Induction of circulating tumor-reactive CD8+ T cells after vaccination of melanoma patients with the gp100 209-2M peptide.

Patients with stage I-III melanoma were vaccinated with the modified HLA-A2-binding gp100(209-2M)-peptide after complete surgical resection of their primary lesion and sentinel node biopsy. Cytoplasmic interferon-gamma production by freshly thawed peripheral blood mononuclear cells (direct ex vivo analysis) or by peripheral blood mononuclear cells subjected to 1 cycle of in vitro sensitization with peptide, interleukin-2, and interleukin-15 was measured following restimulation with the modified and native gp100 peptides, and also A2gp100 melanoma cell lines. Peptide-reactive and tumor-reactive T cells were detected in 79% and 66% of selected patients, respectively. Patients could be classified into 3 groups according to their vaccine-elicited T-cell responses. One group of patients responded only to the modified peptide used for immunization, whereas another group of patients reacted to both the modified and native gp100 peptides, but not to naturally processed gp100 antigen on melanoma cells. In the third group of patients, circulating CD8 T cells recognized A2gp100 melanoma cell lines and also both the modified and native peptides. T cells with a low functional avidity, which were capable of lysing tumor cells only if tumor cells were first pulsed by the exogenous administration of native gp100(209-217) peptide were identified in most patients. These results indicate that vaccination with a modified gp100 peptide induced a heterogeneous group of gp100-specific T cells with a spectrum of functional avidities; however, high avidity, tumor-reactive T cells were detected in the majority of patients.

The course of inflammatory bowel disease during pregnancy and postpartum: a prospective European ECCO-EpiCom Study of 209 pregnant women.

BACKGROUND: The impact of pregnancy on the course of IBD is still controversial. AIM: To investigate the impact of pregnancy on IBD and to search for factors with potential impact on remission. METHODS: Pregnant IBD women from 12 European countries were enrolled between January 2003 and December 2006 and compared at conception (1:1) with nonpregnant IBD women. Data on disease course were prospectively collected at each trimester during pregnancy and in the postpartum (6 months) using a standardised questionnaire. RESULTS: A total of 209 pregnant IBD women were included: 92 with Crohn's disease (CD; median age 31 years, range 17-40) and 117 with ulcerative colitis (UC; median age 32 years, range 19-42). No statistically significant difference in disease course during pregnancy and postpartum was observed between pregnant and nonpregnant CD women. Longer disease duration in CD and immunosuppressive therapy were found to be risk factors for activity during pregnancy. Pregnant UC women were more likely than nonpregnant UC women to relapse both during pregnancy (RR 2.19; 95% CI: 1.25-3.97, 0.004) and postpartum (RR 6.22; 95% CI: 2.05-79.3, P = 0.0004). During pregnancy, relapse was mainly observed in the first (RR 8.80; 95% CI 2.05-79.3, P < 0.0004) and the second trimester (RR 2.84, 95% CI 1.2-7.45, P = 0.0098). CONCLUSIONS: Pregnant women with Crohn's disease had a similar disease course both during pregnancy and after delivery as the nonpregnant women. In contrast, pregnant women with ulcerative colitis were at higher risk of relapse during pregnancy and in the postpartum than nonpregnant ulcerative colitis women.

The Lithium, Boron and Beryllium content of serpentinized peridotites from ODP Leg 209 (Sites 1272A and 1274A): Implications for lithium and boron budgets of oceanic lithosphere

Despite the key importance of altered oceanic mantle as a repository and carrier of light elements (B, Li, and Be) to depth, its inventory of these elements has hardly been explored and quantified. In order to constrain the systematics and budget of these elements we have studied samples of highly serpentinized (>50%) spinel harzburgite drilled at the Mid-Atlantic Ridge (Fifteen-Twenty Fracture zone, ODP Leg 209, Sites 1272A and 1274A). In-situ analysis by secondary ion mass spectrometry reveals that the B, Li and Be contents of mantle minerals (olivine, orthopyroxene, and clinopyroxene) remain unchanged during serpentinization. B and Li abundances largely correspond to those of unaltered mantle minerals whereas Be is close to the detection limit. The Li contents of clinopyroxene are slightly higher (0.44-2.8 mu g g(-1)) compared to unaltered mantle clinopyroxene, and olivine and clinopyroxene show an inverse Li partitioning compared to literature data. These findings along with textural observations and major element composition obtained from microprobe analysis suggest reaction of the peridotites with a mafic silicate melt before serpentinization. Serpentine minerals are enriched in B (most values between 10 and 100 mu g g(-1)), depleted in Li (most values below I mu g g(-1)) compared to the primary phases, with considerable variation within and between samples. Be is at the detection limit. Analysis of whole rock samples by prompt gamma activation shows that serpentinization tends to increase B (10.4-65.0 mu g g(-1)), H2O and Cl contents and to lower Li contents (0.07-3.37 mu g g(-1)) of peridotites, implying that-contrary to alteration of oceanic crust-B is fractionated from Li and that the B and Li inventory should depend essentially on rock-water ratios. Based on our results and on literature data, we calculate the inventory of B and Li contained in the oceanic lithosphere, and its partitioning between crust and mantle as a function of plate characteristics. We model four cases, an ODP Leg 209-type lithosphere with almost no igneous crust, and a Semail-type lithosphere with a thick igneous crust, both at I and 75 Ma, respectively. The results show that the Li contents of the oceanic lithosphere are highly variable (17-307 kg in a column of I m x I m x thickness of the lithosphere (kg/col)). They are controlled by the primary mantle phases and by altered crust, whereas the B contents (25-904 kg/col) depend entirely on serpentinization. In all cases, large quantities of B reside in the uppermost part of the plate and could hence be easily liberated during slab dehydration. The most prominent input of Li into subduction zones is to be expected from Semail-type lithosphere because most of the Li is stored at shallow levels in the plate. Subducting an ODP Leg 209-type lithosphere would mean only very little Li contribution from the slab. Serpentinized mantle thus plays an important role in B recycling in subduction zones, but it is of lesser importance for Li. (C) 2008 Elsevier Ltd. All rights reserved.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Proteasomal cleavage does not determine immunogenicity of gp100-derived peptides gp100 209-217 and gp100 209-217T210M.

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100(209-217) is the most prominent (Kawakami et al., J Immunol 154:3961-3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100(209-217T210M), that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100(209-217) and gp100(209-217T210M) to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100(201-223)) or the modified sequence (gp100(201-223T210M)). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100(201-223T210M) were used to activate an HLA-A*0201-expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100(209-217T210M) seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport.

Determination of displacement, stress- and strain-distribution in the human heart: a FE-model on the basis of MR imaging.

The determination of characteristic cardiac parameters, such as displacement, stress and strain distribution are essential for an understanding of the mechanics of the heart. The calculation of these parameters has been limited until recently by the use of idealised mathematical representations of biventricular geometries and by applying simple material laws. On the basis of 20 short axis heart slices and in consideration of linear and nonlinear material behaviour we have developed a FE model with about 100,000 degrees of freedom. Marching Cubes and Phong's incremental shading technique were used to visualise the three dimensional geometry. In a quasistatic FE analysis continuous distribution of regional stress and strain corresponding to the endsystolic state were calculated. Substantial regional variation of the Von Mises stress and the total strain energy were observed at all levels of the heart model. The results of both the linear elastic model and the model with a nonlinear material description (Mooney-Rivlin) were compared. While the stress distribution and peak stress values were found to be comparable, the displacement vectors obtained with the nonlinear model were generally higher in comparison with the linear elastic case indicating the need to include nonlinear effects.

Temporal and spatial appearance of the membrane cytoskeleton and perineuronal nets in the rat neocortex.

Parvalbumin-immunoreactive interneurons are surrounded by perineuronal nets, containing molecules of the extracellular matrix (e.g. tenascin-R). Furthermore, they seem to have a special cytoskeleton composed of, among others, ankyrinR and beta Rspectrin. In the present developmental study we showed that the intracellular markers parvalbumin, ankyrinR and beta Rspectrin as well as Vicia Villosa agglutinin, an extracellular marker for perineuronal nets, appeared in the second postnatal week. In the third postnatal week, ankyrinR and beta R spectrin were present in the parvalbumin-positive interneurons. Tenascin-R appeared in a similar topographic distribution as the intracellular markers. The adult pattern was established upon the end of the fourth postnatal week. Our results indicate that cytoskeletal maturity maybe a prerequisite for the organization of perineuronal nets of extracellular matrix.

Marbles and carbonate rocks from central Morocco: a petrographic, mineralogical and geochemical study

Petrographic, mineralogical, and stable isotopes (delta C-13, delta O-18 values) compositions were used to characterise marbles and sedimentary carbonate rocks from central Morocco, which are considered to be a likely source of ornamental and building material from Roman time to the present day. This new data set was used in the frame of an archaeometric provenance study on Roman artefacts from the town of Thamusida (Kenitra, north Morocco), to assess the potential employment of these rocks for the manufacture of the archaeological materials. A representative set of samples from marbles and other carbonate rocks (limestone, dolostone) were collected in several quarries and outcrops in the Moroccan Meseta, in a region extending from the Meknes-Khenifra alignment to the Atlantic Ocean. All the samples were studied using a petrographic, mineralogical and geochemical methods. The petrographic and minerological investigations (optical microscopy, electron microscopy, X-ray diffraction) allowed to group the carbonate rocks in limestones, foliated limestone, diagenetic breccias and dolostone. The limestones could be further grouped as mudstones, wackestones-packstones, crinoid grainstones, oolitic grainstone and floatstones. Textural differences allowed to define marbles varieties. The stable carbon and oxygen isotope composition proved to be quite useful in the discrimination of marble sources, with apparently less discriminatory potential for carbonate rocks.

ADOS: an educational primary prevention programme for preventing excess body weight in adolescents.

OBJECTIVE: The prevalence of adolescent obesity has increased considerably over the past decade in Switzerland and has become a serious public health problem in Europe. Prevention of obesity using various comprehensive programmes appears to be very promising, although we must admit that several interventions had generally disappointing results compared with the objectives and target initially fixed. Holistic programmes including nutritional education combined with promotion of physical activity and behaviour modification constitute the key factors in the prevention of childhood and adolescent obesity. The purpose of this programme was to incorporate nutrition/physical education as well as psychological aspects in selected secondary schools (9th grade, 14-17 years). METHODS: The educational strategy was based on the development of a series of 13 practical workshops covering wide areas such as physical inactivity, body composition, sugar, energy density, invisible lipids, how to read food labels, is meal duration important? Do you eat with pleasure or not? Do you eat because you are hungry? Emotional eating. For teachers continuing education, a basic highly illustrated guide was developed as a companion booklet to the workshops. These materials were first validated by biology, physical education, dietician and psychologist teachers as well as school medical officers. RESULTS: Teachers considered the practical educational materials innovative and useful, motivational and easy to understand. Up to now (early 2008), the programme has been implemented in 50 classes or more from schools originating from three areas in the French part of Switzerland. Based on the 1-week pedometer value assessed before and after the 1 school-year programme, an initial evaluation indicated that overall physical placidity was significantly decreased as evidenced by a significant rise in the number of steps per day. CONCLUSION: Future evaluation will provide more information on the effectiveness of the ADOS programme.

Increased activation in Broca's area after cognitive remediation in schizophrenia.

Functional magnetic resonance imaging (fMRI) was used to measure changes in cerebral activity in patients with schizophrenia after participation in the Cognitive Remediation Program for Schizophrenia and other related disorders (RECOS). As RECOS therapists make use of problem-solving and verbal mediation techniques, known to be beneficial in the rehabilitation of dysexecutive syndromes, we expected an increased activation of frontal areas after remediation. Executive functioning and cerebral activation during a covert verbal fluency task were measured in eight patients with schizophrenia before (T1) and after (T2) 14 weeks of RECOS therapy. The same measures were recorded in eight patients with schizophrenia who did not participate in RECOS at the same intervals of time (TAU group). Increased activation in Broca's area, as well as improvements in performance of executive/frontal tasks, was observed after cognitive training. Metacognitive techniques of verbalization are hypothesized to be the main factor underlying the brain changes observed in the present study.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Summary : Internal ribosome entry sites (IRES) are used by viruses as a strategy to bypass inhibition of cap-dependent translation that commonly results from viral infection. IRES are also used in eukaryotic cells to control mRNA translation under conditions of cellular stress (apoptosis, heat shock) or during the G2 phase of the cell cycle when general protein synthesis is inhibited. Variation in cellular expression levels has been shown to be inherited. Expression is controlled, among others, by transcriptional factors and by the efficiency of cap-mediated translation and ribosome activity. We aimed at identifying genomic determinants of variability in IRES-mediated translation of two representative IRES [Encephalomyocarditis virus (EMCV) and X-linked Inhibitor-of-Apoptosis (XIAP) IRES]. We used bicistronic lentiviral constructions expressing two fluorescent reporter transgenes. Lentiviruses were used to transduce seven different laboratory cell lines and B lymphoblastoid cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209); representing an in vitro approach to family structure allowing genome scan analyses. The relative expression of the two markers was assessed by FACS. IRES efficiency varies according to cellular background, but also varies, for a same cell type, among individuals. The control of IRES activity presents an inherited component (h2) of 0.47 and 0.36 for EMCV and XIAP IRES, respectively. A genome scan identified a suggestive Quantitative Trait Loci (LOD 2.35) involved in the control of XIAP IRES activity. Résumé : Les sites internes d'entrée des ribosomes (IRES = internal ribosome entry sites) sont utilisés par les virus comme une stratégie afin d'outrepasser l'inhibition de traduction qui résulte communément d'une infection virale. Les IRES sont également utilisés par les cellules eucaryotes pour contrôler la traduction de l'ARN messager dans des conditions de stress cellulaire (apoptose, choc thermique) ou durant la phase G2 du cycle cellulaire, situations durant lesquelles la synthèse générale des protéines est inhibée. La variation des niveaux d'expression cellulaire de transcription est un caractère héréditaire. L'expression des gènes est contrôlée entre autre par les facteurs de transcription et par l'efficacité de la traduction initiée par la coiffe ainsi que par l'activité des ribosomes. Durant cette étude nous avons eu pour but d'identifier les déterminants génomiques responsables de la variabilité de la traduction contrôlée par l'IRES. Ceci a été effectué en étudiant deux IRES représentatifs : l'IRES du virus de l'encéphalomyocardite (EMCV) et l'IRES de l'inhibiteur de l'apoptose XIAP (X-linked Inhibitor-of-Apoptosis). Nous avons utilisés des lentivirus délivrant un transgène bicistronique codant pour deux gènes rapporteurs fluorescents. Ces lentivirus ont été utilisés pour transduire sept différentes lignées cellulaires de laboratoire et des lignées cellulaires lymphoblastoïdes B du Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209) qui représentent une approche in vitro de la structure familiale et qui permettent des analyses par balayage du génome. L'expression relative des deux marqueurs fluorescents a été analysée par FACS. Nos résultats montrent que l'efficacité des IRES varie en fonction du type de cellules. Il varie aussi, pour le même type de cellules, selon les individus. Le contrôle de l'activité de l'IRES est un caractère héritable (héritabilité h2) de 0.47 et 0.36 pour les IRES de EMCV et XIAP respectivement. Le balayage du génome a permis l'identification d'un locus à effets quantitatifs [QTL Quantitative Trait Loci (LOD 2.35)] impliqué dans le contôle de l'activité de l'IRES de XIAP.

The application of chemometrics on Infrared and Raman spectra as a tool for the forensic analysis of paints

The aim of this work is to evaluate the capabilities and limitations of chemometric methods and other mathematical treatments applied on spectroscopic data and more specifically on paint samples. The uniqueness of the spectroscopic data comes from the fact that they are multivariate - a few thousands variables - and highly correlated. Statistical methods are used to study and discriminate samples. A collection of 34 red paint samples was measured by Infrared and Raman spectroscopy. Data pretreatment and variable selection demonstrated that the use of Standard Normal Variate (SNV), together with removal of the noisy variables by a selection of the wavelengths from 650 to 1830 cm−1 and 2730-3600 cm−1, provided the optimal results for infrared analysis. Principal component analysis (PCA) and hierarchical clusters analysis (HCA) were then used as exploratory techniques to provide evidence of structure in the data, cluster, or detect outliers. With the FTIR spectra, the Principal Components (PCs) correspond to binder types and the presence/absence of calcium carbonate. 83% of the total variance is explained by the four first PCs. As for the Raman spectra, we observe six different clusters corresponding to the different pigment compositions when plotting the first two PCs, which account for 37% and 20% respectively of the total variance. In conclusion, the use of chemometrics for the forensic analysis of paints provides a valuable tool for objective decision-making, a reduction of the possible classification errors, and a better efficiency, having robust results with time saving data treatments.