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Les cellules stromales mésenchymateuses (CSM) possèdent de nombreux atouts pour la thérapie cellulaire du cerveau. Nous avons tout d'abord démontré que les CSM ne migraient pas dans le cerveau de rats sains alors qu'elles étaient attirées par une lésion située à grande distance de leur site d'implantation. Nous avons également confirmé que la faible survie et différenciation neuronale des cellules in vivo constituent les obstacles majeurs à la thérapie cellulaire du cerveau. Par conséquent, nous nous sommes ensuite attachés à améliorer le potentiel de différenciation neuronal des CSM avant transplantation, à l'aide d'un pré-traitement en « epidermal growth factor » (EGF) et « basic fibroblast growth factor » (bFGF) in vitro. Finalement, nous avons associé des CSM à des vecteurs polymériques, les microcarriers pharmacologiquement actifs (MPA), afin de favoriser la survie, la différenciation neuronale et les capacités de réparation tissulaire des cellules après transplantation. Ces microsphères de PLGA ont ainsi été enrobées d'une surface biomimétique de laminine, après en avoir démontré les bénéfices sur la différenciation neuronale des CSM in vitro. Des CSM ont ensuite été mises en contact avec des MPA enrobées de laminine et libérant une neurotrophine, avant transplantation dans un modèle animal de la maladie de Parkinson. D'importants effets fonctionnels ont été observés par rapport à la greffe de cellules seules, et cette stratégie est la première à démontrer l'intérêt de cellules souches adultes associées à des vecteurs polymériques bioactifs pour protéger le système nerveux central dans le contexte de la maladie de Parkinson.

<p>Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor.</p><p>In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m² to 400 mg/m² worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m² worsened the toxicity without any improvement of the results.</p><p>A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study.</p><p>The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%.</p><p>Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence.</p><p>Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.</p>

<p>Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods.</p><p>Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. </p><p>In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF. </p>

Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor. In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m2 to 400 mg/m2 worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m2 worsened the toxicity without any improvement of the results. A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study. The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%. Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence. Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.

Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods. Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF.

Les cellules folliculo-stéllaires (FS) de l'hypophyse antérieure possèdent une forme étoilée et étendent de longues projections cytoplasmiques qui forment des pseudo-follicules entourant les cellules endocrines. Les cellules FS sont connectées entre elles par des jonctions lacunaires (des fois aussi connu sous le nom de jonction communicante) formant ainsi un réseau tridimensionnel continu. Un des rôles principaux des cellules FS est le maintien du microenvironnement de l'hypophyse antérieure, une activité qui est en partie réalisée par la sécrétion de divers facteurs de croissance et de cytokines. Ces messagers chimiques, y compris le bFGF, le VEGF, l’IL-6 et l’IL-1 contrôlent de nombreux processus cellulaires tels que l’expression des gènes d’hormones. Notre intérêt est de déterminer si la communication entre les cellules FS contribue à leur activité régulatrice. Dans notre étude, nous avons utilisé la lignée cellulaire TtT/GF qui partage de nombreuses caractéristiques morphologiques, physiologiques et biochimiques avec les cellules FS. Les jonctions lacunaires/communicantes sont formées par l’association de deux connexons de cellules adjacentes qui unissent le cytoplasme des cellules connectées et permet la diffusion de petites molécules. Chaque connexon est formé par l’oligomérisation de six protéines connexine (Cx) de la famille α, β ou γ. Les connexons, intégrés dans la membrane d’une vésicule du cytoplasme, se migrent vers la membrane cellulaire où ils s’incorporent dans la couche bilipidique. L’expression de la Cx43 (α) par les cellules FS est régulée en réponse à des facteurs de croissance et des cytokines. Des changements dans le microenvironnement de l'hypophyse antérieure causés par des molécules de signalisation sont susceptibles de modifier la Cx43, en particulier l’état de phosphorylation de la protéine. Ces modifications de la Cx43 peuvent ensuite déclencher des changements du comportement de jonctions lacunaires/communicantes formées par la Cx43, comme leur perméabilité et le renouvellement de la protéine Cx43. Les tissus expriment généralement plus d’un type de connexine. Jusqu’aujourd’hui, la Cx43 est la seule connexine à avoir été identifiée dans les cellules FS. Le cristallin exprime les connexines α: Cx43, Cx46 et Cx50. Leur expression est modulée par des facteurs de croissance. Notre hypothèse de travail a été de vérifier si la Cx46 et la Cx50 étaient exprimées par les cellules FS et si celles-ci contribuaient au rôle modulateur des cellules FS hypophysaires. Dans cette étude, nous avons identifié et caractérisé la Cx46 et la Cx50 dans la lignée cellulaire TtT/GF. Nous avons identifié les produits de transcription de Cx46 et de Cx50 par la technique d’analyse northern blot (PCR). Par la suite, les protéines Cx46 et Cx50 ont été identifiées en utilisant des anticorps dans des analyses western blot. Par microscopie confocale, nous avons déterminé la co-localisation de la Cx46 avec certaines marqueurs d’organites : réseau trans-Golgien, endosomes précoces et lysosomes. La Cx50 co-localise avec des marqueurs du réticulum endoplasmique, du réseau cis-Golgien et des endosomes précoces. Un protocole d’isolation des membranes résistantes aux détergents non-ionique a révélé que la Cx46 et la Cx50 n’étaient pas associées à des radeaux lipidiques ni aux cavéoles. Cependant, la microscopie confocale a montré une co-localisation cytoplasmique de la Cx50 et de la flotilline-1. Nous avons poursuivi l’étude sur la localisation de la Cx46 dans le noyau en utilisant une technique d’isolation des fractions enrichies en noyau. Nous avons établi que plusieurs isoformes de la Cx46 sont exclusivement associées au noyau. De plus, avec la microscopie confocale nous avons démontrée une co-localisation de la Cx46 avec un marqueur du nucléole/corps de Cajal. Nous avons démontré un effet du bFGF sur l'expression temporelle de la Cx46 et de la Cx50. L’expression de la Cx46 diminue au cours de longues expositions au bFGF tandis que les niveaux de Cx50 augmentent de façon transitoire au cours du traitement. Dans une autre étude nous avons démontré des changements importants dans les niveaux de la Cx46 et de la Cx50 dans l’hypophyse antérieure des visons durant le cycle de reproduction annuel. Notre étude démontre que les cellules FS expriment la Cx46 et la Cx50. Nous avons aussi établi que la Cx46 et la Cx50 sont localisées dans différentes structures sous-cellulaires, ce qui suggère des rôles différents dans les cellules FS pour ces protéines de jonction lacunaire/communicante. Il est possible que la Cx46 et la Cx50 ne jouent pas un rôle majeur dans la communication intercellulaire dans les cellules FS quiescentes. Nos résultats suggèrent que la Cx46 et la Cx50 peuvent avoir d'autres fonctions : des isoformes de la Cx46 peuvent contribuer à la biogenèse des ribosomes tandis que la Cx50 pourrait avoir un rôle dans la communication dans les cellules stimulées au bFGF. Nos études établissent une base pour des recherches futures. / The folliculo-stellate (FS) cells of the anterior pituitary are star-shaped and extend long cytoplasmic processes forming pseudo-follicles encircling hormone-secreting cells. Dispersed throughout the anterior pituitary gland, FS cells are joined to form a continuous three dimensional network through communicating gap junctions. One of the primary roles of FS cells is the maintenance of the anterior pituitary microenvironment, accomplished through the expression and secretion of various growth factors and cytokines. These chemical messengers, including bFGF, VEGF, IL-6 and IL-1 mediate a range of cellular processes such as hormone gene expression. Our aim is to study whether intercellular communication among FS cells contributes to the modulatory activity of the FS cells within the anterior pituitary gland. To pursue this, we use the TtT/GF cell line that shares many morphological, physiological and biochemical characteristics with FS cells. Gap junctions are formed by the joining of two connexons/hemichanels from adjacent cells that link their cytoplasms allowing for the passive diffusion of small molecules. Connexons/hemichannels are themselves formed by the oligomerization of six connexin (Cx) proteins from the family α, β or γ, which then migrate into the lipid bilayer of the cell membrane. FS cells express Cx43 (α-connexin), which is regulated in response to growth factors and cytokines. Changes in the anterior pituitary microenvironment due to signaling molecules results in modifications to Cx43, particularly in the phosphorylation status of the protein. Such alterations yield alterations in the physiological behaviour of Cx43 gap junctions such as permeability and turnover. Tissues generally express more than one connexin type and to date, Cx43 has been the sole connexin to be identified in FS cells. The ocular lens expresses the α-connexins: Cx43, Cx46 and Cx50, which are modulated by growth factors that are also present in the anterior pituitary. Based on these facts, we hypothesize that Cx46 and Cx50 are also expressed by the FS cells and contribute to the FS modulatory role in the anterior pituitary gland. In the present study, we have identified and characterized Cx46 and Cx50 in the TtT/GF cell line. We identified Cx46 and Cx50 transcripts through northern blots and identified the corresponding protein products using antibodies and western blot analyses. Through confocal microscopy, we determined that Cx46 co-localized with the organelle markers: trans-Golgi, early endosomes and lysosomes. Cx50 co-localized with markers for the ER, cis-Golgi and early endosomes. An isolation procedure using a non-ionic detergent we showed that neither Cx46 nor Cx50 were associated to lipid rafts or caveolae. However, confocal microscopy showed a cytoplasmic co-localization between Cx50 and flotillin-1. We pursued a finding that localized Cx46 to the nucleus and using a nuclear isolation technique, demonstrated that several isoforms of Cx46 are exclusively located in the nuclear compartment. Furthermore, with confocal microscopy we found a co-localization of Cx46 with a nucleolus/coiled body marker. We demonstrated an effect of bFGF on the temporal expression patterns of Cx46 and Cx50 and showed that Cx46 levels decreased over longer exposures to the growth factor while Cx50 levels transiently increased. Lastly, drastic changes were noted in an in situ study of Cx46 and Cx50 in the male and female mink anterior pituitary during the annual reproductive cycle. Our study indicates that addition to Cx43, FS cells also express Cx46 and Cx50. We also demonstrated that Cx46 and Cx50 localize to different sub-cellular structures, suggesting different roles in the FS cells. While they may not play a major role in intercellular communication in quiescent FS cells, our results suggest that Cx46 and Cx50 may serve other functions: Cx46 isoforms may contribute to ribosome biogenesis and Cx50 may have communication-related responsibilities in stimulated cells. Importantly, our identification and characterization studies provide a foundation on which future studies can be built.