Time-Course In Vivo Trafficking Pattern and Effector Function of Donor-Specific Regulatory T Cells in Response to an Allograft.

Background: Experimental data have suggested that adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs), capable of controlling immune responses to specifi c auto- or alloantigens, could be used as a therapeutic strategy to promote specifi c tolerance in T-cell mediated diseases and in organ transplantation (Tx). However, before advocating the application of immunotherapy with Tregs in Tx, we need to improve our understanding of their in vivo homeostasis, traffi cking pattern and effector function in response to alloantigens. Methods : Donor-antigen specifi c murine Tregs were generated and characterized in vitro following our described protocols. Using an adoptive transfer and skin allotransplantation model, we have analyzed the in vivo expansion and homing of fl uorescent-labeled effector T cells (Teff) and Tregs, at different time-points after Tx, using fl ow-cytometry as well as fl uorescence microscopy techniques. Results: Tregs expressed CD62L, CCR7 and CD103 allowing their homing into lymphoid and non-lymphoid tissues (gut, skin) after intravenous injection. While hyporesponsive to TCR stimulation in vitro, transferred Tregs survived, migrated to secondary lymphoid organs and preferentially expanded within the allograft draining lymph nodes. Furthermore, Foxp3+ cells could be detected inside the allograft as early as day 3-5 after Tx. At a much later time-point (day 60 after Tx), graft-infi ltrating Foxp3+ cells were also detectable in tolerant recipients. When transferred alone, CD4+CD25- Teff cells expanded within secondary lymphoid organs and infi ltrated the allograft by day 3-5 after Tx. The co-transfer of Tregs limited the expansion of alloreactive Teff cells as well as their recruitment into the allograft. The promotion of graft survival observed in the presence of Tregs was in part mediated by the inhibition of the production of effector cytokines by CD4+CD25- T cells. Conclusion: Taken together, our results suggest that the suppression of allograft rejection and the induction of Tx tolerance are in part dependant on the alloantigendriven homing and expansion of Tregs. Thus, the appropriate localization of Tregs may be critical for their suppressive function in vivo.

Effect of diets high or low in unavailable and slowly digestible carbohydrates on the pattern of 24-h substrate oxidation and feelings of hunger in humans.

BACKGROUND: The pattern of substrate utilization with diets containing a high or a low proportion of unavailable and slowly digestible carbohydrates may constitute an important factor in the control, time course, and onset of hunger in humans. OBJECTIVE: We tested the hypothesis that isoenergetic diets differing only in their content of unavailable carbohydrates would result in different time courses of total, endogenous, and exogenous carbohydrate oxidation rates. DESIGN: Two diets with either a high (H diet) or a low (L diet) content of unavailable carbohydrates were fed to 14 healthy subjects studied during two 24-h periods in a metabolic chamber. Substrate utilization was assessed by whole-body indirect calorimetry. In a subgroup of 8 subjects, endogenous and exogenous carbohydrate oxidation were assessed by prelabeling the body glycogen stores with [(13)C]carbohydrate. Subjective feelings of hunger were estimated with use of visual analogue scales. RESULTS: Total energy expenditure and substrate oxidation did not differ significantly between the 2 diets. However, there was a significant effect of diet (P: = 0.03) on the carbohydrate oxidation pattern: the H diet elicited a lower and delayed rise of postprandial carbohydrate oxidation and was associated with lower hunger feelings than was the L diet. The differences in hunger scores between the 2 diets were significantly associated with the differences in the pattern of carbohydrate oxidation among diets (r = -0.67, P: = 0. 006). Exogenous and endogenous carbohydrate oxidation were not significantly influenced by diet. CONCLUSIONS: The pattern of carbohydrate utilization is involved in the modulation of hunger feelings. The greater suppression of hunger after the H diet than after the L diet may be helpful, at least over the short term, in individuals attempting to better control their food intake.

High-resolution numerical modelling of flow-vegetation interactions

In this paper, we present and apply a new three-dimensional model for the prediction of canopy-flow and turbulence dynamics in open-channel flow. The approach uses a dynamic immersed boundary technique that is coupled in a sequentially staggered manner to a large eddy simulation. Two different biomechanical models are developed depending on whether the vegetation is dominated by bending or tensile forces. For bending plants, a model structured on the Euler-Bernoulli beam equation has been developed, whilst for tensile plants, an N-pendula model has been developed. Validation against flume data shows good agreement and demonstrates that for a given stem density, the models are able to simulate the extraction of energy from the mean flow at the stem-scale which leads to the drag discontinuity and associated mixing layer.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.

Prognostic accuracy of cerebral blood flow measurement by perfusion computed tomography, at the time of emergency room admission, in acute stroke patients.

The purpose of this study was to determine the prognostic accuracy of perfusion computed tomography (CT), performed at the time of emergency room admission, in acute stroke patients. Accuracy was determined by comparison of perfusion CT with delayed magnetic resonance (MR) and by monitoring the evolution of each patient's clinical condition. Twenty-two acute stroke patients underwent perfusion CT covering four contiguous 10mm slices on admission, as well as delayed MR, performed after a median interval of 3 days after emergency room admission. Eight were treated with thrombolytic agents. Infarct size on the admission perfusion CT was compared with that on the delayed diffusion-weighted (DWI)-MR, chosen as the gold standard. Delayed magnetic resonance angiography and perfusion-weighted MR were used to detect recanalization. A potential recuperation ratio, defined as PRR = penumbra size/(penumbra size + infarct size) on the admission perfusion CT, was compared with the evolution in each patient's clinical condition, defined by the National Institutes of Health Stroke Scale (NIHSS). In the 8 cases with arterial recanalization, the size of the cerebral infarct on the delayed DWI-MR was larger than or equal to that of the infarct on the admission perfusion CT, but smaller than or equal to that of the ischemic lesion on the admission perfusion CT; and the observed improvement in the NIHSS correlated with the PRR (correlation coefficient = 0.833). In the 14 cases with persistent arterial occlusion, infarct size on the delayed DWI-MR correlated with ischemic lesion size on the admission perfusion CT (r = 0.958). In all 22 patients, the admission NIHSS correlated with the size of the ischemic area on the admission perfusion CT (r = 0.627). Based on these findings, we conclude that perfusion CT allows the accurate prediction of the final infarct size and the evaluation of clinical prognosis for acute stroke patients at the time of emergency evaluation. It may also provide information about the extent of the penumbra. Perfusion CT could therefore be a valuable tool in the early management of acute stroke patients.

A stable and 40Ar/39Ar isotope study of a major thrust in the Helvetic nappes (Swiss Alps) - Evidence for fluid-flow and constraints on nappe kinematics

Stable isotope and Ar-40/Ar-39 measurements,were made on samples associated with a major tectonic discontinuity in the Helvetic Alps, the basal thrust of the Diablerets nappe (external zone of the Alpine Belt) in order to determine both the importance of fluids in this thrust zone and the timing of thrusting. A systematic decrease in the delta(18)O values (up to 6 parts per thousand) of calcite, quartz, and white mica exists within a 10- to 70-m-wide zone over a distance of 37 km along the thrust, and they become more pronounced toward the root of the nappe. A similar decrease in the delta(13)C values of calcite is observed only in the deepest sections (up to 3 parts per thousand). The delta D-SMOW (SMOW = standard mean ocean water) values of white mica are -54 parts per thousand +/- 8 parts per thousand (n = 22) and are independent of the distance from the thrust. These variations are interpreted to reflect syntectonic solution reprecipitation during fluid passage along the thrust. The calculated delta(18)O and delta D values (versus SMOW) for the fluid in equilibrium with the analyzed minerals is 12 parts per thousand to 16 parts per thousand and -30 parts per thousand to +5 parts per thousand, respectively, for assumed temperatures of 250 to 450 degrees C. The isotopic and structural data are consistent with fluids derived from the deep-seated roots of the Helvetic nappes where large volumes of Mesozoic sediments were metamorphosed to the amphibolite facies, It is suggested that connate and metamorphic waters, overpressured by rapid tectonic burial in a subductive system escaped by upward infiltration along moderately dipping pathways until they reached the main shear zone at the base of the moving pile, where they were channeled toward the surface, This model also explains the mechanism by which large amounts of fluid were removed from the Mesozoic sediments during Alpine metamorphism. White mica Ar-49/Ar-39 ages vary from 27 Ma far from the Diablerets thrust to 15 Ma along the thrust. An older component is observed in micas far from the thrust, interpreted as a detrital signature, and indicates that regional metamorphic temperatures were less than about 350 degrees C. The;plateau and near plateau ages nearest the thrust are consistent with either neocrystallization of white mica or argon loss by recrystallization during thrusting, which may have been enhanced in the zones of highest fluid flow. The 15 Ma Ar-40/Ar-39 age plateau measured on white mica sampled exactly on the thrust surface dates the end of both fluid flow and tectonic transport.

Inversion prepared coronary MR angiography: direct visualization of coronary blood flow.

PURPOSE: Visualization of coronary blood flow by means of a slice-selective inversion pre-pulse in concert with bright-blood coronary MRA. MATERIALS AND METHODS: Coronary magnetic resonance angiography (MRA) of the right coronary artery (RCA) was performed in eight healthy adult subjects on a 1.5 Tesla MR system (Gyroscan ACS-NT, Philips Medical Systems, Best, NL) using a free-breathing navigator-gated and cardiac-triggered 3D steady-state free-precession (SSFP) sequence with radial k-space sampling. Imaging was performed with and without a slice-selective inversion pre-pulse, which was positioned along the main axis of the coronary artery but perpendicular to the imaging volume. Objective image quality parameters such as SNR, CNR, maximal visible vessel length, and vessel border definition were analyzed. RESULTS: In contrast to conventional bright-blood 3D coronary MRA, the selective inversion pre-pulse provided a direct measure of coronary blood flow. In addition, CNR between the RCA and right ventricular blood pool was increased and the vessels had a tendency towards better delineation. Blood SNR and CNR between right coronary blood and epicardial fat were comparable in both sequences. CONCLUSION: The combination of a free-breathing navigator-gated and cardiac-triggered 3D SSFP sequence with a slice-selective inversion pre-pulse allows for direct and directional visualization of coronary blood flow with the additional benefit of improved contrast between coronary and right ventricular blood pool.

Three Generation Family With Pattern Dystrophy and a Successful Treatment of Subfoveal Cnv Related to Pattern Dystrophy With Anti-vegf Intravitreal Injections

Purpose: To phenotype a large 3 generation Swiss family with pattern dystrophy and to report a successful result of treatment with ranibizumab of a subfoveal choroidal neovascularisation (CNV) associated with pattern dystrophy in 1 patient Patients and methods: 4 affected and 3 unaffected patients (3 female 4 male, age range: 19 - 80 years) were assessed with a complete ophthalmologic examination. AF images were taken using Heidelberg Retina Angiograph and the digital color photos, fluorescein angiogragraphy (FFA) using the same TOPCON 501 camera. Electroretinogram (full-field and multifocal) was performed in 1 affected patient. One 48 years old patient developed a subfoveal CNV, which was treated with 2 injections of ranibizumab, at 3 months interval. Blood sample was taken for molecular analysis (screening of the gene RDS). Results: Two patients had a typical fundoscopic appearance of pattern dystrophy with butterfly shaped deposit at the fovea and some peripheral flecks, as shown with AF imaging.. Two others affected patients had a more unusual appearance with some macular atrophy in one or both eyes, surrounded by flecks. The visual acuity ranged from 1.0 to 0.1 according to Snellen EDTRS chart. The patient with subfoveal CNV presented a drop of vision form 1.0 to 0.6 within 10 days prior to the diagnosis and also reported some metamorphopsia. FFA and optical computerized tomography (OCT) confirmed a classic CNV. After the 1st injection her vision improved to 1.0 but persistent metamorphopsia and fluid on OCT motivated a second injection. One month after the second injection the OCT was flat and the patient had no symptoms. The results of RDS screening will be presented at the meeting. Conclusion: We present a family with pattern dystrophy, with some members having an unusual fundus appearance, which was mistaken for an early onset dry AMD. The AF imaging is a useful tool in diagnosing this condition. A CNV associated with pattern dystrophy a rare. This is the first report of a successful treatment of the CNV with anti-VEGF intravitreal injections.

Conservation genetics and population monitoring of the Alpine Salamander (Salamandra Atra) :

SUMMARYIn the context of the biodiversity crisis, amphibians are experiencing the most severe worldwide decline of all vertebrates and are in urgent need of better management. Efficient conservation strategies rely on sound knowledge of the species biology and of the genetic and demographic processes that might impair their welfare. Nonetheless, these processes are poorly understood in amphibians. Delineating population boundaries remains consequently problematic for these species, while it is of critical importance to define adequate management units for conservation. In this study, our attention focused on the alpine salamander (Salamandra atra), a species that deserves much interest in terms of both conservation biology and evolution. This endemic alpine species shows peculiar life-history traits (viviparity, reduced activity period, slow maturation) and has a slow population turnover, which might be problematic for its persistence in a changing environment. Due to its elusive behaviour (individuals spend most of their time underground and are unavailable for sampling), dynamic processes of gene and individuals were poorly understood for that species. Consequently, its conservation status could hardly be reliably assessed. Similarly the fire salamander (Salamandra salamandra) also poses special challenges for conservation, as no clear demarcation of geographical populations exists and dispersal patterns are poorly known. Through a phylogeographic analysis, we first studied the evolutionary history of the alpine salamander to better document the distribution of the genetic diversity along its geographical range. This study highlighted the presence of multiple divergent lineages in Italy together with a clear genetic divergence between populations from Northern and Dinaric Alps. These signs of cryptic genetic differentiation, which are not accounted for by the current taxonomy of the species, should not be neglected for further definition of conservation units. In addition, our data supported glacial survival of the species in northern peripheral glacial réfugia and nunataks, a pattern rarely documented for long-lived species. Then, we evaluated the level of gene flow between populations at the local scale and tested for asymmetries in male versus female dispersal using both field-based (mark-recapture) and genetic approaches. This study revealed high level of gene flow between populations, which stems mainly from male dispersal. This corroborated the idea that salamanders are much better dispersers than hitherto thought and provided a well- supported example of male-biased dispersal in amphibians. In a third step, based on a mark- recapture survey, we addressed the problem of sampling unavailability in alpine salamanders and evaluated its impact on two monitoring methods. We showed that about three quarters of individuals were unavailable for sampling during sampling sessions, a proportion that can vary with climatic conditions. If not taken into account, these complexities would result in false assumptions on population trends and misdirect conservation efforts. Finally, regarding the daunting task of delineating management units, our attention was drawn on the fire salamander. We conducted a local population genetic study that revealed high levels of gene flow among sampling sites. Management units for this species should consequently be large. Interestingly, despite the presence of several landscape features often reported to act as barriers, genetic breaks occurred at unexpected places. This suggests that landscape features may rather have idiosyncratic effects on population structure. In conclusion, this work brought new insights on both genetic and demographic processes occurring in salamanders. The results suggest that some biological paradigms should be taken with caution when particular species are in focus. Species- specific studies remain thus fundamental for a better understanding of species evolution and conservation, particularly in the context of current global changes.RESUMEDans le contexte de la crise de la biodiversité actuelle, les amphibiens subissent le déclin le plus important de tous les vertébrés et ont urgemment besoin d'une meilleure protection. L'établissement de stratégies de conservation efficaces repose sur des connaissances solides de la biologie des espèces et des processus génétiques et démographiques pouvant menacer leur survie. Ces processus sont néanmoins encore peu étudiés chez les amphibiens.Dans cette étude, notre attention s'est portée sur la salamandre noire (Salamandra atra), une espèce endémique des Alpes dont les traits d'histoire de vie atypiques (viviparité, phase d'activité réduite, lent turnover des populations) pourraient la rendre très vulnérable face aux changements environnementaux. Par ailleurs, en raison de son comportement cryptique (les individus passent la plupart de leur temps sous terre) la dynamique des gènes et des individus est mal comprise chez cette espèce. Il est donc difficile d'évaluer son statut de conservation de manière fiable. La salamandre tachetée {Salamandra salamandra), pour qui il n'existe aucune démarcation géographique apparente des populations, pose également des problèmes en termes de gestion. Dans un premier temps, nous avons étudié l'histoire évolutive de la salamandre noire afin de mieux décrire la distribution de sa diversité génétique au sein de son aire géographique. Cela a permis de mettre en évidence la présence de multiples lignées en Italie, ainsi qu'une nette divergence entre les populations du nord des Alpes et des Alpes dinariques. Ces résultats seront à prendre en compte lorsqu'il s'agira de définir des unités de conservation pour cette espèce. D'autre part, nos données soutiennent l'hypothèse d'une survie glaciaire dans des refuges nordiques périglaciaires ou dans des nunataks, fait rarement documenté pour une espèce longévive. Nous avons ensuite évalué la différentiation génétique des populations à l'échelle locale, ce qui a révélé d'important flux de gènes, ainsi qu'une asymétrie de dispersion en faveur des mâles. Ces résultats corroborent l'idée que les amphibiens dispersent mieux que ce que l'on pensait, et fournissent un exemple robuste de dispersion biaisée en faveur des mâles chez les amphibiens. Nous avons ensuite abordé le problème de Γ inaccessibilité des individus à la capture. Nous avons montré qu'environ trois quarts des individus sont inaccessibles lors des échantillonnages, une proportion qui peut varier en fonction des conditions climatiques. Ignoré, ce processus pourrait entraîner une mauvaise interprétation des fluctuations de populations ainsi qu'une mauvaise allocation des efforts de conservation. Concernant la définition d'unités de gestion pour la salamandre tachetée, nous avons pu mettre en évidence un flux de gènes important entre les sites échantillonnés. Les unités de gestion pour cette espèce devraient donc être étendues. Etonnamment, malgré la présence de nombreuses barrières potentielles au flux de gènes, les démarcations génétiques sont apparues à des endroits inattendus. En conclusion, ce travail a apporté une meilleure compréhension des processus génétiques et démographiques en action chez les salamandres. Les résultats suggèrent que certains paradigmes biologiques devraient être considérés avec précaution quand il s'agit de les appliquer à des espèces particulières. Les études spécifiques demeurent donc fondamentales pour une meilleure compréhension de l'évolution des espèces et leur conservation, tout particulièrement dans le contexte des changements globaux actuels.

Ambient vertical flow in long-screen wells: a case study in the Fontainebleau Sands Aquifer (France)

A tritium (H-3) profile was constructed in a long-screened well (LSW) of the Fontainebleau Sands Aquifer (France), and the data were combined with temperature logs to gain insight into the potential effects of the ambient vertical flow (AVF) of water through the well on the natural aquifer stratification. AVF is commonly taken into account in wells located in fracture aquifers or intercepting two different aquifers with distinct hydraulic heads. However, due to the vertical hydraulic gradient of the flow lines intercepted by wells, AVF of groundwater is a common process within any type of aquifer. The detection of 3H in the deeper parts of the studied well ( approximate depth 50m), where H-3-free groundwater is expected, indicates that shallow young water is being transported downwards through the well itself. The temperature logs show a nearly zero gradient with depth, far below the mean geothermal gradient in sedimentary basins. The results show that the age distribution of groundwater samples might be biased in relation to the age distribution in the surroundings of the well. The use of environmental tracers to investigate aquifer properties, particularly in LSWs, is then limited by the effects of the AVF of water that naturally occurs through the well.

Study of gene flow through a hybrid zone in the common shrew (Sorex araneus) using microsatellites

The common shrew (Sorer araneus) is subdivided into several chromosomal races. As hybrid zones between them have been characterized, this organism is of particular interest in studying the role of chromosomes in speciation. Six microsatellite loci were used to evaluate the level of gene how in the S. araneus hybrid zone between the Cordon and Valais races. Most of these loci were very polymorphic, the total number of alleles detected per locus ranging from 3 to 20. Using Mantel tests, we showed that the effect of rivers as barriers to gene flow is less important at this sampling scale. The effect of the chromosomal race is of particular importantance in diminishing gene flow.

The effects of sevoflurane and halothane anesthesia on cerebral blood flow velocity in children.

We compared cerebral blood flow velocity during anesthesia with sevoflurane and halothane in 23 children admitted for elective surgery (age, 0.4-9.7 yr; median age, 1.9 yr; ASA physical status I-II). Inhaled induction was performed in a randomized sequence with sevoflurane or halothane. Under steady-state conditions, cerebral blood flow velocity (systolic [V(s)], mean [V(mn)], and diastolic [VD]) were measured by a blinded investigator using transcranial pulsed Doppler ultrasonography. The anesthetic was then changed. CBFV measurements were repeated after washout of the first anesthetic and after steady-state of the second (equivalent minimal alveolar concentration to first anesthetic). The resistance index was calculated. VD and V(mn) were significantly lower during sevoflurane (V(mn) 1.35 m/s) than during halothane (V(mn) 1.50 m/s; P = 0.001), whereas V(s) was unchanged. The resistance index was lower during halothane (P < 0.001). Our results indicate lower vessel resistance and higher mean velocity during halothane than during sevoflurane. IMPLICATIONS: The mean cerebral blood flow velocity is significantly decreased in children during inhaled anesthesia with sevoflurane than during halothane. This might be relevant for the choice of anesthetic in children with risk of increased intracranial pressure, neurosurgery, or craniofacial osteotomies.

Oligo-Miocene extensional tectonics and fluid flow across the Northern Snake Range detachment system, Nevada

The Northern Snake Range (Nevada) represents a spectacular example of a metamorphic core complex and exposes a complete section from the mylonitic footwall into the hanging wall of a fossil detachment system. Paired geochronological and stable isotopic data of mylonitic quartzite within the detachment footwall reveal that ductile deformation and infiltration of meteoric fluids occurred between 27 and 23 Ma. Ar-40/Ar-39 ages display complex recrystallization-cooling relationships but decrease systematically from 26.9 +/- 0.2 Ma at the top to 21.3 +/- 0.2 Ma at the bottom of footwall mylonite. Hydrogen isotope (delta D) values in white mica are very low (-150 to -145 %) within the top 80-90 m of detachment footwall, in contrast to values obtained from the deeper part of the section where values range from -77 to -64 %, suggesting that time-integrated interaction between rock and meteoric fluid was restricted to the uppermost part of the mylonitic footwall. Pervasive mica-water hydrogen isotope exchange is difficult to reconcile with models of Ar-40 loss during mylonitization solely by volume diffusion. Rather, we interpret the Ar-40/Ar-39 ages of white mica with low-delta D values to date syn-mylonitic hydrogen and argon isotope exchange, and we conclude that the hydrothermal system of the Northern Snake Range was active during late Oligocene (27-23 Ma) and has been exhumed by the combined effects of ductile strain, extensional detachment faulting, and erosion.

HIV-associated neurocognitive disorders (HAND): a changing pattern

Combination antiretroviral therapy has dramatically decreased the incidence of HIV-related mortality and serious opportunistic diseases, among which is HIV- associated dementia. However, minor forms of cognitive dysfunction have not disappeared and may even have increased in frequency. Aging of HIV+ patients, insufficient penetration of antiretroviral drugs into the brain with continuous low- grade viral production and inflammation may play a role. A putative neurotoxicity of combination antiretroviral therapy is controversial. In this article, we will discuss these aspects, as well as clinical and pathophysiological features shared by HIV-associated neurocognitive disorders and other neurodegenerative diseases, especially Alzheimer's disease. This article will briefly summarize the current clinical trials on neuroprotective agents, and the management of patients with neurocognitive disorders will be discussed