Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2⁺-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.     

Post‐last glacial maximum expansion from Iberia to North Africa revealed by fine characterization of mtDNA H haplogroup in Tunisia

The first large‐scale fine characterization of Tunisian H lineages clarifies that the post‐Last glacial maximum expansion originating in Iberia not only led to the resettlement of Europe but also of North Africa. We found that 46% of 81 Tunisian H lineages subscreened for 1,580 bp in mtDNA coding region were affiliated with H1 and H3 subhaplogroups, which are known to have originated in Iberia. Although no signs of local expansion were detected, which would allow a clear dating of their introduction, the younger and less diverse Tunisian H1 and H3 lineages indicate Iberia as the radiating centre. Major contributions from historical migrations to this Iberian genetic imprint in Tunisia were ruled out by the mtDNA gene pool similarity between Berber/Arab/cosmopolitan samples and some “Andalusian” communities, settled by the descendents of the “Moors” who once lived in Iberia for 10 centuries (between 8th and 17th centuries), before being expelled to Tunisia. Am J Phys Anthropol, 2009. © 2008 Wiley‐Liss, Inc.     

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.     

Selective probing of a NADPH site controlled light‐induced enzymatic catalysis

Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO‐synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light‐driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH‐mediated enzyme. In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO‐synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse. Copyright © 2009 John Wiley & Sons, Ltd.     

Placebo-Suggestion Modulates Conflict Resolution in the Stroop Task

Here, we ask whether placebo-suggestion (without any form of hypnotic induction) can modulate the resolution of cognitive conflict. Naïve participants performed a Stroop Task while wearing an EEG cap described as a “brain wave” machine. In Experiment 1, participants were made to believe that the EEG cap would either enhance or decrease their color perception and performance on the Stroop task. In Experiment 2, participants were explicitly asked to imagine that their color perception and performance would be enhanced or decreased (non-hypnotic imaginative suggestion). We observed effects of placebo-suggestion on Stroop interference on accuracy: interference was decreased with positive suggestion and increased with negative suggestion compared to baseline. Intra-individual variability was also increased under negative suggestion compared to baseline. Compliance with the instruction to imagine a modulation of performance, on the other hand, did not influence accuracy and only had a negative impact on response latencies and on intra-individual variability, especially in the congruent condition of the Stroop Task. Taken together, these results demonstrate that expectations induced by a placebo-suggestion can modulate our ability to resolve cognitive conflict, either facilitating or impairing response accuracy depending on the suggestion’s contents. Our results also demonstrate a dissociation between placebo-suggestion and non-hypnotic imaginative suggestion.     

Impact d’une méthode originale de synchronisation respiratoire sur la détection des lésions hépatiques en TEP/TDM au F-FDG

In ¹⁸F-Fluoro-Desoxyglucose (¹⁸F-FDG) positron emission tomography/computed tomography (PET/CT), respiratory motion induces bias in image interpretations. These movements can introduce organs misregistration between both modalities yielding erroneous attenuation correction and thus wrong maximum standardized uptake values (SUV_max). We present here the results of a clinical study which aims to assess the benefits of a novel respiratory gating method (CT-based) for liver lesions detection. Forty-nine patients planed to undergo hepatic surgery were addressed to our department for PET/CT examination before surgery. Each patient had both standard and CT-based protocols. Hepatic lesions described by two observers on PET images were compared with pathological analysis and intra-operative ultrasound. Sensitivities calculated for observer 1 were 60 and 64% for standard and CT-based, respectively. For the second observer, sensitivities were 58.7 and 72%. CT-based showed a significant increase (P < 0.01) of sensitivity on a per-lesion basis for one observer. CT-based did not improve inter-observer variability. At last, SUV_max were significantly higher with CT-based method (P < 0.001). Respiratory gating CT-based method is easily bearable by the patients. This procedure ensures good matching between both modalities and reduces motion-blurring effect in PET data. CT-based method improves liver lesions detectability and allows more accurate quantitation compared to non-gated FDG-PET/CT examinations.     

Outcomes of pregnant patients with Pap smears classified as atypical glandular cells

J. Slama, P. Freitag, P. Dundr, J. Duskova, D. Fischerova, M. Zikan, I. Pinkavova and D. Cibula
Outcomes of pregnant patients with Pap smears classified as atypical glandular cells Objectives: The incidence of cervical cancers increases with age. Due to the trend of increasing age of first pregnancy, abnormal Pap smears including those classified as atypical glandular cells (AGC) are being found more often in early pregnancy. Once invasive cancer is excluded, conservative management of squamous intraepithelial lesions (SIL) in pregnancy is considered safe; however, optimal management of AGC is not well established. The aim of our study was to evaluate the outcome of patients with AGC diagnosed from smears during pregnancy. Methods: The study included 17 patients referred to us in early pregnancy with Pap smears reported as AGC: 11 not otherwise specified (AGC‐NOS), five favour neoplasia (AGC‐FN) and one adenocarcinoma in situ (AIS). Thirty‐one with high‐grade SIL (HSIL) Pap smears confirmed on punch biopsy in early pregnancy comprised a control group. Human papillomavirus (HPV) positivity was found in seven patients with persistent AGC‐NOS (including all four who had CIN3 postpartum). All the women were initially examined by expert colposcopy and those with AGC‐FN or AIS smears also by transrectal ultrasound to exclude invasive endocervical cancer. Follow‐up controls were carried out every 8–12 weeks and, if there were no signs of progression, revaluation was scheduled 6–8 weeks after delivery. Results: The mean age of the women was 31.4 years. Conization in one patient in the study group was performed in the 16th week of pregnancy due to colposcopic signs of microinvasive squamous cell cancer confirmed on histology. Progression to invasive cancer was not found in any of the other 16 patients in the study group or in the control group. Cervical intraepithelial neoplasia or AIS was confirmed postpartum by conization or punch biopsy in 47.1% (8/17) of patients in the study group and, in 77.4% (24/31) of patients in the control group. Conclusions: Conservative management of women with AGC in pregnancy is safe where invasive cancer is excluded. As histological verification of glandular pre‐cancerous lesions by punch biopsy is not reliable and the postpartum regression rate cannot be determined precisely, conization should be performed in all cases with AGC‐FN or AIS. Triage of persistent AGC‐NOS with HPV testing is useful in distinguishing significant underlying lesions.     

Two Tunisian patients with Peters plus syndrome harbouring a novel splice site mutation in the B3GALTL gene that modulates the mRNA secondary structure

Peters plus syndrome is an autosomal recessive rare disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities, distinctive facial features, and often other major/minor additional defects. Peters plus syndrome is related to mutations in the B3GALTL gene with only seven recently reported mutations, leading to the inactivation of the B1, 3-glucosyltransferase. In this study, we screened the B3GALTL gene in two unrelated patients with typical Peters plus syndrome. A novel homozygous c.597-2A>G mutation was identified in both patients. Bioinformatic analyses showed that this mutation modulates the pre mRNA secondary structure of the gene, and decreases the score value related to the formation of splicing loops. Moreover, the c.597-2A>G mutation is located in a CpG Island of the B3GALTL gene, suggesting a potential epigenetic role of this position including gene's methylation and regulation. These data confirm an important role of the B3GALTL gene test that provides diagnosis confirmation and improves genetic counseling for the families.     

HIV Controllers Maintain a Population of Highly Efficient Th1 Effector Cells in Contrast to Patients Treated in the Long Term

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.