Desenvolupament d'hidrogels superficials que contenen motius d'adhesió cel·lular

Projecte de recerca elaborat a partir d’una estada al Departament d’Enginyeria Química del Massachusetts Institute of Technology entre abril i octubre del 2006. S’ha dissenyat i sintetitzat uns nous films polimèrics, amb aplicacions en l’àmbit de l’enginyeria de teixits, utilitzant la tècnica anomenada iCVD (initiated Chemical Vapor Deposition), prèviament desenvolupada pel grup receptor. Es tracta d’uns hidrogels superficials de gruix controlable, que incorporen un monòmer fluorat, el qual s’havia estudiat extensament en el grup d’origen. Aquest monòmer es caracteritza per reaccionar molt fàcilment amb pèptids, de manera que aquests queden units covalentment a la superfície. Diferents estratègies pel desenvolupament d’aquests copolímers han estat avaluades, tant des del punt de vista purament sintètic com de la pròpia aplicació. Les condicions de polimerització han estat optimitzades i els hidrogels s’han caracteritzat químicament per tècniques espectroscòpiques (FTIR, XPS), i físicament per angle de contacte i el·lipsometria. D’aquesta manera, s’ha estudiat la capacitat dels hidrogels d’absorbir aigua i alhora augmentar el seu gruix, depenent de la quantitat d’agent reticulant introduït i de la incorporació del nou monòmer. A continuació, s’han optimitzat les condicions de reacció d’aquestes superfícies amb pèptids que incorporen una molècula fluorescent, la qual permet detectar fàcilment per microscòpia de fluorescència si la reacció ha tingut lloc. Una vegada la plataforma ha estat posada a punt, s’han iniciat assajos cel·lulars tant amb fibroblasts embriònics de ratolí com amb cèl·lules humanes umbilicals. Els resultats preliminars suggereixen una morfologia diferent de les cèl·lules segons si es cultiven sobre films modificats amb pèptids que promouen l’adhesió cel·lular o sobre les seves seqüències permutades no actives. Però, el més interessant és que també s’han observat certes diferències depenent si els films contenen el component hidrogel o no, fet que suggeriria un paper actiu d’aquests noves superfícies en el comportament cel·lular.

Rôle de l'activation de c-Jun N-terminal kinase (JNK) dans un modèle de glaucome chez le rat et neuroprotection par inhibition de la voie de JNK

RESUME : Dans ce travail effectué chez le rat adulte, l'excitotoxicité rétinienne est élicitée par injection intravitréenne de NMDA. Les lésions en résultant sont localisées dans la rétine interne. Elles prennent la forme de pycnoses dans la couche des cellules ganglionnaires (corps cellulaires des cellules ganglionnaires et amacrines déplacées) et dans la partie interne de la couche nucléaire interne (cellules amacrines). Cette localisation est liée à la présence de récepteurs au glutamate de type NMDA sur ces cellules. L'activation de ces récepteurs entraîne un influx calcique et l'activation de diverses enzymes (phospholipase A, calpaïnes, calmoduline, synthase d'oxyde nitrique). La signalisation se poursuit en aval en partie par les voies des Mitogen Activated Protein Kinase (MAPK) : ERK, p38, ]NK. Dans les expériences présentées, toutes trois sont activées après l'injection de NMDA. Dans les cascades de signalisation de JNK, trois kinases s'ancrent sur une protéine scaffold. Les MAPKKK phosphorylent MKK4 et MKK7, qui phosphorylent JNK. JNK a de nombreuses cibles nucléaires (dont le facteur de transcription c-Jun) et cytoplasmiques. La voie de JNK est bloquée par l'inhibiteur peptidique D-JNKI-1 en empêchant l'interaction de la kinase avec son substrat. L'inhibiteur est formé de 20 acides aminés du domaine de liaison JBD et de 10 acides aminés de la partie TAT du virus HIV. L'injection intravitréenne de D-JNKI-1 permet une diminution des taux de JNK et c-Jun phosphorylés dans les lysats de rétine. L'effet prépondérant est la restriction importante des altérations histologiques des couches internes de la rétine. L'évaluation par électrorétinogramme met en sus en évidence une sauvegarde de la fonction cellulaire. Ce travail a ainsi permis d'établir la protection morphologique et fonctionnelle des cellules de la rétine interne par inhibition spécifique de la voie de JNK lors d'excitotoxicité. SUMMARY Excitotoxicity in the retina associates with several pathologies like retinal ischemia, traumatic optic neuropathy and glaucoma. In this study, excitotoxicity is elicited by intravitreal NMDA injection in adult rats. Lesions localise in the inner retina. They present as pyknotic cells in the ganglion cell layer (ganglion cells and displaced amacrines) and the inner nuclear layer (amacrine cells). These cells express NMDA glutamate receptors. The receptor activation leads to a calcium flow into the cell and hence enzyme activation (phospholipase, calpains, calmodulin, nitric oxide synthase). The subsequent signaling pathways can involve the Mitogen Activated Protein Kinases (MAPK): ERK, p38 end JNK. These were all activated in our experiments. The signaling cascade organises around several scaffold proteins. The various MAPKKK phosphorylate MKK4 and MKK7, which phosphorylate JNK. JNK targets are of nuclear (c-Jun transcription factor) or cytoplasmic localisation. The peptidic inhibitor D-JNKI-1, 20 amino acids from the JNK binding domain JBD coupled to 10 amino acids of the TAT transporter, disrupts the binding of JNK with its substrate. Intravitreal injection of the inhibitor lowers phosphorylated forms of JNK and c-Jun in retinal extracts. It protects strongly against histological lesions in the inner retina and allows functional rescue.

Desenvolupament de vacunes peptídiques y dendrímers basats en polietilenglicol per al tractament del càncer

El principal problema de les teràpies actuals contra el càncer es la baixa especificitat envers les cèl•lules tumorals, cosa que comporta gran quantitat d’efectes secundaris. Per això es important el desenvolupament de nous tipus de teràpies i sistemes d’alliberament efectius per als fàrmacs ja existents al mercat. En la immunoteràpia contra el càncer es pretén estimular el sistema immunològic per a eliminar les cèl•lules canceroses de manera selectiva. En aquest projecte s’han sintetitzat derivats de l’antigen peptídic de melanoma NY-ESO1 i s’ha estudiat la seva capacitat per a estimular el sistema immunològic com a vacunes contra el càncer. També s’han encapsulat el antígens peptídics en liposomes com a adjuvants i sistemes d’alliberament. De totes les variants peptídiques la que resultà més immunogènica fou la que contenia el grup palmitoil i el fragment toxoide tetànic en la seva estructura. La utilització de liposomes com a sistema adjuvant sembla una estratègia interessant per al disseny de vacunes contra el càncer donat que l’encapsulació del pèptid en liposomes va augmentar notablement la resposta immunològica de l’antigen. Per altra banda, s’han desenvolupat dendrímers basats en polietilenglicol com a sistemes alliberadors de fàrmacs per al tractament de tumors. El polietilenglicol és àmpliament utilitzat com a sistema d’alliberament de fàrmacs degut a les seves interessants propietats, augment de la solubilitat i dels temps de residència en plasma, entre d’altres. La metodologia química descrita permet la diferenciació controlada de varies posicions en la superfície del dendrímer a més del creixement del dendrímer fins a una segona generació. S’ha sintetitzat la primera generació del dendrímer contenint el fàrmac antitumoral 5-fluorouracil i s’han realitzat estudis de citotoxicitat comprovant que l’activitat del nanoconjugat és del mateix ordre de magnitud que el 5-fluorouracil sense conjugar.

Glioprotective impact of cell-permeable peptides in preclinical models of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by progressive degeneration of upper and lower motor neurons. It is mostly sporadic, but about 2% of cases are associated with mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1). A major constraint to the comprehension of the pathogenesis of ALS has been long represented by the conviction that this disorder selectively affects motor neurons in a cell-autonomous manner. However, the failure to identify the events underlying the neurodegenerative process and the increased knowledge of the complex cellular interactions necessary for the correct functioning of the CNS has recently focused the attention on the contribution to neurodegeneration of glial cells, including astrocytes. Astrocytes can hurt motor neurons directly by secreting neurotoxic factors, but they can also play a deleterious role indirectly by losing functions that are supportive for neurons. Recently, we reported that a subpopulation of astrocytes degenerates in the spinal cord of hSOD1G93A transgenic mouse model of ALS. Mechanistic studies in cultured astrocytes revealed that such effect is mediated by the excitatory amino acid glutamate.On the bsis of these observations, we next used the established cell culture model as a tool to screen the glioprotective effect of innovative drugs, namely cell-permeable therapeutics. These consist of peptidic effector moieties coupled to the selective intracellular peptide transporter TAT protein. We initially validated the usefulness of these molecules demonstrating that a control fluorescent peptide enters astrocytes in culture and is retained within the cells up to 24-48 h, according to the timing of our cytotoxicity experiments. We then tested the impact of specific intracellular peptides with antiapoptotic properties on glutamate-treated hSOD1G93A- expressing astrocytes and we identified one molecule that protects the cells from death. Chronic treatment of ALS mice with this peptide had a positive impact on the outcome of the disease.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Gut-derived signaling molecules and vagal afferents in the control of glucose and energy homeostasis.

PURPOSE OF REVIEW: The control of glucose and energy homeostasis, including feeding behaviour, is tightly regulated by gut-derived peptidic and nonpeptidic endocrine mediators, autonomic nervous signals, as well as nutrients such as glucose. We will review recent findings on the role of the gastrointestinal tract innervation and of portal vein glucose sensors; we will review selected data on the action of gastrointestinally released hormones. RECENT FINDINGS: The involvement of mechanosensory vagal afferents in postprandial meal termination has been clarified using mouse models with selective impairments of genes required for development of mechanosensory fibres. These activate central glucogen-like peptide-1/glucogen-like peptide-2 containing ascending pathways linking the visceroceptive brainstem neurons to hypothalamic nuclei. Mucosal terminals comprise the chemosensory vagal afferents responsive to postprandially released gastrointestinal hormones. The mechanism by which the hepatoportal glucose sensor stimulates glucose utilization by muscles was demonstrated, using genetically modified mice, to be insulin-independent but to require GLUT4 and AMP-kinase. This sensor is a key site of glucogen-like peptide-1 action and plays a critical role in triggering first phase insulin secretion. PeptideYY and ghrelin target intracerebral receptors as they are bidirectionally transported across the blood brain barrier. The anorectic functions of peripherally released peptideYY may however be mediated both via vagal afferents and intracerebral Y2 receptors in the brainstem and arcuate nucleus. SUMMARY: These recent findings demonstrate that the use of improved anatomical and physiological techniques and animal models with targeted gene modifications lead to an improved understanding of the complex role of gastrointestinal signals in the control of energy homeostasis.

N-cadherin as a therapeutic target in cancer.

During tumor progression, cancer cells undergo dramatic changes in the expression profile of adhesion molecules resulting in detachment from original tissue and acquisition of a highly motile and invasive phenotype. A hallmark of this change, also referred to as the epithelial-mesenchymal transition, is the loss of E- (epithelial) cadherin and the de novo expression of N- (neural) cadherin adhesion molecules. N-cadherin promotes tumor cell survival, migration and invasion, and a high level of its expression is often associated with poor prognosis. N-cadherin is also expressed in endothelial cells and plays an essential role in the maturation and stabilization of normal vessels and tumor-associated angiogenic vessels. Increasing experimental evidence suggests that N-cadherin is a potential therapeutic target in cancer. A peptidic N-cadherin antagonist (ADH-1) has been developed and has entered clinical testing. In this review, the authors discuss the biochemical and functional features of N-cadherin, its potential role in cancer and angiogenesis, and summarize the preclinical and clinical results achieved with ADH-1.

Targeting Angiogenesis in Glioma - Challenges and Pitfalls

Malignant gliomas, notably glioblastoma are among the most vascularized and angiogenic cancers, and microvascular proliferation is one of the hallmarks for the diagnosis of glioblastoma. Angiogenesis is regulated by a balance of pro- and antiangiogenic signals; overexpression of VEGF and activation of its receptors, most notable VEGFR-2 and -3, results in endothelial cell proliferation and leaky vasculature. Heterogeneous perfusion and oxygenation, peritumoral edema and increased interstitial pressure are the consequence. Both endothelial and tumour cells are strongly dependent on integrin-mediated adhesion for cell proliferation, survival, migration and invasion.Strategies aiming at inhibition of cell signaling and angiogenesis, including integrin inhibitors, have been clinically investigated in gliomas over the last 5 years. Radiological responses, a decreased requirement of corticosteroids and temporary improvement in performance status have repeatedly been observed. Toxicity was mild-moderate and manageable, notably there was no evidence for a substantially increased incidence of intracranial bleeding. However definitive comparative (randomized !) investigation has failed to demonstrate improved outcome with singleagent inhibition of EGFR, or PDGFR or VEGF/VEGFRs pathways in recurrent glioblastoma. Definitive phase III trials combining the anti- VEGF monoclonal antibody bevacizumab, or cilengitide, a peptidic integrininhibitor, together with temozolomide and radiotherapy are ongoing (accrual completed).The integration of anti-angiogenic strategies in the management of malignant glioma also poses entirely new challenges in patient management: 1) Many agents are known for increasing the risk of thrombosis, embolism and intracranial bleeding. 2) Evaluation of treatment efficacy is difficult and new biomarkers of activity, including functional, metabolic or molecular imaging techniques are urgently needed. Normalization of vasculature leads to decrease in contrast enhancement without necessarily reflecting tumour shrinkage. Tumour heterogeneity, putative prognostic or predictive factors require early controlled trials, novel trial designs and endpoints.3) Activation of alternate pathways and tumour escape mechanisms may require combination of multiple agents, which is often not feasible due to regulatory restrictions and potential complex toxicities. Emerging clinical and experimental evidence suggests that anti-angiogenic drugs might need to be combined with drugs targeting tumour adaptive mechanisms in addition to cytotoxic chemotherapy and irradiation for a maximal antitumour effect.

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes.

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.

Promises of apoptosis-inducing peptides in cancer therapeutics.

Until recently, most research efforts aimed at developing anti-cancer tools were focusing on small molecules. Alternative compounds are now being increasingly assessed for their potential anti-cancer properties, including peptides and their derivatives. One earlier limitation to the use of peptides was their limited capacity to cross membranes but this limitation was alleviated with the characterization of cell-permeable sequences. Additionally, means are designed to target peptides to their malignant targets. Most anti-cancer peptidic compounds induce apoptosis of tumor cells by modulating the activity of Bcl-2 family members that control the release of death factors from the mitochondria or by inhibiting negative regulators of caspases, the proteases that mediate the apoptotic response in cells. Some of these peptides have been shown to inhibit the growth of tumors in mouse models. Hopefully, pro-apoptotic anti-tumor peptides will soon be tested for their efficacy in patients with cancers.

Síntesis de dendrímeros fosforados funcionalizados con quinina y su utilización como organocatalizadores soportados

En este trabajo nos hemos propuesto preparar organocatalizadores dendriméricos conteniendo en superficie un compuesto del pool chiral, como es la quinina. Ésta es un alcaloide derivado de la familia de la cinchona que presenta la ventaja de ser barato, comercialmente asequible y que ha demostrado ser un buen organocatalizador en varias reacciones químicas. Para llevar a cabo el objetivo propuesto, se preparó un compuesto derivado de la quinina a través de una modificación del grupo vinilo, el cual presentaba un grupo fenol en su estructura que sirvió para el posterior anclaje de la molécula resultante a los dendrímeros fosforados de primera y cuarta generación.

Excitotoxicity-induced endocytosis confers drug targeting in cerebral ischemia.

OBJECTIVE: Targeting neuroprotectants specifically to the cells that need them is a major goal in biomedical research. Many peptidic protectants contain an active sequence linked to a carrier such as the transactivator of transcription (TAT) transduction sequence, and here we test the hypothesis that TAT-linked peptides are selectively endocytosed into neurons stressed by excitotoxicity and focal cerebral ischemia. METHODS: In vivo experiments involved intracerebroventricular injection of TAT peptides or conventional tracers (peroxidase, fluorescein isothiocyanate-dextran) in young rats exposed to occlusion of the middle cerebral artery at postnatal day 12. Cellular mechanisms of uptake were analyzed in dissociated cortical neuronal cultures. RESULTS: In both models, all tracers were taken up selectively into stressed neurons by endocytosis. In the in vivo model, this was neuron specific and limited to the ischemic area, where the neurons displayed enhanced immunolabeling for early endosomal antigen-1 and clathrin. The highly efficient uptake of TAT peptides occurred by the same selective mechanism as for conventional tracers. All tracers were targeted to the nucleus and cytoplasm of neurons that appeared viable, although ultimately destined to die. In dissociated cortical neuronal cultures, an excitotoxic dose of N-methyl-D-aspartate induced a similar endocytosis. It was 100 times more efficient with TAT peptides than with dextran, because the former bound to heparan sulfate proteoglycans at the cell surface, but it depended on dynamin and clathrin in both cases. INTERPRETATION: Excitotoxicity-induced endocytosis is the main entry route for protective TAT peptides and targets selectively the neurons that need to be protected.

Encapsulated, genetically engineered cells, secreting glucagon-like peptide-1 for the treatment of non-insulin-dependent diabetes mellitus.

Non-insulin-dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose-induced insulin secretion by pancreatic beta cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon-like peptide-1 (GLP-1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP-1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP-1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP-1 which has a longer half-life in vivo but which is as potent as the wild-type peptide. The genetically engineered cells are then encapsulated in semi-permeable hollow fibers for implantation in diabetic hosts for constant, long-term, in situ delivery of the peptide. This approach may be a novel therapy for type II diabetes.

Effect of SR 49059, a V1a vasopressin receptor antagonist, in Raynaud's phenomenon.

OBJECTIVE: To assess whether vasopressin V1a receptor blockade reduces the abnormal vasoactive response to cold in patients suffering from Raynaud's phenomenon (RP). METHODS: SR 49059, an orally active, non-peptidic vasopressin V1a receptor antagonist, was given orally (300 mg once daily) to 20 patients with RP in a single-centre, double-blind, placebo-controlled, randomized cross-over study with two 7-day periods of treatment separated by 21 days of washout. Bilateral finger systolic blood pressure and skin temperature were assessed before and after immersion of the hand in cold water for 3 min (15 degrees C) during the screening phase and three times (before and 2 and 4 h after drug intake) on days 1 and 7 of each of the two treatment periods. Recovery of digital pressure and skin temperature was measured 0, 10, 20 and 32 min after the end of the cold immersion test. RESULTS: SR 49059 significantly attenuated the cold-induced fall in systolic pressure by 14.5% (95% confidence interval 0-29; P = 0.045) on the most affected hand on day 7 compared with placebo. Temperature recovery after the end of the cold test showed a trend to enhancement 2 and 4 h after SR 49059 on day 7 (P = 0.060 and P = 0.062 respectively). The beneficial effects on finger pressure and temperature recovery were obtained without changes in supine blood pressure or in heart rate. CONCLUSION: SR 49059 given orally once a day for 7 days to patients with RP showed favourable effects compared with placebo on finger systolic pressure and temperature recovery after cold immersion, without inducing side-effects.