Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system.

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telencephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5-50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5-5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The two PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.

Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Soluble MHC-peptide complexes induce rapid death of CD8+ CTL.

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.

Development and evaluation of antibody-MHC/peptide conjugates as a new form of cancer immunotherapy

Summary One of the major goals of cancer immunotherapy is the induction of a specific and effective antitumor cytotoxic T lymphocyte (CTL) response. However, the downregulation of Class I Major Histocompatibility Complexes (MHC) expression and the low level of tumor peptide presentation on tumor cell surface, ás well as the low immunogenicity of tumor specific antigens, limit the effectiveness of anti-tumor CTL responses. On the other hand, monoclonal antibodies, which bind with high affinity to tumor cell surface markers, are powerful tumor targeting tools. However, their capacity to .kill cancer cells is limited and mAb cancer treatments usually require the addition of different form of chemotherapy. The new cancer immunotherapy strategy described herein combines the advantage of the high tumor targeting capacity of monoclonal antibodies (mAb) with the powerful cytotoxicity of CD8 T lymphocytes directed against highly antigenic peptide-MHC complexes. Monoclonal antibody Fab fragments directed against a cell surface tumor associated antigen (TAA) are chemically coupled to soluble MHC class I complexes carrying a highly antigenic peptide. Antibody guided targeting and oligomerization of numerous antigenic class IMHC/peptide complexes on tumor cell surfaces can redirect the cytotoxicity of peptide-specific CD8 T cells towards target cancer cells. After the description of the production of murine anti-tumor xMHC/peptide conjugates in the first part of this thesis, the therapeutic potential of such conjugates were sequentially investigated in different syngeneic tumor mouse models. As a first proof of principle, transgenic OT-1 mice and later CEA transgenic C57BL/6 (B6) mice, adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, were used as a model of high frequency of ova peptide specific T cells. In these mice, growth inhibition and regression of palpable colon carcinoma expressing CEA, were obtained by systemic injection of anti-CEA Fab/H-2Kb/ova peptide conjugates. Next, LCMV virus and influenza virus infection of B6 mice were used as viral models to redirect natural antiviral CTL responses to tumors via conjugates loaded with viral peptides. We showed that in mice infected with the LCMV virus, subcutaneous CEA-expressing tumor cells were inhibited by the H2Db/GP33 restricted anti-viral CTL response when preincubated before grafting with anti-CEA Fab-H-2Db/GP33 peptide conjugates. In mice infected with the influenza virus, lung metastases expressing the HER2 antigen were inhibited by the H-2Db/NP366 restricted CTLs response when preincubated before injection with anti-Her2 Fab-H-2Db/NP366 peptide conjugates. In the last chapter, the stability of the peptide in the anti-CEA Fab-H-2Db/GP33 conjugates was improved by the covalent photocross-link of the GP33 peptide in the H-2Db MHC groove. Thus, LCMV immune mice could reject CEA expressing tumors when treated with systemic injections of anti-CEA FabH-2Db/GP33 cross-linked conjugates. These results are encouraging for the potential application of this strategy in clinic. Such conjugates could be used alone in patients boosted by the relevant virus, or used in combination with existing T cell based ìmmunotherapy. Résumé Une des principales approches utilisées dans l'immunothérapie contre le cancer consiste en l'induction d'une réponse T cytotoxique (CTL) spécifiquement dirigée contre la tumeur. Cependant, le faible niveau d'expression des complexes majeurs d'histocompatibilité de classe I (CMH I) et de présentation des peptides tumoraux à la surface des cellules cancéreuses ainsi que la faible immunogenicité des antigens tumoraux, limitent l'efficacité de la réponse CTL. D'autre part,. l'injection d'anticorps monoclonaux (mAb), se liant avec une haute affinité aux marqueurs de surface des cellules tumorales, a fourni des résultats cliniques encourageant. Cependant l'efficacité de ces mAbs contre des tumeur solides reste limitée et necessite souvent l'addition de chimiotherapie. La nouvelle stratégie thérapeutique décrite dans ce travail associe le fort pouvoir de localisation des anticorps monoclonaux et le fort pouvoir cytotoxique des lymphocytes T CD8+. Des fragments Fab d'anticorps monoclonaux, dirigés contre des antigènes surexprimés à la surface de cellules tumorales, ont été chimiquement couplés à des CMH I solubles, portant un peptide fortement antigénique. Le ciblage et l'oligomérisation à la surface des cellules tumorales de nombreux CMH I présentant un peptide antigénique, va réorienter la cytotoxicité des cellules T CD8+ spécifiques du peptide présenté, vers les cellules tumorales cibles. Après une description de la production de conjugé anti-tumeur x CMH Upeptide dans la première partie de cette thèse, le potentiel thérapeutique de tels conjugés a été successivement étudiés in vivo dans différents modèles de tumeur syngénéiques. Tout d'abord, des souris OT-1 transgéniques, puis des souris C57BL/6 (B6) transférées avec des cellules de rate OT-1 puis immunisées avec l'ovalbumine, ont été employées comme modèle de haute fréquence de cellules T CD8+ spécifiques du peptide ova. Chez ces souris, l'inhibition de la croissance et la régression de nodules palpables de carcinomes exprimant l'antigène caccino embryonaire (ACE), ont été obtenues par l'injection systémique de conjugés anti-ACE Fab/H-2Kb/ova. Par la suite, l'infection de souris B6 par le virus LCMV et par le virus de la grippe, ont été utilisés comme modèles viraux pour redirigées des réponses anti-virales naturelles vers les tumeurs, en utilisant des conjugés chargés avec des peptides viraux. Nous avons montré que .chez les souris infectées par le LCMV, la croissance de carcinome sous-cutané est empêchée par la réponse anti-virale, spécifique du complexe H2Db/GP33, lorsque les cellules tumorales greffées sont pré-incubées avec des conjugés anti-CEA Fab-H-2Db/GP33. Dans le cas de souris infectées par le virus de la grippe, la métastatisation de mélanomes pulmonaires exprimant l'antigène HER-2 est inhibée par la réponse anti-virale spécifique du complexe H-2Db/NP366, après pré-incubation des cellules tumorales avec des conjugés anti-Her2 FabxH-2Db/NP366. Dans le dernier chapitre, la liaison covalente du peptide GP33 dans le complexe H-2Db a amélioré la stabilité des conjugés correspondants et a permis le traitement systémique de souris greffées avec des tumeurs exprimant l'ACE et infectées par le LCMV. L'ensemble de ces résultats sont encourageant pour l'application de cette strategie en clinique. De tels conjugués pourraient être employés seuls ou en combinaison avec des protocols d'immunisation peptidique anti-tumoral. Résumé pour un large public Dans les pays industrialisés, le cancer se situe au deuxième rang des causes de mortalité après les maladies cardiovasculaires. Les principaux traitement de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. L'immunothérapie est l'une des nouvelles approches mises en oeuvre pour la lutte contre le cancer. Elle peut être humorale, et s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la proliferation des cellules tumorales exprimant l'antigène. Certains sont utilisés en clinique pour le traitement de lymphomes, de carcinomes de l'ovaire et du sein ou encore de carcinomes metastatiques du côlon. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements exigent souvent d'être combiner avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et reposer sur une démarche de type vaccinal, consistant à générer des lymphocytes T cytotoxiques (cytotoxic T lymphocytes :CTL) capables de détruire spécifiquement les cellules malignes. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I. Or les cellules tumorales ne presentent pas efficacement les peptides antigèniques, car elles se caractérisent par une diminution ou une absence d'expression des antigènes d'histocompatibilité de classe I, des molécules d'adhésion et des cytokines costimulatrices, et par une faible expression des antigènes associés aux tumeurs. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL specifiquement dirigés contre des antigens tumoraux, les régressions tumorales obtenus grace à ces vaccinations sont relativement rares. Alors que chez les personnes atteintes du cancer on observe l'instauration d'une tolérance immunitaire vis-à-vis de la tumeur, à l'inverse, notre systeme immunitaire reste parfaitement capable de combattre des infection virales classiques, tels que la grippe, qui font aussi appel à une réponse T cytotoxique. Notre groupe de recherche a donc eu l'idee de développer une nouvelle approche thérapeutique où une réponse immunitaire anti-virale très efficace serait redirigée vers les tumeurs par des anticorps monoclonaux. Concrètement, nous avons chimiquement couplés des fragments d'anticorps monoclonaux dirigés contre des antigènes surexprimés à la surface de cellules tumorales, à des CMH I portant un peptide viral antigénique. Les cellules tumorales, ciblées par le fragment anticorps et couvertes d' antigènes viraux présentés par des molécules de CMH I, peuvent ainsi tromper les lymphocytes cytotoxiques anti-viraux qui vont détruire les cellules tumorales comme si elles étaient infectées par le virus. Suite à des résultats prometteurs obtenus in vitro avec différents conjugués anticorps-CMH humain de type HLA.A2/peptide Flu, le but du projet était de tester in vivo des conjugués anticorps-CMH I murins sur des modèles expérimentaux de souris. Tout d'abord, des souris transgéniques pour un recepteur T specifique du peptide ova, puis des transferts adoptifs de ces cellules T specifiques dans des souris immunocompétentes, ont été choisi comme modèle de haute fréquence des cellules T spécifiques, et ont permi de valider le principe de la strategie in vivo. Puis, deux modèles viraux ont été elaboré avec le virus LCMV et le virus Influenza, pour réorienter des réponses antivirales naturelles vers les tumeurs grâce à des conjugés chargés avec des peptides viraux. Nous avons montré la grande capacité de nos conjugués à rediriger des réponses cytotoxiques vers les tumeurs et inhiber la croissance de tumeurs syngénéiques sous cutanés et pulmonaires. Ces résultats d'inhibition tumorales obtenus dans des souris immunocompétentes, grâce à l'injection de conjugués anticorps xCMH/peptide et réorientant deux réponses antivirales différentes vers deux modèles tumoraux syngeneiques, sont encourageant pour l'application de cette nouvelle stratégie en clinique.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts.

The concept of antibody-mediated targeting of antigenic MHC/peptide complexes on tumor cells in order to sensitize them to T-lymphocyte cytotoxicity represents an attractive new immunotherapy strategy. In vitro experiments have shown that an antibody chemically conjugated or fused to monomeric MHC/peptide can be oligomerized on the surface of tumor cells, rendering them susceptible to efficient lysis by MHC-peptide restricted specific T-cell clones. However, this strategy has not yet been tested entirely in vivo in immunocompetent animals. To this aim, we took advantage of OT-1 mice which have a transgenic T-cell receptor specific for the ovalbumin (ova) immunodominant peptide (257-264) expressed in the context of the MHC class I H-2K(b). We prepared and characterized conjugates between the Fab' fragment from a high-affinity monoclonal antibody to carcinoembryonic antigen (CEA) and the H-2K(b) /ova peptide complex. First, we showed in OT-1 mice that the grafting and growth of a syngeneic colon carcinoma line transfected with CEA could be specifically inhibited by systemic injections of the conjugate. Next, using CEA transgenic C57BL/6 mice adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, we demonstrated that systemic injections of the anti-CEA-H-2K(b) /ova conjugate could induce specific growth inhibition and regression of well-established, palpable subcutaneous grafts from the syngeneic CEA-transfected colon carcinoma line. These results, obtained in a well-characterized syngeneic carcinoma model, demonstrate that the antibody-MHC/peptide strategy can function in vivo. Further preclinical experimental studies, using an anti-viral T-cell response, will be performed before this new form of immunotherapy can be considered for clinical use.

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.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

Proliferative and Osteogenic Differentiation Potentials of Human Fetal Bone Cells

BACKGROUND. Human primary fetal bone cells (hFBC) are being characterized for use in bone tissue regeneration. Unlike human mesenchymal stem cells (hMSC), hFBC are partially differentiated with high expansion and regeneration potential. To date, proliferative and osteoblastic differentiation capacities of fetal bone cells remain poorly examined. The goal of this study was to define an environmental culture conditions for optimal proliferation and production of extracellular bone matrix leading to efficient bone repair. METHODS. Human primary FBC derived from our dedicated, consistent banks of bone cells comprising several fetal donors. For proliferation study, monolayer cultures of both cell types were expanded in DMEM or α- MEM media. Osteoblastic differentiation potentials of both hFBC and hMSC were evaluated through RT-PCR. Regulation of osteogenic differentiation by protein ligands Wnt3a and Wnt5a was studied by ALP enzymatic activity measurement. RESULTS. Evaluation of the proliferation rate demonstrated that hFBC proliferated more rapidly in α-MEM medium. Regarding growth factors that could stimulate cell proliferation rate, we observed that PDGF, FGF2 and Wnt3a had positive effects on proliferation of hFBC. Gene expression analysis demonstrated a higher expression of runx2 in hFBC cultured in basal conditions, which was was similar than that was observed in hMSC in osteoinductive culture conditions. Expression of sox9 was very low in hBFC and hMSC, compared to expression observed in fetal cartilage cells. Looking at osteogenic differentiation capacity, ALP activity was positively regulated byWnt5awhen hFBCwere cultured inα-MEM, but not in DMEM. Conversely, Wnt3a was shown to block the effect of osteogenic inductors on differentiation of both cell types. CONCLUSION. Data presented in this study indicate that the proliferation and differentiation of fetal and mesenchymal stem cells is optimal in α- MEM. Evidence for a pre-differentiated state of hBFC was given by extracellular matrix spontaneous mineralization as well as by higher ALP activity levels observed for these cells in baseline culture conditions, in comparison with hMSC. As we showed that, in vitro, hFBC express a higher capacity to differentiate in osteoblasts, they represent an attractive and promising prospect for fundamental research, and specifically for a new generation of skeletal tissue engineering.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Pharmacological Therapy in the Heart as an Alternative to Cellular Therapy: A Place for the Brain Natriuretic Peptide?

The discovery that stem cells isolated from different organs have the ability to differentiate into mature beating cardiomyocytes has fostered considerable interest in developing cellular regenerative therapies to treat cardiac diseases associated with the loss of viable myocardium. Clinical studies evaluating the potential of stem cells (from heart, blood, bone marrow, skeletal muscle, and fat) to regenerate the myocardium and improve its functional status indicated that although the method appeared generally safe, its overall efficacy has remained modest. Several issues raised by these studies were notably related to the nature and number of injected cells, as well as the route and timing of their administration, to cite only a few. Besides the direct administration of cardiac precursor cells, a distinct approach to cardiac regeneration could be based upon the stimulation of the heart's natural ability to regenerate, using pharmacological approaches. Indeed, differentiation and/or proliferation of cardiac precursor cells is controlled by various endogenous mediators, such as growth factors and cytokines, which could thus be used as pharmacological agents to promote regeneration. To illustrate such approach, we present recent results showing that the exogenous administration of the natriuretic peptide BNP triggers "endogenous" cardiac regeneration, following experimental myocardial infarction.

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis

Novel mechanisms of growth hormone regulation: growth hormone-releasing peptides and ghrelin

Growth hormone secretion is classically modulated by two hypothalamic hormones, growth hormone-releasing hormone and somatostatin. A third pathway was proposed in the last decade, which involves the growth hormone secretagogues. Ghrelin is a novel acylated peptide which is produced mainly by the stomach. It is also synthesized in the hypothalamus and is present in several other tissues. This endogenous growth hormone secretagogue was discovered by reverse pharmacology when a group of synthetic growth hormone-releasing compounds was initially produced, leading to the isolation of an orphan receptor and, finally, to its endogenous ligand. Ghrelin binds to an active receptor to increase growth hormone release and food intake. It is still not known how hypothalamic and circulating ghrelin is involved in the control of growth hormone release. Endogenous ghrelin might act to amplify the basic pattern of growth hormone secretion, optimizing somatotroph responsiveness to growth hormone-releasing hormone. It may activate multiple interdependent intracellular pathways at the somatotroph, involving protein kinase C, protein kinase A and extracellular calcium systems. However, since ghrelin has a greater ability to release growth hormone in vivo, its main site of action is the hypothalamus. In the current review we summarize the available data on the: a) discovery of this peptide, b) mechanisms of action of growth hormone secretagogues and ghrelin and possible physiological role on growth hormone modulation, and c) regulation of growth hormone release in man after intravenous administration of these peptides.