Marx-Type solid-state bipolar modulator topologies: Performance comparison

The operation of generalized Marx-type solid-state bipolar modulators is discussed and compared with simplified Marx-derived circuits, to evaluate their capability to deal with various load conditions. A comparative analysis on the number of switches per cell, fiber optic trigger count, losses, and switch hold-off voltages has been made. A circuit topology is obtained as a compromise in terms of operating performance, trigger simplicity, and switching losses. A five-stage laboratory prototype of this circuit has been assembled using 1200 V insulated gate bipolar transistors (IGBTs) and diodes, operating with 1000 V dc input voltage and 1 kHz frequency, giving 5 kV bipolar pulses, with 2.5 mu s pulse width and 5 mu s relaxation time into resistive, capacitive, and inductive loads.

Comprehensive comparison of a current-source and a voltage-source converter for three-phase EV fast battery chargers

This paper presents a comprehensive comparison of a current-source converter and a voltage-source converter for three-phase electric vehicle (EV) fast battery chargers. Taking into account that the current-source converter (CSC) is a natural buck-type converter, the output voltage can assume a wide range of values, which varies between zero and the maximum instantaneous value of the power grid phase-to-phase voltage. On the other hand, taking into account that the voltage-source converter (VSC) is a natural boost-type converter, the output voltage is always greater than the maximum instantaneous value of the power grid phase-to-phase voltage, and consequently, it is necessary to use a dc-dc buck-type converter for applications as EV fast battery chargers. Along the paper is described in detail the principle of operation of both the CSC and the VSC for EV fast chargers, as well as the main equations of the power theory and current control strategies. The comparison between both converters is mainly established in terms of the total harmonic distortion of the grid current and the estimated efficiency for a range of operation between 10 kW and 50 kW.

The relation of the concentration of macronutrients in the substrate and in the foliage to cell wall thickness and cellulose concentration in the xylem of slash pine (Pinus elliotti)

Sand culture experiments, using a sub-irrigation technique, were installed in order to find out the effects of the macronutrients N, P, K, Ca, Mg and S on growth, aspect, mineral composition, length of fibers, thickness of cell wall and cellulose concentration in slash pine. The aim was to obtain, under controlled conditions, basic information which could eventually lead to practical means designed to increase the rate of growth and to make of slash pine a richer source of cellulose. Nitrogen, Phosphorus, Potassium Experiment A 3 x 3 x 3 factorial design with two replicates was used. Nitrogen was supplied initially at the levels of 25, 50 and 100 ppm; phosphorus was given at the rates of 5, 10 and 20 ppm; potassium was supplied at the rates of 25, 50 and 100 ppm; six months after the experiment was started the first level for each element was dropped to zero. Others macro and all micronutrients were supplied at uniform rates. Fifteen hours of illumination per day were provided. The experimental technique for growing the slash pine seedlings proved quite satisfactory. Symptoms of deficiency of nitrogen, phosphorus and potassium were observed, described and recorded in photographs and water colors. These informations will help to identify abnormalities which may appear under field conditions. Chemical analysis of the several plant parts, on the other hand, give a valuable means to assess the nutritional status of slash pine, thus confirming when needed, the visual diagnosis. The correctness of manurial pratices, on the other hand, can be judged with the help of the analytical data tabulated. Under the experimental conditions nitrogen caused the highest increases on growth, as measured by increments in height and dry weights, whereas the effects of phosphorus and potassium were less marked. Cellulose concentration was not significantly affected by the treatments used. Higher levels of N seemed to decrease both length of fiber elements and the thickness of cell wall. The effects of P and K were not well defined. Calcium, Magnesium, Sulfur Experiment A 3 x 3 x 3 factorial design with two replicates was used. Calcium was supplied initially at the levels of 12.5, 25 and 50 ppm; magnesium and sulfur were given at the rates of 6, 12.5 and 25 ppm. Other macro and micronutrients were supplied at uniform rates, common to all treatments. Three months after starting the experiment the first level for each element was dropped to zero. Symptoms of deficiency of calcium, magnesium and sulfur were observed, described and recorded as in the case of the previous experiment. Chemical analysis were made, both for mineral content and cellulose concentration. Length of fibers and thickness of cell wall were measured. Both calcium and magnesium increase height, sulfur failing to give significant response. Dry weight was beneficially affected by calcium and sulfur. The levels of calcium, magnesium and sulfur in the needles associated with deficiency and maximum growth are comparable with those found in the literature. Cellulose concentration increased when the level of sulfur in the substrate was raised. The thickness of cell wall was negatively affected by the treatments; no effect was observed with regards to length of fibers.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Effect of rufinamide on gating properties of voltage-gated sodium channel Na(v)1.7

Background: Voltage-gated sodium channels (Nav1.x) are important players in chronic pain. A particular interest has grown in Nav1.7, expressed in nociceptors, since mutations in its gene are associated to two inherited pain syndromes or insensitivity to pain. Rufinamide, a drug used to treat refractory epilepsy such as the Lennox-Gastaut syndrome, has been shown to reduce the number of action potentials in cortical neurons without completely blocking Na channels. Aim: The goal of this study was to investigate the effect of rufinamide on Nav1.7 current. Methods and results: Whole-cell patch clamp experiments were performed using HEK293 cells stably expressing Nav1.7. Rufinamide significantly decreased peak sodium current by 28.3, 21.2 and 12.5% at concentrations of 500, 100 and 50μM respectively (precise EC50 could not be calculated since higher rufinamide concentrations could not be achieved in physiological buffer solution). No significant difference on the V1/2 of voltage-dependence of activation was seen; however a shift in the steady-state inactivation curve was observed (-82.6 mV to -88.8 mV and -81.8 to -87.6 mV for 50 and 100 μM rufinamide respectively, p <0.005). Frequency-dependent inhibition of Nav1.7 was also influenced by the drug. One hundred μM rufinamide reduced the peak sodium current (in % of the peak current taken at the first sweep of a train of 50) from 90.8 to 80.8% (5Hz), 88.7 to 71.8% (10 Hz), 69.1 to 49.2% (25 Hz) and 22.3 to 9.8% (50 Hz) (all p <0.05). Onset of fast inactivation was not influenced by the drug since no difference in the time constant of current decay was observed. Conclusion: In the concentration range of plasma level in human treated for epilepsy, 15 μM, rufinamide only minimally blocks Nav1.7. However, it stabilizes the inactivated state and exerts frequencydependent inhibition of Nav1.7. These pharmacological properties may be of use in reducing ectopic discharges as a causal and symptom related contributor of neuropathic pain syndrome.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Reverse-transcriptase-associated RNaseH activity mediates template switching during reverse transcription in vitro.

During the first steps of reverse transcription of the retroviral genome, sequences present at the extremities of the RNA are used to reconstitute a host cell PolII promoter. The assembly of the promoter occurs by template switching, which takes advantage of a direct repeat at the ends of the RNA molecule. These steps are catalysed by the viral reverse transcriptase, which carries an intrinsic RNaseH activity that is probably also involved therein. To study the role of the RNaseH activity in this first template-switching event, an in vitro system has been developed based on primer extensions of synthetic RNAs. When an RNA was reverse transcribed with wild-type reverse transcriptase in the presence of a second RNA the 3' part of which was repeated at the 5' end of the first one, extension products could be observed corresponding to a chimeric cDNA comprising both RNA species. This template switching could not be detected when a mutant reverse transcriptase lacking the RNaseH activity was used. The results show that the RNaseH activity is needed to remove the 5' RNA sequences from the cDNA:RNA hybrid thereby enabling its translocation to another RNA containing an appropriate complementary target sequence.

FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases.

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.

Regulation of the voltage-gated sodium channel Nav1.7 by ubiquitin ligase Nedd4-2

Background and aim: Neuropathic pain (NP) is a frequent and disabling disorder occurring as a consequence of a direct lesion of the nervous system and recurrently associated with a positive shift toward nervous system excitability. Peripheral nerve activity is mainly carried by voltage-gated sodium channels (VGSC), with Nav1.7 isoform being an important candidate since loss of function mutations of its gene is associated with congenital inability to experience pain. Interestingly, ubiquitin ligases from the Nedd4 family are well known proteins that regulate the turnover of many membrane proteins such as VGSC and we showed Nedd2-2 is downregualted in experimental models of chronic pain. The aim of this study was to investigate the importance of Nedd4-2 in the modulation of Nav1.7 at the membrane. Methods: In vitro: whole cell patch clamp on HEK293 cell line stably expressing Nav1.7 was used to record sodium currents (INa), where the peak current of INa reflects the quantity of functional Nav1.7 expressed at the membrane. The possibility that Nedd4-2 modulates the currents was assessed by investigating the effect of its cotransfection on INa. Biotinylation of cell surface was used to isolate membrane-targeted Nav1.7. Furthermore, as the interaction between Nedd4-2 and Nav isoforms was previously reported to rely on an xPPxYx sequence (PY-motif), we mutated this latter to study its impact in the specific interaction between Nav1.7 and Nedd4-2. GST-fusion proteins composed of the Nav1.7 c terminal 66 amino acids (wild-type or PY mutated) and GST were used to pull-down Nedd4-2 from lysates. Results: Co-transfection of Nav1.7 with Nedd4-2 reduced the Nav1.7 current amplitude by ~80% (n = 36, p <0.001), without modifying the biophysical properties of INa. In addition, we show that the quantity of Nav1.7 at the membrane was decreased when Nedd4-2 was present. This effect was dependent on the PY-motif since mutations in this sequence abolished the down-regulatory effect of Nedd4-2. The importance of this motif was further confirmed by pull down experiments since the PY mutant completely eliminate the interaction with Nedd4-2. Perspectives: Altogether, these results point to the importance of Nedd4-2 as a Nav1.7 regulator through cell surface modulation of this sodium channel. Further experiments in freshly dissociated neurons from wild type and Scn1bflox/Nedd4-2Cre mice are needed to confirm in vivo these preliminary data.

p.L1612P, a novel voltage-gated sodium channel Nav1.7 mutation inducing a cold sensitive paroxysmal extreme pain disorder.

BACKGROUND: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. METHODS: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. RESULTS: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady-state inactivation curve (V1/2 from -61.8 ± 4.5 mV to -30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady-state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. CONCLUSIONS: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

Estimation of zero-sequence impedance of undergrounds cables for single-phase fault location in distribution systems with electric arc

This paper focus on the problem of locating single-phase faults in mixed distribution electric systems, with overhead lines and underground cables, using voltage and current measurements at the sending-end and sequence model of the network. Since calculating series impedance for underground cables is not as simple as in the case of overhead lines, the paper proposes a methodology to obtain an estimation of zero-sequence impedance of underground cables starting from previous single-faults occurred in the system, in which an electric arc occurred at the fault location. For this reason, the signal is previously pretreated to eliminate its peaks voltage and the analysis can be done working with a signal as close as a sinus wave as possible

CNS safety at 48-week of switching to ATV/r plus 3TC or two nucleos(t)ides in HIV-suppressed patients on stable ART: the SALT neurocognitive sub-study.

INTRODUCTION Due to their low CNS penetrance, there are concerns about the capacity of non-conventional PI-based ART (monotherapy and dual therapies) to preserve neurocognitive performance (NP). METHODS We evaluated the NP change of aviremic participants of the SALT clinical trial (1) switching therapy to dual therapy (DT: ATV/r+3TC) or triple therapy (TT: ATV/r+2NRTI) who agreed to perform an NP assessment (NPZ-5) at baseline and W48. Neurocognitive impairment and NP were assessed using AAN-2007 criteria (2) and global deficit scores (GDS) (3). Neurocognitive change (GDS change: W48 - baseline) and the effect of DT on NP evolution crude and adjusted by significant confounders were determined using ANCOVA. RESULTS A total of 158 patients were included (Table 1). They had shorter times because HIV diagnosis, ART initiation and HIV-suppression and their virologic outcome at W48 by snapshot was higher (79.1% vs 72.7%; p=0.04) compared to the 128 patients not included in the sub-study. By AAN-2007 criteria, 51 patients in each ART group (68% vs 63%) were neurocognitively impaired at baseline (p=0.61). Forty-seven patients were not reassessed at W48: 30 lost of follow-up (16 DT-14 TT) and 17 had non-evaluable data (6 DT-11 TT). Patients retested were more likely to be men (78.9% vs 61.4%) and had neurological cofounders (9.6% vs 0%) than patients non-retested. At W48, 3 out of 16 (5.7%) patients on DT and 6 out of 21 (10.5%) on TT who were non-impaired at baseline became impaired (p=0.49) while 10 out of 37 (18.9%) on DT and 7 out of 36 (12.3%) on TT who were neurocognitively impaired at baseline became non-impaired (p=0.44). Mean GDS changes (95% CI) were: Overall -0.2 (-0.3 to -0.04): DT -0.26 (-0.4 to -0.07) and TT -0.08 (-0.2 to 0.07). NP was similar between DT and TT (0.15). This absence of differences was also observed in all cognitive tests. Effect of DT: -0.16 [-0.38 to 0.06]) (r(2)=0.16) on NP evolution was similar to TT (reference), even after adjusting (DT: -0.11 [-0.33 to 0.1], TT: reference) by significant confounders (geographical origin, previous ATV use and CD4 cell count) (r(2)=0.25). CONCLUSIONS NP stability was observed after 48 weeks of follow up in the majority of patients whether DT or TT was used to maintain HIV-suppression. Incidence rates of NP impairment or NP impairment recovery were also similar between DT and TT.

Monitoring of innate and adaptive immune responses in the context of human immunotherapy

Summary1 SummaryCancer patients have a better clinical outcome when their tumours display marked infiltration by memory Τ cells. Moreover, the overrepresentation of Th1 gene signatures in primary tumours correlates with favourable prognosis. Thus, vaccination to induce Τ cells capable of infiltrating and eradicating the tumour seems a promising strategy for the treatment of cancer. Here, I monitored CD4 Τ cell responses in melanoma patients vaccinated with the long synthetic peptides Melan- A16-35(A27L) and NY-ESO-179.108. Most of the patients developed strong and diverse peptide antigen specific CD4 Τ cell responses. Analysis of the fine specificity of CD4 Τ cell antigen recognition led to the identification of two new epitopes. The peptide Melan-A16_35(A27L) was delivered by virus-like particles (VLPs) derived from bacteriophage Οβ, which themselves displayed strong immunogenicity. I show evidence for induction of Οβ- and Melan-A specific CD4 Τ cell responses that developed a Th1 functional profile after repeated vaccination cycles. They also specifically released the chemokines CCL-3 and CCL-4, which play important roles in attracting CD8 Τ cells to the APC surface for priming and formation of Τ cell memory. We further found induction of robust humoral IgG responses upon VLP vaccination, and the lgG1-lgG4 isotype composition depended on the adjuvant used. Since heavy chain class switching largely dépends on the presence of CD4 Τ cell help, this result suggests that the adjuvant can influence the differentiation of elicited CD4 Τ cells, thereby contributing to the quality and function of both Β cells and CD8 Τ cells. The nature of the inflammatory processes in the tumour microenvironment can modulate CD8 Τ cell function. A collaboration was established for the investigation regulation of inflammasome activation in human primary monocytes. We identified IL- 4 and TGF-β as strong inhibitors of IL-1 β secretion, Indicating some level of regulation from effector Th2 and Treg responses. We further found a potent inhibition of inflammasome activation by type I interferon, and demonstrated in vivo inhibition of IL-1 β responses in monocytes from active multiple sclerosis patients under IFN-β therapy. This finding further offers a possible explanation for its success, which mechanism of action is still largely unclear. Interestingly, type I interferon is also being used as adjuvant treatment for tumour free metastatic cutaneous melanoma patients. While its clinical benefit has remained controversial, recent data suggest that the subset of patients with ulcerated primary melanoma lesions can benefit from this therapy. Future investigations will shed light on the implication of the inflammasome in this context, and may offer new strategies for improved adjuvant treatments of melanoma.2 RésuméLes patients atteints de cancer ont une meilleure chance de survie si leurs tumeurs s'avèrent être largement infiltrées par des cellules Τ mémoires. De plus, la surreprésentation d'une signature génique Th1 est en corrélation avec un pronostic favorable. Ainsi, la vaccination visant à induire des cellules Τ capables d'infiltrer et de détruire la tumeur parait être une stratégie prometteuse pour le traitement du cancer. Dans ce travail, j'ai procédé au monitoring de la réponse des cellules Τ CD4 dans des patients atteints de mélanome vaccinés avec les longs peptides synthétiques Melan-A16_35(A27L) et NY-ESO-179_108. Ces peptides représentent des antigènes tumoraux reconnus par des lymphocytes T. La majorité des patients a développé une réponse forte et diversifiée des cellules Τ CD4 spécifiques contre les peptides. L'analyse de la spécificité fine de la reconnaissance antigénique des cellules Τ CD4 nous a conduits à l'identification de deux nouveaux épitopes. Le peptide Melan-Aie. 35(A27L) a été délivré par des particules de type viral (VLPs) dérivés de bactériophages Qβ, qui ont eux-mêmes démontré une forte immunogénicité. Mon travail montre les preuves d'une induction de réponses spécifiques des cellules Τ CD4 contre les Qβ et Melan-A développant un profil fonctionnel Th1 après plusieurs cycles de vaccination. Elles secrètent aussi spécifiquement les chimiokines CCL-3 et CCL-4, qui jouent un rôle important dans l'attraction des cellules Τ CD8 à la surface des cellules présentatrices d'antigènes et contribuent ainsi à induire et former la mémoire cellulaire Τ CD8. Nous avons également remarqué une induction de fortes réponses humorales IgG après vaccination avec les VLPs, et que la composition des isotypes lgG1-lgG4 dépendait de l'adjuvant utilisé. Etant donné qu'une commutation de classe de la chaîne lourde dépend largement ùie l'aide des cellules Τ CD4, ce résultat suggère que l'adjuvant puisse influencer la différeritiation de cellules Τ CD4 en différent types, contribuant ainsi à la qualité et à la fonction des cellules Β et des cellules Τ CD8.La nature des processus d'inflammation dans le microenvironnement tumoral peut moduler la fonction des cellules Τ CD8. Une collaboration a été établie pour investiguer la régulation de l'activation de l'inflammasome dans des monocytes primaires humains. Nous avons identifié l'IL-4 et le TGF-β comme étant de puissants inhibiteurs de la sécrétion de IL-Ιβ, indiquant une certaine régulation de la réponse inflammatoire induite par les cellules Th2 et Τ régulatrices. Nous avons également trouvé une forte inhibition de l'activation de l'inflammasome par l'interféron type I, et nous avons démontré une inhibition in vivo de la réponse IL-1 β dans des monocytes de patients atteints d'une sclérose en plaque active sous traitement IFN-β. Ce résultat nous offre une possible explication du succès de cette thérapie, dont le mécanisme reste à ce jour encore largement obscur. Il est intéressant de noter que l'interféron de type I est également utilisé pour le traitement de patients atteints de mélanome cutané métastasique sans tumeurs. Bien que le bénéfice clinique de ce traitement reste controversé, des études récentes montrent qu'une partie des patients atteints de mélanome primaire ulcéré peut tirer bénéfice de cette thérapie. De futures investigations pourront mieux nous renseigner sur l'implication de l'inflammasome dans ce contexte et offrir de nouvelles stratégies pour améliorer les traitements adjuvants du mélanome.