Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection.

Curative vs. preventive management of nitrogen transfers in rural areas: lessons from the case of the Orgeval watershed (Seine River basin, France).

The Orgeval watershed (104 km(2)) is a long-term experimental observatory and research site, representative of rural areas with intensive cereal farming of the temperate world. Since the past few years, we have been carrying out several studies on nitrate source, transformation and transfer of both surface and groundwaters in relation with land use and agriculture practices in order to assess nitrate (NO3(-)) leaching, contamination of aquifers, denitrification processes and associated nitrous oxide (N2O) emissions. A synthesis of these studies is presented to establish a quantitative diagnosis of nitrate contamination and N2O emissions at the watershed scale. Taking this watershed as a practical example, we compare curative management measures, such as pond introduction, and preventive measures, namely conversion to organic farming practices, using model simulations. It is concluded that only preventive measures are able to reduce the NO3(-) contamination level without further increasing N2O emissions, a result providing new insights for future management bringing together water-agro-ecosystems.

Psychological effect of exercise in women with breast cancer receiving adjuvant therapy: what is the optimal dose needed?

BACKGROUND: Several meta-analyses have examined the role of exercise interventions in improving psychological outcomes in cancer survivors but most did not focus on adjuvant therapy period and did not investigate the optimal dose of exercise needed. The present meta-analysis examines the impact of exercise interventions delivered at this particular period on fatigue, anxiety, depression, and quality of life (QoL) as well as dose-response relationships between volume of prescribed exercise and these psychological outcomes. MATERIALS AND METHODS: Randomized, controlled trials that proposed an exercise intervention to patients with breast cancer undergoing chemotherapy and/or radiotherapy were systematically identified and coded. Psychological outcomes effect sizes were calculated and analyzed for trends using linear and quadratic regressions. RESULTS: Pooled effects of the 17 included studies revealed improvement for all outcomes, significant for fatigue, depression, and QoL with pooled estimates ranging from 0.2 to 0.5 favoring intervention. Significant inverse associations of the volume of prescribed exercise with fatigue and QoL were observed. CONCLUSIONS: Exercise intervention improved fatigue, depression, and QoL in patients with breast cancer receiving adjuvant therapy. Prescription of relatively low doses of exercise (<12 MET h/week) consisting in ∼90-120 min of weekly moderate physical exercise seems more efficacious in improving fatigue and QoL than higher doses.

Detection of over 98% cystic fibrosis mutations in a Celtic population.

We have conducted a large systematic study of 365 cystic fibrosis (CF) chromosomes in a Celtic population from Brittany, France, in which we have been able to identify more than 98% of the cystic fibrosis gene mutations. We detected 19 different CFTR mutations located in 9 exons. Eleven of these mutations have not been described previously and nine of them are presented in this study. The denaturing gradient gel electrophoresis strategy we have used, can be applied to other populations suggesting that population screening for CF on a large scale might be possible.

Bubble production mechanism in a microfluidic foam generator.

We present the design and characterization of a microfluidic bubble generator that has the potential of producing monodisperse bubbles in 256 production channels that can operate in parallel. For a single production channel we demonstrate a production rate of up to 4 kHz with a coefficient of variation of less than 1%. We observe a two-stage bubble production mechanism: initially the gas spreads onto a shallow terrace, and then overflows into a larger foam collection channel; pinning of the liquid-gas meniscus is observed at the terrace edge, the result being an asymmetric pinch-off. A semiempirical physical model predicts the scaling of bubble size with fluid viscosity and gas pressure from measurements of the pinned meniscus width.

Zoonotic bacteria, antimicrobial use and antimicrobial resistance in ornamental fish: a systematic review of the existing research and survey of aquaculture-allied professionals.

Using systematic review methodology, global research reporting the frequency of zoonotic bacterial pathogens, antimicrobial use (AMU) and antimicrobial resistance (AMR) in ornamental fish, and human illness due to exposure to ornamental fish, was examined. A survey was performed to elicit opinions of aquaculture-allied personnel on the frequency of AMU and AMR in ornamental fish. The most commonly reported sporadic human infections were associated with Mycobacterium marinum, while Salmonella Paratyphi B var. Java was implicated in all reported outbreaks. Aeromonas spp. were most frequently investigated (n=10 studies) in 25 studies surveying ornamental fish from various sources. High levels of resistance were reported to amoxicillin, penicillin, tetracycline and oxytetracycline, which was also in agreement with the survey respondents' views. Studies on AMU were not found in our review. Survey respondents reported frequent use of quinolones, followed by tetracyclines, nitrofurans, and aminoglycosides. Recommendations for future surveillance and public education efforts are presented.

Microfluidic capillary separation and real-time spectroscopic analysis of specific components from multiphase mixtures.

We present a technique of phase separation suitable for microfluidic systems and demonstrate its efficient integration with a microfluidic optical cell for performing real-time spectrometric measurements on one specific phase from a mixture. We demonstrate that efficient and robust phase separation based on capillarity is possible within a microfluidic chip using either microfabricated capillary channels in polydimethylsiloxane (PDMS) or oil-wet fluoropolymer membranes, allowing for extraction of either the continuous or of the dispersed phases from a multiphase mixture. We analyze the dependence of phase separation efficiency on the operating parameters of the device and observe the presence of a hysteresis cycle during pressure sweeps above a water breakthrough pressure (P(b)); we also observe and analyze the reversibility of the oil-wet state of the membrane upon pressure reduction below a reset pressure (P(r) < P(b)). We test the capillary separation method extensively with several types of organic/water mixtures and emulsions and derive criteria for design and operation of a robust microfluidic capillary separator. As an example of monitoring application we describe the design and manufacturing of a microfluidic spectrometer cell optimized for fast response time, which was used to analyze the oil extracted from an oil/water emulsion using a capillary separator. The complete separator-sensor system is characterized in terms of response and cleanup times to instantaneous changes in the dye concentration of the phase of interest.

Repeated therapeutic dosing selects macrolide-resistant Campylobacter spp. in a turkey facility.

AIMS: This study assessed the effects of the therapeutic use of Tylan® in a large-scale turkey production facility on the selection of macrolide-resistant Campylobacter. METHODS AND RESULTS: A flock of production turkeys (c. 30,000 birds) was followed from brooding to slaughter, and the effects of macrolide application was assessed in one half of the flock from finishing stage to final product and compared against the control barn where no macrolide was used. Overall, Campylobacter prevalence in turkeys was almost 100% by 4 weeks of age. When Campylobacter prevalence was assessed in relation to treatment, high levels of macrolide resistance were evident in this group following treatment, with Campylobacter coli becoming the dominant strain type. Over time, and in the absence of a selection agent, the population of resistant strains decreased suggesting that there was a fitness cost associated with macrolide resistance carriage and persistence. Macrolide resistance was detected in the control barn at a very low level (four isolates recovered during the study), suggesting that the creation or selection of macrolide-resistant Campylobacter was correlated with the treatment regime used. Molecular analysis of a selection of macrolide-resistant Campylobacter recovered was assessed using PCR, RFLP and sequence analysis of the 23S rRNA. The majority of isolates displaying high-level macrolide resistance (>256 µg ml(-1)) possessed an A2075G transition mutation in the 23S rRNA and the CmeABC efflux pump. CONCLUSIONS: These studies suggest that macrolide resistance can be promoted through the application of treatment during the grow-out phase and once established in a production facility has the potential to persist and be transferred to final product. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights the prudent use of antimicrobials in treatment of disease in poultry. Of significance is the presence of macrolide-resistant Campylobacter in poultry production and finished product as a consequence of macrolide usage.

CFTR mutations in the Algerian population.

The nature and frequency of the major CFTR mutations in the North African population remain unclear, although a small number of CFTR mutation detection studies have been done in Algeria and Tunisia, showing largely European mutations such as F508del, G542X and N1303K, albeit at different frequencies, which presumably emerged via population admixture with Caucasians. Some unique mutations were identified in these populations. This is the first study that includes a genetic and clinical evaluation of CF patients living in Algeria. In order to offer an effective diagnostic service and to make accurate risk estimates, we decided to identify the CFTR mutations in 81 Algerian patients. We carried out D-HPLC, chemical-clamp denaturing gradient gel electrophoresis, multiplex amplification analysis of the CFTR gene and automated direct DNA sequencing. We identified 15 different mutations which account for 58.5% of the CF chromosomes. We used a quantitative PCR technique (quantitative multiplex PCR short fragment fluorescence analysis) to screen for deletion/duplication in the 27 exons of the gene. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients. As CFTR mutations have been detected in males with infertility, 46 unrelated Algerian individuals with obstructive azoospermia were also investigated.

Vitamin K epoxide reductase genetic polymorphism is associated with venous thromboembolism: results from the EDITH Study.

BACKGROUND: The vitamin K epoxide reductase complex subunit 1 (VKORC1) recycles endogenous vitamin K, a cofactor for vitamin K-dependent coagulation factor synthesis. Common polymorphisms in VKORC1, the gene coding for VKORC1, have been found to affect the dose response to vitamin K antagonists, and to confer an increased risk of vascular diseases in a Chinese population. The aim of this study was to evaluate the association between the VKORC1 1173C > T polymorphism and venous thromboembolism (VTE). METHODS: We report the results of a case-control study designed to evaluate interactions between acquired and inherited risk factors of VTE. We studied 439 cases hospitalized with a first venous thromboembolic event that was not related to a major acquired risk factor for VTE, and 439 matched controls. The VKORC1 1173C > T polymorphism was selected for genotyping as the tagging single-nucleotide polymorphism for previously identified VKORC1 haplotypes. RESULTS: The relationship between VTE and the VKORCI 1173C > T polymorphism was consistent with a recessive model. The frequency of the VKORCI TT genotype was lower in cases than in controls. The odds ratio (OR) (95% CI) was 0.62 (0.41-0.94) for the TT genotype as compared to CT/CC genotypes. Adjustment on cardiovascular diseases, body mass index, factor V (FV) and prothrombin gene mutations did not alter the results. CONCLUSIONS: In this case-control study, the frequency of the VKORCI TT genotype was lower in patients with VTE than in matched controls. The clinical consequence of these results remains to be determined, but gives new perspectives for exploration of the role of VKORCI polymorphism in the pathogenesis of VTE.

TP53 gene mutation profile in esophageal squamous cell carcinomas.

Esophageal squamous cell carcinoma is a form of cancer occurring most commonly in males, particularly those living in some areas of Asia, Africa, and western Europe. In some of these tumors, a sequence alteration has been identified in the coding region of the TP53 gene which is known to inactivate the tumor suppressor function of its product. Using a GC clamp (i.e., a GC rich domain) denaturing gradient gel electrophoresis assay we have been able to identify sequence modifications in 27 of the 32 tumor samples analyzed (84%). Most of the mutations occur in exon 6, a region of the gene which has not previously been reported as being a hot spot for the mutations of other cancers. Twelve of the mutations reported here have not been described in other types of tumors and these consist mostly of frameshift or splice mutations. The distribution of mutations [transitions (45%), transversions (34%), and frameshift (21%)] suggests that the etiological contribution of genotoxic factors might be complex and might associate different exogenous and endogenous mutagen exposures.

Risk factors and outcome of graft failure after HLA matched and mismatched unrelated donor hematopoietic stem cell transplantation: a study on behalf of SFGM-TC and SFHI.

Graft failure remains a severe complication of hematopoietic stem cell transplantation (HSCT). Several risk factors have already been published. In this study, we re-evaluated them in a large cohort who had the benefit of the recent experience in HSCT (2006-2012). Data from 4684 unrelated donor HSCT from 2006 to 2012 were retrospectively collected from centers belonging to the French Society for Stem Cell Transplantation. Among the 2716 patients for whom HLA typing was available, 103 did not engraft leading to a low rate of no engraftment at 3.8%. In univariate analysis, only type of disease and status of disease at transplant for malignant diseases remained significant risk factors (P=0.04 and P<0.0001, respectively). In multivariate analysis, only status of disease was a significant risk factor (P<0.0001). Among the 61 patients who did not engraft and who were mismatched for 1 HLA class I and/or HLA-DP, 5 donor-specific antibodies (DSAs) were detected but only 1 was clearly involved in graft failure, for the others their role was more questionable. Second HSCT exhibited a protective although not statistically significant effect on OS (hazard ratio=0.57 [0.32-1.02]). In conclusion, only one parameter (disease status before graft) remains risk factor for graft failure in this recent cohort.

Transgene expression kinetics after transfection with cationic phosphonolipids in hematopoietic non adherent cells.

Cationic lipids are considered to be capable of efficiently and safely mediating DNA transfer into cells, although expression is transient. A new family of cationic lipids, called phosphonolipids, has been developed, with the relationship between the hydrophobic domain of the lipid molecules and the significant enhancement of transduction efficiency in a non-adherent cell line characterised in the present study. The kinetics of transfection efficiency were also investigated. Our results demonstrate that the peak of the transient expression of these reporter genes mediated by cationic lipids occurred within 3 to 14 days, depending on the aliphatic chain length of the complex used and on its formulation in the presence or absence of DOPE. Furthermore, the kinetics of transgene expression were found to differ in adherent and non-adherent cells. These results were obtained using three different techniques: CPRG, luminescence, and FACS-gal, and were in agreement with electron microscopy studies. We thus hypothesized that the plasma membrane composition of cells could affect the efficiency of transfection with cationic lipids. Our results suggest that phosphonolipids constitute a promising class of compounds for gene transfer protocols, and that galenic optimization should improve and modify the transfection efficiency of these DNA-lipid complexes.

Cationic phosphonolipids as nonviral gene transfer agents in the lungs of mice.

With the aim of developing new gene transfer tools for treating CF with gene therapy, we have synthesized a novel family of molecules named cationic phosphonolipids. The most efficient among them were selected by in vitro screening to compare their activities in vivo in mouse lungs. We used a reporter gene whose activity was measured cytofluorimetrically (FACS-Gal assay) and by means of a chemiluminescence technique. These tests allowed us to identify the percentage of transfected cells and to quantify total beta-galactosidase in the lungs. This enabled us to identify two molecules, significantly efficient in comparison with DNA alone: GLB73 (p = 0.0015) and GLB253 (p = 0.007). Their use resulted in a time lag between transfection and maximum efficiency: maximum efficiency was observed 4 days after transfection with GLB73, whereas it was noticeable only on day 7 with GLB253. Moreover, from toxicity studies carried out in vivo, GLB73 seems to be nontoxic. In vivo results were correlated with in vitro results obtained with CF epithelial cell lines. Consequently, GLB73 is a potential candidate for phase I clinical trials in humans.

Novel mutations in the duplicated region of PKD1 gene.

Autosomal dominant polycystic kidney disease (ADPKD) exhibits a genetically heterogeneous transmission involving at least three different genes. PKD1 gene linked mutations are responsible for the disease in about 85% of ADPKD cases. The search for mutations is a very important step in understanding the molecular mechanisms underlying ADPKD. We undertook this study using denaturing gradient gel electrophoresis (DGGE), after a stage of long range PCR, to scan for mutations in the duplicated region of the PKD1 gene in French ADPKD families. This allowed us to identify eight novel mutations and several polymorphisms: among the mutations, three are nonsense mutations, two are deletions in the coding sequence leading to frameshift mutations, one is a splice mutation and two are highly probable missense mutations. In this paper, we also provide a review of the mutations reported so far which are widespread throughout the gene. Although no clear hot spot for mutation is apparent, we will focus on some clustering observed.

Corticopontine projection in the rat: the distribution of labelled cortical cells after large injections of horseradish peroxidase in the pontine nuclei.

The distribution of cortical cells projecting to the pontine nuclei in rats was studied by making large injections of horseradish peroxidase that filled the basilar pons and measuring the density of labelled cells in each cortical area. All retrogradely labelled cells were layer V pyramidal cells. The highest densities of labelled cells were observed in the motor areas. The lowest densities were in temporal association cortex and perirhinal cortex. Visual cortical areas, including the primary visual cortex, provided a major source of pontine projections. The distribution of corticopontine cells within the primary visual cortex was studied in more detail. In all cases the highest density of labelled cells was observed in the region of cortex that represents the nasal visual field. Control injections into brainstem regions adjacent to the pontine nuclei produced a much lower absolute density of retrogradely labelled cortical cells and the distribution of those cells was different from that observed following pontine injections. We conclude that every area of the rat's cerebral cortex projects to the pontine nuclei and that there are consistent variations in the density of the projections both between and within areas.

Corticopontine projection in the macaque: the distribution of labelled cortical cells after large injections of horseradish peroxidase in the pontine nuclei.

We studied the distribution of corticopontine cells in the monkey cerebral cortex. Horseradish peroxidase (HRP) was injected into the brainstem of monkeys in an attempt to fill the pontine nuclei on one or both sides. In control animals we injected the medullary pyramids or varied the route, size, or location of pontine injections. All retrograde filled corticopontine neurons were layer V pyramidal cells. Corticopontine cells were distributed within a largely continuous area of cortex which extended from the cingulate cortex medially to the sylvian fissure laterally; from the superior temporal fissure caudally to the medial part of the frontal granular cortex rostrally. Areas 4 and 6 of Brodmann (1905) contained the highest density of filled cells. In the primary visual cortex, area 17, there were a few labelled cells restricted to the rostral portion of the upper bank of the calcarine fissure, in a region representing the lower periphery of the visual field. The results are discussed in relation to the possible functions of the corticopontine system, especially the role of the extrastriate visual areas in visually guided movement.

Visual pontocerebellar projections in the macaque.

The cerebellum plays an important role in the visual guidance of movement. In order to understand the anatomical basis of visuomotor control, we studied the projection of pontine visual cells onto the cerebellar cortex of monkeys. Wheat germ agglutinin horseradish peroxidase was injected into the dorsolateral pons two monkeys. Retrogradely labelled cells were mapped in the cerebral cortex and superior colliculus, and orthogradely labelled fibers in the cerebellar cortex. The largest number of retrogradely labelled cells in the cerebral cortex was in a group of medial extrastriate visual areas. The major cerebellar target of these dorsolateral pontine cells is the dorsal paraflocculus. There is a weaker projection to the uvula, paramedian lobe, and Crus II, and a sparse but definite projection to the ventral paraflocculus. There are virtually no projections to the flocculus. There are sparse ipsilateral pontocerebellar projections to these same regions of cerebellar cortex. In nine monkeys, we made small injections of the tracer into the cerebellar cortex and studied the location of retrogradely filled cells in the pontine nuclei and inferior olive. Injections into the dorsal paraflocculus or rostral folia of the uvula retrogradely labelled large numbers of cells in the dorsolateral region of the contralateral pontine nuclei. Labelled cells were found ipsilaterally, but in reduced numbers. Injections outside of these areas in ventral paraflocculus or paramedian lobule labelled far fewer cells in this region of the pons. We conclude that the principal source of cerebral cortical visual information arises from a medial group of extrastriate visual areas and is relayed through cells in the dorsolateral pontine nuclei. The principal target of pontine visual cells is the dorsal paraflocculus.