Segmentation and grid generation for numerical simulations of vad connections with patient-specific data

Objectives: We are interested in the numerical simulation of the anastomotic region comprised between outflow canula of LVAD and the aorta. Segmenta¬tion, geometry reconstruction and grid generation from patient-specific data remain an issue because of the variable quality of DICOM images, in particular CT-scan (e.g. metallic noise of the device, non-aortic contrast phase). We pro¬pose a general framework to overcome this problem and create suitable grids for numerical simulations.Methods: Preliminary treatment of images is performed by reducing the level window and enhancing the contrast of the greyscale image using contrast-limited adaptive histogram equalization. A gradient anisotropic diffusion filter is applied to reduce the noise. Then, watershed segmentation algorithms and mathematical morphology filters allow reconstructing the patient geometry. This is done using the InsightToolKit library (www.itk.org). Finally the Vascular Model¬ing ToolKit (www.vmtk.org) and gmsh (www.geuz.org/gmsh) are used to create the meshes for the fluid (blood) and structure (arterial wall, outflow canula) and to a priori identify the boundary layers. The method is tested on five different patients with left ventricular assistance and who underwent a CT-scan exam.Results: This method produced good results in four patients. The anastomosis area is recovered and the generated grids are suitable for numerical simulations. In one patient the method failed to produce a good segmentation because of the small dimension of the aortic arch with respect to the image resolution.Conclusions: The described framework allows the use of data that could not be otherwise segmented by standard automatic segmentation tools. In particular the computational grids that have been generated are suitable for simulations that take into account fluid-structure interactions. Finally the presented method features a good reproducibility and fast application.

Developmental, metabolic and immunological costs of flea infestation in the common vole

Parasites use resources from their hosts, which can indirectly affect a number of host functions because of trade-offs in resource allocation. In order to get a comprehensive view of the costs imposed by blood sucking parasites to their hosts, it is important to monitor multiple components of the development and physiology of parasitized hosts over long time periods. The effect of infestation by fleas on body mass, body length growth, haematocrit, resistance to oxidative stress, resting metabolic rate and humoral immune response were experimentally evaluated. During a 3-month period, male common voles, Microtus arvalis, were either parasitized by rat fleas (Nosopsyllus fasciatus), which are naturally occurring generalist ectoparasites of voles, or reared without fleas. Then voles were challenged twice by injecting Keyhole Limpet Haemocyanin (KLH) to assess whether the presence of fleas affects the ability of voles to produce antibodies against a novel antigen. During the immune challenge we measured the evolution of body mass, haematocrit, resistance to oxidative stress and antibody production. Flea infestation negatively influenced the growth of voles. Moreover, parasitized voles had reduced haematocrit, higher resting metabolic rate and lower production of antibodies against the KLH. Resistance to oxidative stress was not influenced by the presence of fleas. During the immune challenge with KLH, body mass decreased in both groups, while the resistance to oxidative stress remained stable. In contrast, the haematocrit decreased only in parasitized voles. Our experiment shows that infestation by a haematophageous parasite negatively affects multiple traits like growth, energy consumption and immune response. Fleas may severely reduce the survival probability and reproductive success of their host in natural conditions.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

Fécondation in vitro: intérêt et désavantage du remboursement par l'assurance maladie en Suisse [In-vitro fertilization: advantage and disadvantage of covering the costs of IVF/ICSI by the health insurance in Switzerland]

In-vitro fertilization: advantage and disadvantage of covering the costs of IVF/CSI by the health insurance in Switzerland The reimbursement of certain infertility treatments (stimulation with/without insemination) whereas IVF/ICSI is not leads patients with an indication of IVF to prefer treatments of low efficacy. The costs of multiple pregnancies issued by reimbursed or non-reimbursed fertility treatments are paid by the society. There should be measures to reduce these costs and to take the money used today to pay the complications of infertility treatments to reimburse IVF. The efficacy of such a system (single embryo transfer) has been proven in Belgium since several years. The dangers of complete reimbursement (IVF treatment in cases without any chances of success, only because it is for free) can be avoided by an Efficacy and Safety Board.

Towards a new combination therapy for tuberculosis with next generation benzothiazinones.

The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.

Generation of reconstituted human plasma-derived secretory-like IgA and IgM and their protective effect on intestinal epithelium

Les muqueuses sont les membranes tapissant les cavités du corps, tel que le tube digestif, et sont en contact direct avec l'environnement extérieur. Ces surfaces subissent de nombreuses agressions pouvant être provoquées par des agents pathogènes (bactéries, toxines ou virus). Cela étant, les muqueuses sont munies de divers mécanismes de protection dont notamment deux protéines-clés permettant de neutraliser les agents pathogènes : les anticorps ou immunoglobulines sécrétoires A (SIgA) et M (SIgM). Ces anticorps sont, d'une part, fabriqués au niveau de la muqueuse sous forme d'IgA et IgM. Lorsqu'ils sont sécrétés dans l'intestin, ils se lient à une protéine appelée pièce sécrétoire et deviennent ainsi SIgA et SïgM. La présence de la pièce sécrétoire est essentielle pour que les anticorps puissent fonctionner au niveau de la muqueuse. D'autre part, ces anticorps sont également fabriqués dans d'autres parties du corps en général et se retrouvent dans le sang sous forme d'IgA et IgM Chez l'homme, des thérapies basées sur l'injection d'anticorps donnent de bons résultats depuis de nombreuses années notamment dans le traitement des infections. Bien qu'un certain nombre d'études ont montré le rôle protecteur des anticorps de type IgA et IgM, ceux-ci ne sont que rarement utilisés dans les thérapies actuelles. La principale raison de cette faible utilisation réside dans la production ou la purification des IgA/IgM ou SIgA/SIgM (la forme active au niveau des muqueuses) qui est difficile à réaliser à large échelle. Ainsi, le but de la thèse était (1) d'étudier la possibilité d'employer des IgA et des IgM provenant du sang humain pour générer des SIgA et SIgM et (2) de voir si ces anticorps reconstitués pouvaient neutraliser certains agents pathogènes au niveau des muqueuses. Tout d'abord, une analyse biochimique des IgA et des IgM issues du sang a été effectuée. Nous avons observé que ces anticorps avaient des caractéristiques similaires aux anticorps naturellement présents au niveau des muqueuses. De plus, nous avons confirmé que ces anticorps pouvaient être associés à une pièce sécrétoire produite en laboratoire pour ainsi donner des SIgA et SIgM reconstituées. Ensuite, la fonctionnalité des anticorps reconstitués a été testée grâce à un modèle de couche unique de cellules intestinales différenciées (monocouches) en laboratoire imitant la paroi de l'intestin. Ces monocouches ont été infectées par une bactérie pathogène, Shigella flexneri, responsable de la shigellose, une maladie qui provoque des diarrhées sanglantes chez l'homme. L'infection des monocouches par les bactéries seules ou combinées aux SIgA et SIgM reconstituées a été analysée. Nous avons observé que les dommages des cellules étaient moins importants lorsque les SIgA étaient présentes. Il apparaît que les SIgA neutralisent les bactéries en se fixant dessus, ce qui provoque leur agrégation, et diminuent l'inflammation des cellules. La protection s'est montrée encore plus efficace avec les SIgM. De plus, nous avons vu que les SIgA et SIgM pouvaient diminuer la sécrétion de facteurs nocifs produits par les bactéries. Utilisant le même modèle des monocouches, la fonctionnalité des IgA issues du sang humain a aussi été testée contre une toxine sécrétée par une bactérie appelée Clostridium diffìcile. Cette bactérie peut être présente naturellement dans l'intestin de personnes saines, cependant elle peut devenir pathogène dans certaines conditions et être à l'origine de diarrhées et d'inflammations de l'intestin via la sécrétion de toxines. Des préparations d'anticorps contenant une certaine proportion de SIgA reconstituées ont amené à une diminution des dommages et de l'inflammation des monocouches causés par la toxine. L'ensemble de ces résultats prometteurs, montrant que des SIgA et SIgM reconstituées peuvent protéger la paroi de l'intestin des infections bactériennes, nous conduisent à approfondir la recherche sur ces anticorps dans des modèles animaux. L'aboutissement de ce type de recherche permettrait de tester, par la suite, l'efficacité sur l'homme de traitements des infections des muqueuses par injection d'anticorps de type SIgA et SIgM reconstituées. Les muqueuses, telle que la muqueuse gastrointestinale, sont des surfaces constamment exposées à l'environnement et leur protection est garantie par une combinaison de barrières mécaniques, physicochimiques et immunologiques. Parmi les divers mécanismes de protection immunologiques, la réponse humorale spécifique joue un rôle prépondérant et est assurée par les immunoglobulines sécrétoires de type A (SIgA) et M (SIgM). Les thérapies basées sur l'administration d'IgG apportent d'importants bénéfices dans le domaine de la santé. Bien que des études sur les animaux aient montré que l'administration par voie muqueuse d'IgA polymérique (plgA) ou SIgA pouvaient protéger des infections, des IgA/SIgA n'ont été utilisées qu'occasionnellement dans les thérapies. De plus, des études précliniques et cliniques ont démontré que l'administration par voie systémique de préparations enrichies en IgM pouvait aussi protéger des infections. Cependant, l'administration par voie muqueuse d'IgM/SIgM purifiées n'a pas été examinée jusqu'à présent. La principale raison est que la purification ou là production des IgA/SIgA et IgM/SIgM est difficile à réaliser à large échelle. Le but de ce travail de thèse était d'examiner la possibilité d'associer des IgA et IgM polyclonals purifiées à partir du plasma humain avec une pièce sécrétoire recombinante humaine afin de générer des SIgA et SIgM reconstituées fonctionnelles. Tout d'abord, une analyse biochimique des IgA et IgM issues du plasma humain a été effectuée par buvardage de western et Chromatographie. Ces molécules avaient des caractéristiques biochimiques similaires à celles des immunoglobulines issues de la muqueuse. L'association entre plgA ou IgM issues du plasma humain et la pièce sécrétoire recombinante humaine a été confirmée, ainsi que la stoechiométrie 1:1 de l'association. Comme dans les conditions physiologiques, cette association permettait de retarder la dégradation des SIgA et SIgM reconstituées exposées à des protéases intestinales. Ensuite, la fonctionnalité et le mode d'action des IgA et IgM issues du plasma humain, ainsi que des SIgA et SIgM reconstituées, ont été explorés grâce à un modèle in vitro de monocouches de cellules intestinales épithéliales polarisées de type Caco-2, qui imite l'épithélium intestinal. Les monocouches ont été infectées par un pathogène entérique, Shigella flexneri, seul ou combiné aux immunoglobulines issues du plasma humain ou aux immunoglobulines sécrétoires reconstituées. Bien que les dommages des monocouches aient été retardés par les plgA et SIgA reconstituées, les IgM et SIgM reconstituées se sont montrées supérieures dans le maintien de l'intégrité des cellules. Une agrégation bactérienne et une diminution de l'inflammation des monocouches ont été observées avec les plgA et SIgA reconstituées. Ces effets étaient augmentés avec les IgM et SIgM reconstituées. De plus, il s'est révélé que les deux types d'immunoglobulines de type sécrétoire reconstituées agissaient directement sur la virulence des bactéries en réduisant leur sécrétion de facteurs de virulence. La fonctionnalité des IgA issues du plasma humain a aussi été testée contre la toxine A de Clostridium difficile grâce au même modèle de monocouches de cellules épithéliales. Nous avons démontré que des préparations enrichies en IgA provenant du plasma humain pouvaient diminuer les dommages et l'inflammation des monocouches induits par la toxine. L'ensemble de ces résultats démontrent que des IgA et IgM de type sécrétoire peuvent être générées à partir d'IgA et IgM issues du plasma humain en les associant à la pièce sécrétoire et que ces molécules protègent l'épithélium intestinal contre des bactéries pathogènes. Ces molécules pourraient dès lors être testées dans des modèles in vivo. Le but final serait de les utiliser chez l'homme à des fins d'immunisation passive dans le traitement de pathologies associées à la muqueuse telles que les infections. - Mucosal surfaces, such as gastrointestinal mucosa, are constantly exposed to the external environment and their protection is ensured by a combination of mechanical, physicochemical and immunological barriers. Among the various immunological defense mechanisms, specific humoral mucosal response plays a crucial role and is mediated by secretory immunoglobulins A (SIgA) and M (SIgM). Immunoglobulin therapy based on the administration of IgG molecules leads important health benefits. Even though animal studies have shown that mucosal application of polymeric IgA (plgA) or SIgA provided protection against infections, IgA/SIgA have been only used occasionally for therapeutic application. Moreover, preclinical and clinical studies have demonstrated that systemic administration of IgM-enriched preparations could also afford protection against infections. Nevertheless, mucosal application of purified IgM/SIgM has not been examined. The main reason is that the purification or production of IgA/SIgA and IgM/SIgM at large scale is difficult to achieve. The aim of this PhD project was to examine the possibility to associate polyclonal human plasma-derived IgA and IgM with recombinant human secretory component (SC) to generate functional secretoiy-like IgA and IgM. First, biochemical analysis of human plasma IgA and IgM was performed by western blotting and chromatography. These molecules exhibited the same biochemical features as mucosa-derived antibodies (Abs). The association between human plasma plgA or IgM and recombinant human SC was confirmed, as well as the 1:1 stoichiometry of association. Similarly to physiological conditions, this association delayed the degradation of secretory-like IgA or IgM by intestinal proteases. Secondly, the function activity and the mode of action of human plasma IgA and IgM, as well as secretory-like IgA and IgM were explored using an in vitro model of polarized intestinal epithelial Caco-2 cell monolayers mimicking intestinal epithelium. Cell monolayers were infected with an enteropathogen, Shigella flexneri, alone or in combination to plasma Abs or secretory-like Abs. Even though plasma plgA and secretoiy-like IgA resulted in a delay of bacteria-induced damages of cell monolayers, plasma IgM and secretory-like IgM were shown to be superior in maintenance of cell integrity. Polymeric IgA and secretory-like IgA induced bacterial aggregation and decreased cell monolayer inflammation, effects further amplified with IgM and secretory-like IgM. In addition, both secretory-like Abs directly impacted on bacterial virulence leading to a reduction in secretion of virulence factors by bacteria. The functionality of human plasma IgA was also tested against Clostridium difficile toxin A using Caco-2 cell monolayers. Human plasma IgA- enriched preparations led to a diminution of cell monolayer damages and a decrease of cellular inflammation induced by the toxin. The sum of these results demonstrates that secretory-like IgA and IgM can be generated from purified human plasma IgA and IgM associated to SC and that these molecules are functional to protect intestinal epithelium from bacterial infections. These molecules could be now tested using in vivo models. The final goal would be to use them by passive immunization in the treatment of mucosa-associated pathologies like infections in humans.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs.

The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sensitivity [0.78, 95 % confidence interval (CI) 0.73-0.82] and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.

Controlled dynamic generation of standard 1,6-hexamethylene diisocyanate aerosols

A procedure for the dynamic generation of 1,6-hexamethylene diisocyanate (HDI) aerosol atmospheres of 70 micrograms m-3 (0.01 ppm) to 1.75 mg m-3 (0.25 ppm), based on the precise control of the evaporation of pure liquid HDI and subsequent dilution with air, was developed. The apparatus consisted of a home-made glass nebulizer coupled with a separation stage to exclude non-respirable droplets (greater than 10 microns). The aerosol concentrations were achieved by passing air through the nebulizer at 1.5-4.5 l. min-1 to generate dynamically 0.01-0.25 ppm of diisocyanate in an experimental chamber of 8.55 m3. The distribution of the liquid aerosol was established with an optical counter and the diisocyanate concentration was determined from samples collected in impingers by a high-pressure liquid chromatographic method. The atmospheres generated were suitable for the evaluation both of sampling procedures full scale, and of analytical methods: at 140 micrograms m-3 (0.02 ppm) they remained stable for 15-min provocation tests in clinical asthma, as verified by breath-zone sampling of exposed patients.

Co-evolution between sociality and dispersal: The role of synergistic cooperative benefits.

Explaining the evolution of sociality is challenging because social individuals face disadvantages that must be balanced by intrinsic benefits of living in a group. One potential route towards the evolution of sociality may emerge from the avoidance of dispersal, which can be risky in some environments. Although early studies found that local competition may cancel the benefits of cooperation in viscous populations, subsequent studies have identified conditions, such as the presence of kin recognition or specific demographic conditions, under which altruism will still spread. Most of these studies assume that the costs of cooperating outweigh the direct benefits (strong altruism). In nature, however, many organisms gain synergistic benefits from group living, which may counterbalance even costly altruistic behaviours. Here, we use an individual based model to investigate how dispersal and social behaviour co-evolve when social behaviours result in synergistic benefits that counterbalance the relative cost of altruism to a greater extent than assumed in previous models. When the cost of cooperation is high, selection for sociality responds strongly to the cost of dispersal. In particular, cooperation can begin to spread in a population when higher cooperation levels become correlated with lower dispersal tendencies within individuals. In contrast, less costly social behaviours are less sensitive to the cost of dispersal. In line with previous studies, we find that mechanisms of global population control also affect this relationship: when whole patches (groups) go extinct each generation, selection favours a relatively high dispersal propensity, and social behaviours evolve only when they are not very costly. If random individuals within groups experience mortality each generation to maintain a global carrying capacity, on the other hand, social behaviours spread and dispersal is reduced, even when the latter is not costly.

Life expectancy with and without disability

Average life expectancy reached 78.8 years in Europe in 2002 (WHO 2003); most Europeans can, therefore, now anticipate living well past 75 years of age. Projections in industrialized nations suggest a continuing mortality decline in the next decades 1 while birth rates will probably continue to decline, resulting in further ageing of these nations. As those aged 80 years and over are the fastest expanding segment of the older population, concerns are growing about a potential dramatic increase in the number of disabled persons. The ageing of the population and the related increase in chronic disease burden have already had major impacts on most Western health-care systems, and will probably further affect these systems in the future as the baby-boom generation becomes older. For instance, in Switzerland, it is estimated that costs due to long-term care could more than double by 2030, from 6.5 to 15.3 billion SFr.2 Similar trends are expected in most European countries. As a consequence, postponement of the onset of disability, with a compression of functional dependency into a shorter period towards the end of life, is becoming a major goal. To successfully achieve this goal and improve the control of growing health and social care expenditures, various strategies of health promotion and disease prevention are developed and tested. Although several of these experiences had some effects on functional decline and institutional placement, they have not been shown to be cost-effective. Additional strategies are, therefore, needed to prevent or delay the onset of disability in older persons, reduce functional impairment, and face the challenge of an increasing disabled elderly population.

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.