Establishment of models to study mouse and human retinogenesis "in vitro" : isolation and characterization of retinal stem cells

SUMMARY IN FRENCH Les cellules souches sont des cellules indifférenciées capables a) de proliférer, b) de s'auto¬renouveller, c) de produire des cellules différenciées, postmitotiques et fonctionnelles (multipotencialité), et d) de régénérer le tissu après des lésions. Par exemple, les cellules de souches hematopoiétiques, situées dans la moelle osseuse, peuvent s'amplifier, se diviser et produire diverses cellules différenciées au cours de la vie, les cellules souches restant dans la moelle osseuse et consentant leur propriété. Les cellules souches intestinales, situées dans la crypte des microvillosités peuvent également régénérer tout l'intestin au cours de la vie. La rétine se compose de six classes de neurones et d'un type de cellule gliale. Tous ces types de cellules sont produits par un progéniteur rétinien. Le pic de production des photorécepteurs se situe autour des premiers jours postnatals chez la souris. A cette période la rétine contient les cellules hautement prolifératives. Dans cette étude, nous avons voulu analyser le phénotype de ces cellules et leur potentiel en tant que cellules souches ou progénitrices. Nous nous sommes également concentrés sur l'effet de certains facteurs épigéniques sur leur destin cellulaire. Nous avons observé que toutes les cellules prolifératives isolées à partir de neurorétines postnatales de souris expriment le marqueur de glie radiaire RC2, ainsi que des facteurs de transcription habituellement trouvés dans la glie radiaire (Mash1, Pax6), et répondent aux critères des cellules souches : une capacité élevée d'expansion, un état indifférencié, la multipotencialité (démontrée par analyse clonale). Nous avons étudié la différentiation des cellules dans différents milieux de culture. En l'absence de sérum, l'EGF induit l'expression de la β-tubulin-III, un marqueur neuronal, et l'acquisition d'une morphologie neuronale, ceci dans 15% des cellules présentes. Nous avons également analysé la prolifération de cellules. Seulement 20% des cellules incorporent le bromodéoxyuridine (BrdU) qui est un marqueur de division cellulaire. Ceci démontre que l'EGF induit la formation des neurones sans une progression massive du cycle cellulaire. Par ailleurs, une stimulation de 2h d'EGF est suffisante pour induire la différentiation neuronale. Certains des neurones formés sont des cellules ganglionnaires rétiniennes (GR), comme l'indique l'expression de marqueurs de cellules ganglionnaires (Ath5, Brn3b et mélanopsine), et dans de rare cas d'autres neurones rétiniens ont été observés (photorécepteurs (PR) et cellules bipolaires). Nous avons confirmé que les cellules souches rétiniennes tardives n'étaient pas restreintes au cours du temps et qu'elles conservent leur multipotencialité en étant capables de générer des neurones dits précoces (GR) ou tardifs (PR). Nos résultats prouvent que l'EGF est non seulement un facteur contrôlant le développement glial, comme précédemment démontré, mais également un facteur efficace de différentiation pour les neurones rétiniens, du moins in vitro. D'autre part, nous avons voulu établir si l'oeil adulte humain contient des cellules souches rétiniennes (CSRs). L'oeil de certains poissons ou amphibiens continue de croître pendant l'âge adulte du fait de l'activité persistante des cellules souches rétiniennes. Chez les poissons, le CSRs se situe dans la marge ciliaire (CM) à la périphérie de la rétine. Bien que l'oeil des mammifères ne se développe plus pendant la vie d'adulte, plusieurs groupes ont prouvé que l'oeil de mammifères adultes contient des cellules souches rétiniennes également dans la marge ciliaire plus précisément dans l'épithélium pigmenté et non dans la neurorétine. Ces CSRs répondent à certains critères des cellules souches. Nous avons identifié et caractérisé les cellules souches rétiniennes résidant dans l'oeil adulte humain. Nous avons prouvé qu'elles partagent les mêmes propriétés que leurs homologues chez les rongeurs c.-à-d. auto-renouvellement, amplification, et différenciation en neurones rétiniens in vitro et in vivo (démontré par immunocoloration et microarray). D'autre part, ces cellules peuvent être considérablement amplifiées, tout en conservant leur potentiel de cellules souches, comme indiqué par l'analyse de leur profil d'expression génique (microarray). Elles expriment également des gènes communs à diverses cellules souches: nucleostemin, nestin, Brni1, Notch2, ABCG2, c-kit et son ligand, aussi bien que cyclin D3 qui agit en aval de c-kit. Nous avons pu montré que Bmi1et Oct4 sont nécessaires pour la prolifération des CSRs confortant leur propriété de cellules souches. Nos données indiquent que la neurorétine postnatale chez la souris et l'épithélium pigmenté de la marge ciliaire chez l'humain adulte contiennent les cellules souches rétiniennes. En outre, nous avons développé un système qui permet d'amplifier et de cultiver facilement les CSRs. Ce modèle permet de disséquer les mécanismes impliqués lors de la retinogenèse. Par exemple, ce système peut être employé pour l'étude des substances ou des facteurs impliqués, par exemple, dans la survie ou dans la génération des cellules rétiniennes. Il peut également aider à disséquer la fonction de gènes ou les facteurs impliqués dans la restriction ou la spécification du destin cellulaire. En outre, dans les pays occidentaux, la rétinite pigmentaire (RP) touche 1 individu sur 3500 et la dégénérescence maculaire liée à l'âge (DMLA) affecte 1 % à 3% de la population âgée de plus de 60 ans. La génération in vitro de cellules rétiniennes est aussi un outil prometteur pour fournir une source illimitée de cellules pour l'étude de transplantation cellulaire pour la rétine. SUMMARY IN ENGLISH Stem cells are defined as undifferentiated cells capable of a) proliferation, b) self maintenance (self-renewability), c) production of many differentiated functional postmitotic cells (multipotency), and d) regenerating tissue after injury. For instance, hematopoietic stem cells, located in bone marrow, can expand, divide and generate differentiated cells into the diverse lineages throughout life, the stem cells conserving their status. In the villi crypt, the intestinal stem cells are also able to regenerate the intestine during their life time. The retina is composed of six classes of neurons and one glial cell. All these cell types are produced by the retinal progenitor cell. The peak of photoreceptor production is reached around the first postnatal days in rodents. Thus, at this stage the retina contains highly proliferative cells. In our research, we analyzed the phenotype of these cells and their potential as possible progenitor or stem cells. We also focused on the effect of epigenic factor(s) and cell fate determination. All the proliferating cells isolated from mice postnatal neuroretina harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash 1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after the transfer of the cells in different culture media. In the absence of serum, EGF led to the expression of the neuronal marker β-tubulin-III, and the acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating massively cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro. On the other hand, we wanted to find out if the adult human eye contains retina stem cells. The eye of some fishes and amphibians continues to grow during adulthood due to the persistent activity of retinal stem cells (RSCs). In fish, the RSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina. Although, the adult mammalian eye does not grow during adult life, several groups have shown that the adult mouse eye contains retinal stem cells in the homologous zone (i.e. the ciliary margin), in the pigmented epithelium and not in the neuroretina. These RSCs meet some criteria of stem cells. We identified and characterized the human retinal stem cells. We showed that they posses the same features as their rodent counterpart i.e. they self-renew, expand and differentiate into retinal neurons in vitro and in vivo (indicated by immunostaining and microarray analysis). Moreover, they can be greatly expanded while conserving their sternness potential as revealed by the gene expression profile analysis (microarray approach). They also expressed genes common to various stem cells: nucleostemin, nestin, Bmil , Notch2, ABCG2, c-kit and its ligand, as well as cyclin D3 which acts downstream of c-kit. Furthermore, Bmil and Oct-4 were required for RSC proliferation reinforcing their stem cell identity. Our data indicate that the mice postnatal neuroretina and the adult pigmented epithelium of adult human ciliary margin contain retinal stem cells. We developed a system to easily expand and culture RSCs that can be used to investigate the retinogenesis. For example, it can help to screen drugs or factors involved, for instance, in the survival or generation of retinal cells. This could help to dissect genes or factors involved in the restriction or specification of retinal cell fate. In Western countries, retinitis pigmentosa (RP) affects 1 out of 3'500 individuals and age-related macula degeneration (AMD) strikes 1 % to 3% of the population over 60. In vitro generation of retinal cells is thus a promising tool to provide an unlimited cell source for cellular transplantation studies in the retina.

How can we teach EBM in clinical practice? : an analysis of barriers to implementation of on-the-job EBM teaching and learning.

Introduction: Evidence-based medicine (EBM) improves the quality of health care. Courses on how to teach EBM in practice are available, but knowledge does not automatically imply its application in teaching. We aimed to identify and compare barriers and facilitators for teaching EBM in clinical practice in various European countries. Methods: A questionnaire was constructed listing potential barriers and facilitators for EBM teaching in clinical practice. Answers were reported on a 7-point Likert scale ranging from not at all being a barrier to being an insurmountable barrier. Results: The questionnaire was completed by 120 clinical EBM teachers from 11 countries. Lack of time was the strongest barrier for teaching EBM in practice (median 5). Moderate barriers were the lack of requirements for EBM skills and a pyramid hierarchy in health care management structure (median 4). In Germany, Hungary and Poland, reading and understanding articles in English was a higher barrier than in the other countries. Conclusion: Incorporation of teaching EBM in practice faces several barriers to implementation. Teaching EBM in clinical settings is most successful where EBM principles are culturally embedded and form part and parcel of everyday clinical decisions and medical practice.

A systematic review of longitudinal population-based studies on the predictors of smoking cessation in adolescent and young adult smokers.

OBJECTIVE: To describe the determinants of self-initiated smoking cessation of duration of at least 6 months as identified in longitudinal population-based studies of adolescent and young adult smokers. METHODS: A systematic search of the PubMed and EMBASE databases using smoking, tobacco, cessation, quit and stop as keywords was performed. Limits included articles related to humans, in English, published between January 1984 and August 2010, and study population aged 10-29 years. A total of 4502 titles and 871 abstracts were reviewed independently by 2 and 3 reviewers, respectively. Nine articles were retained for data abstraction. Data on study location, timeframe, duration of follow-up, number of data collection points, sample size, age/grade of participants, number of quitters, smoking status at baseline, definition of cessation, covariates and analytic method were abstracted from each article. The number of studies that reported a statistically significant association between each determinant investigated and cessation were tabulated, from among all studies that assessed the determinant. RESULTS: Despite heterogeneity in methods across studies, five factors robustly predicted quitting across studies in which the factor was investigated: not having friends who smoke, not having intentions to smoke in the future, resisting peer pressure to smoke, being older at first use of cigarette and having negative beliefs about smoking. CONCLUSIONS: The literature on longitudinal predictors of cessation in adolescent and young adult smokers is not well developed. Cessation interventions for this population will remain less than optimally effective until there is a solid evidence base on which to develop interventions.

2013 Update in addiction medicine for the generalist.

Increasingly, patients with unhealthy alcohol and other drug use are being seen in primary care and other non-specialty addiction settings. Primary care providers are well positioned to screen, assess, and treat patients with alcohol and other drug use because this use, and substance use disorders, may contribute to a host of medical and mental health harms. We sought to identify and examine important recent advances in addiction medicine in the medical literature that have implications for the care of patients in primary care or other generalist settings. To accomplish this aim, we selected articles in the field of addiction medicine, critically appraised and summarized the manuscripts, and highlighted their implications for generalist practice. During an initial review, we identified articles through an electronic Medline search (limited to human studies and in English) using search terms for alcohol and other drugs of abuse published from January 2010 to January 2012. After this initial review, we searched for other literature in web-based or journal resources for potential articles of interest. From the list of articles identified in these initial reviews, each of the six authors independently selected articles for more intensive review and identified the ones they found to have a potential impact on generalist practice. The identified articles were then ranked by the number of authors who selected each article. Through a consensus process over 4 meetings, the authors reached agreement on the articles with implications for practice for generalist clinicians that warranted inclusion for discussion. The authors then grouped the articles into five categories: 1) screening and brief interventions in outpatient settings, 2) identification and management of substance use among inpatients, 3) medical complications of substance use, 4) use of pharmacotherapy for addiction treatment in primary care and its complications, and 5) integration of addiction treatment and medical care. The authors discuss each selected articles' merits, limitations, conclusions, and implication to advancing addiction screening, assessment, and treatment of addiction in generalist physician practice environments.

Team Effectiveness in Patient Health Management: An Overview of Reviews

Background: The desire to improve the quality of health care for an aging population with multiple chronic diseases is fostering a rapid growth in inter-professional team care, supported by health professionals, governments, businesses and public institutions. However, the weight of evidence measuring the impact of team care on patient and health system outcomes has not, heretofore, been clear. To address this deficiency, we evaluated published evidence for the clinical effectiveness of team care within a chronic disease management context in a systematic overview. Methods: A search strategy was built for Medline using medical subject headings and other relevant keywords. After testing for perform- ance, the search strategy was adapted to other databases (Cinhal, Cochrane, Embase, PsychInfo) using their specific descriptors. The searches were limited to reviews published between 1996 and 2011, in English and French languages. The results were analyzed by the number of studies favouring team intervention, based on the direction of effect and statistical significance for all reported outcomes. Results: Sixteen systematic and 7 narrative reviews were included. Diseases most frequently targeted were depression, followed by heart failure, diabetes and mental disorders. Effective- ness outcome measures most commonly used were clinical endpoints, resource utilization (e.g., emergency room visits, hospital admissions), costs, quality of life and medication adherence. Briefly, while improved clinical and resource utilization endpoints were commonly reported as positive outcomes, mixed directional results were often found among costs, medication adherence, mortality and patient satisfaction outcomes. Conclusions: We conclude that, although suggestive of some specific benefits, the overall weight of evidence for team care efficacy remains equivocal. Further studies that examine the causal interactions between multidisciplinary team care and clinical and economic outcomes of disease management are needed to more accurately assess its net program efficacy and population effectiveness.

Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology.

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

Contrast : a concept in language and its development in Medieval Spanish

The purpose of this PhD thesis is to investigate a semantic relation present in the connection of sentences (more specifically: propositional units). This relation, which we refer to as contrast, includes the traditional categories of adversatives - predominantly represented by the connector but in English and pero in Modern Spanish - and concessives, prototypically verbalised through although / aunque. The aim is to describe, analyse and - as far as possible - to explain the emergence and evolution of different syntactic schemes marking contrast during the first three centuries of Spanish (also referred to as Castilian) as a literary language, i.e., from the 13th to the 15th century. The starting point of this question is a commonplace in syntax, whereby the semantic and syntactic complexity of clause linkage correlates with the degree of textual elaboration. In historical linguistics, i.e., applied to the phylogeny of a language, it is commonly referred to as the parataxis hypothesis A crucial part of the thesis is dedicated by the definition of contrast as a semantic relation. Although the label contrast has been used in this sense, mainly in functional grammar and text linguistics, mainstream grammaticography and linguistics remain attached to the traditional categories adversatives and concessives. In opposition to this traditional view, we present our own model of contrast, based on a pragma-semantic description proposed for the analysis of adversatives by Oswald Ducrot and subsequently adopted by Ekkehard König for the analysis of concessives. We refine and further develop this model in order for it to accommodate all, not just the prototypical instances of contrast in Spanish, arguing that the relationship between adversatives and concessives is a marked opposition, i.e., that the higher degree of semantic and syntactic integration of concessives restricts some possible readings that the adversatives may have, but that this difference is almost systematically neutralised by contextual factors, thus justifying the assumption of contrast as a comprehensive onomasiological category. This theoretical focus is completed by a state-of-the-question overview attempting to account for all relevant forms in which contrast is expressed in Medieval Spanish, with the aid of lexicographic and grammaticographical sources, and an empirical study investigating the expression of corpus in a corpus study on the textual functions of contrast in nine Medieval Spanish texts: Cantar de Mio Cid, Libro de Alexandre, Milagros de Nuestra Sehora, Estoria de Espana, Primera Partida, Lapidario, Libro de buen amor, Conde Lucanor, and Corbacho. This corpus is analysed using quantitative and qualitative tools, and the study is accompanied by a series of methodological remarks on how to investigate a pragma-semantic category in historical linguistics. The corpus study shows that the parataxis hypothesis fails to prove from a statistical viewpoint, although a qualitative analysis shows that the use of subordination does increase over time in some particular contexts.

Validity of silhouette showcards as a measure of body size and obesity in a population in the African region : a practical research tool for general-purpose surveys.

BACKGROUND: The purpose of this study is to validate the Pulvers silhouette showcard as a measure of weight status in a population in the African region. This tool is particularly beneficial when scarce resources do not allow for direct anthropometric measurements due to limited survey time or lack of measurement technology in face-to-face general-purpose surveys or in mailed, online, or mobile device-based surveys. METHODS: A cross-sectional study was conducted in the Republic of Seychelles with a sample of 1240 adults. We compared self-reported body sizes measured by Pulvers' silhouette showcards to four measurements of body size and adiposity: body mass index (BMI), body fat percent measured, waist circumference, and waist to height ratio. The accuracy of silhouettes as an obesity indicator was examined using sex-specific receiver operator curve (ROC) analysis and the reliability of this tool to detect socioeconomic gradients in obesity was compared to BMI-based measurements. RESULTS: Our study supports silhouette body size showcards as a valid and reliable survey tool to measure self-reported body size and adiposity in an African population. The mean correlation coefficients of self-reported silhouettes with measured BMI were 0.80 in men and 0.81 in women (P < 0.001). The silhouette showcards also showed high accuracy for detecting obesity as per a BMI ≥ 30 (Area under curve, AUC: 0.91/0.89, SE: 0.01), which was comparable to other measured adiposity indicators: fat percent (AUC: 0.94/0.94, SE: 0.01), waist circumference (AUC: 0.95/0.94, SE: 0.01), and waist to height ratio (AUC: 0.95/0.94, SE: 0.01) amongst men and women, respectively. The use of silhouettes in detecting obesity differences among different socioeconomic groups resulted in similar magnitude, direction, and significance of association between obesity and socioeconomic status as when using measured BMI. CONCLUSIONS: This study highlights the validity and reliability of silhouettes as a survey tool for measuring obesity in a population in the African region. The ease of use and cost-effectiveness of this tool makes it an attractive alternative to measured BMI in the design of non-face-to-face online- or mobile device-based surveys as well as in-person general-purpose surveys of obesity in social sciences, where limited resources do not allow for direct anthropometric measurements.

Identification of the regions involved in phonological assembly using a novel paradigm.

Here we adopt a novel strategy to investigate phonological assembly. Participants performed a visual lexical decision task in English in which the letters in words and letterstrings were delivered either sequentially (promoting phonological assembly) or simultaneously (not promoting phonological assembly). A region of interest analysis confirmed that regions previously associated with phonological assembly, in studies contrasting different word types (e.g. words versus pseudowords), were also identified using our novel task that controls for a number of confounding variables. Specifically, the left pars opercularis, the superior part of the ventral precentral gyrus and the supramarginal gyrus were all recruited more during sequential delivery than simultaneous delivery, even when various psycholinguistic characteristics of the stimuli were controlled. This suggests that sequential delivery of orthographic stimuli is a useful tool to explore how readers, with various levels of proficiency, use sublexical phonological processing during visual word recognition.

Early Modern Persian, Urdu, and English Historiography and the Imagination of Islamic India under British Rule

This paper analyses the early modern transformations of South Asian literary cultures through the production of historiography in Persian, English, and Urdu. In the 18th-19th centuries, South Asian communities experienced and participated in a major restructuring of the languages of the subcontinent. Urdu and English were institutionalized as governmental languages and utilized in new literary productions as Persian was gradually marginalized from the centre of literary and governmental polities. Three interrelated colonial policies reshaped the historical consciousness of South Asia and Britain: the production of new Persian histories commissioned under British patronage, the initiation of Urdu historiography through the translation of Persian and English histories, and the construction of the British history of India written in English. This article explores the historical and social dynamics of these events and situates the origins and evolution of the colonial historiographical project. Major works discussed are the Tārīkh-i Bangālah of Salīm Allāh Munshī (fl. 1763), James Mill's (1773-1836) The History of British India first published in 1817, Mīr Sher ʿAlī Afsos' the Ārāʾish-i mahfil, as well as the production of original Urdu histories such as Muḥammad Zakāʾ-Allāh's (1832-1910) the Tārīkh-i Hindustān.