Mechanisms of functional T-cell deficiency in melanoma patients

SUMMARYAs a result of evolution, humans are equipped with an intricate but very effective immune system with multiple defense mechanisms primarily providing protection from infections. This system comprises various cell types, including T-lymphocytes, which are able to recognize and directly kill infected cells. T-cells are not only able to recognize cells carrying foreign antigens, such as virus-infected cells, but also autologous cells. In autoimmune diseases, e.g. multiple sclerosis, T- cells attack autologous cells and cause the destruction of healthy tissue. To prevent aberrant immune reactions, but also to prevent damage caused by an overreacting immune response against foreign targets, there are multiple systems in place that attenuate T-cell responses.By contrast, anti-self immune responses may be highly welcome in malignant diseases. It has been demonstrated that activated T-cells are able to recognize and lyse tumor cells, and may even lead to successful cure of cancer patients. Through vaccination, and especially with the help of powerful adjuvants, frequencies of tumor-reactive T-cells can be augmented drastically. However, the efficacy of anti-tumor responses is diminished by the same checks and balances preventing the human body from harm induced by overly activated T-cells in infections.In the context of my thesis, we studied spontaneous and vaccination induced T-cell responses in melanoma patients. The aim of my studies was to identify situations of T-cell suppression, and pinpoint immune suppressive mechanisms triggered by malignant diseases. We applied recently developed techniques such as multiparameter flow cytometry and gene arrays, allowing the characterization of tumor-reactive T-cells directly ex vivo. In our project, we determined functional capabilities, protein expression, and gene expression profiles of small numbers of T- cells from metastatic tissue and blood obtained from healthy donors and melanoma patients. We found evidence that tumor-specific T-cells were functionally efficient effector cells in peripheral blood, but severely exhausted in metastatic tissue. Our molecular screening revealed the upregulation of multiple inhibitory receptors on tumor-specific T-cells, likely implied in T-cell exhaustion. Functional attenuation of tumor-specific T-cells via inhibitory receptors depended on the anatomical location and immune suppressive mechanisms in the tumor microenvironment, which appeared more important than self-tolerance and anergy mechanisms. Our data reveal novel potential targets for cancer therapy, and contribute to the understanding of cancer biology.RÉSUMÉAu cours de l'évolution, les êtres humains se sont vus doter d'un système immunitaire complexe mais très efficace, avec de multiples mécanismes de défense, principalement contre les infections. Ce système comprend différents types de cellules, dont les lymphocytes Τ qui sont capables de reconnaître et de tuer directement des cellules infectées. Les cellules Τ reconnaissent non seulement des cellules infectées par des virus, mais également des cellules autologues. Dans le cas de maladies auto-immunes, comme par exemple la sclérose en plaques, les cellules Τ s'attaquent à des cellules autologues, ce qui engendre la destruction des tissus sains. Il existe plusieurs systèmes de contrôle des réponses Τ afin de minimiser les réactions immunitaires aberrantes et d'empêcher les dégâts causés par une réponse immunitaire trop importante contre une cible étrangère.Dans le cas de maladies malignes en revanche, une réponse auto-immune peut être avantageuse. Il a été démontré que les lymphocytes Τ étaient également capables de reconnaître et de tuer des cellules tumorales, pouvant même mener à la guérison d'un patient cancéreux. La vaccination peut augmenter fortement la fréquence des cellules Τ réagissant contre une tumeur, particulièrement si elle est combinée avec des adjuvants puissants. Cependant, l'efficacité d'une réponse antitumorale est atténuée par ces mêmes mécanismes de contrôle qui protègent le corps humain des dégâts causés par des cellules Τ activées trop fortement pendant une infection.Dans le cadre de ma recherche de thèse, nous avons étudié les réponses Τ spontanées et induites par la vaccination dans des patients atteints du mélanome. Le but était d'identifier des conditions dans lesquelles les réponses des cellules Τ seraient atténuées, voire inhibées, et d'élucider les mécanismes de suppression immunitaire engendrés par le cancer. Par le biais de techniques nouvelles comprenant la cryométrie de flux et l'analyse globale de l'expression génique à partir d'un nombre minimal de cellules, il nous fut possible de caractériser des cellules Τ réactives contre des tumeurs directement ex vivo. Nous avons examiné les profiles d'expression de gènes et de protéines, ainsi que les capacités fonctionnelles des cellules Τ isolées à partir de tissus métastatiques et à partir du sang de patients. Nos résultats indiquent que les cellules Τ spécifiques aux antigènes tumoraux sont fonctionnelles dans le sang, mais qu'elles sont épuisées dans les tissus métastatiques. Nous avons découvert dans les cellules Τ antitumorales une augmentation de l'expression des récepteurs inhibiteurs probablement impliqués dans l'épuisement de ces lymphocytes T. Cette expression particulière de récepteurs inhibiteurs dépendrait donc de leur localisation anatomique et des mécanismes de suppression existant dans l'environnement immédiat de la tumeur. Nos données révèlent ainsi de nouvelles cibles potentielles pour l'immunothérapie du cancer et contribuent à la compréhension biologique du cancer.

Safety and functional outcome of thrombolysis in dissection-related ischemic stroke: a meta-analysis of individual patient data.

Background and Purpose-The safety and efficacy of thrombolysis in cervical artery dissection (CAD) are controversial. The aim of this meta-analysis was to pool all individual patient data and provide a valid estimate of safety and outcome of thrombolysis in CAD.Methods-We performed a systematic literature search on intravenous and intra-arterial thrombolysis in CAD. We calculated the rates of pooled symptomatic intracranial hemorrhage and mortality and indirectly compared them with matched controls from the Safe Implementation of Thrombolysis in Stroke-International Stroke Thrombolysis Register. We applied multivariate regression models to identify predictors of excellent (modified Rankin Scale=0 to 1) and favorable (modified Rankin Scale=0 to 2) outcome.Results-We obtained individual patient data of 180 patients from 14 retrospective series and 22 case reports. Patients were predominantly female (68%), with a mean +/- SD age of 46 +/- 11 years. Most patients presented with severe stroke (median National Institutes of Health Stroke Scale score=16). Treatment was intravenous thrombolysis in 67% and intra-arterial thrombolysis in 33%. Median follow-up was 3 months. The pooled symptomatic intracranial hemorrhage rate was 3.1% (95% CI, 1.3 to 7.2). Overall mortality was 8.1% (95% CI, 4.9 to 13.2), and 41.0% (95% CI, 31.4 to 51.4) had an excellent outcome. Stroke severity was a strong predictor of outcome. Overlapping confidence intervals of end points indicated no relevant differences with matched controls from the Safe Implementation of Thrombolysis in Stroke-International Stroke Thrombolysis Register.Conclusions-Safety and outcome of thrombolysis in patients with CAD-related stroke appear similar to those for stroke from all causes. Based on our findings, thrombolysis should not be withheld in patients with CAD. (Stroke. 2011;42:2515-2520.)

Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways.

The PHO1 family comprises 11 members in Arabidopsis thaliana. In order to decipher the role of these genes in inorganic phosphate (Pi) transport and homeostasis, complementation of the pho1 mutant, deficient in loading Pi to the root xylem, was determined by the expression of the PHO1 homologous genes under the control of the PHO1 promoter. Only PHO1 and the homologue PHO1;H1 could complement pho1. The PHO1;H1 promoter was active in the vascular cylinder of roots and shoots. Expression of PHO1;H1 was very low in Pi-sufficient plants, but was strongly induced under Pi-deficient conditions. T-DNA knock-out mutants of PHO1;H1 neither showed growth defects nor alteration in Pi transport dynamics, or Pi content, compared with wild type. However, the double mutant pho1/pho1;h1 showed a strong reduction in growth and in the capacity to transfer Pi from the root to the shoot compared with pho1. Grafting experiments revealed that phenotypes associated with the pho1 and pho1/pho1;h1 mutants were linked to the lack of gene expression in the root. The increased expression of PHO1;H1 under Pi deficiency was largely controlled by the transcription factor PHR1 and was suppressed by the phosphate analogue phosphite, whereas the increase of PHO1 expression was independent of PHR1 and was not influenced by phosphite. Together, these data reveal that although transfer of Pi to the root xylem vessel is primarily mediated by PHO1, the homologue PHO1;H1 also contributes to Pi loading to the xylem, and that the two corresponding genes are regulated by Pi deficiency by distinct signal transduction pathways.

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatic islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9-39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9-39) was an antagonist of the receptor, inhibiting GLP-1-induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.

Family impact in intellectual disability, severe mental health disorders and mental health disorders in ID. A comparison

Family impact (or family burden) is a concept born in the field of mental health that has successfully been exported to the ambit of intellectual disability (ID). However, differences in family impact associated with severe mental health disorders (schizophrenia), to ID or to mental health problems in ID should be expected. Seventy-two adults with intellectual disability clients of the Carmen Pardo-Valcarce Foundation's sheltered workshops and vocational employment programmes in Madrid (Spain), 203 adults diagnosed with schizophrenia from four Spanish Community Mental Health Services (Barcelona, Madrid, Granada and Navarra) and 90 adults with mental health problems in ID (MH-ID) from the Parc Sanitari Sant Joan de Déu Health Care Site in Sant Boi de Llobregat, Barcelona (Spain) were asked to participate in the present study along with their main caregivers. Family impact experienced by caregivers was assessed with the ECFOS-II/SOFBI-II scale (Entrevista de Carga Familiar Objetiva y Subjetiva/Objective and Subjective Family Burden Interview). In global terms, results showed that the higher family impact was found between caregivers to people with MH-ID. The interaction of both conditions (ID and mental health problems) results in a higher degree of burden on families than when both conditions are presented separately. There was also an impact in caregivers to people with schizophrenia, this impact being higher than the one detected in caregivers to people with intellectual disability. Needs of caregivers to people with disability should be addressed specifically in order to effectively support families.

Functional analysis of HLA-A*0201/Melan-A peptide multimer+ CD8+ T cells isolated from an HLA-A*0201- donor: exploring tumor antigen allorestricted recognition.

Recent studies in mouse models have suggested that genetic transfer of tumor antigen-specific high affinity T cell receptors (TCR) into host lymphocytes could be a viable strategy for the rapid induction of tumor-specific immunity. A previously proposed approach for the isolation of such TCRs consists in circumventing tolerance to self-restricting HLA/peptide complexes by deriving them from PMBCs of allogenic donors. Towards this aim, we used fluorescent HLA-A2 class-I/peptide soluble multimers to isolate A2-restricted CD8+ T cells specific for a previously described Melan-A peptide enhanced analog (Melan-A 26-35 A27L) from an HLA-A*0201 (A2) negative donor. We isolated two distinct groups of Melan-A 26-35 A27L-specific clones. Clones from the first group recognized the analog peptide with high avidity but showed very low recognition of Melan-A parental peptides. In contrast, clones from the second group efficiently recognized Melan-A parental peptides. Surprisingly however, most clones recognized not only A2+ Melan-A+ targets, but also A2+ Melan-A- targets suggesting that they can also recognize endogenous peptides other than Melan-A. In addition, one clone showed full cross-recognition of an antigenically unrelated peptide. Together, our data show that HLA-A2/peptide multimers can be successfully used for the isolation of allorestricted CD8+ T cells reactive with tumor antigen-derived peptides. However, as the cross-reactivity of these apparently peptide-specific allorestricted TCRs is presently unpredictable, a careful in vitro analysis of their reactivity to the host's normal cells is recommended.

Functional homogenization of bumblebee communities in alpine landscapes under projected climate change

Background: Bumblebees represent an active pollinator group in mountain regions and assure the pollination of many different plant species from low to high elevations. Plant-pollinator interactions are mediated by functional traits. Shift in bumblebee functional structure under climate change may impact plant-pollinator interactions in mountains. Here, we estimated bumblebee upward shift in elevation, community turnover, and change in functional structure under climate change. Method: We sampled bumblebee species at 149 sites along the elevation gradient. We used stacked species distribution models (S-SDMs) forecasted under three climate change scenarios (A2, A1B, RCP3PD) to model the potential distribution of the Bombus species. Furthermore, we used species proboscis length measurements to assess the functional change in bumblebee assemblages along the elevation gradient. Results: We found species-specific response of bumblebee species to climate change. Species differed in their predicted rate of range contraction and expansion. Losers were mainly species currently restricted to high elevation. Under the most severe climate change scenarios (A2), we found a homogenization of proboscis length structure in bumblebee communities along the elevation gradient through the upward colonization of high elevation by species with longer proboscides. Conclusions: Here, we show that in addition to causing the shift in the distribution of bumblebee species, climate change may impact the functional structure of communities. The colonization of high elevation areas by bumblebee species with long proboscides may modify the structure of plant-pollination interaction networks by increasing the diversity of pollination services at high elevation.

CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression.

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160+ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160+ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.