An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

Alpha beta lineage-committed thymocytes can be rescued by the gamma delta T cell receptor (TCR) in the absence of TCR beta chain.

Commitment of the alpha beta and gamma delta T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCR gamma delta-transgenic or TCR beta knockout mice, both of which are unable to generate TCR alpha beta-positive T cells, develop phenotypically alpha beta-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCR beta protein, the gamma delta TCR can promote the development of alpha beta-like thymocytes, which, however, do not expand significantly and do not mature into gamma delta T cells. These results show that commitment to the alpha beta lineage can be determined independently of the isotype of the TCR, and suggest that alpha beta versus gamma delta T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCR gamma and delta gene rearrangements on alpha beta T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.

Colonoscopy is the preferred colorectal cancer screening method in a population-based program.

BACKGROUND AND STUDY AIMS: Various screening methods for colorectal cancer (CRC) are promoted by professional societies; however, few data are available about the factors that determine patient participation in screening, which is crucial to the success of population-based programs. This study aimed (i) to identify factors that determine acceptance of screening and preference of screening method, and (ii) to evaluate procedure success, detection of colorectal neoplasia, and patient satisfaction with screening colonoscopy. PATIENTS AND METHODS: Following a public awareness campaign, the population aged 50 - 80 years was offered CRC screening in the form of annual fecal occult blood tests, flexible sigmoidoscopy, a combination of both, or colonoscopy. RESULTS: 2731 asymptomatic persons (12.0 % of the target population) registered with and were eligible to take part in the screening program. Access to information and a positive attitude to screening were major determinants of participation. Colonoscopy was the method preferred by 74.8 % of participants. Advanced colorectal neoplasia was present in 8.5 %; its prevalence was higher in males and increased with age. Significant complications occurred in 0.5 % of those undergoing colonoscopy and were associated with polypectomy or sedation. Most patients were satisfied with colonoscopy and over 90 % would choose it again for CRC screening. CONCLUSIONS: In this population-based study, only a small proportion of the target population underwent CRC screening despite an extensive information campaign. Colonoscopy was the preferred method and was safe. The determinants of participation in screening and preference of screening method, together with the distribution of colorectal neoplasia in different demographic categories, provide a rationale for improving screening procedures.

Inter-informant agreement and prevalence estimates for substance use disorders: direct interview versus family history method.

OBJECTIVES: Family studies typically use multiple sources of information on each individual including direct interviews and family history information. The aims of the present study were to: (1) assess agreement for diagnoses of specific substance use disorders between direct interviews and the family history method; (2) compare prevalence estimates according to the two methods; (3) test strategies to approximate prevalence estimates according to family history reports to those based on direct interviews; (4) determine covariates of inter-informant agreement; and (5) identify covariates that affect the likelihood of reporting disorders by informants. METHODS: Analyses were based on family study data which included 1621 distinct informant (first-degree relatives and spouses) - index subject pairs. RESULTS: Our main findings were: (1) inter-informant agreement was fair to good for all substance disorders, except for alcohol abuse; (2) the family history method underestimated the prevalence of drug but not alcohol use disorders; (3) lowering diagnostic thresholds for drug disorders and combining multiple family histories increased the accuracy of prevalence estimates for these disorders according to the family history method; (4) female sex of index subjects was associated with higher agreement for nearly all disorders; and (5) informants who themselves had a history of the same substance use disorder were more likely to report this disorder in their relatives, which entails the risk of overestimation of the size of familial aggregation. CONCLUSION: Our findings have important implications for the best-estimate procedure applied in family studies.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Analysis of a finite volume element method for the Stokes problem

In this paper we propose a stabilized conforming finite volume element method for the Stokes equations. On stating the convergence of the method, optimal a priori error estimates in different norms are obtained by establishing the adequate connection between the finite volume and stabilized finite element formulations. A superconvergence result is also derived by using a postprocessing projection method. In particular, the stabilization of the continuous lowest equal order pair finite volume element discretization is achieved by enriching the velocity space with local functions that do not necessarily vanish on the element boundaries. Finally, some numerical experiments that confirm the predicted behavior of the method are provided.

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate.

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5°C or at room temperature for one hour, or by storage at -80°C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k(obs) varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.

Transcriptional profiling reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver.

Little is known about the role of the transcription factor peroxisome proliferator-activated receptor (PPAR) beta/delta in liver. Here we set out to better elucidate the function of PPARbeta/delta in liver by comparing the effect of PPARalpha and PPARbeta/delta deletion using whole genome transcriptional profiling and analysis of plasma and liver metabolites. In fed state, the number of genes altered by PPARalpha and PPARbeta/delta deletion was similar, whereas in fasted state the effect of PPARalpha deletion was much more pronounced, consistent with the pattern of gene expression of PPARalpha and PPARbeta/delta. Minor overlap was found between PPARalpha- and PPARbeta/delta-dependent gene regulation in liver. Pathways upregulated by PPARbeta/delta deletion were connected to innate immunity and inflammation. Pathways downregulated by PPARbeta/delta deletion included lipoprotein metabolism and various pathways related to glucose utilization, which correlated with elevated plasma glucose and triglycerides and reduced plasma cholesterol in PPARbeta/delta-/- mice. Downregulated genes that may underlie these metabolic alterations included Pklr, Fbp1, Apoa4, Vldlr, Lipg, and Pcsk9, which may represent novel PPARbeta/delta target genes. In contrast to PPARalpha-/- mice, no changes in plasma free fatty acid, plasma beta-hydroxybutyrate, liver triglycerides, and liver glycogen were observed in PPARbeta/delta-/- mice. Our data indicate that PPARbeta/delta governs glucose utilization and lipoprotein metabolism and has an important anti-inflammatory role in liver. Overall, our analysis reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver.

Postmortem computed tomography angiography vs. conventional autopsy: advantages and inconveniences of each method.

PURPOSE: Postmortem computed tomography angiography (PMCTA) was introduced into forensic investigations a few years ago. It provides reliable images that can be consulted at any time. Conventional autopsy remains the reference standard for defining the cause of death, but provides only limited possibility of a second examination. This study compares these two procedures and discusses findings that can be detected exclusively using each method. MATERIALS AND METHODS: This retrospective study compared radiological reports from PMCTA to reports from conventional autopsy for 50 forensic autopsy cases. Reported findings from autopsy and PMCTA were extracted and compared to each other. PMCTA was performed using a modified heart-lung machine and the oily contrast agent Angiofil® (Fumedica AG, Muri, Switzerland). RESULTS: PMCTA and conventional autopsy would have drawn similar conclusions regarding causes of death. Nearly 60 % of all findings were visualized with both techniques. PMCTA demonstrates a higher sensitivity for identifying skeletal and vascular lesions. However, vascular occlusions due to postmortem blood clots could be falsely assumed to be vascular lesions. In contrast, conventional autopsy does not detect all bone fractures or the exact source of bleeding. Conventional autopsy provides important information about organ morphology and remains the only way to diagnose a vital vascular occlusion with certitude. CONCLUSION: Overall, PMCTA and conventional autopsy provide comparable findings. However, each technique presents advantages and disadvantages for detecting specific findings. To correctly interpret findings and clearly define the indications for PMCTA, these differences must be understood.

Experimental Uveitis can be Maintained in Rabbits for a Period of Six Weeks After a Safe Sensitization Method.

Abstract Purpose: New treatments against long-lasting uveitis need to be tested. Our aim was to develop a six-week model of uveitis in rabbits. Methods: Rabbits were presensitized with an s.c. injection of Mycobacterium tuberculosis H37RA emulsified with TiterMax® Gold adjuvant. Uveitis was induced at day 28 and 50, by intravitreal challenges of antigen suspension. Ocular inflammation was assessed till euthanasia at day 71 after s.c. injection of M. tuberculosis H37RA by: (a) the number of inflammatory cells in aqueous humor (AH); (b) the protein concentration in AH; (c) the clinical score (mean of conjunctival hyperaemia, conjunctival chemosis, oedema and secretion); (d) the microscopical score (mean presence of fibrin and synechiae, aqueous cell density and aqueous flare grade, as scored by slit lamp). Results: At the sites of presensitization injection, rabbits presented flat nodules which progressively vanished. The first challenge induced a significant increase in the four parameters (p < 0.05 the Wilcoxon/Kruskal-Wallis test). The AH contained 764 ± 82 cells/µl and 32 ± 0.77 mg protein/ml. During the following days, inflammatory parameters decreased slightly. The second intravitreal challenge increased inflammation (3564 ± 228 cells/µl AH and 31 ± 1 mg protein/ml), which remained at a high level for a longer period of time. Conclusion: We developed a model of long-term uveitis, which could be maintained in rabbits for at least six weeks. Such a model could be used to test the efficacy of either new drugs or various drug delivery systems intended to deliver active agents during a few months.

Parent-child agreement and prevalence estimates of diagnoses in childhood: direct interview versus family history method.

Diagnostic information on children is typically elicited from both children and their parents. The aims of the present paper were to: (1) compare prevalence estimates according to maternal reports, paternal reports and direct interviews of children [major depressive disorder (MDD), anxiety and attention-deficit and disruptive behavioural disorders]; (2) assess mother-child, father-child and inter-parental agreement for these disorders; (3) determine the association between several child, parent and familial characteristics and the degree of diagnostic agreement or the likelihood of parental reporting; (4) determine the predictive validity of diagnostic information provided by parents and children. Analyses were based on 235 mother-offspring, 189 father-offspring and 128 mother-father pairs. Diagnostic assessment included the Kiddie-schedule for Affective Disorders and Schizophrenia (K-SADS) (offspring) and the Diagnostic Interview for Genetic Studies (DIGS) (parents and offspring at follow-up) interviews. Parental reports were collected using the Family History - Research Diagnostic Criteria (FH-RDC). Analyses revealed: (1) prevalence estimates for internalizing disorders were generally lower according to parental information than according to the K-SADS; (2) mother-child and father-child agreement was poor and within similar ranges; (3) parents with a history of MDD or attention deficit hyperactivity disorder (ADHD) reported these disorders in their children more frequently; (4) in a sub-sample followed-up into adulthood, diagnoses of MDD, separation anxiety and conduct disorder at baseline concurred with the corresponding lifetime diagnosis at age 19 according to the child rather than according to the parents. In conclusion, our findings support large discrepancies of diagnostic information provided by parents and children with generally lower reporting of internalizing disorders by parents, and differential reporting of depression and ADHD by parental disease status. Follow-up data also supports the validity of information provided by adolescent offspring.

A new method for geoelectrical investigations underwater

A new electrical method is proposed for determining the apparent resistivity of multi-earth layers located underwater. The method is based on direct current geoelectric sounding principles. A layered earth model is used to simulate the stratigraphic target. The measurement array is of pole-pole type; it is located underwater and is orientated vertically. This particular electrode configuration is very useful when conventional electrical methods cannot be used, especially if the water depth becomes very important. The calculated apparent resistivity shows a substantial quality increase in the measured signal caused by the underwater targets, from which little or no response is measured using conventional surface electrode methods. In practice, however, different factors such as water stratification, underwater streams or meteorological conditions complicate the interpretation of the field results. A case study is presented, where field surveys carried out on Lake Geneva were interpreted using the calculated apparent resistivity master-curves.

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom