Molecular mechanisms of follicular associated epithelium differentiation

Les muqueuses respiratoires, genitales et digestives sont continuellement exposées aux antigènes de l?alimentation, à la flore intestinale et aux pathogènes. Cela implique une activité immunologique intense et finement régulée dans ces tissus. On admet que la modulation de ces réponses immunitaires muqueuses s?effectue dans des organes sentinels spécifiques appelés o-MALT (organized mucosal associated lymphoid tissues). Ces processus de modulation et la biologie de ces sites immuno-inducteurs sont peu connus. Ceci est pourtant d?une grande relevance si l?on veut faire un design rationnel de drogues et de vaccins muqueux. Dans l?intestin grèle, ces organes sont composés de follicules multiples et sont appelés plaques de Peyer. Ils sont constitués de follicules enrichis en cellules B comprenant ou non un centre germinatif, de regions interfolliculaires comprenant des cellules T, et d?une région en d ome riche en cellules dendritiques, cellules B naives et cellules T CD4+, surmontée par un epithelium specialisé, le FAE (epithelium associé aux follicules). Le FAE contient des cellules M spécialisées dans le transport de macromolécules et micro-organismes de la lumière intestinale au tissu lymphoide sous-jacent. Ce transport des antigènes est une condition obligatoire pour induire une réponse immunitaire. Les cellules du FAE, outre les cellules M, expriment un programme de différenciation distinct de celui des cellules associées aux villosités. Ceci est characterisé par une baisse des fonctions digestives et de défenses, et l?expression constitutive des chimiokines: CCL20 et CCL25. Le but de l?étude présentée ici est de rechercher les facteurs cellulaires et/ou moléculaire responsables de cette différenciation. Certaines études ont démontré l?importance du contact entre le compartiment mésenchymateux et l?épithelium pour la morphogenèse de ce dernier. En particulier, les molécules de la matrice extracellulaire peuvent activer des gènes clefs qui, à leur tour, vont controler l?adhésion et la differenciation cellulaire. Dans l?intestin, les cellules mésenchymateuses différencient en myofibroblastes qui participent à l?élaboration de la matrice extracellulaire. Dans cette étude, nous avons décrit les différences d?expression de molécules de la matrices sous le FAE et les villosités. Nous avons également montré une absence de myofibroblastes sous le FAE. Suite à plusieurs évidences expérimentales, certains ont proposé une influence des composés présents dans la lumière sur la différenciation et/ou la maturation des plaques de Peyer. La chimiokine CCL20, capable de recruter des cellules initiatrices de la réponse immunitaire, constitue notre seul marqueur positif de FAE. Nous avons pu montrer que la flagelline, un composé du flagelle bactérien, était capable d?induire l?expression de CCL20 in vitro et in vivo. Cet effet n?est pas limité aux cellules du FAE mais est observé sur l?ensemble de l?épithelium intestinal. Molecular mechanisms of FAE differenciation. La signalement induit par la lymphotoxine ß est critique pour l?organogenèse des plaques de Peyer, car des souris déficientes pour cette molécules ou son récepteur n?ont ni plaque de Peyer, ni la plupart des ganglions lymphatiques. Nous avons obtenus plusieurs évidences que la lymphotoxine ß était impliquée dans la régulation du gène CCL20 in vitro et in vivo.<br/><br/>Mucosal surfaces of the respiratory, genital and digestive systems are exposed to food antigens, normal bacterial flora and oral pathogens. This justifies an intense and tuned immunological activity in mucosal tissues. The modulation of immune responses in the mucosa is thought to occur in specific sentinel sites, the organized mucosa associated lymphoid tissues (o-MALT). This immune modulation and the biology of these immune-inductive sites are poorly understood but highly important and relevant in the case of drugs and vaccines design. In the small intestine, these organs (gut associated lymphoid tissue : GALT) consists of single or multiple lymphoid follicles, the so-called Peyer?s patches (PP), with typical B cell-enriched follicles and germinal centers, inter-follicular T cell areas, and a dome region enriched in dendritic cells, naive B cells, and CD4+ T cells under a specialized follicle associated epithelium (FAE). To trigger protective immunity, antigens have to cross the mucosal epithelial barrier. This is achieved by the specialized epithelial M cells of the FAE that are able to take up and transport macromolecules and microorganisms from the environment into the underlying organized lymphoid tissue. The ontogeny of M cells remains controversial: some data are in favor of a distinct cell lineage, while others provide evidence for the conversion of differentiated enterocytes into M cells. In this study we mapped the proliferative, M cells and apoptotic compartments along the FAE. Enterocytes acquire transient M cell features as they leave the crypt and regain enterocyte properties as they move towards the apoptotic compartment at the apex of the FAE, favouring the hypothesis of a plastic phenotype. The follicle-associated epithelium (FAE) is found exclusively over lymphoid follicles in mucosal tissues, including Peyer?s patches. The enterocytes over Peyer?s patches express a distinct phenotype when compared to the villi enterocytes, characterized by the down regulation of digestive and defense functions and the constitutive expression of chemokines, i.e. CCL20 and CCL25. The purpose of this study was to investigate and identify the potential cells and/or molecules instructing FAE differentiation. Contact between the epithelial and the mesenchymal cell compartment is required for gut morphogenesis. Extracellular matrix molecules (ECM) can activate key regulatory genes which in turn control cell adhesion and differentiation. In the gut, mesenchymal cells differentiate into myofibroblats that participate to the elaboration of ECM. We have described a differential expression of extracellular matrix components under the FAE, correlating with the absence of subepithelial myofibroblats. Molecular mechanisms of FAE differenciation. Different studies proposed an influence of the luminal compartment in the differentiation and/or the maturation of PP. CCL20, a chemokine able to recruit cells that initiate adaptive immunity constitutes our first positive FAE molecular marker. We have shown that CCL20 gene expression is inducible in vitro and in vivo in intestinal epithelium by flagellin, a component of bacterial flagella. This effect was not restricted to the FAE. Lymphotoxin ß (LTß) signaling is critical for PPs organogenesis as LT deficient mice as well as LTß-receptor-/- mice lack PPs and most of the lymph nodes (LN). The continuous signaling via LTßR-expressing cells appears necessary for the maintenance throughout the life of PP architecture. We obtained in vitro and in vivo evidence that LTß signalling is involved in CCL20 gene expression.

Development of a hepatocyte hollow fiber bioreactor for the study of contrast agents by magnetic resonance imaging

Thanks to the continuous progress made in recent years, medical imaging has become an important tool in the diagnosis of various pathologies. In particular, magnetic resonance imaging (MRI) permits to obtain images with a remarkably high resolution without the use of ionizing radiation and is consequently widely applied for a broad range of conditions in all parts of the body. Contrast agents are used in MRI to improve tissue discrimination. Different categories of contrast agents are clinically available, the most widely used being gadolinium chelates. One can distinguish between extracellular gadolinium chelates such as Gd-DTPA, and hepatobiliary gadolinium chelates such as Gd-BOPTA. The latter are able to enter hepatocytes from where they are partially excreted into the bile to an extent dependent on the contrast agent and animal species. Due to this property, hepatobiliary contrast agents are particularly interesting for the MRI of the liver. Actually, a change in signal intensity can result from a change in transport functions signaling the presence of impaired hepatocytes, e.g. in the case of focal (like cancer) or diffuse (like cirrhosis) liver diseases. Although the excretion mechanism into the bile is well known, the uptake mechanisms of hepatobiliary contrast agents into hepatocytes are still not completely understood and several hypotheses have been proposed. As a good knowledge of these transport mechanisms is required to allow an efficient diagnosis by MRI of the functional state of the liver, more fundamental research is needed and an efficient MRI compatible in vitro model would be an asset. So far, most data concerning these transport mechanisms have been obtained by MRI with in vivo models or by a method of detection other than MRI with cellular or sub-cellular models. Actually, no in vitro model is currently available for the study and quantification of contrast agents by MRI notably because high cellular densities are needed to allow detection, and no metallic devices can be used inside the magnet room, which is incompatible with most tissue or cell cultures that require controlled temperature and oxygenation. The aim of this thesis is thus to develop an MRI compatible in vitro cellular model to study the transport of hepatobiliary contrast agents, in particular Gd-BOPTA, into hepatocytes directly by MRI. A better understanding of this transport and especially of its modification in case of hepatic disorder could permit in a second step to extrapolate this knowledge to humans and to use the kinetics of hepatobiliary contrast agents as a tool for the diagnosis of hepatic diseases.

Simultaneous irradiation of fibroblasts and carcinoma cells repress the secretion of soluble factors able to stimulate carcinoma cell migration.

Stroma mediated wound healing signals may share similarities with the ones produced by tumor's microenvironment and their modulation may impact tumor response to the various anti-cancer treatments including radiation therapy. Therefore we conducted this study, to assess the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts irrespective of their RhoB status do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms which are TGF-β1 and MMP-mediated respectively. Lastly, we found that simultaneous irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This last result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response.

Mechanisms of action of brief alcohol interventions remain largely unknown - a narrative review.

A growing body of evidence has shown the efficacy of brief intervention (BI) for hazardous and harmful alcohol use in primary health care settings. Evidence for efficacy in other settings and effectiveness when implemented at larger scale are disappointing. Indeed, BI comprises varying content; exploring BI content and mechanisms of action may be a promising way to enhance efficacy and effectiveness. Medline and PsychInfo, as well as references of retrieved publications were searched for original research or review on active ingredients (components or mechanisms) of face-to-face BIs [and its subtypes, including brief advice and brief motivational interviewing (BMI)] for alcohol. Overall, BI active ingredients have been scarcely investigated, almost only within BMI, and mostly among patients in the emergency room, young adults, and US college students. This body of research has shown that personalized feedback may be an effective component; specific MI techniques showed mixed findings; decisional balance findings tended to suggest a potential detrimental effect; while change plan exercises, advice to reduce or stop drinking, presenting alternative change options, and moderation strategies are promising but need further study. Client change talk is a potential mediator of BMI effects; change in norm perceptions and enhanced discrepancy between current behavior and broader life goals and values have received preliminary support; readiness to change was only partially supported as a mediator; while enhanced awareness of drinking, perceived risks/benefits of alcohol use, alcohol treatment seeking, and self-efficacy were seldom studied and have as yet found no significant support as such. Research is obviously limited and has provided no clear and consistent evidence on the mechanisms of alcohol BI. How BI achieves the effects seen in randomized trials remains mostly unknown and should be investigated to inform the development of more effective interventions.

Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies.

The evolution of alternative splicing patterns and regulatory mechanisms

Alternative splicing produces multiple isoforms from the same gene, thus increasing the number of transcripts of the species. Alternative splicing is a virtually ubiquitous mechanism in eukaryotes, for example more than 90% of protein-coding genes in human are alternatively spliced. Recent evolutionary studies showed that alternative splicing is a fast evolving and highly species- specific mechanism. The rapid evolution of alternative splicing was considered as a contribution to the phenotypic diversity between species. However, the function of many isoforms produced by alternative splicing remains unclear and they might be the result of noisy splicing. Thus, the functional relevance of alternative splicing and the evolutionary mechanisms of its rapid divergence among species are still poorly understood. During my thesis, I performed a large-scale analysis of the regulatory mechanisms that drive the rapid evolution of alternative splicing. To study the evolution of alternative splicing regulatory mechanisms, I used an extensive RNA-sequencing dataset comprising 12 tetrapod species (human, chimpanzee and bonobo, gorilla, orangutan, macaque, marmoset, mouse, opossum, platypus, chicken and frog) and 8 tissues (cerebellum, brain, heart, kidney, liver, testis, placenta and ovary). To identify the catalogue of alternative splicing eis-acting regulatory elements in the different tetrapod species, I used a previously defined computational approach. This approach is a statistical analysis of exons/introns and splice sites composition and relies on a principle of compensation between splice sites strength and the presence of additional regulators. With an evolutionary comparative analysis of the exonic eis-acting regulators, I showed that these regulatory elements are generally shared among primates and more conserved than non-regulatory elements. In addition, I showed that the usage of these regulatory elements is also more conserved than expected by chance. In addition to the identification of species- specific eis-acting regulators, these results may explain the rapid evolution of alternative splicing. I also developed a new approach based on evolutionary sequence changes and corresponding alternative splicing changes to identify potential splicing eis-acting regulators in primates. The identification of lineage-specific substitutions and corresponding lineage-specific alternative splicing changes, allowed me to annotate the genomic sequences that might have played a role in the alternative splicing pattern differences among primates. Finally, I showed that the identified splicing eis-acting regulator datasets are enriched in human disease-causing mutations, thus confirming their biological relevance.

Extra Forces induced by wide-pulse, high-frequency electrical stimulation: Occurrence, magnitude, variability and underlying mechanisms.

OBJECTIVE: In contrast to conventional (CONV) neuromuscular electrical stimulation (NMES), the use of "wide-pulse, high-frequencies" (WPHF) can generate higher forces than expected by the direct activation of motor axons alone. We aimed at investigating the occurrence, magnitude, variability and underlying neuromuscular mechanisms of these "Extra Forces" (EF). METHODS: Electrically-evoked isometric plantar flexion force was recorded in 42 healthy subjects. Additionally, twitch potentiation, H-reflex and M-wave responses were assessed in 13 participants. CONV (25Hz, 0.05ms) and WPHF (100Hz, 1ms) NMES consisted of five stimulation trains (20s on-90s off). RESULTS: K-means clustering analysis disclosed a responder rate of almost 60%. Within this group of responders, force significantly increased from 4% to 16% of the maximal voluntary contraction force and H-reflexes were depressed after WPHF NMES. In contrast, non-responders showed neither EF nor H-reflex depression. Twitch potentiation and resting EMG data were similar between groups. Interestingly, a large inter- and intrasubject variability of EF was observed. CONCLUSION: The responder percentage was overestimated in previous studies. SIGNIFICANCE: This study proposes a novel methodological framework for unraveling the neurophysiological mechanisms involved in EF and provides further evidence for a central contribution to EF in responders.

Cell wall maturation of Arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition.

Arabidopsis (Arabidopsis thaliana) leaf trichomes are single-cell structures with a well-studied development, but little is understood about their function. Developmental studies focused mainly on the early shaping stages, and little attention has been paid to the maturation stage. We focused on the EXO70H4 exocyst subunit, one of the most up-regulated genes in the mature trichome. We uncovered EXO70H4-dependent development of the secondary cell wall layer, highly autofluorescent and callose rich, deposited only in the upper part of the trichome. The boundary is formed between the apical and the basal parts of mature trichome by a callose ring that is also deposited in an EXO70H4-dependent manner. We call this structure the Ortmannian ring (OR). Both the secondary cell wall layer and the OR are absent in the exo70H4 mutants. Ecophysiological aspects of the trichome cell wall thickening include interference with antiherbivore defense and heavy metal accumulation. Ultraviolet B light induces EXO70H4 transcription in a CONSTITUTIVE PHOTOMORPHOGENIC1-dependent way, resulting in stimulation of trichome cell wall thickening and the OR biogenesis. EXO70H4-dependent trichome cell wall hardening is a unique phenomenon, which may be conserved among a variety of the land plants. Our analyses support a concept that Arabidopsis trichome is an excellent model to study molecular mechanisms of secondary cell wall deposition.