Immunologic and virologic characterization of an ART-treated HIV-I patients cohort with long-term control of viremia

Background Long-term treatment of primary HIV-1 infection (PHI) may allow the immune reconstitution of responses lost during the acute viremic phase and decrease of peripheral reservoirs. This in turn may represent the best setting for the use of therapeutic vaccines in order to lower the viral set-point or control of viral rebound upon ART discontinuation. Methods We investigated a cohort of 16 patients who started ART at PHI, with treatment duration of ≥4 years and persistent aviremia (<50 HIV-1 copies/ml). The cohort was characterized in terms of viral subtype, cell-associated RNA, proviral DNA and HLA genotype. Secretion of IFN-γ, IL-2 and TNF-α by CD8 T-cells was analysed by polychromatic flowcytometry using a panel of 192 HIV-1-derived epitopes. Results This cohort is highly homogenous in terms of viral subtype: 81% clade B. We identified 44 epitope-specific responses: all patients had detectable responses to >1 epitope and the mean number of responding epitopes per patient was 3. The mean frequency of cytokines-secreting CD8 T-cells was 0.32%. CD8 T-cells secreting simultaneously IFN-γ, IL-2 and TNF-α made up for about 40% of the response and cells secreting at least 2 cytokines for about 80%, consistent with a highly polyfunctional CD8 T-cell profile. There was no difference in term of polyfunctionality when HLA restriction, or recognized viral regions and epitopes were considered. Proviral DNA was detectable in all patients but at low levels (mean = 108 copies/1 million PBMCs) while cell-associated mRNA was not detectable in 19% of patients (mean = 11 copies/1 million PBMCs when detectable). Conclusion Patients with sustained virological suppression after initiation of ART at PHI show polyfunctional CD8 T-cell and low levels of proviral DNA with an absence of residual replication in a substantial percentage of patients. The use of therapeutic vaccines in this population may promote low level of rebound viremia or control of viral replication upon ART cessation.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Renal response to the angiotensin II receptor subtype 1 antagonist irbesartan versus enalapril in hypertensive patients.

OBJECTIVE: To compare the acute and sustained renal hemodynamic effects on hypertensive patients of 100 mg irbesartan and 20 mg enalapril each once daily. PATIENTS: Twenty patients (aged 35-70 years) with uncomplicated, mild-to-moderate essential hypertension and normal serum creatinine levels completed this study. STUDY DESIGN: After random allocation to treatment (n=10 per group), administration schedule (morning or evening) was determined by further random allocation, with crossover of schedules after 6 weeks' therapy. Treatment and administration assignments were double-blind. Twenty-four-hour ambulatory blood pressure was monitored before and after 6 and 12 weeks of therapy. Renal hemodynamics were determined on the first day of drug administration and 12 and 24 h after the last dose during chronic treatment. RESULTS: Administration of each antihypertensive agent induced a renal vasodilatation with no significant change in glomerular filtration rate. However, the time course appeared to differ: irbesartan had no significant acute effect 4 h after the first dose, but during chronic administration a renal vasodilatory response was found 12 and 24 h after the dose; enalapril was effective acutely and 12 h after administration, but no residual effect was found 24 h after the dose. Both antihypertensive agents lowered mean ambulatory blood pressure effectively, with no significant difference between treatments or between administration schedules (morning versus evening). CONCLUSIONS: Irbesartan and enalapril have comparable effects on blood pressure and renal hemodynamics in hypertensive patients with normal renal functioning. However, the time profiles of the renal effects appear to differ, which might be important for long-term renoprotective effects.

Dose-dependent acute and sustained renal effects of the endothelin receptor antagonist avosentan in healthy subjects.

The endothelin receptor antagonist avosentan may cause fluid overload at doses of 25 and 50 mg, but the actual mechanisms of this effect are unclear. We conducted a placebo-controlled study in 23 healthy subjects to assess the renal effects of avosentan and the dose dependency of these effects. Oral avosentan was administered once daily for 8 days at doses of 0.5, 1.5, 5, and 50 mg. The drug induced a dose-dependent median increase in body weight, most pronounced at 50 mg (0.8 kg on day 8). Avosentan did not affect renal hemodynamics or plasma electrolytes. A dose-dependent median reduction in the fractional renal excretion of sodium was found (up to 8.7% at avosentan 50 mg); this reduction was paralleled by a dose-related increase in proximal sodium reabsorption. It is suggested that avosentan dose-dependently induces sodium retention by the kidney, mainly through proximal tubular effects. The potential clinical benefits of avosentan should therefore be investigated at doses of <or= 5 mg.

Impaired expression of the inducible cAMP early repressor accounts for sustained adipose CREB activity in obesity.

OBJECTIVEIncrease in adipose cAMP response binding protein (CREB) activity promotes adipocyte dysfunction and systemic insulin resistance in obese mice. This is achieved by increasing the expression of activating transcription factor 3 (ATF3). In this study we investigated whether impaired expression of the inducible cAMP early repressor (ICER), a transcriptional antagonist of CREB, is responsible for the increased CREB activity in adipocytes of obese mice and humans.RESEARCH DESIGN AND METHODSTotal RNA and nuclear proteins were prepared from visceral adipose tissue (VAT) of human nonobese or obese subjects, and white adipose tissue (WAT) of C57Bl6-Rj mice that were fed with normal or high-fat diet for 16 weeks. The expression of genes was monitored by real-time PCR, Western blotting, and electromobility shift assays. RNA interference was used to silence the expression of Icer.RESULTSThe expression of Icer/ICER was reduced in VAT and WAT of obese humans and mice, respectively. Diminution of Icer/ICER was restricted to adipocytes and was accompanied by a rise of Atf3/ATF3 and diminution of Adipoq/ADIPOQ and Glut4/GLUT4. Silencing the expression of Icer in 3T3-L1 adipocytes mimicked the results observed in human and mice cells and hampered glucose uptake, thus confirming the requirement of Icer for appropriate adipocyte function.CONCLUSIONSImpaired expression of ICER contributes to elevation in CREB target genes and, therefore, to the development of insulin resistance in obesity.

Sustained 24-hour blockade of the renin-angiotensin system: a high dose of a long-acting blocker is as effective as a lower dose combined with an angiotensin-converting enzyme inhibitor.

Whether a higher dose of a long-acting angiotensin II receptor blocker (ARB) can provide as much blockade of the renin-angiotensin system over a 24-hour period as the combination of an angiotensin-converting enzyme inhibitor and a lower dose of ARB has not been formally demonstrated so far. In this randomized double-blind study we investigated renin-angiotensin system blockade obtained with 3 doses of olmesartan medoxomil (20, 40, and 80 mg every day) in 30 normal subjects and compared it with that obtained with lisinopril alone (20 mg every day) or combined with olmesartan medoxomil (20 or 40 mg). Each subject received 2 dose regimens for 1 week according to a crossover design with a 1-week washout period between doses. The primary endpoint was the degree of blockade of the systolic blood pressure response to angiotensin I 24 hours after the last dose after 1 week of administration. At trough, the systolic blood pressure response to exogenous angiotensin I was 58% +/- 19% with 20 mg lisinopril (mean +/- SD), 58% +/- 11% with 20 mg olmesartan medoxomil, 62% +/- 16% with 40 mg olmesartan medoxomil, and 76% +/- 12% with the highest dose of olmesartan medoxomil (80 mg) (P = .016 versus 20 mg lisinopril and P = .0015 versus 20 mg olmesartan medoxomil). With the combinations, blockade was 80% +/- 22% with 20 mg lisinopril plus 20 mg olmesartan medoxomil and 83% +/- 9% with 20 mg lisinopril plus 40 mg olmesartan medoxomil (P = .3 versus 80 mg olmesartan medoxomil alone). These data demonstrate that a higher dose of the long-acting ARB olmesartan medoxomil can produce an almost complete 24-hour blockade of the blood pressure response to exogenous angiotensin in normal subjects. Hence, a higher dose of a long-acting ARB is as effective as a lower dose of the same compound combined with an angiotensin-converting enzyme inhibitor in terms of blockade of the vascular effects of angiotensin.

Effects of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase on the vasopressor response to exogenous angiotensin I and angiotensin II challenges in healthy volunteers.

MDL 100,240, a dual inhibitor of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), was administered intravenously to two panels of four healthy males in a four-period, dose-increasing (0, 1.56, 6.25, and 25 mg, and 0, 3.13, 12.5, and 50 mg, respectively) double-blind, placebo-controlled study. Plasma ACE activity and blood-pressure response to exogenous angiotensin I and angiotensin II i.v. challenges and safety and tolerance were assessed over a 24-h period. MDL 100,240 induced a rapid, dose-related, and sustained inhibition of ACE (>70% over 24 h at doses > or =12.5 mg). The time integral of ACE inhibition was related to the dose but with near-maximal values already attained at doses > or =12.5 mg. Systolic and diastolic blood-pressure responses to exogenous angiotensin I challenges were inhibited in a dose-dependent fashion, whereas the effects of angiotensin II remained unaffected. Mean supine blood pressure decreased transiently (3 h) at doses > or =3.125 mg and < or =24 h with the 25- and 50-mg doses, but not significantly. MDL 100,240 was well tolerated. In healthy subjects, MDL 100,240 exerts a dose-dependent and long-lasting ACE-blocking activity, also expressed by the inhibition of the pressor responses to exogenous angiotensin I challenges. The baroreceptor reflex, assessed by the response to exogenous angiotensin II challenge, remains unaltered.

Response of embryonic heart to hypoxia and reoxygenation: an in vitro model

OBJECTIVES: To define properly the consequences of oxygen deprivation and readmission for the functioning of the developing heart. METHODS: Spontaneously beating hearts excised from three-day-old chick embryos were loaded with a drop of viscous nontoxic silicone oil and cultured in a special chamber in which variations of PO2 at the tissue level could be strictly controlled. All parts of the hearts were simultaneously submitted to identical changes in PO2. Instantaneous heart rate, myocardial shortening, velocities of contraction and relaxation, and mechanical propagation along the heart tube were determined photometrically. RESULTS: The hearts, submitted to a PO2 ramp (0 to 9.3 kPa) or absolute anoxia, reacted rapidly, reversibly and reproducibly. Under sustained anoxia, ventricular activity stopped after 3.8±0.7 mins (n=4) and then resumed intermittently in the form of tachycardic bursts. Brief anoxia (1 min) provoked tachycardia followed by bradycardia, induced contracture, depressed contractility and retarded atrioventricular propagation. Upon reoxygenation, ventricular contractions ceased suddently for 20±11 s (n=5), whereas a residual atrial activity could persist. The duration of this arrest and the rate of recovery depended on duration of the preceding anoxia. Such a dysfunction constitutes the embryonic analogue of the oxygen paradox observed in adult hearts. Initial impulses, including arrhythmic activity, originated exclusively from the atrium, and no ventricular ectopic beats were detected whatever the conditions of oxygenation. CONCLUSIONS: This in vitro model seems promising for studying the pathophysiological mechanisms associated with hypoxia and reoxygenation in the developing heart.

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy

Neutrophils and TNF: two key players in the immune response induced by "Leishmania major" infection

Summary Resolution of the inflammation is as important as its induction. In this thesis, we investigated the contributions of two prominent factors involved in inflammation, Tumour Necrosis Factor (TNF) and neutrophils. We studied their role in the resolution óf the inflammatory lesion induced by the infection with the protozoan parasite Leishmania major. In mice susceptible to infection with L. major, unhealing lesions are characterized by an elevated number and sustained presence of inflammatory neutrophils in the infected tissue, illustrating an acute inflammatory process. In contrast, mice from resistant strains, which resolve their lesions, can control the presence of neutrophils at the site of infection. Neutrophil persistence in the infected tissue may result from several events including an increased survival of neutrophils mediated by factors produced by the pathogen or the microenvironment. Following infection with L. major, the cellular composition of the inflammatory lesion differs significantly between susceptible and resistant mice and a higher proportion of macrophages is present in the lesions of resistant strains. In an attempt to clarify the factors involved in neutrophil persistence, we investigated the mechanisms modulating neutrophil cell death. We demonstrated that macrophages could induce neutrophil apoptosis in a process involving TNF. TNF is an essential cytokine with pro- and anti-inflammatory properties, which is expressed as a transmembrane protein that can be cleaved releasing the secreted form. Our data show the essential role of the transmembrane form of TNF (mTNF) in the induction of neutrophil apoptosis by macrophages, revealing macrophages and mTNF as important regulators of neutrophil apoptosis. TNF is critical in the resolution of the inflammatory lesion induced by L. major infection, and in L. major resistant strains its absence results in increased swelling of the lesions. We investigated the contribution of mTNF in the outcome of L. major infection. Our data demonstrate that following infection with L. major, mTNF is sufficient to support the resolution of the inflammatory lesion and optimal parasite killing. In addition, we show that the presence of mTNF is essential to induce neutrophil clearance in the infected tissue. While the persistence of neutrophils is deleterious for the host, we could demonstrate an early anti-inflammatory role of neutrophils. Altogether, this study demonstrates the importance of mTNF in the induction of neutrophil apoptosis, a process involved in the resolution of the inflammatory lesion induced by L. major infection. Résumé La résolution de l'inflammation est toute aussi importante que son initiation. Durant ce travail de thèse, nous avons étudié les contributions de deux facteurs importants impliqués dans l'inflammation, le TNF (Facteur Nécrosant des Tumeurs) et les neutrophiles, dans la résolution de la lésion inflammatoire induite par l'infection avec le parasite protozoaire Leishmania major. Chez les souris sensibles à l'infection avec L. major, des lésions importantes qui ne guérissent pas se développent ; celles-ci sont caractérisées par un nombre élevé et une présence soutenue de neutrophiles dans les tissus infectés, ce qui illustre un processus inflammatoire aigu. Au contraire, les souris résistantes à l'infection qui guérissent leurs lésions, sont capables de contrôler la présence des neutrophiles au site d'infection. La persistance des neutrophiles dans la lésion inflammatoire peut être la conséquence de plusieurs événements, dont une augmentation de la survie des neutrophiles induite par des facteurs produits par le pathogène ou le micro-environnement. Suite à l'infection avec L. major, la composition cellulaire de la lésion inflammatoire est significativement différente entre les souris sensibles et résistantes à l'infection, et une plus grande proportion de macrophages est présente dans les lésions des souris résistantes. Dans l'objectif de clarifier les facteurs impliqués dans la persistance des neutrophiles dans les tissus infectés par L. major, nous avons étudié les mécanismes de régulation de la mort des neutrophiles. Nous avons démontré que les macrophages pouvaient induire l'apoptose des neutrophiles dans un procédé impliquant le TNF. Le TNF est une cytokine aux propriétés pro- et anti-inflammatoires, exprimée sous une forme transmembranaire qui peut être clivée pour relâcher la forme sécrétée. Nos expériences illustrent le rôle essentiel de la forme transmembranaire du TNF (mTNF) dans l'induction de l'apoptose des neutrophiles par les macrophages. Lé TNF est une cytokine importante dans la résolution de la réaction inflammatoire induite par L. major, et chez les souris résistantes l'absence de TNF provoque des lésions inflammatoires plus importantes. Nous avons étudié la contribution du mTNF dans la résolution de l'infection avec L. major. Nos résultats démontrent que suite à une infection avec le parasite, la présence du mTNF est suffisante pour guérir la lésion inflammatoire et contrôler efficacement la réplication du parasite. De plus, le mTNF joue un rôle essentiel dans l'élimination des neutrophiles du tissu infecté. Alors que la persistance des neutrophiles est nocive pour l'hôte, nous avons montré que les neutrophiles avaient un rôle précoce anti-inflammatoire. En résumé, cette étude révèle l'importance du mTNF dans l'induction de l'apoptose des neutrophiles par les macrophages, un procédé impliqué dans la résolution de la lésion inflammatoire induite par l'infection avec L. major.

Characterization of the role of AKAP-Lbc/p38 complex in the hypertrophic response of the heart

In response to chronic stress the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy, increased cellular apoptosis and fibrosis, which ultimately causes cardiac dysfunction and heart failure. Increasing evidence suggest the role of scaffolding and anchoring proteins in coordinating different signaling pathways that mediate the hypertrophic response of the heart. In this context, the family of Α-kinase anchoring proteins (AKAPs) emerged as important regulators of the cardiac function. During my thesis work I have conducted two independent projects, both of them aiming at elucidating the role of AKAPs in the heart. It has been shown that AKAP-Lbc, an anchoring protein that possesses an intrinsic Rho- specific exchange factor activity, organizes a signaling complex that links AKAP-Lbc- dependent activation of RhoA with the mitogen activated protein kinase (MAPK) p38. The first aim of my thesis was to study the role of this novel transduction pathway in the context of cardiac hypertrophy. Here we show that transgenic mice overexpressing in cardiomyocytes a competitor fragment of AKAP-Lbc, which specifically disrupts endogenous AKAP-Lbc / p38 complexes, developed early dilated cardiomyopathy in response to two weeks of transverse aortic constriction (TAC) as compared to controls. Interestingly, inhibition of the AKAP-Lbc / p38 transduction pathway significantly reduced the hypertrophic growth of single cardiomyocytes induced by pressure overload. Therefore, it appears that the AKAP- Lbc / p38 complex is crucially involved in the regulation of stress-induced cardiomyocyte hypertrophy and that disruption of this signaling pathway is detrimental for the heart under conditions of sustained hemodynamic stress. Secondly, in order to identify new AKAPs involved in the regulation of cardiac function, we followed a proteomic approach which allowed us to characterize AKAP2 as a major AKAP in the heart. Importantly, here we show that AKAP2 interacts with several proteins known to be involved in the control of gene transcription, such as the nuclear receptor coactivator 3 (NCoA3) or the ATP-dependent SWI/SNF chromatin remodeling complex. Thus, we propose AKAP2 as a novel mediator of cardiac gene expression through its interaction with these transcriptional regulators.

Low-replicating viruses and strong anti-viral immune response associated with prolonged disease control in a superinfected HIV-1 LTNP elite controller.

OBJECTIVE: To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient.¦METHODOLOGY AND PRINCIPAL FINDINGS: We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs.¦CONCLUSIONS: The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.

IL28B polymorphisms, IP-10 and viral load predict virological response to therapy in chronic hepatitis C.

Aliment Pharmacol Ther 2011; 33: 1162-1172 SUMMARY: Background  Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma and the identification of the predictors of response to antiviral therapy is an important clinical issue. Aim  To determine the independent contribution of factors including IL28B polymorphisms, IFN-gamma inducible protein-10 (IP-10) levels and the homeostasis model assessment of insulin resistance (HOMA-IR) score in predicting response to therapy in chronic hepatitis C (CHC). Methods  Multivariate analysis of factors predicting rapid (RVR) and sustained (SVR) virological response in 280 consecutive, treatment-naive CHC patients treated with peginterferon alpha and ribavirin in a prospective multicentre study. Results  Independent predictors of RVR were HCV RNA <400 000 IU/mL (OR 11.37; 95% CI 3.03-42.6), rs12980275 AA (OR 7.09; 1.97-25.56) and IP-10 (OR 0.04; 0.003-0.56) in HCV genotype 1 patients and lower baseline γ-glutamyl-transferase levels (OR = 0.02; 0.0009-0.31) in HCV genotype 3 patients. Independent predictors of SVR were rs12980275 AA (OR 9.68; 3.44-27.18), age <40 years (OR = 4.79; 1.50-15.34) and HCV RNA <400 000 IU/mL (OR 2.74; 1.03-7.27) in HCV genotype 1 patients and rs12980275 AA (OR = 6.26; 1.98-19.74) and age <40 years (OR 5.37; 1.54-18.75) in the 88 HCV genotype 1 patients without a RVR. RVR was by itself predictive of SVR in HCV genotype 1 patients (OR 33.0; 4.06-268.32) and the only independent predictor of SVR in HCV genotype 2 (OR 9.0, 1.72-46.99) or genotype 3 patients (OR 7.8, 1.43-42.67). Conclusions  In HCV genotype 1 patients, IL28B polymorphisms, HCV RNA load and IP-10 independently predict RVR. The combination of IL28B polymorphisms, HCV RNA level and age may yield more accurate pre-treatment prediction of SVR. HOMA-IR score is not associated with viral response.

Short-term and sustained renal effects of angiotensin II receptor blockade in healthy subjects.

We investigated the short-term and sustained hormonal and renal effects of angiotensin II (Ang II) receptor blockade in normotensive healthy volunteers. Twenty-four subjects maintained on a fixed sodium diet were randomized to receive for 8 days a placebo or 10 or 50 mg doses of the Ang II antagonist irbesartan (SR 47436, BMS 186295) according to a double-blind, parallel group design. Plasma renin activity, plasma immunoreactive Ang II and aldosterone levels, blood pressure, renal hemodynamics, and urinary electrolyte excretion were measured for 8 hours after the first and eighth administration of each dose of irbesartan or placebo. Ang II receptor blockade with irbesartan induced a dose-dependent compensatory increase in plasma renin activity and plasma angiotensin levels and a significant decrease in plasma aldosterone levels. The compensatory rise in plasma renin activity and Ang II levels was more pronounced on day 8, reflecting a long duration of the blocking effect of irbesartan. Irbesartan induced small changes in blood pressure and did not significantly modify renal blood flow and glomerular filtration rate. However, a significant decrease in filtration fraction was observed during receptor blockade on days 1 and 8. The tubular effects of irbesartan were characterized by a dose-dependent increase in sodium and chloride excretions. Interestingly, the cumulative natriuretic response to Ang II receptor blockade was similar on days 1 and 8, suggesting that in these subjects, renal Ang II receptors are not blocked over 24 hours during repeated administration even though this antagonist has a long duration of action (t1/2 of 15 to 17 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of indomethacin on the renal response to angiotensin II receptor blockade in healthy subjects.

BACKGROUND: Non-steroidal anti-inflammatory drugs are known to promote sodium retention and to blunt the blood pressure lowering effects of several classes of antihypertensive agents including beta-blockers, diuretics and angiotensin converting enzyme (ACE) inhibitors. The purpose of the present study was to investigate the acute and sustained effects of indomethacin on the renal response to the angiotensin II receptor antagonist valsartan and to the ACE inhibitor enalapril. METHODS: Twenty normotensive subjects maintained on fixed sodium intake (100 mmol sodium/day) were randomized to receive for one week: valsartan 80 mg o.d., enalapril 20 mg o.d., valsartan 80 mg o.d. + indomethacin 50 mg bid and enalapril 20 mg o.d. + indomethacin 50 mg bid. This single-blind study was designed as a parallel (valsartan vs. enalapril) and cross-over trial (valsartan or enalapril vs. valsartan + indomethacin or enalapril + indomethacin). Renal hemodynamics and urinary electrolyte excretion were measured for six hours after the first and seventh administration of each treatment regimen. RESULTS: The results show that valsartan and enalapril have comparable renal effects characterized by no change in glomerular filtration rate and significant increases in renal plasma flow and sodium excretion. The valsartan- and enalapril-induced renal vasodilation is not significantly blunted by indomethacin. However, indomethacin similarly abolishes the natriuresis induced by the angiotensin II antagonist and the ACE inhibitor. CONCLUSIONS: This observation suggests that although angiotensin receptor antagonists do not affect prostaglandin metabolism, the administration of a non-steroidal anti-inflammatory drug blunts the natriuretic response to angiotensin receptor blockade.