DC) migration out of the skin. To assess whether CpG motifs may act directly on LC/DC to induce their emigration we next analyzed the effects of CpG motifs in vitro on the expression of adhesion molecules involved in LC/DC migration. The results of these experiments show that alpha(6) integrins, E-Cadherin, ICAM-1, CD11b and CD11c were differentially regulated upon CpG-oligo treatment of immortalized DC. CpG treatment (10 microg/ml for 8 h) resulted in a 100% increase in ICAM-1 staining intensity, a 50% decrease in E-Cadherin staining and a 25% decrease in alpha(6) integrins staining, while no changes in the levels of CD11b and CD11c expression were recorded. Changes in adhesion molecule expression were mirrored by concomitant changes in the cell morphology that included cell depolarization, the appearance of filopods and loss of adherence. This study provides the first in vivo evidence that CpG motifs signal the migration of LC from the epidermis.

In vivo immunomodulation following intradermal injection with DNA encoding IL-18.

IL-18, a recently identified cytokine synthesized by different cell types, including Kupffer cells, activated macrophages, and keratinocytes, induces IFN-gamma production by T cells and NK cells. The cDNA encoding IL-18 with its natural signal peptide was cloned under control of the CMV promoter and injected into the skin of mice. A single intradermal injection of this construction led to efficient in vivo expression of IL-18 in cutaneous dermal cells and induced IFN-gamma mRNA production, indicating that it was produced in a biologically active form. In addition, a massive cellular infiltrate was observed in the skin 2 days after injection. When the mice were subsequently infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG), they produced lower levels of anti-BCG Abs than control animals. However, in contrast to their lowered humoral immune response, the mice produced higher amounts of Ag-specific IFN-gamma after in vitro restimulation, as compared with the controls. Therefore, injection of DNA encoding IL-18 into the skin modulates both Ag-specific humoral and T cell responses upon mycobacterial infection. It increases the Th1 type response, which may be particularly useful for the development of new immunotherapeutic or immunoprotective approaches against infections by intracellular parasites, such as mycobacteria.