A novel and divergent role of granzyme a and B in resistance to helminth infection.

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA×B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

Résumé La plupart des cellules issues du sang ont une durée de vie limitée. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des télomères diminue progressivement à chaque division cellulaire, pouvant aboutir à des instabilités chromosomiques. L'expression ectopique du gène de la transcriptase réverse de la télomérase (hTERT) dans les cellules restaure l'activité de la télomérase, et permet un rallongement de leur vie réplicative. Malgré l'absence de signes caractéristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines études récentes proposent que la télomérase joue plusieurs rôles additionnels dans d'autres phénomènes biologiques tels que la réparation de l'ADN, la survie et la croissance des cellules. Dans notre étude, nous avons utilisé des clones issus de lymphocytes T cytotoxiques surexprimant la télomérase afin d'étudier les mécanismes moléculaires qui règlent leur prolifération et leur sénescence. Nous avons montré que les «jeunes » cellules T exprimant ou non hTERT révèlent des taux de croissance identiques suite à des réponses de stimulation induites par des mitogènes. De plus, aucun changement global dans leur expression des gènes n'a pu être mis en évidence. Curieusement, nous avons observé des réponses réduites dans la prolifération des cellules transduites avec la télomérase qui présentaient une élongation des télomères et une durée de vie prolongée. Ces cellules, malgré le maintien d'un niveau élevé de l'expression de gènes impliqués dans la progression du cycle cellulaire, ont également montré une expression accrue de plusieurs gènes trouvés en commun avec nos lymphocytes T vieillissants n'exprimant pas de télomérase. En particulier, les cellules ayant une durée de vie prolongée grâce à l'expression de la télomérase accumulaient également certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associés à l'arrêt de la croissance cellulaire. En résumé, nos résultats indiquent la présence fonctionnelle de mécanismes alternatifs pouvant contrôler la croissance réplicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation à moindre risque de lymphocytes T «immortalisés » à des fins thérapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

The Bcl-2/Bcl-XL inhibitor ABT-737 promotes death of retinoblastoma cancer cells.

Purpose: Retinoblastoma is a malignant tumor that usually develops in early childhood. During retinoblastoma spreading, RB1 gene inactivation is followed by additional genomic modifications which progressively lead to resistance of tumor cells to death. Drugs that act at downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. ABT-737, a BH3 mimetic molecule effective at the mitochondrial level, has been shown to induce apoptosis in different human tumoral cell lines as well as in primary patient-derived cells, and in a mouse xenograph model. Methods: In this report, we analyzed the pro-death effect of ABT-737 on two human retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the mouse photoreceptor cell line 661W. Results: We observed that ABT-737 was very effective as a single agent in inducing human WERI-Rb cells apoptosis without affecting the mouse 661W photoreceptor cells. However human Y79 cells were resistant to ABT-737, as a probable consequence of the absence of Bax. The high sensitivity of WERI-Rb to ABT-737 can be increased by downregulating Mcl-1 using the proteasome inhibitor MG-132. Preliminary analysis in primary mouse retinoblastoma tumoral cell lines predicts high sensitivity to ABT-737. Conclusion: Our data suggest that ABT-737 or related compounds could be a highly effective drug in the treatment of some retinoblastomas.

Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice.

BACKGROUND: Food allergy has reached an epidemic level in westernized countries and although central mechanisms have been described, the variability associated with genetic diversity underscores the still unresolved complexity of these disorders. OBJECTIVE: To develop models of food allergy and oral tolerance, both strictly induced by the intestinal route, and to compare antigen-specific responses. METHODS: BALB/c mice were mucosally sensitized to ovalbumin (OVA) in the presence of the mucosal adjuvant cholera toxin, or tolerized by intra-gastric administrations of OVA alone. Antibody titres and cytokines were determined by ELISA, and allergic status was determined through several physiologic parameters including decline in temperature, diarrhoea, mast cell degranulation and intestinal permeability. RESULTS: OVA-specific antibodies (IgE, IgGs and IgA in serum and feces) were produced in sensitized mice exclusively. Upon intra-gastric challenge with OVA, sensitized mice developed anaphylactic reactions associated with a decline of temperature, diarrhoea, degranulation of mast cells, which were only moderately recruited in the small intestine, and increased intestinal permeability. Cytokines produced by immune cells from sensitized mice included T-helper type 2 cytokines (IL-5, IL-13), but also IL-10, IFN-gamma and IL-17. In contrast, all markers of allergy were totally absent in tolerized animals, and yet the latter were protected from subsequent sensitization, demonstrating that oral tolerance took place efficiently. CONCLUSION: This work allows for the first time an appropriate comparison between sensitized and tolerized BALB/c mice towards OVA. It highlights important differences from other models of allergy, and thus questions some of the generally accepted notions of allergic reactions, such as the protective role of IFN-gamma, the importance of antigen-specific secretory IgA and the role of mucosal mast cells in intestinal anaphylaxis. In addition, it suggests that IL-17 might be an effector cytokine in food allergy. Finally, it demonstrates that intestinal permeability towards the allergen is increased during challenge.

Expression of common acute lymphoblastic leukemia antigen (CALLA) on human malignant melanoma cell lines.

Fifteen human melanoma cells lines were tested by an antibody-binding radioimmunoassay using a monoclonal antibody (A12) directed against the common acute lymphoblastic leukemia antigen (CALLA). Cells from six melanoma lines were found to react with this antibody. The level of antigen and the percentage of positive cells in these six melanoma lines showed wide variation, as demonstrated by analysis in the fluorescence-activated cell sorter (FACS). Immunoprecipitation of solubilized 125I-labeled membrane proteins from CALLA positive melanoma cells with A12 monoclonal antibody revealed a major polypeptide chain with an apparent m.w. of 100,000 daltons, characteristic for CALLA as determined on SDS-polyacrylamide gel electrophoresis. The expression of CALLA on MP-6 melanoma cells was modulated when the cells were cultured in the presence of A12 antibody. Reexpression of CALLA on these cells occurred within 5 days after transfer of the modulated cells into medium devoid of monoclonal antibody.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

Transformed and nontransformed human T lymphocytes migrate to skin in a chimeric human skin/SCID mouse model.

To study human T cell migration to human skin in vivo, we grafted severe combined immunodeficient mice with 500-microm thick human skin. Two weeks after grafting, epidermal and dermal structures in the grafts were of human origin. When we intraperitoneally injected grafted mice with clones of the human HUT-78 T cell line derived from a patient with cutaneous T cell lymphoma and Sézary syndrome, we detected in the grafts the rare Vbeta23-Jbeta1.2 T cell receptor transcripts characteristic for the HUT-78 clones. These signals were found 2-6 d after cell injection in about 40% of the grafted and HUT-78 cell injected mice but not in grafts from mice that received no exogenous T cells. In contrast to HUT-78 cells, which only accumulate in low number, grafts topically challenged with nickel sufate in vaseline from mice that were injected with autologous nickel-reactive T cell lines led to massive accumulation of T cells within 3 d. Only scattered T cells accumulated in the skin when grafted mice received vaseline plus T cells, nickel sulfate alone, T cells alone, or nickel sulfate plus an allogeneic nickel-nonreactive T cell clone. When the T cell lines were labeled with the fluorochrome PKH-26 before cell injection, spots of fluorescent label in the size and shape of cells were found in the grafts challenged with nickel. Together, these results clearly demonstrate that human T cells can migrate to human skin in this chimeric human/mouse model.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Systemic mastocytosis with associated myeloproliferative disease and precursor B lymphoblastic leukaemia with t(13;13)(q12;q22) involving FLT3.

Systemic mastocytoses represent neoplastic proliferations of mast cells. In about 20% of cases systemic mastocytoses are accompanied by clonal haematopoietic non-mast cell-lineage disorders, most commonly myeloid neoplasms. A case of systemic mastocytosis carrying the characteristic mutation at codon 816 (D816V) in the KIT gene of mast cells, with two concurrent accompanying clonal haematopoietic non-mast cell-lineage disorders, chronic myeloproliferative disease, unclassifiable and precursor B lymphoblastic leukaemia is documented. Both accompanying clonal haematopoietic non-mast cell-lineage disorders carried the wild-type KIT gene, but had a novel t(13;13)(q12;q22) involving the FLT3 locus at 13q12. The chronic myeloproliferative disease, unclassifiable and the precursor B lymphoblastic leukaemia were cured by syngenous stem cell transplantation, but the systemic mastocytosis persisted for more than 10 years. The additional impact of molecular techniques on the correct diagnosis in haematological malignancies is highlighted, and evidence is provided that, apart from internal tandem duplications and mutations, FLT3 can be activated by translocations.

Interactions between cells and functionalized nanoparticles

In this present thesis Superparamagnetic Iron Oxide Nanoparticles (SPIONs) with 9 nm in diameter were selected as nanocarriers in order to study their potential application as drug delivery systems. Therefore the aim of the study was to demonstrate the proof of concept by establishing an efficient system of drug delivery, which would be a valuable tool in biomedical applications, such as the treatement of cancer, by reducing the side effects due to administration of a high concentration of therapeutic agents. As demonstrated in a previous study, the uptake of SPIONs by tumoral human cells was enhanced by the presence of amino groups on their surface. The stabilization of SPIONs were then performed and optimized by the coating of poly(vinylalcohol) and poly(vinylalcohol/vinylamine). Such nanoparticles were known as aminoPVA-SPIONs. The toxicity and the inflammatory reaction of aminoPVA-SPIONs were evaluated in order to establish their potentiel use in the human body. The results demonstrated that the human cells were able to invaginate aminoPVA-SPIONS without revealing any toxicity and inflammatory reaction. The analysis by transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-TEM, confocal microscopy and histological staining (i.e. Prussian Blue) showed that the iron oxide core of SPIONs were located in the cytoplasm of cells and concentrated in vesicles. The evaluation of the mechanism of uptake of aminoPVA-SPIONs revealed that their uptake by monolayer cell culture was performed via an active mechanism, which was achieved by a clathrin-mediated endocytosis. Consequently, it was suggested that aminoPVA-SPIONs were good candidates as nanocarriers in drug delivery systems, which were able to reach the cytoplasm of cells. Their incubation with three-dimensional models mimicing tissues, such as differentiated rat brain cell-derived aggregates and spheroids, revealed that aminoPVA-SPIONs were able to invade into deep cell layers according to the stage of growth of these models. In the view of these promising results, drug-SPIONs were prepared by the functionalization of aminoPVA-SPIONs via a biological labile chemical bond by one of these three antineoplastic agents, which are widely used in clinical practice: 5-fluorourdine (Fur) (an antimetabolite), or camptothecin (CPT) (a topoisomerase inhibitor) or doxorubicin (DOX) (an anthracycline which interfere with DNA). The results shown that drug-SPIONs were internalized by human melanoma cells, as it was expected due the previous results with aminoPVA-SPIONs, and in addition they were active as anticancer agents, suggesting the efficient release of the drug from the drug-SPIONs. The results with CPT-SPIONs were the most promising, whereas DOX- SPIONs did not demonstrate a prononced activity of DOX. In conclusion, the results demonstrated that functionalized iron oxide nanoparticles are a promising tool in order to deliver therapeutic agents. - Dans le cadre de ce travail de thèse, les nanoparticules superparamagnétiques d'oxyde de fer (SPIONs) ayant un diamètre de 9 nm ont été choisies, afin d'étudier leur éventuelle utilisation dans un système de délivrance d'agents thérapeutiques. Ainsi le but de la thèse est de démontrer la faisabilité de fabriquer un système efficace de délivrance d'agents thérapeutiques, qui serait un outil intéressant dans le cadre d'une utilisation biomédicale, par exemple lors du traitement du cancer, qui pourrait réduire les effets secondaires provoqués par le dosage trop élevé de médicaments. Comme il a été démontré dans une précédente étude, l'invagination des SPIONs par des cellules humaines cancéreuses est améliorée par la présence de groupes fonctionnels amino à leur surface. La stabilisation des SPIONs est ainsi effectuée et optimisée par l'enrobage de poly(vinylalcool) et de (poly(vinylalcool/vinylamine), qui sont connues sous le nom de aminoPVA-SPIONs. La toxicité et la réaction inflammatoire des aminoPVA-SPIONs ont été évaluées dans le but de déterminer leur potentielle utilisation dans le corps humain. Les résultats démontrèrent que les cellules humaines sont capables d'invaginer les aminoPVAS-SPIONs sans induire une réaction toxique ou inflammatoire. L'analyse par la microscopie électronique en transmission électronique (TEM), la microscopie électronique à balayage (SEM), le cryo-microscopie électronique (SEM), la microscopie confocale et la coloration histologique (par ex, le bleu de Prusse) a montré que l'oxyde de fer des SPIONs est localisé dans le cytoplasme des cellules et est concentré dans des vesicules. L'évaluation du méchanisme d'invagination des aminoPVA-SPIONs ont révélé que leur invagination par des monocultures de cellules est effectué par un méchanisme actif, contrôlé par une endocytose induite par les clathrins. Par conséquent, les aminoPVA-SPIONs sont de bons candidats en tant que transporteurs (nanocamers) dans un système de délivrance d'agents thérapeuthique, capable d'atteindre le cytoplasme des cellules. Leur incubation avec des modèles tridimenstionnels imitant les tissues, tels que les aggrégats de cellules de cerveau différenciées et les sphéroïdes, a montré que les aminoPVA-SPIONs sont capable de pénétrer dans les couches profondes des modèles, selon l'état d'avancement de leur croissance. En vue de ces résultats prometteurs, les drug-SPIONs ont été préparés en fonctionalisant les aminoPVA-SPIONs par le biai d'une liaison chimique labile par un des trois agents thérapeutiques, déjà utilisé en pratique : 5-fluorourdine (Fur) (un antimétabolite), or camptothecin (CPT) (un inhibiteur de la topoisomerase) or doxorubicin (DOX) (un anthracycline qui interfère avec le DNA). Les résultats ont montré que les drug-SPIONs sont capable d'être internalisés par les mélanomes, comme il a été attendu d'après les résultats obtenus précédemment avec les aminoPVA-SPIONs, et de plus, les drug-SPIONs sont actifs, ce qui suggère un relargage efficace de l'agent thérapeutique du drug-SPIONs. Les résultats obtenus avec les CPT-SPIONs sont les plus prometteurs, tandis que ceux avec les DOX-SPIONs, ce n'est pas le cas, dont l'activité thérapeutique de DOX n'a pas été aussi efficace. En conclusion, les résultats ont pu démontrer que les nanoparticules d'oxyde de fer fonctionnalisées sont un outil prometteur dans la délivrance d'agents thérapeutiques.

Role of Tyk-2 in Th9 and Th17 cells in allergic asthma.

In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.

A Cell Permeable Rasgap-derived Peptide Sensitizes Cancer Cells To Radiotherapy

Purpose/Objective(s): Radiotherapy is an effective treatment modality against cancer. Despite recent technical progresses in radiation delivery precision, toxicity to healthy tissues remains the main limiting factor. RasGAP is a regulator of the Ras and Rho pathway; it has either a pro- or anti-apoptotic activity depending on the level of caspase expressed in the cell. The RasGAP derived peptide: TAT-RasGAP317 - 326 is the minimal sequence known to sensitize cancer cells, but not healthy cells, to genotoxin-induced apoptosis. In this study the TAT-RasGAP317 - 326 radio-sensitizing effect was tested in vitro and in vivo.Materials/Methods: Two weeks clonogenic forming assays with 5 human cancer cells (PANC-1, HCT116, U87, U251 and HeLa) and a non tumorigenic cell line (HaCaT) were performed. Cells were exposed to 0, 1, 2 and 4 Gy with or without 20 mMTAT-RasGAP317 - 326. Twenty mMTAT peptide was also used as control. TAT-RasGAP317 - 326 effect was also tested in tumor xenograft mouse models. Mice bearing HCT116 tumors (WT or p53 mutant) received 1.65 mg/kg TAT-RasGAP317 - 326 i.p. injected and were locally irradiated for 10 days with 3 Gy. Tumor volume was then followed during a minimum of 20 days. Control mice were treated with a single modality, either with TAT-RasGAP317 - 326 or with radiotherapy.Results: At all the tested radiation doses TAT-RasGAP317 - 326 showed a significant supra additive radio-sensitizing effect on all the tested tumor cell lines. Furthermore, it showed no sensitizing effect on the non tumorigenic cell line. In vivo, TAT-RasGAP317 - 326 also showed a significantly radio-sensitizing effect as shown by a significant higher reduction in tumor volume as much as by a significant tumor growth delay.Conclusions: Taken together our data suggest that TAT-RasGAP317 - 326 has a radio-sensitizing effect on in vivo and in vitro tumors without any effect on healthy tissues. Therefore TAT-RasGAP317 - 326 should be considered as a novel and attractive sensitizer compound allowing an improvement of the therapeutic interval.

The long-term culture of porcine urothelial cells and induction of urothelial stratification.

OBJECTIVE: To assess porcine urothelial cell cultures and the in vitro induction of urothelial stratification in long-term cultures, to study their morphological, functional and genetic behaviour, and thus provide potential autologous urothelium for tissue-engineered substitutes for demucosalized gastric or colonic tissue. MATERIALS AND METHODS: Primary cultures of porcine urothelium were established and the cells passaged thereafter. Cell specificity was confirmed by cytokeratin analysis, cell membrane stability assessed using lactate dehydrogenase leakage, cell de-differentiation by gamma-glutamyl transferase activity and genomic stability by karyotype investigations. Histology and scanning electron microscopy were performed to study the cultured cells and the stratified constructs. Furthermore, collagen matrices were tested as cell scaffolds. RESULTS: The cells were cultured for 180 days; 10 subcultures were established during this period. Stratification was induced in a culture flask and on a collagen matrix. Cytokeratins 7, 8, 17 and 18 were expressed in all cultures, and cell membranes were stable, with no evident de-differentiation. The cultures were stable in their genotype and no chromosomal aberrations were found. The histology and immunohistochemistry of the stratified porcine constructs, and cell membrane stability and cell de-differentiation, were compared with those in the human system. CONCLUSION: Pig and human urothelial cells can be cultured over a long period with no signs of senescence. Urothelial stratification can be induced in vitro. The collagen matrix seems to be an excellent scaffold, allowing cell adherence and growth.

Chemical Functionalization of Bioceramics To Enhance Endothelial Cells Adhesion for Tissue Engineering.

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8+ T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.