Transmyocardial laser revascularisation in acutely ischaemic myocardium.

OBJECTIVE: Although recent experience suggests that transmyocardial laser revascularisation (TMLR) relieves angina, its mechanism of action remains undefined. We examined its functional effects and analysed its morphological features in an animal model of acute ischaemia. METHODS: A total of 15 pigs were randomised to ligation of left marginal arteries (infarction group, n = 5), to TMLR of the left lateral wall using a holmium:yttrium-aluminium garnet (Ho:YAG) laser (laser group, n = 5), and to both (laser-infarction group, n = 5). All the animals were sacrificed 1 month after the procedure. Haemodynamics and echocardiography with segmental wall motion score were carried out at both time intervals (scale 0-3: 0, normal; 1, hypokinesia; 2, akinesia; 3, dyskinesia). Histology of the involved area was analysed. RESULTS: Laser group showed no change of the segmental wall motion score of the involved area 30 min after the laser channels were made (score: 0 +/- 0). Infarction and laser infarction groups both showed a persistent and definitive increase of the segmental wall motion score (at 30 min: 1.6 +/- 0.3 and 2 +/- 0, respectively; at 1 month: 1.8 +/- 0.2 and 1.8 +/- 0.4, respectively). These increases were all statistically significant in comparison with baseline values (P < 0.5), however comparison between infarction and laser-infarction groups showed no significant difference. On macroscopic examination of the endocardial surface, no channel was opened. On histology, there were signs of neovascularisation around the channels in the laser group, whereas in the laser-infarction group the channels were embedded in the infarction scar. CONCLUSIONS: In this acute pig model, TMLR did not provide improvement of contractility of the ischaemic myocardium. To the degree that the present study pertains to the clinical setting, the results suggest that mechanisms other than blood flow through the channels should be considered, such as a laser-induced triggering of neovascularisation or neural destruction.

Heritability and quantitative genetic divergence of serotiny, a fire-persistence plant trait.

BACKGROUND AND AIMS: Although it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (QST-FST comparison). METHODS: A common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h(2)) and quantitative genetic differentiation among populations for serotiny (QST) were estimated by means of an 'animal model' fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, QST estimates for serotiny were compared with FST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance. KEY RESULTS: Serotiny showed a significant narrow-sense heritability (h(2)) of 0·20 (credible interval 0·09-0·40). Quantitative genetic differentiation among provenances for serotiny (QST = 0·44) was significantly higher than expected under a neutral process (FST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites. CONCLUSIONS: Serotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.

Bayesian genetic parameters for body weight and survival of Nile tilapia farmed in Brazil

The objective of this study was to estimate genetic parameters for survival and weight of Nile tilapia (Oreochromis niloticus), farmed in cages and ponds in Brazil, and to predict genetic gain under different scenarios. Survival was recorded as a binary response (dead or alive), during harvest time in the 2008 grow-out period. Genetic parameters were estimated using a Bayesian mixed linear-threshold animal model via Gibbs sampling. The breeding population consisted of 2,912 individual fish, which were analyzed together with the pedigree of 5,394 fish. The heritabilities estimates, with 95% posterior credible intervals, for tagging weight, harvest weight and survival were 0.17 (0.09-0.27), 0.21 (0.12-0.32) and 0.32 (0.22-0.44), respectively. Credible intervals show a 95% probability that the true genetic correlations were in a favourable direction. The selection for weight has a positive impact on survival. Estimated genetic gain was high when selecting for harvest weight (5.07%), and indirect gain for tagging weight (2.17%) and survival (2.03%) were also considerable.

HIV-1 vaccines and adaptive trial designs.

Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although nonhuman primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, improving the characterization of promising vaccine candidates and the identification of correlates of immune protection.

Exploration of the visual system: Part 1: Dissection of the mouse eye for RNA, protein, and histological analyses

Due to the power of genetics, the mouse has become a widely used animal model in vision research. However, its eyeball has an axial length of only about 2 mm. The present protocol describes how to easily dissect the small rodent eye post mortem. This allows collecting different tissues of the eye, i.e., cornea, lens, iris, retina, optic nerve, retinal pigment epithelium (RPE), and sclera. We further describe in detail how to process these eye samples in order to obtain high‐quality RNA for RNA expression profiling studies. Depending on the eye tissue to be analyzed, we present appropriate lysis buffers to prepare total protein lysates for immunoblot and immuno‐precipitation analyses. Fixation, inclusion, embedding, and cryosectioning of the globe for routine histological analyses (HE staining, DAPI staining, immunohistochemistry, in situ hybridization) is further presented. These basic protocols should allow novice investigators to obtain eye tissue samples rapidly for their experiments.

Hepatitis C virus infection: in vivo and in vitro models.

Major advances in the understanding of the molecular biology of hepatitis C virus (HCV) have been made recently. While the chimpanzee is the only established animal model of HCV infection, several in vivo and in vitro models have been established that allow us to study various aspects of the viral life cycle. In particular, the replicon system and the production of recombinant infectious virions revolutionized the investigation of HCV-RNA replication and rendered all steps of the viral life cycle, including entry and release of viral particles, amenable to systematic analysis. In the following we will review the different in vivo and in vitro models of HCV infection.

Moderate hypercapnia exerts beneficial effects on splanchnic energy metabolism during endotoxemia.

PURPOSE: Low tidal volume ventilation and permissive hypercapnia are required in patients with sepsis complicated by ARDS. The effects of hypercapnia on tissue oxidative metabolism in this setting are unknown. We therefore determined the effects of moderate hypercapnia on markers of systemic and splanchnic oxidative metabolism in an animal model of endotoxemia. METHODS: Anesthetized rats maintained at a PaCO(2) of 30, 40 or 60 mmHg were challenged with endotoxin. A control group (PaCO(2) 40 mmHg) received isotonic saline. Hemodynamic variables, arterial lactate, pyruvate, and ketone bodies were measured at baseline and after 4 h. Tissue adenosine triphosphate (ATP) and lactate were measured in the small intestine and the liver after 4 h. RESULTS: Endotoxin resulted in low cardiac output, increased lactate/pyruvate ratio and decreased ketone body ratio. These changes were not influenced by hypercapnia, but were more severe with hypocapnia. In the liver, ATP decreased and lactate increased independently from PaCO(2) after endotoxin. In contrast, the drop of ATP and the rise in lactate triggered by endotoxin in the intestine were prevented by hypercapnia. CONCLUSIONS: During endotoxemia in rats, moderate hypercapnia prevents the deterioration of tissue energetics in the intestine.

In conditions of limited chromophore supply rods entrap 11-cis-retinal leading to loss of cone function and cell death.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.

Immunology of viral disease, how to curb persistent infection

1. 1. Summaries 1.1. Preamble and extended abstract The present thesis dissertation addresses the question of antiviral immunity from the particular standpoint of the adaptive T cell-mediated immune response. The experimental work is presented in the form of three published articles (two experimental articles and one review article, see sections 4.1, 4.2 and 4.3 on pages 73, 81 and 91, respectively), describing advances both in our understanding of viral control by CD8 T lymphocytes, and in vaccine development against the Human Immunodeficiency Virus Type 1 (HIV-1). Because the articles focus on rather specialized areas of antiviral immunity, the article sections are preceded by a general introduction (section 3) on the immune system in general, and on four viruses that were addressed in the experimental work, namely HIV-1, Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and Influenzavirus (Flu). This introduction section is aimed at providing a glimpse on viral molecular biology and immunity, to help the hypothetical non-expert reader proceeding into the experimental part. For this reason, each section is presented as individual entity and can be consulted separately. The four viruses described are of peculiar relevance to immunity because they induce an array of opposite host responses. Flu causes a self limiting disease after which the virus is eradicated. CMV and EBV cause pauci-symptomatic or asymptomatic diseases after which the viruses establish lifelong latency in the host cells, but are kept in check by immunity. Eventually, HIV-1 establishes both latency - by inserting its genome into the host cell chromosome - and proceeds in destroying the immune system in a poorly controlled fashion. Hence, understanding the fundamental differences between these kinds of viral host interactions might help develop new strategies to curb progressive diseases caused by viruses such as HIV-1. Publication #1: The first article (section 4.1, page 73) represents the main frame of my laboratory work. It analyses the ability of CD8 T lymphocytes recovered from viral-infected patients to secrete interferon γ (IFN-γ) alone or in conjunction with interleukin 2 (IL-2) when exposed in vitro to their cognate viral antigens. CD8 T cells are instrumental in controlling viral infection. They can identify infected cells by detecting viral antigens presented at the surface of the infected cells, and eliminate both the cell and its infecting virus by triggering apoptosis and/or lysis of the infected cell. Recognition of these antigens triggers the cognate CD8 cells to produce cytokines, including IFN-γ and IL-2, which in turn attract and activate other pro-inflammatory cells. IFN-γ triggers both intrinsic antiviral activity of the infected cells and distant activation of pro-inflammatory cells, which are important for the eradication of infection. IL-2 is essential for clonal expansion of the antigen (Ag)-specific CD8 T cell. Hence the existence of Ag-specific CD8 cells secreting both IFN-γand IL-2 should be beneficial for controlling infection. In this first work we determined the percentage of IFN-y/IL-2 double positive and single IFN-γsecreting CD8 T cells against antigens HIV-1, CMV, EBV and Flu in three groups of subjects: (i) HIV-1 infected patients progressing to disease (progressors), (ii) HIV-1-infected subjects not progressing to disease (long-term non progressors or LTNP), and (iii) HIV negative blood donors. The results disclosed a specific IFN-y/IL-2 double positive CD8 response in all subjects able to control infection. In other words, IFN-y/IL-2 double positive CD8 cells were present in virus-specific CD8 T cells against Flu, CMV and EBV as well against HIV-1 in LTNP. In contrast, progressors only had single IFN-γsecreting CD8 T cells. Hence, the ability to develop an IFN-y/IL-2 double positive response might be critical to control infection, independently of the nature of the virus. Additional experiments helped identify the developmental stage of the missing cells (using different markers such as CD45RA and CCR7) and showed a correlation between the absence of IL-2 secreting CD8 T cells and a failure in the proliferation capacity of virus-specific CD8 T cells. Addition of exogenous IL-2 could restore clonal expansion of HIV-1 specific CD8 T cells, at least in vitro. It could further been shown, that IL-2 secreting CD8 T cells are sufficient to support proliferation even in absence of CD4 help. However, the reason for the missing IFN-y/IL-2 double positive CD8 T cell response in HIV-1 progessors has yet to be determined. Publication #2: The second article (section 4.2, page 81) explores new strategies to trigger CD8 T cell immunity against specific HIV-1 proteins believed to be processed and exposed as "infection signal" at the surface of infected cells. Such signals consist of peptide fragments (8- 13 amino acids) originating from viral proteins and presented to CD8 T cells in the frame of particular cell surface molecules of the major histocompatibility complex class I* (MHC I). To mimic "natural" viral infection, the HIV-1 polyprotein Gagpolnef was inserted and expressed in either of two attenuated viruses i.e. vaccinia virus (MVA) or poxvirus (NYVAC). Mice were infected with these recombinant viruses and specific CD8 T cell response to Gagpolnef peptides was sought. Mice could indeed mount a CD8 T cell response against the HIV-1 antigens, indicating that the system worked, at least in this animal model. To further test whether peptides from Gagpolnef could also be presented in the frame of the human MHC class I proteins, a second round of experiments was performed in "humanized" transgenic mice expressing human MHC molecules. The transgenic mice were also able to load Gagpolnef peptides on their human MHC molecule, and these cells could be detected and destroyed by Ag-specific CD8 T cells isolated from HIV-1-infected patients. Therefore, expressing Gagpolnef on attenuated recombinant viruses might represent a valid strategy for anti-HIV-1 immunization in human. Publication #3: This is a review paper (section 4.3, page 91) describing the immune response to CMV and newly developed methods to detect this cellular immune response. Some of it focuses on the detection of T cells by using in vitro manufactured tetramers. These consist of four MHC class I molecules linked together and loaded with the appropriate antigenic peptide. The tetramer can be labeled with a fluorochrome and analyzed with a fluorescence-activated cell sorter. Taken together, the work presented indicates that (i) an appropriate CD8 T cell response consisting of IFN-y/IL-2 double positive effectors, can potentially control viral infection, including HIV-1 infection, (ii) such a response might be triggered by recombinant viral vaccines, and (iii) CD8 T cell response can be monitored by a variety of techniques, including recently-developed MHC class I tetramers. 1. 2. Préambule et résumé élargi Le présent travail de thèse s'intéresse à l'immunité antivirale du point de vue particulier de la réponse adaptative des cellules T. Le travail expérimental est présenté sous la forme de trois articles publiés (2 articles expérimentaux et 1 article de revue, voir sections 4.1, 4.2 et 4.3, pages 58, 66 et 77, respectivement), décrivant des progrès dans la compréhension du contrôle de l'infection virale par les lymphocytes T CD8, ainsi que dans le développement de nouveaux vaccins contre le Virus d'Immunodéficience de Humaine de type 1 (VIH-1). En raison du caractère spécialisé de l'immunité antivirale de type cellulaire, les articles sont précédés par une introduction générale (section 3), dont le but est de pourvoir le lecteur non avisé avec des bases nécessaire à une meilleure appréhension du travail expérimental. Cette introduction présente les grandes lignes du système immunitaire, et décrit de façon générale les 4 virus utilisés dans le travail expérimental: à savoir le virus VIH-1, le Cytomégalovirus (CMV), le virus Epstein Barr (EBV) et le virus Influenza A (Flu). Toutes les sections sont présentées de façon individuelle et peuvent être consultées séparément. La description des 4 virus a une pertinence particulière quant à leur interaction avec le système immun. En effet, ils induisent une panoplie de réponses immunitaires s'étendant aux extrêmes de la réaction de l'hôte. Influenza A est à l'origine d'une maladie cytopathique aiguë, au décours de laquelle le virus est éradiqué par l'hôte. CMV et EBV sont classiquement à l'origine d'infections pauci-symptomatiques, voire asymptomatiques, après lesquelles les virus persistent de façon latente dans la cellule hôte. Cependant, ils restent sous le contrôle du système immun, qui peut prévenir une éventuelle réactivation. Enfin, VIH-1 s'établit à la fois en infection latente - par l'insertion de son génome dans le chromosome des cellules hôtes - et en infection productive et cytopathique, échappant au contrôle immunitaire et détruisant ses cellules cibles. La compréhension des différences fondamentales entre ces différents types d'interactions virus-hôte devraient faciliter le développement de nouvelles stratégies antivirales. Article 1: Le premier article (section 4.1 Page 58) représente l'objet principal de mon travail de laboratoire. Il analyse la capacité des lymphocytes T CD8 spécifiques de différent virus à sécréter de l'interféron gamma (IFN-y) et/ou de l'interleukine 2 (IL-2) après stimulation par leur antigène spécifique. Les cellules T CD8 jouent un rôle crucial dans le contrôle des infections virales. Elles identifient les cellules infectées en détectant des antigènes viraux présentés à la surface de ces mêmes cellules, et éliminent à la fois les cellules infectées et les virus qu'elles contiennent en induisant l'apoptose et/ou la lyse des cellules cibles. Parallèlement, l'identification de l'antigène par la cellule T CD8 la stimule à sécréter des cytokines. L'IFN-γen est un exemple. L'IFN-γ stimule les cellules infectées à développer une activé antivirale intrinsèque. De plus, il attire sur place d'autres cellules de l'inflammation, et active leur fonction d'éradication des pathogènes. L'IL-2 est un autre exemple. L'IL-2 est essentielle à l'expansion clonale des cellules T CD8 spécifiques à un virus donné. Elle est donc essentielle à augmenter le pool de lymphocytes antiviraux. En conséquence, la double capacité de sécréter de l'IFN-γ et de IL-2 pourrait être un avantage pour le contrôle antiviral par les cellules T CD8. Dans ce travail nous avons comparé les proportions de lymphocytes T CD8 doubles positifs (IFN-γ/IL-2) et simples positifs (IFN-γ) chez trois groupes de sujets: (i) des patients infectés par VIH-1 qui ne contrôlent pas l'infection (progresseurs), (ii) des patients infectés par VIH-1, mais contrôlant l'infection malgré l'absence de traitement ("long term non progressors" [LTNP]) et (iii) des donneurs de sang négatifs pour l'infection à VIH-1. Les résultats ont montré que les individus capables de contrôler une infection possédaient des cellules T CD8 doubles positifs (IFN-γ/IL-2), alors que les patients ne contrôlant pas l'infection procédaient prioritairement des CD8 simples positifs (IFN-γ). Spécifiquement, les lymphocytes T spécifiques pour Flu, CMV, EBV, et VII-1-1 chez les LTNP étaient tous IFN-γ/IL-2 doubles positifs. Au contraire, les lymphocytes T CD8 spécifique à VIH-1 étaient IFN-γ simples positifs chez les progresseurs. La capacité de développer une réponse IFN-γ/IL-2 pourraient être primordiale pour le contrôle de l'infection, indépendamment de la nature du virus. En effet, il a été montré que l'absence de sécrétion d'IL2 par les lymphocytes T CD8 corrélait avec leur incapacité de proliférer. Dans nos mains, cette prolifération a pu être restaurée in vitro par l'adjonction exogène d'IL-2. Toutefois, la faisabilité de ce type de complémentation in vivo n'est pas claire. Des expériences additionnelles ont permis de préciser de stade de développement des lymphocytes doubles positifs et simples positifs par le biais des marqueurs CD45RA et CCR7. Il reste maintenant à comprendre pourquoi certains lymphocytes T CD8 spécifiques sont incapables à sécréter de l'IL-2. Article 2: Le deuxième article explore des nouvelles stratégies pour induire une immunité T CD8 spécifique aux protéines du VIH-1, qui sont édités et exposés à la surface des cellules infectées. Ces signaux consistent en fragments de peptide de 8-13 acide aminés provenant de protéines virales, et exposées à la surface des cellules infectées dans le cadre des molécules spécialisées d'histocompatibilité de classe I (en anglais "major histocompatibility class I" ou MHC I). Pour mimer une infection virale, la polyprotéine Gagpolnef du VIH-1 a été insérée et exprimée dans deux vecteurs viraux atténués, soit MVA (provenant de vaccinia virus) ou NYVAC (provenant d'un poxvirus). Ensuite des souris ont été infectées avec ces virus recombinants et la réponse T CD8 aux peptides issus de Gagpolnef a été étudiée. Les souris ont été capables de développer une réponse de type CD8 T contre ces antigènes du VIH-1. Pour tester si ces antigènes pouvaient aussi être présentés par dans le cadre de molécules MHC humaines, des expériences supplémentaires ont été faites avec des souris exprimant un MHC humain. Les résultats de ces manipulations ont montré que des cellules T CD8 spécifique aux protéines du VIH pouvaient être détectées. Ce travail ouvre de nouvelles options quant à l'utilisation des virus recombinants exprimant Gagpolnef comme stratégie vaccinale contre le virus VIH-I chez l'homme. Article 3: Ces revues décrivent la réponse immunitaire à CMV ainsi que des nouvelles méthodes pouvant servir à sa détection. Une partie du manuscrit décrit la détection de cellule T à l'aide de tétramères. Il s'agit de protéines chimériques composées de 4 quatre molécules MHC liées entre elles. Elles sont ensuite "chargées" avec le peptide antigénique approprié, et utilisée pour détecter les cellules T CD8 spécifiques à ce montage. Elles sont aussi marquées par un fluorochrome, qui permet une analyse avec un cytomètre de flux, et l'isolement ultime des CD8 d'intérêt. En résumé, le travail présenté dans cette thèse indique que (i) une réponse T CD8 appropriée - définie par la présence des cellules effectrices doublement positives pour l'IFN-γ et l'IL-2 - semble indispensable pour le contrôle des infections virales, y compris par le VIH-1, (ii) une telle réponse peut être induite par des vaccin viral recombinant, et (iii) la réponse T CD8 peut être analysée et suivie grâce à plusieurs techniques, incluant celle des tétramères de MHC class I. 1.3. Résumé pour un large public Le système immunitaire humain est composé de différents éléments (cellules, tissus et organes) qui participent aux défenses de l'organisme contre les pathogènes (bactéries, virus). Parmi ces cellules, les lymphocytes T CD8, également appelés cellules tueuses, jouent un rôle important dans la réponse immunitaire et le contrôle des infections virales. Les cellules T CD8 reconnaissent de manière spécifique des fragments de protéines virales qui sont exposés à la surface des cellules infectées par le virus. Suite à cette reconnaissance, les cellules T CD8 sont capables de détruire et d'éliminer ces cellules infectées, ainsi que les virus qu'elles contiennent. Dans le contexte d'une infection par le virus de l'immunodéficience humaine (VIH), le virus responsable du SIDA, il a pu être montré que la présence des cellules T CD8 est primordiale. En effet, en l'absence de ces cellules, les individus infectés par le VIH progressent plus rapidement vers le SIDA. Au cours de la vie, l'Homme est exposé à plusieurs virus. Mais à l'opposé du VIH, certains d'entre eux ne causent pas des maladies graves : par exemple le virus de la grippe (Influenza), le cytomégalovirus ou encore le virus d'Epstein-Barr. Certains de ces virus peuvent être contrôlés et éliminés de l'organisme (p. ex. le virus de la grippe), alors que d'autres ne sont que contrôlés par notre système immunitaire et restent présents en petite quantité dans le corps sans avoir d'effet sur notre santé. Le sujet de mon travail de thèse porte sur la compréhension du mécanisme de contrôle des infections virales par le système immunitaire : pourquoi certains virus peuvent être contrôlés ou même éliminés de l'organisme alors que d'autres, et notamment le VIH, ne le sont pas. Ce travail a permis de démontrer que les cellules T CD8 spécifiques du VIH ne sécrètent pas les mêmes substances, nécessaires au développement d'une réponse antivirale efficace, que les cellules T CD8 spécifiques des virus contrôlés (le virus de la grippe, le cytomégalovirus et le virus d'Epstein-Barr). Parallèlement nous avons également observé que les lymphocytes T CD8 spécifiques du VIH ne possèdent pas la capacité de se diviser. Ils sont ainsi incapables d'être présents en quantité suffisante pour assurer un combat efficace contre le virus du SIDA. La (les) différence(s) entre les cellules T CD8 spécifiques aux virus contrôlés (grippe, cytomégalovirus et Epstein-Barr) et au VIH pourront peut-être nous amener à comprendre comment restaurer une immunité efficace contre ce dernier.

Local Salmonella immunostimulation recruits vaccine-specific CD8 T cells and increases regression of bladder tumor.

NlmCategory="UNASSIGNED">The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in complement component 5a, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

Potential Microbiological Effects of Higher Dosing of Echinocandins.

The antifungal "paradoxical effect" has been described as the reversal of growth inhibition at high doses of echinocandins, most usually caspofungin. This microbiological effect appears to be a cellular compensatory response to cell wall damage, resulting in alteration of cell wall content and structure as well as fungal morphology and growth. In vitro studies demonstrate this reproducible effect in a certain percentage of fungal isolates, but animal model and clinical studies are less consistent. The calcineurin and Hsp90 cell signaling pathways appear to play a major role in regulating these cellular and structural changes. Regardless of the clinical relevance of this paradoxical growth effect, understanding the specific actions of echinocandins is paramount to optimizing their use at either standard or higher dosing schemes, as well as developing future improvements in our antifungal arsenal.

Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor-α (TNFα). To date, however, no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here, we show that local increase of TNFα in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning-memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS, as well as in other CNS conditions accompanied by inflammatory states or infections.

Environmental factors in multiple sclerosis [Environmental factors in multiple sclerosis]

Although multiple sclerosis (MS) is recognized as a disorder involving the immune system, the interplay of environmental factors and individual genetic susceptibility seems to influence MS onset and clinical expression, as well as therapeutic responsiveness. Multiple human epidemiological and animal model studies have evaluated the effect of different environmental factors, such as viral infections, vitamin intake, sun exposure, or still dietary and life habits on MS prevalence. Previous Epstein-Barr virus infection, especially if this infection occurs in late childhood, and lack of vitamin D (VitD) currently appear to be the most robust environmental factors for the risk of MS, at least from an epidemiological standpoint. Ultraviolet radiation (UVR) activates VitD production but there are also some elements supporting the fact that insufficient UVR exposure during childhood may represent a VitD-independent risk factor of MS development, as well as negative effect on the clinical and radiological course of MS. Recently, there has been a growing interest in the gut-brain axis, a bidirectional neuro-hormonal communication system between the intestinal microbiota and the central nervous system (CNS). Indeed, components of the intestinal microbiota may be pro-inflammatory, promote the migration of immune cells into the CNS, and thus be a key parameter for the development of autoimmune disorders such as MS. Interestingly most environmental factors seem to play a role during childhood. Thus, if childhood is the most fragile period to develop MS later in life, preventive measures should be applied early in life. For example, adopting a diet enriched in VitD, playing outdoor and avoiding passive smoking would be extremely simple measures of primary prevention for public health strategies. However, these hypotheses need to be confirmed by prospective evaluations, which are obviously difficult to conduct. In addition, it remains to be determined whether and how VitD supplementation in adult life would be useful in alleviating the course of MS, once this disease has already started. A better knowledge of the influence of various environmental stimuli on MS risk and course would certainly allow the development of add-on therapies or measures in parallel to the immunotherapies currently used in MS.

Diffantom: Whole-Brain Diffusion MRI Phantoms Derived from Real Datasets of the Human Connectome Project.

Food allergies are believed to be on the rise and currently management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low allergenic foods for egg allergic patients to extent their diet. This article is protected by copyright. All rights reserved.

DWI and complex brain network analysis predicts vascular cognitive impairment in spontaneous hypertensive rats undergoing executive function tests

The identification of biomarkers of vascular cognitive impairment is urgent for its early diagnosis. The aim of this study was to detect and monitor changes in brain structure and connectivity, and to correlate them with the decline in executive function. We examined the feasibility of early diagnostic magnetic resonance imaging (MRI) to predict cognitive impairment before onset in an animal model of chronic hypertension: Spontaneously Hypertensive Rats. Cognitive performance was tested in an operant conditioning paradigm that evaluated learning, memory, and behavioral flexibility skills. Behavioral tests were coupled with longitudinal diffusion weighted imaging acquired with 126 diffusion gradient directions and 0.3 mm(3) isometric resolution at 10, 14, 18, 22, 26, and 40 weeks after birth. Diffusion weighted imaging was analyzed in two different ways, by regional characterization of diffusion tensor imaging (DTI) indices, and by assessing changes in structural brain network organization based on Q-Ball tractography. Already at the first evaluated times, DTI scalar maps revealed significant differences in many regions, suggesting loss of integrity in white and gray matter of spontaneously hypertensive rats when compared to normotensive control rats. In addition, graph theory analysis of the structural brain network demonstrated a significant decrease of hierarchical modularity, global and local efficacy, with predictive value as shown by regional three-fold cross validation study. Moreover, these decreases were significantly correlated with the behavioral performance deficits observed at subsequent time points, suggesting that the diffusion weighted imaging and connectivity studies can unravel neuroimaging alterations even overt signs of cognitive impairment become apparent.