Absence of association between specific common variants of the obesity-related FTO gene and psychological and behavioral eating disorder phenotypes.

Extensive population-based genome-wide association studies have identified an association between the FTO gene and BMI; however, the mechanism of action is still unknown. To determine whether FTO may influence weight regulation through psychological and behavioral factors, seven single-nucleotide polymorphisms (SNPs) of the FTO gene were genotyped in 1,085 individuals with anorexia nervosa (AN) and 677 healthy weight controls from the international Price Foundation Genetic Studies of Eating Disorders. Each SNP was tested in association with eating disorder phenotypes and measures that have previously been associated with eating behavior pathology: trait anxiety, harm-avoidance, novelty seeking, impulsivity, obsessionality, compulsivity, and concern over mistakes. After appropriate correction for multiple comparisons, no significant associations between individual FTO gene SNPs and eating disorder phenotypes or related eating behavior pathology were identified in cases or controls. Thus, this study found no evidence that FTO gene variants associated with weight regulation in the general population are associated with eating disorder phenotypes in AN participants or matched controls. © 2011 Wiley-Liss, Inc.

Mycobacterium tuberculosis-specific CD8 T cells are functionally and phenotypically different between latent infection and active disease

Purpose/Objective: Tuberculosis (TB) is the second worldwide leading cause of death from an infectious disease after HIV infection. Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8 T-cells is controversial. We performed comprehensive functional and phenotypic characterizations of Mtb-specific CD8 T-cell responses in 273 subjects with either latent Mtb infection (LTBI) or active TB disease (TB) to assess their profile and relevance in TB. Materials and methods: Using multi-parametric flow cytometry, we assessed Mtb-specific CD8 T-cell functional (production of IFNgamma, IL-2 and TNF-alpha; proliferation capacity and cytotoxicity) and phenotypic (T-cell differentiation and exhaustion) profiles in cells isolated from peripheral blood and correlated these profiles with distinct clinical presentations. Results: Mtb-specific CD8 T-cells were detected in most TB patients and few LTBI subjects (65% and 15%, respectively; P < 0.00001) and were of similar magnitude with a comparable cytokines profile (IFNg+TNFa+IL2-) in both groups. Mtb-specific CD8 T-cells were mostly TEMRA (CD45RA+ CCR7-) co-expressing 2B4 and CD160 in LTBI subjects and mostly TEM (CD45RA-CCR7-) lacking PD-1/ CD160/2B4 in TB patients. Furthermore, Mtb-specific CD8 T-cells mostly expressed very little perforin and granulysin but contained granzymes A and B or lacked all these cytotoxic markers in TB and LTBI subjects, respectively. However, in vitro expanded Mtb-specific CD8 T-cells acquired perforin, granulysin and granzymes. Finally, Mtb-specific CD8 T-cell responses were more robust and prone to proliferate in patients with extrapulmonary compared to pulmonary TB. Conclusions: The clinical status and TB presentation are associated to specific profiles of Mtb-specific CD8 T-cell responses, thus indicating distinct dynamics between the mycobacteria, the CD8 T-cell response and the clinical outcome. Our data shed light on the controversial reached by studies performed in human and animal models, thus advancing the current knowledge on the complex dynamic of TB immunity.

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.

Direct identification as well as isolation of antigen-specific T cells became possible since the development of "tetramers" based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.

Can hTERT peptide (540-548) -specific CD8 T cells recognize and kill tumor cells?

This commentary reviews the data on HLA-A2-restricted CD8 T cells specific for peptide (540-548) derived from hTERT (human telomerase reverse transcriptase). Several studies have reported the successful generation of such T cells (1, 2, 3). However, tumor recognition was observed in some, but not all, studies. More data are required to elucidate whether hTERT peptide (540-548) -specific T cells can indeed recognize and destroy tumor cells. It would be highly useful if telomerase would emerge as a universal tumor antigen that can be targeted in the cancer immunotherapy of HLA-A2 positive patients.

Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR.

Efficient immune attack of malignant disease requires the concerted action of both CD8+ CTL and CD4+ Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the alpha3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8+ T lymphocytes with broad tumor-specific CTL activity and turned CD4+ T cells into potent tumor-reactive, p53A2.1-specific Th cells. Both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The intentional redirection of both CD4+ Th cells and CD8+ CTL by the same high-affinity, CD8-independent, tumor-specific TCR could provide the basis for novel broad-spectrum cancer immunotherapeutics.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Context specific affinity thresholds for CD8 T cell activation

After antigen driven activationnaïve CD8 T cells develop intocytolytic effector cells and subsequentlyinto memory cells. The molecularinteractions orchestrating Tcell activation are complex and we sofar have a limited understanding howindividual signals impact the Tcell response.Using OT-1 TCR transgeniccells and Listeria monocytogenesstrains expressing a set of altered peptideligands (APL) for the OT-1 TCRwe have recently studied how thelevel of TCR stimulation impacts theT cell response in vivo. We therebyobserved that even very low levels ofTCR stimulation are sufficient forfunctional effector and memoryT celldifferentiation. In order to addresshow much further the level of TCRstimulation can be reduced until the Tcells do not become activated anymore,we generated additional OT-1APL expressing Listeria strains. TheAPLused in our present study cover arange of potency down to the level ofpositive selection. Using all our APLListeria strains we can demonstratethat the threshold of peripheral T cellactivation is above the level of positiveselection but far below the levelthat is thought to be required for negativeselection. Furthermore, we characterizedthe thresholds of activatingmemory T cells and found them intrinsicallyto be very similar to thoseof naïve T cells. However, we observedthat T cell competition at thelevel of antigen presenting cells criticallyraises the activation threshold ofmemory CD8 T cells. Taken togetherour data indicate that the threshold foractivating T cells critically dependson the context and the environment inwhich T cells respond to antigen.

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.

K-r/K-c but not d(N)/d(S) correlates positively with body mass in birds, raising implications for inferring lineage-specific selection

Background: The ratio of the rates of non-synonymous and synonymous substitution (d(N)/d(S)) is commonly used to estimate selection in coding sequences. It is often suggested that, all else being equal, d(N)/d(S) should be lower in populations with large effective size (Ne) due to increased efficacy of purifying selection. As N-e is difficult to measure directly, life history traits such as body mass, which is typically negatively associated with population size, have commonly been used as proxies in empirical tests of this hypothesis. However, evidence of whether the expected positive correlation between body mass and d(N)/d(S) is consistently observed is conflicting. Results: Employing whole genome sequence data from 48 avian species, we assess the relationship between rates of molecular evolution and life history in birds. We find a negative correlation between dN/dS and body mass, contrary to nearly neutral expectation. This raises the question whether the correlation might be a method artefact. We therefore in turn consider non-stationary base composition, divergence time and saturation as possible explanations, but find no clear patterns. However, in striking contrast to d(N)/d(S), the ratio of radical to conservative amino acid substitutions (K-r/K-c) correlates positively with body mass. Conclusions: Our results in principle accord with the notion that non-synonymous substitutions causing radical amino acid changes are more efficiently removed by selection in large populations, consistent with nearly neutral theory. These findings have implications for the use of d(N)/d(S) and suggest that caution is warranted when drawing conclusions about lineage-specific modes of protein evolution using this metric.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Landmark detection for fusion of fundus and MRI toward a patient-specific multimodal eye model.

Ophthalmologists typically acquire different image modalities to diagnose eye pathologies. They comprise, e.g., Fundus photography, optical coherence tomography, computed tomography, and magnetic resonance imaging (MRI). Yet, these images are often complementary and do express the same pathologies in a different way. Some pathologies are only visible in a particular modality. Thus, it is beneficial for the ophthalmologist to have these modalities fused into a single patient-specific model. The goal of this paper is a fusion of Fundus photography with segmented MRI volumes. This adds information to MRI that was not visible before like vessels and the macula. This paper contributions include automatic detection of the optic disc, the fovea, the optic axis, and an automatic segmentation of the vitreous humor of the eye.

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)