Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Identification by a monoclonal antibody of a glycolipid highly expressed by cells from the human myeloid lineage.

A monoclonal antibody (MAb) HL-C5, which bound selectively to cells of the myeloid lineage tested, was derived from a fusion between P3/NS2/1-AG8 myeloma cells and splenocytes from a mouse immunized with cells of the promyelocytic leukemia line HL-60. Among a panel of 29 human cell lines derived from either hematopoietic or solid tumors, MAb HL-C5 was found to react exclusively with cells from the five differentiated acute myeloid leukemia lines, HL-60, ML1, ML2, ML3, KG-1B and not with the less differentiated myeloid lines. Fluorescence-activated cell sorter analysis of normal bone marrow samples confirmed that the reactivity of MAb HL-C5 was limited to myeloid cells, from the promyelocytic stage of differentiation to the mature granulocytes. Indirect immunoperoxidase staining of cytocentrifuge preparations of normal bone marrow and peripheral blood leukocytes confirmed these results and showed that MAb HL-C5 stained neutrophils but not eosinophils or basophils. The antigen recognized by HL-C5 was recovered in the upper phase of chloroform-methanol-water lipid extracts prepared from HL-60 cells. By competitive binding experiments, it was found that MAb HL-C5 recognizes the same antigenic determinant as MAb WGHS 29-1, which has been reported to react with glycolipids containing the sugar sequence lacto-N-fucopentaose 111. Autoradiographs of thin layer chromatograms of HL-60 glycolipid extracts which were revealed by incubation with MAb HL-C5 or WGHS 29-1 followed by the addition of 125I-labelled rabbit anti-mouse immunoglobulin antibody confirmed that the two MAbs reacted with the same or structurally very similar glycolipids.

Ten-year trends in overweight and obesity in the adult Portuguese population, 1995 to 2005.

ABSTRACT: BACKGROUND: There is little information regarding the trends in body mass index (BMI) and obesity in the overall Portuguese population, namely if these trends are similar according to educational level. In this study, we assessed the trends in the prevalence of overweight and obesity in the Portuguese population, overall and by educational level. METHODS: Cross-sectional national health interview surveys conducted in 1995-6 (n=38,504), 1998-9 (n=38,688) and 2005-6 (n=25,348). Data were derived from the population and housing census of 1991 and two geographically-based strata were defined. The sampling unit was the house, and all subjects living in the sampling unit were surveyed. Height and weight were self-reported; the effects of gender, age group and educational level were also assessed by self-reported structured questionnaires. Bivariate comparisons were performed using Chi-square or analysis of variance (ANOVA). Trends in BMI levels were assessed by linear regression analysis, while trends in the prevalence of obesity were assessed by logistic regression. RESULTS: Mean (+/-standard deviation) BMI increased from 25.2+/-4.0 in 1995-6 to 25.7+/-4.5 kg/m2 in 2005-6. Prevalence of overweight remained stable (36.1% in 1995-6 and 36.4% in 2005) while prevalence of obesity increased (11.5% in 1995-6 and 15.1% in 2005-6). Similar findings were observed according to age group. Mean age-adjusted BMI increase (expressed in kg/m2/year and 95% confidence interval) was 0.073 (0.062, 0.084), 0.016 (0.000, 0.031) and 0.073 (0.049, 0.098) in men with primary, secondary and university levels, respectively; the corresponding values in women were 0.085 (0.073, 0.097), 0.052 (0.035, 0.069) and 0.062 (0.038, 0.084). Relative to 1995-6, obesity rates increased by 48%, 41% and 59% in men and by 40%, 75% and 177% in women with primary, secondary and university levels, respectively. The corresponding values for overweight were 6%, 1% and 23% in men and 5%, 7% and 65% in women. CONCLUSION: Between 1995 and 2005, obesity increased while overweight remained stable in the adult Portuguese population. Although higher rates were found among lesser educated subjects, the strong increase in BMI and obesity levels in highly educated subjects is of concern.

Self-Development of Competences for Social Inclusion Using the TENCompetence Infrastructure

This paper describes a pilot study centred on the technology-enhanced self-development of competences in lifelong learning education carried out in the challenging context of the Association of Participants Àgora. The pilot study shows that the use of the TENCompetence infrastructure, i.e. in this case the Personal Development Planner tool, provides various kinds of benefits for adult participants with low educational profiles and who are traditionally excluded from the use of innovative learning technologies and the knowledge society. The selforganized training supported by the PDP tool aims at allowing the learners to create and control their own learning plans based on their interests and educational background including informal and non-formal experiences. In this sense, the pilot participants had the opportunity to develop and improve their competences in English language (basic and advanced levels) and ICT competence profiles which are mostly related to functional and communicative skills. Besides, the use of the PDP functionalities, such as the self-assessment, the planning and the self-regulating elements allowed the participants to develop reflective skills. Pilot results also provide indications for future developments in the field of technology support for self-organized learners. The paper introduces the context and the pilot scenario, indicates the evaluation methodology applied and discusses the most significant findings derived from the pilot study.

Free-and open source software for a course on network management: authoring and enactment of scripts based on collaborative learning strategies

This paper describes a Computer-Supported Collaborative Learning (CSCL) case study in engineering education carried out within the context of a network management course. The case study shows that the use of two computing tools developed by the authors and based on Free- and Open-Source Software (FOSS) provide significant educational benefits over traditional engineering pedagogical approaches in terms of both concepts and engineering competencies acquisition. First, the Collage authoring tool guides and supports the course teacher in the process of authoring computer-interpretable representations (using the IMS Learning Design standard notation) of effective collaborative pedagogical designs. Besides, the Gridcole system supports the enactment of that design by guiding the students throughout the prescribed sequence of learning activities. The paper introduces the goals and context of the case study, elaborates onhow Collage and Gridcole were employed, describes the applied evaluation methodology, anddiscusses the most significant findings derived from the case study.

New insight in aetiopathogenesis of aortic diseases.

BACKGROUND: Knowledge in the aetiopathogeny of aortic disease helps to characterise aortic lesions better and determine the risk of evolution and therapeutic strategies as well. This article focusses on aneurysms and dissections, and excludes causes related to infection, systemic inflammatory diseases and trauma. METHODS AND RESULTS: The biomedical literature of the past 10 years has been reviewed here. Aortic diseases are heterogeneous along the aorta as far as their genetic determinants, contribution of smooth muscle cells, inflammation and thrombus formation are concerned. Degradation of extracellular matrix by proteases causing aortic disease is a 'terminal' event, modulated by genetic background, haemodynamic strain, cellular events and thrombus formation. New genetic determinants of aortic disease have been identified. Proteases degrading the aortic wall are derived from a variety of cell types in addition to macrophages, including neutrophils on the luminal thrombus, mesenchymal and endothelial cells in the wall. Smooth muscle cells contribute to aortic wall homeostasis against inflammation and proteolysis. The degradation of the wall is followed by, or paralleled with, a failure of aortic reconstruction. CONCLUSIONS: Aortic diseases are diverse, and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Future research needs to unravel distinct cellular and molecular mechanisms causing the clinical events, in particular, dissection, expansion of already formed aneurysms and rupture.

Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

Spirometry reference equations for central European populations from school age to old age.

BACKGROUND: Spirometry reference values are important for the interpretation of spirometry results. Reference values should be updated regularly, derived from a population as similar to the population for which they are to be used and span across all ages. Such spirometry reference equations are currently lacking for central European populations. OBJECTIVE: To develop spirometry reference equations for central European populations between 8 and 90 years of age. MATERIALS: We used data collected between January 1993 and December 2010 from a central European population. The data was modelled using "Generalized Additive Models for Location, Scale and Shape" (GAMLSS). RESULTS: The spirometry reference equations were derived from 118'891 individuals consisting of 60'624 (51%) females and 58'267 (49%) males. Altogether, there were 18'211 (15.3%) children under the age of 18 years. CONCLUSION: We developed spirometry reference equations for a central European population between 8 and 90 years of age that can be implemented in a wide range of clinical settings.

Amoeba/amoebal symbiont genetic transfers: lessons from giant virus neighbours.

Free-living amoebae serve as hosts for a variety of amoebae-resisting microorganisms, including giant viruses and certain bacteria. The latter include symbiotic bacteria as well as bacteria exhibiting a pathogenic phenotype towards amoebae. Amoebae-resisting bacteria have been shown to be widespread in water and to use the amoebae as a reservoir, a replication niche, a protective armour as well as a training ground to select virulence traits allowing survival in the face of microbicidal effects of macrophages, the first line of defense against invading pathogens. More importantly, amoebae play a significant role as a melting pot for genetic exchanges. These ecological and evolutionary roles of amoebae might also be at play for giant viruses and knowledge derived from the study of amoebae-resisting bacteria is useful for the study and understanding of interactions between amoebae and giant viruses. This is especially important since some genes have spread in all domains of life and the exponential availability of eukaryotic genomes and metagenomic sequences will allow researchers to explore these genetic exchanges in a more comprehensive way, thus completely changing our perception of the evolutionary history of organisms. Thus, a large part of this review is dedicated to report current known gene exchanges between the different amoebae-resisting organisms and between amoebae and the internalized bacteria.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Bands width, credibility and exchange risk: Lessons from the EMS experience

This paper presents an analysis of the credibility of the EMScurrencies that covers the period before and after the increase in thebands of fluctuation. Our credibility indicator is based on the inferredprobabilities derived from the estimation of a Markov-switching model(Hamilton (1989)) applied to the expected rate of depreciation. Theresults show that, for most of the currencies, credibility has improved,at least transitorily, after the increase in the bands. However, for allcurrencies, the credibility measured by the indicator proposed in thispaper has been eroded recently even with the widened bands.

Improving the sensitivity of the sequence profile method.

The sequence profile method (Gribskov M, McLachlan AD, Eisenberg D, 1987, Proc Natl Acad Sci USA 84:4355-4358) is a powerful tool to detect distant relationships between amino acid sequences. A profile is a table of position-specific scores and gap penalties, providing a generalized description of a protein motif, which can be used for sequence alignments and database searches instead of an individual sequence. A sequence profile is derived from a multiple sequence alignment. We have found 2 ways to improve the sensitivity of sequence profiles: (1) Sequence weights: Usage of individual weights for each sequence avoids bias toward closely related sequences. These weights are automatically assigned based on the distance of the sequences using a published procedure (Sibbald PR, Argos P, 1990, J Mol Biol 216:813-818). (2) Amino acid substitution table: In addition to the alignment, the construction of a profile also needs an amino acid substitution table. We have found that in some cases a new table, the BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc Natl Acad Sci USA 89:10915-10919), is more sensitive than the original Dayhoff table or the modified Dayhoff table used in the current implementation. Profiles derived by the improved method are more sensitive and selective in a number of cases where previous methods have failed to completely separate true members from false positives.

The international development of the RGHQoL: a quality of life measure for recurrent genital herpes.

This paper describes the international development and psychometric testing of the Recurrent Genital Herpes Quality of Life Questionnaire (RGHQoL), a condition-specific quality of life (QoL) instrument. The theoretical foundation for the measure is the needs-based model of QoL and the content of the instrument was derived from in-depth qualitative interviews with relevant patients in the UK. Versions of the RGHQoL were required for the UK, USA, Italy, Germany, France and Denmark for use in international clinical trials. The results indicate that the final 20 item measure has good reliability, internal consistency and validity for all language versions. A small responsiveness study in Denmark suggested that the measure is sensitive to changes in QoL associated with the initiation of suppression treatment for recurrent genital herpes (RGH). It is concluded that the RGHQoL is a valuable instrument for inclusion in clinical trials. The psychometric properties of the instrument are such that it may also be used to monitor the progress of individual patients.

A integração das TIC no ensino secundário em Cabo Verde : um estudo de caso

Actualmente, um desafio permanente se coloca ao sistema educativo em todo o mundo devido aos impulsos decorrentes das incomensuráveis possibilidades que as TIC oferecem ao processo de ensino e aprendizagem e na mudança que elas acarretam nas atitudes dos agentes educativos e na própria estrutura escolar. Em Cabo Verde as TIC têm despertado interesse dos responsáveis da educação no concernente ao seu uso em contexto pedagógico e a existência de projectos voltados para sua utilização no ensino secundário é uma realidade. Com efeito, iniciou-se no ano lectivo 2009/2010 a experiência de integração das TIC em duas escolas secundárias do país. Porém, volvidos cinco anos, importa saber como estão sendo vivenciadas estas experiências, particularmente no que diz respeito à disponibilidade e acessibilidade das TIC por parte dos principais agentes educativos, a forma como se organiza a escola para colocá-las à disposição destes, o grau de satisfação dos professores com relação à sua formação em TIC, as atitudes dos professores e alunos face as TIC e o nível de sua utilização em contexto de sala de aula. De modo que, o problema em estudo centra-se em saber como está a decorrer o processo de integração das TIC no ensino secundário, no âmbito das atuais políticas de incentivo ao uso das tecnologias na educação. Do ponto de vista metodológico optamos por um estudo de caso de natureza exploratória (Yin, 1993; Gil, 1994; Morgado, 2012) e para recolher os dados aplicamos dois inquéritos por questionário, em formato papel, um dirigido a alunos, (uma turma por ano) e outro a todos os professores da escola em estudo. Foram também realizadas duas entrevistas a dois dirigentes da escola. A escolha de fontes quantitativas (inquéritos) e qualitativas (entrevistas) tem base de sustentação nos argumentos apresentados por Yin (1994) e Coutinho (2005). As conclusões do estudo apontam para o reconhecimento de que há ainda muito por fazer no tocante à integração pedagógica das TIC, principalmente no que tange à disponibilidade das TIC (a maioria dos alunos não tem acesso aos computadores e não existem softwares), na organização da escola (falta de técnicos qualificados, e inexistência de projecto pedagógico para uso das TIC), na formação dos professores (necessidade de formação continua) e na ausência das TIC nas práticas pedagógicas, conforme nos ensina a literatura especializada sobre a integração das TIC (Pelgrum, 2001; Paiva, 2002; Silva & Miranda, 2005; Amante, 2007; OIE, 2008, Sunkel, 2009; Barbosa & Loureiro, 2011).

Relationships of Scincid Lizards (Mabuya spp; Reptilia: Scincidae) from the Cape Verde Islands Based on Mitochondrial and Nuclear DNA Sequences

Partial DNA sequences from two mitochondrial (mt) and one nuclear gene (cytochrome b, 12S rRNA, and C-mos) were used to estimate the phylogenetic relationships among the six extant species of skinks endemic to the Cape Verde Archipelago. The species form a monophyletic unit, indicating a single colonization of the islands, probably from West Africa. Mabuya vaillanti and M. delalandii are sister taxa, as indicated by morphological characters. Mabuya fogoensis and M. stangeri are closely related, but the former is probably paraphyletic. Mabuya spinalis and M. salensis are also probably paraphyletic. Within species, samples from separate islands always form monophyletic groups. Some colonization events can be hypothesized, which are in line with the age of the islands. C-mos variation is concordant with the topology derived from mtDNA.