Terapia celular com células mesenquimais por via intracoronária: contributos do estudo invasivo da microcirculação coronária

RESUMO: Introdução - A utilização de células e das suas propriedades para o tratamento das doenças cardiovasculares, é uma promessa para o futuro e talvez a única forma de ultrapassar algumas das insuficiências das terapêuticas atuais. A via de entrega das células mais utilizada na investigação tem sido a intracoronária, ganhando a microcirculação especial relevância, por ser onde ocorre a primeira interação com o tecido nativo. As células estaminais mesenquimais (CEM) têm propriedades que as tornam particularmente aptas para a Terapia Celular, mas as suas dimensões, superiores ao diâmetro dos capilares, tem motivado controvérsia quanto à sua entrega intracoronária. A cardiologia de intervenção tem atualmente técnicas que permitem a avaliação em tempo real e in vivo do estado da microcirculação coronária. A determinação do índice da resistência da microcirculação (IRM) fornece informação sobre a circulação dos pequenos vasos, de forma independente da circulação coronária e do estado hemodinâmico, mas a aplicabilidade clínica deste conhecimento encontra-se ainda por definir. Objectivos Esclarecer o potencial do IRM no estudo dos efeitos do transplante de CEM por via intracoronária. População e Métodos . Estudo pré-clínico com modelo animal (suíno) desenvolvido em 3 fases. Na Primeira Fase foram utilizados 8 animais saudáveis para estudar e validar a técnica de determinação de estudo da microcirculação. Efetuou-se a determinação do IRM com duas doses diferentes de papaverina para a indução da resposta hiperémica máxima (5 e 10 mg) e após a disfunção da microcirculação com injeção intracoronária de microesferas de embozene com 40 μm de diâmetro. Na Segunda Fase foram utilizados 18 animais saudáveis, randomizados em grupo controlo e grupo recetor de 30 x 106 CEM por via intracoronária. Foram avaliados de forma cega o IRM, a pressão aórtica, o fluxo coronário epicárdico e a ocorrência de alterações electrocardiográficas. Na Terceira Fase foram utilizados 18 animais, com enfarte agudo do miocárdio provocado (EAM), randomizados em grupo controlo, grupo recetor de CEM expandidas de forma convencional e grupo recetor de CEM expandidas com metodologia inovadora e de menores dimensões. Foi realizada uma exploração da dose/efeito com infusão faseada de 10 x 106, 15 x 106 e 20 x 106 CEM, com determinação do IRM, da pressão aórtica, do fluxo coronário epicárdico e da ocorrência de alterações eletrocardiográficas. Quatro semanas após a entrega das células foi novamente avaliado o IRM e foi efetuado o estudo anatomopatológico dos animais na procura de evidência de neoangiogénese e de regeneração miocárdica, ou de um efeito positivo da resposta reparadora após o enfarte. Resultados Nas 3 fases todos os animais mantiveram estabilidade hemodinâmica e eletrocardiográfica, com exceção da elevação de ST de V1-V3 verificada após a injeção das microesferas. Na Primeira Fase as duas doses de papaverina induziram uma resposta hiperémica eficaz, sem tradução com significado na determinação do IRM (variação da pressão distal de - 11,4 ± 5 e de - 10,6± 5 mmHg com as doses de 5 e 10 mg respetivamente (p=0,5). Com a injeção das microesferas o IRM teve uma elevação média de 310 ± 190 %, para um valor médio de 41,3 ± 16 U (p = 0,001). Na Segunda Fase não houve diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os dois grupos. O IRM de base foi semelhante e após a infusão intracoronária observou-se uma elevação expressiva do IRM nos animais que receberam células em comparação com o grupo controlo (8,8 U ± 1 vs. 14,2 U ± 1,8, P=0,02) e quanto ao seu valor de base (aumento de 112%, p=0,008). Na terceira Fase não houve novamente diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os três grupos. Houve uma elevação do IRM nos animais que receberam células a partir da 2ª dose (72% nas células convencionai e 108% nas células inovadoras) e que se manteve com a 3ª dose (100% nas células convencionais e 88% nas inovadoras) com significado estatístico em comparação com o grupo controlo (p=0,034 com a 2ªdose e p=0,024 com a 3ª dose). Quatro semanas após a entrega das CEM observou-se a descida do IRM nos dois grupos que receberam células, para valores sobreponíveis aos do grupo controlo e aos valores pós-EAM. Na avaliação anatomopatológica e histológica dos corações explantados não houve diferenças entre os três grupos. Conclusões O IRM permite distinguir alterações da microcirculação coronária motivadas pela entrega intracoronária de CEM, na ausência de alterações de outros parâmetros clínicos da circulação coronária utilizados em tempo real. As alterações do IRM são progressivas e passíveis de avaliar o efeito/dose, embora não tenha sido possível determinar diferenças com os dois tipos de CEM. No nosso modelo a injeção intracoronária não se associou a evidência de efeito benéfico na reparação ou regeneração miocárdica após o EAM.---------------------------- ABSTRACT: ABSTRACT Introduction The use of cells for the treatment of cardiovascular disease is a promise for the future and perhaps the only option to overcome some of the shortcomings of current therapies. The strategy for the delivery of cells most often used in current research has been the intracoronary route and due to this microcirculation gains special relevance, mainly because it is the first interaction site of transplanted cells with the native tissue. Mesenchymal stem cells (MSC) have properties that make them suitable for Cell Therapy, but its dimensions, larger than the diameter of capillaries, have prompted controversy about the safety of intracoronary delivery. The interventional cardiology currently has techniques that allow for real-time and in vivo assessment of coronary microcirculation state. The determination of the index of microcirculatory resistance index (IMR) provides information about small vessels, independently of the coronary circulation and hemodynamic status, but the clinical applicability of this knowledge is yet to be defined. Objectives To clarify the potential use of IMR in the study of the effects of MSC through intracoronary transplantation. Population and Methods Preclinical study with swine model developed in three phases. In Phase One 8 healthy animals were used to study and validate the IMR assessment in our animal model. IMR was assessed with two different doses of papaverine for inducing the maximal hyperaemic response (5 and 10 mg) and microcirculation dysfunction was achieved after intracoronary injection with embozene microspheres with 40 μm in diameter. In Phase Two we randomized 18 healthy animals divided between the control group and the one receiving 30 x 106 MSC through an intracoronary infusion. There we blindly evaluated IMR, the aortic pressure, the epicardial coronary flow and the occurrence of ECG changes. In Phase Three we used 18 animals with a provoked acute myocardial infarction (AMI), randomized into a control group, a MSC expanded conventionally receiver group and a MSC expanded with an innovative methodology receiver group. There was a stepwise infusion with doses of 10 x 106, 15 x 106 and 20 x 106 MSC with determination of IMR, the aortic pressure, the epicardial coronary flow and occurrence of electrocardiographic abnormalities. Four weeks after cell delivery we again measured the IMR and proceeded with the pathological study of animals in the search for evidence of neoangiogenesis and myocardial regeneration, or a positive effect in the reparative response following the infarction. Results All animals remained hemodynamically stable and with no electrocardiographic abnormalities, except for the ST elevation in V1-V3 observed after injection of the microspheres. In Phase One the two doses of papaverine achieved an hyperemic and effective response without significant differences in IMR (variation of the distal pressure -11.4 ± 5 and -10.6 ± 5 mmHg with the doses of 5 and 10 mg respectively (p = 0.5). With the injection of the microspheres the IMR had an average increase of 310 ± 190% for an average value of 41.3 ± 16 U (p = 0.001). In the second phase there were no significant differences in hemodynamic parameters, epicardial flow and electrocardiographic assessment between the two groups. The baseline IMR was similar and after intracoronary infusion there was a significant increase in animals receiving cells compared with the control group (8.8 ± U 1 vs. 14.2 ± 1.8, p = 0.02) and with their baseline (112% increase, p = 0.008). In the third phase again there were no significant differences in hemodynamic parameters, the epicardial flow and electrocardiographic evaluation between the three groups. There was a significant increase in IMR in animals that received cells from the 2nd dose (72% in conventional cells and 108% in the innovative cells) that remained with the 3rd dose (100% in conventional cells and 88% in the innovative) with statistical significance compared with the control group (p = 0.034 with 2nd dose, p = 0.024 with 3rd dose). Four weeks after delivery of the MSC we observed the fall of the IMR in the two groups that received cells with values overlapping those of the control group. In pathological and histological evaluation of removed hearts there were no differences among the three groups. Conclusions The IMR allows for the differentiation of changes in coronary microcirculation motivated by intracoronary delivery of MSC in the absence of modification in other clinical parameters. IMR changes are progressive and enable the evaluation of the effect / dose, though it has not been possible to determine differences in the two types of MSC. In our model, intracoronary injection of MSC was not associated with evidence of repair or myocardial regeneration after AMI.

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischemia, inflammation, and infection costing $7.5 billion/year in the US alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization, and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA microparticles that provides a sustained release of bioactive insulin for >20days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring, healing. Using a heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04mg insulin/cm2, every three days for 9 days, have faster closure, faster rate of disintegration of dead tissue, and decreased oxidative stress.In addition, in insulin-treated wounds the pattern of neutrophil inflammatory response suggests faster clearing of the burn dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibers organized more like a basket weave (normal skin) than aligned and crosslinked (scar tissue). In summary , application of ASD-containing insulin-loaded PLGA particles on burns every three days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Search for resonant diboson production in the ℓℓqq¯ final state in pp collisions at s√=8 TeV with the ATLAS detector

This paper reports on a search for narrow resonances in diboson production in the ℓℓqq¯ final state using pp collision data corresponding to an integrated luminosity of 20fb−1 collected at s√=8 TeV with the ATLAS detector at the Large Hadron Collider. No significant excess of data events over the Standard Model expectation is observed. Upper limits at the 95% confidence level are set on the production cross section times branching ratio for Kaluza--Klein gravitons predicted by the Randall--Sundrum model and for Extended Gauge Model W' bosons. These results lead to the exclusion of mass values below 740 GeV and 1590 GeV for the graviton and W' boson respectively.

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Searches for Higgs boson pair production in the hh→bbττ,γγWW∗,γγbb,bbbb channels with the ATLAS detector

Searches for both resonant and non-resonant Higgs boson pair production are performed in the hh→bbττ,γγWW∗ final states using 20.3 fb−1 of pp collision data at a center-of-mass energy of 8 TeV recorded with the ATLAS detector at the Large Hadron Collider. No evidence of their production is observed and 95% confidence level upper limits on the production cross sections are set. These results are then combined with the published results of the hh→γγbb,bbbb analyses. An upper limit of 0.69 (0.47) pb on the non-resonant Standard Model like hh production is observed (expected), corresponding to 70 (48) times of the SM gg→hh cross section. For production via narrow resonances, cross section limits of hh production from a heavy Higgs boson decay are set as a function of the heavy Higgs boson mass. The observed (expected) limits range from 2.1 (1.1) pb at 260 GeV to 0.011 (0.018) pb at 1000 GeV. These results are interpreted in the context of two simplified scenarios of the Minimal Supersymmetric Standard Model.

Modelação, simulação e implementação de agitação acústica para aplicações em microfluídica

Tese de Doutoramento (Programa Doutoral em Engenharia Biomédica)

“Fantasia Lusitana”, de João Canijo: O Portugal ficcional vs. o país real. O Estado Novo e a ‘portugalidade’. A construção da identidade

ISBN: 978-989-96858-3-3

A imprensa desportiva do papel ao online: o exemplo do jornal Record

Dissertação de mestrado em Ciências da Comunicação (área de especialização em Informação e Jornalismo)

A critical evaluation of methods for the reconstruction of tissue-specific models

Under the framework of constraint based modeling, genome-scale metabolic models (GSMMs) have been used for several tasks, such as metabolic engineering and phenotype prediction. More recently, their application in health related research has spanned drug discovery, biomarker identification and host-pathogen interactions, targeting diseases such as cancer, Alzheimer, obesity or diabetes. In the last years, the development of novel techniques for genome sequencing and other high-throughput methods, together with advances in Bioinformatics, allowed the reconstruction of GSMMs for human cells. Considering the diversity of cell types and tissues present in the human body, it is imperative to develop tissue-specific metabolic models. Methods to automatically generate these models, based on generic human metabolic models and a plethora of omics data, have been proposed. However, their results have not yet been adequately and critically evaluated and compared. This work presents a survey of the most important tissue or cell type specific metabolic model reconstruction methods, which use literature, transcriptomics, proteomics and metabolomics data, together with a global template model. As a case study, we analyzed the consistency between several omics data sources and reconstructed distinct metabolic models of hepatocytes using different methods and data sources as inputs. The results show that omics data sources have a poor overlapping and, in some cases, are even contradictory. Additionally, the hepatocyte metabolic models generated are in many cases not able to perform metabolic functions known to be present in the liver tissue. We conclude that reliable methods for a priori omics data integration are required to support the reconstruction of complex models of human cells.

Synchronous reproduction may facilitate introgression in a hybrid mussel (Mytilus) population

Mussel populations on the Irish Atlantic coast comprise an interbreeding mixture of the blue mussel, Mytilus edulis (L.) and the Mediterranean mussel, Mytilus galloprovincialis (Lmk.). The occurrence of hybrid genotypes varies between sites but can be as high 80%. This study compares the reproductive cycle of M. edulis, M. galloprovincialis and their hybrids to determine if the extensive hybridisation observed at Irish Atlantic coast sites is linked to spawning synchrony between the two taxa. Mussels (40-45 mm size class) were collected monthly from a sheltered shore in Galway Bay from January to December 2005. Two major spawning events (March- June and September-October) were observed and gametogenesis took place throughout the year. The spawning cycles of the three taxa were largely overlapping. Small differences were observed in the timing of peak spawning which occurred in March and October in M. galloprovincialis and in May-June and September in M.edulis. Spawning of hybrid individuals was intermediate between the parental genotypes. Fecundity was slightly higher in M. galloprovincialis females compared to the other taxa (up to 30% difference, p<0.05). This apparent advantage is not shared by the sexes and is likely being offset by high numbers of hybrid genotypes releasing gametes during peak spawning of M. galloprovincialis. There was no evidence for increased mortality in hybrid males; sex ratios did not deviate from the 1:1 ratio. The results show that in this region of the hybrid zone the timing of reproduction does not present a barrier to gene flow between M. edulis and M. galloprovincialis. Nonetheless, small differences in the timing of peak spawning may increase the likelihood of conspecific fertilisation at certain times of the year. Hybrids outnumber the parental genotypes, undergo complete gametogenesis and show no evidence of depressed fitness (i.e. hybrids are reproductively competent suggesting a high degree of introgression.

Morphometric variability in populations of Palaemonetes spp. (Crustacea, Decapoda, Palaemonidae) from the Peruvian and Brazilian Amazon Basin

Morphometric variability among shrimp populations of the genus Palaemonetes Heller, 1869 from seven lakes (Huanayo and Urcococha, in Peru; Amanã, Mamirauá, Camaleão, Cristalino e Iruçanga, in Brasil) in the Amazon Basin, presumably belonging to Palaemonetes carteri Gordon, 1935 and Palaemonetes ivonicus Holthuis, 1950, were studied. The morphometric studies were carried out from the ratios obtained from the morphometric characters. Multivariated analysis (Principal Components Analysis-PCA, Discriminant Function Analysis and Cluster Analysis) were applied over the ratios. Intra- and interpopulation variations of the rostrum teeth, and the number of spines in the male appendix, were analyzed through descriptive statistics and bivariate analysis (Spearman Rank Correlation test). Results indicated a wide plasticity and overlapping in the studied ratios between populations. The Principal Components Analysis was not able to separate different populations, revealing a large intrapopulation plasticity and strong interpopulation similarity in the studied ratios. Although the Discriminant Functions Analysis was not able to fully discriminate populations, they could be allocated in three subgroups: 1) Cristalino and Iruçanga; 2) Huanayo, Urcococha and Camaleão and 3) Mamirauá and Amanã. The first two groups were morphometrically separated from each other, whereas the third one presented a strong overlap with the former two. The Cluster Analysis confirmed the first two subgroups separation, and indicated that the first and third groups were closely related. Rostrum teeth and number of spines in the appendix masculina showed a large intrapopulation variation and a strong overlapping among the studied populations, regardless of the species.

Population ecology of the multivoltine Neotropical gall midge Eugeniamyia dispar (Diptera, Cecidomyiidae)

Our understanding of the population ecology of insect gallers is largely built on examples from temperate zones, but tropical and subtropical gallers may present distinct patterns of abundance and distribution across time. Eugeniamyia dispar Maia, Mendonça & Romanowski, 1996 is a multivoltine Neotropical cecidomyiid that induces spongy leaf galls on Eugenia uniflora (Myrtaceae). Galls were censused in the urban area of Porto Alegre, southern Brazil on six plants at two sites, for two years, at roughly weekly intervals. Overall 9,694 eggs, galling attempts and galls were counted. New galls continuously appear on developing leaves, but galls with live inducers are absent from June to at least early August. Galls on a same shoot develop synchronically, thus the shoot is probably the unit for oviposition. Given the also synchronic appearance of galls on different plants on a site, it seems midges can disperse and attack close-by plants. Gall cohorts varied in abundance by two orders of magnitude; there were more galls during summer than for spring and autumn, in a wave-like pattern. Host plant leaf production was seasonal, with low production during winter and cyclic production during the rest of the year. Perhaps because of this very variable pattern, gall abundance did not follow leaf production: this galler is not synchronised with its host at least during most of the year. This multivoltine gall inducer with "labile" galls, short development time, partially overlapping generations and low host synchronisation differs from the commonly studied species of the temperate regions providing a subject for ecological comparisons.

Social capital and government in the production of public goods

As a response to the rapidly growing empirical literature on social capital and the evidence of its correlation with government performance, we build a theoretical framework to study the interactions between social capital and government's action. This paper presents a model of homogeneous agents in an overlapping generations framework incorporating social capital as the values transmitted from parent to child. The government's role is to provide public goods. First, government expenditure is exogenously given. Then, it will be chosen at the preferred level of the representative agent. For both setups the equilibrium outcomes are characterized and the resulting dynamics studied. Briefly we include an analysis of the effect of productivity growth on the evolution of social capital. The results obtained caution caution against both the crowding out effect of the welfare state and the impact of sustained economic growth on social capital.

Un diàleg de sords: intentant donar sentit al debat sobre la publicació electrònica

Aquest article se centra en les implicacions de la difusió electrònica per al sistema de publicació de revistes basat en la revisió per parells [peer-reviewed]. Per donar sentit a un assumpte tan complex, és de molt ajut mirar-s'ho des de la perspectiva dels orígens del sistema i de les seves tres funcions nuclears: el rànquing en la recerca, facilitar la comunicació interactiva entre els estudiosos i crear un arxiu global del coneixement científic. Cadascuna d’aquestes funcions principals té requeriments diferents que, en certa mesura, se sobreposen però que també entren, d'alguna manera, en conflicte. Internet obre la possibilitat de desenvolupar una varietat de models distints de comunicació científica modulant la intensitat de cadascun d'aquests tres rols que les revistes en paper han desenvolupat i, possiblement, d'altres funcions que no eren ni tan sols imaginables abans del desenvolupament de les xarxes electròniques d'abast planetari. Les implicacions de la distribució electrònica per a la propietat i accés a la literatura científica són profundes i tendeixen a agreujar la ja seriosa crisi dels preus de les revistes que està frenant l'accés a la informació científica. La comunitat d'estudiosos, que és autora del material que aquestes publicacions contenen i, al mateix temps, n'és el principal consumidor, està en possessió de la clau per a solucionar aquesta crisi tot permetent a Internet ser un vehicle que faciliti la difusió d’una recerca finançada des del sector públic en comptes de crear una situació de propietat privada d'aquesta recerca.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.