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INTRODUCTION: The quest for novel MS therapies focuses on promoting remyelination and neuroprotection, necessitating innovative drug design paradigms and robust preclinical validation methods to ensure efficient clinical translation. The complexity of new drugs action mechanisms is strengthening the need for solid biological validation attempting to address all possible pitfalls and biases precluding access to efficient and safe drugs. AREAS COVERED: In this review, the authors describe the different in vitro and in vivo models that should be used to create an integrated approach for preclinical validation of novel drugs, including the evaluation of the action mechanism. This encompasses 2D, 3D in vitro models and animal models presented in such a way to define the appropriate use in a global process of drug screening and hit validation. EXPERT OPINION: None of the current available tests allow the concomitant evaluation of anti-inflammatory, immune regulators or remyelinating agents with sufficient reliability. Consequently, the collaborative efforts of academia, industry, and regulatory agencies are essential for establishing standardized protocols, validating novel methodologies, and translating preclinical findings into clinically meaningful outcomes.
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Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease leading to demyelination and axonal loss. Current treatments are immunomodulatory or immunosuppressive drugs acting on the inflammatory component. However, these treatments do not adequately address the crucial aspect of neuroprotection. Recently, an association between an altered balance of adipokines and MS has been proposed as both a risk factor for developing MS and a chronic disease aggravating factor. Specifically, a decrease of apelin plasma levels in MS patients compared to controls correlates with the number of relapses and disease severity. Here we report a dramatic downregulation of apelin levels in the CNS of EAE mice which is also detected in MS patients brain samples compared to controls. Exploiting innovative design and synthesis techniques, we engineered a novel fluorinated apelin-13 peptide characterized by enhanced plasmatic stability compared to its native counterpart. With this peptide, we assessed the potential therapeutic benefits of apelin preventive supplementation in the EAE mouse model. We show that the fluorinated Apelin-13 peptide ameliorates EAE clinical score and preserves myelin content in the EAE MOG model recapitulating the progressive form of disease. These results combined with ex-vivo experiments in brain organotypic slices and in vitro studies in neurons and primary microglia and macrophages suggest that apelin has neuroprotective effects and influences the microglia/macrophages function.
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Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Esclerosis Múltiple , Fármacos Neuroprotectores , Animales , Fármacos Neuroprotectores/farmacología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Ratones , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Microglía/efectos de los fármacos , Microglía/metabolismo , Apelina/metabolismo , Apelina/farmacologíaRESUMEN
Interleukin 10 (IL-10) exerts anti-inflammatory and immune regulatory roles through its fixation to the IL-10 receptor (IL-10R). The two subunits (IL-10Rα and IL-10Rß) organise themselves to form a hetero-tetramer to induce the activation of the transcription factor STAT3. We analysed the activation patterns of the IL-10R, especially the contribution of the transmembrane (TM) domain of the IL-10Rα and IL-10Rß subunits, as evidence accumulates that this short domain has tremendous implications in receptor oligomerisation and activation. We also addressed whether targeting the TM domain of IL-10R with peptides mimicking the TM sequences of the subunits translates into biological consequences. The results illustrate the involvement of the TM domains from both subunits in receptor activation and feature a distinctive amino acid crucial for the interaction. The TM peptide targeting approach also appears to be suitable for modulating the activation of the receptor through its action on the dimerization capabilities of the TM domains and thereby constitutes a potential new strategy for the modulation of the inflammation in pathologic contexts.
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Regulación de la Expresión Génica , Factores de Transcripción , Receptores de Interleucina-10 , Transducción de Señal , AminoácidosRESUMEN
Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system associated with chronic inflammation, demyelination, and axonal damage. MS is a highly heterogeneous disease that leads to discrepancies regarding the clinical appearance, progression, and therapy response of patients. Therefore, there is a strong unmet need for clinically relevant biomarkers capable of recapitulating the features of the disease. Experimental autoimmune encephalomyelitis (EAE) is a valuable model for studying the pathophysiology of MS as it recapitulates the main hallmarks of the disease: inflammation, blood-brain barrier (BBB) disruption, gliosis, myelin damage, and repair mechanisms. In this study, we used the EAE-PLP animal model and established a molecular RNA signature for each phase of the disease (onset, peak, remission). We compared variances of expression of known biomarkers by RT-qPCR in the brain and spinal cord of sham and EAE animals monitoring each of the five hallmarks of the disease. Using magnetic cell isolation technology, we isolated microglia and oligodendrocytes of mice of each category, and we compared the RNA expression variations. We identify genes deregulated during a restricted time frame, and we provide insight into the timing and interrelationships of pathological disease processes at the organ and cell levels.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Ratones , Esclerosis Múltiple/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación , Biomarcadores , ARNRESUMEN
Multiple Sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system which is characterized by demyelinating lesions and axonal damage. MS is a complex disease characterized by important pathophysiological heterogeneity affecting the clinical appearance, progression and therapeutic response for each patient. Therefore, there is a strong unmet need to define specific biomarkers that will reflect the different features of the disease. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for the study of MS, as it resembles the pathological features of human MS in many aspects and has allowed for the elucidation of pathogenesis pathways and the validation of certain targets for MS therapies. In this review, we discuss clinically relevant MS molecular biomarkers, divided into five groups based on the key pathological hallmarks of MS: inflammation, blood-brain barrier disruption, myelin and axonal damage, gliosis and, ultimately, repair mechanisms. To address the feasibility of translation between the animal model and human disease, we present an overview of several molecular biomarkers of each category and compare their respective deregulation patterns. We conclude that, like any disease animal model, EAE models can sometimes fail to mimic the entire spectrum of human disease, but they can nonetheless recapitulate the disease's primary hallmarks. We show that the EAE model is a valuable tool for understanding MS physiopathological mechanisms and for identifying biomarkers fundamental for drug development.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Biomarcadores , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Humanos , Esclerosis Múltiple/patología , Vaina de Mielina/patologíaRESUMEN
Macrophages and microglia are implicated in several diseases with divergent roles in physiopathology. This discrepancy can be explained by their capacity to endorse different polarization states. Theoretical extremes of these states are called M1 and M2. M1 are pro-inflammatory, microbicidal, and cytotoxic whereas M2 are anti-inflammatory, immunoregulatory cells in favor of tumor progression. In pathological states, these polarizations are dysregulated, thus restoring phenotypes could be an interesting treatment approach against diseases. In this review, we will focus on compounds targeting macrophages and microglia polarization in two very distinctive pathologies: multiple sclerosis and glioblastoma. Multiple sclerosis is an inflammatory disease characterized by demyelination and axon degradation. In this case, macrophages and microglia endorse a M1-like phenotype inducing inflammation. Promoting the opposite M2-like polarization could be an interesting treatment strategy. Glioblastoma is a brain tumor in which macrophages and microglia facilitate tumor progression, spreading, and angiogenesis. They are part of the tumor associated macrophages displaying an anti-inflammatory phenotype, thereby inhibiting anti-tumoral immunity. Re-activating them could be a method to limit and reduce tumor progression. These two pathologies will be used to exemplify that targeting the polarization of macrophages and microglia is a promising approach with a broad spectrum of applications deserving more attention.
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Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation.
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Apoptosis/genética , Dominios Proteicos/genética , Multimerización de Proteína/genética , Receptor fas/genética , Diferenciación Celular/genética , Homeostasis/genética , Humanos , Ligandos , Mutación/genética , Receptores de Muerte Celular/genética , Transducción de Señal/genéticaRESUMEN
A better understanding of tumor development is crucial for treating cancer. Polarimetric imaging is an interesting alternative for monitoring subcutaneous tumors as it is non-invasive. In this study, a Mueller spectro-polarimeter is used to monitor tumor development on mice injected with non-pigmented breast cancer cells or with pigmented murine melanoma cells. Three stages of non-pigmented tumor development are revealed with three polarimetric parameters. These stages also appear for pigmented tumors, although less clearly. A halo of high depolarization surrounding the non-pigmented tumor in the first stage allows the outlining of the tumor. Considering polarimetric parameters, a biological interpretation is proposed.
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In demyelinated lesions, astrocytes, activated microglia and infiltrating macrophages secrete several factors regulating oligodendrocyte precursor cells' behaviour. What appears to be the initiation of an intrinsic mechanism of myelin repair is only leading to partial recovery and inefficient remyelination, a process worsening over the course of the disease. This failure is largely due to the concomitant accumulation of inhibitory cues in and around the lesion sites opposing to growth promoting factors. Here starts a complex game of interactions between the signalling pathways controlling oligodendrocytes migration or differentiation. Receptors of positive or negative cues are modulating Ras, PI3K or RhoGTPases pathways acting on oligodendrocyte cytoskeleton remodelling. From the description of this intricate signalling network, this review addresses the extent to which the modulation of the global response to inhibitory cues may pave the route towards novel therapeutic approaches for myelin repair.
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Diferenciación Celular , Esclerosis Múltiple/terapia , Oligodendroglía/citología , Regeneración , Remielinización , Animales , Humanos , Oligodendroglía/fisiologíaRESUMEN
Microglial cells are key players in neural pathogenesis and microglial function regulation appears to be pivotal in controlling neuroinflammatory/neurological diseases. Here, we investigated the effects and mechanism of action of neurosteroid allopregnanolone (ALLO) on murine microglial BV-2 cells and primary microglia in order to determine ALLO-induced immunomodulatory potential and to provide new insights for the development of both natural and safe neuroprotective strategies targeting microglia. Indeed, ALLO-treatment is increasingly suggested as beneficial in various models of neurological disorders but the underlying mechanisms have not been elucidated. Therefore, the microglial cells were cultured with various serum concentrations to mimic the blood-brain-barrier rupture and to induce their activation. Proliferation, viability, RT-qPCR, phagocytosis, and morphology analyzes, as well as migration with time-lapse imaging and quantitative morphodynamic methods, were combined to investigate ALLO actions on microglia. BV-2 cells express subunits of GABA-A receptor that mediates ALLO activity. ALLO (10µM) induced microglial cell process extension and decreased migratory capacity. Interestingly, ALLO modulated the phagocytic activity of BV-2 cells and primary microglia. Our results, which show a direct effect of ALLO on microglial morphology and phagocytic function, suggest that the natural neurosteroid-based approach may contribute to developing effective strategies against neurological disorders that are evoked by microglia-related abnormalities.
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Forma de la Célula , Microglía/citología , Microglía/metabolismo , Neuroprotección , Neuroesteroides/metabolismo , Fagocitosis , Pregnanolona/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Modelos Biológicos , Neuroprotección/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Fagocitosis/efectos de los fármacos , Pregnanolona/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , SueroRESUMEN
BACKGROUND: Central nervous system damage in multiple sclerosis (MS) is responsible for serious deficiencies. Current therapies are focused on the treatment of inflammation; however, there is an urgent need for innovative therapies promoting neuroregeneration, particularly myelin repair. It is demonstrated that testosterone can act through neural androgen receptors and several clinical observations stimulated an interest in the potential protective effects of testosterone treatment for MS. Here, we sought to demonstrate the effects of a testosterone supplementation in testosterone-deficient men with relapsing-remitting MS. METHODS/DESIGN: This report presents the rationale and methodology of TOTEM RRMS, a French, phase 2, multicenter, randomized, placebo-controlled, and double-blind trial, which aims to prevent the progression of MS in men with low testosterone levels by administration of testosterone undecanoate, who were kept under natalizumab (Tysabri®) to overcome the anti-inflammatory effect of testosterone. Forty patients will be randomized into two groups receiving either a testosterone treatment (Nebido®) or a matching placebo. The intervention period for each group will last 66 weeks (treatment will be injected at baseline, week 6, and then every 12 weeks). The main objective is to determine the neuroprotective and remyelinating effects of testosterone using tensor diffusion imaging techniques and thalamic atrophy analyses. As secondary objectives, impacts of the testosterone supplementation will be studied using other conventional and unconventional MRI parameters and with clinical outcomes. DISCUSSION: The action of testosterone is observed in different experimental autoimmune encephalomyelitis models and epidemiological studies in humans. However, despite several preclinical data and some small clinical trials in MS, clear evidence for a therapeutic effect of hormone therapy is still missing. Therefore, our goal is to demonstrate the effects of testosterone therapies in MS. As there is no effective treatment currently available on fatigue in MS, careful attention should also be paid to secondary endpoints: fatigue, cognitive functions, and other symptoms that may improve life quality. Assuming a positive outcome of the trial, this treatment could be considered as a new neuroprotective and remyelinating therapy in relapsing-remitting MS and could be applicable to other demyelinating diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT03910738. Registered on 10 April 2019.
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Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Vaina de Mielina/efectos de los fármacos , Testosterona/uso terapéutico , Progresión de la Enfermedad , Método Doble Ciego , Humanos , Imagen por Resonancia Magnética , Masculino , Estudios Multicéntricos como Asunto , Neuroprotección , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del TratamientoRESUMEN
Cellular functions are regulated by extracellular signals such as hormones, neurotransmitters, matrix ligands, and other chemical or physical stimuli. Ligand binding on its transmembrane receptor induced cell signaling and the recruitment of several interacting partners to the plasma membrane. Nowadays, it is well-established that the transmembrane domain is not only an anchor of these receptors to the membrane, but it also plays a key role in receptor dimerization and activation. Indeed, interactions between transmembrane helices are associated with specific biological activity of the proteins as cell migration, proliferation, or differentiation. Overexpression or constitutive dimerization (due notably to mutations) of these transmembrane receptors are involved in several physiopathological contexts as cancers. The transmembrane domain of tyrosine kinase receptors as ErbB family proteins (implicated in several cancers as HER2 in breast cancer) or other receptors as Neuropilins has been described these last years as a target to inhibit their dimerization/activation using several strategies. In this review, we will focus on the strategy which consists in using peptides to disturb in a specific manner the interactions between transmembrane domains and the signaling pathways (induced by ligand binding) of these receptors involved in cancer. This approach can be extended to inhibit other transmembrane protein dimerization as neuraminidase-1 (the catalytic subunit of elastin receptor complex), Discoidin Domain Receptor 1 (a tyrosine kinase receptor activated by type I collagen) or G-protein coupled receptors (GPCRs) which are involved in cancer processes.
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The presence of stromal cells in tumors is altering the significance of molecular profiling when using standard methods of gene expression quantification. We developed a novel normalization method to rank target gene expression in tumor samples by comparisons with reference samples representing the different cell types found in a tumor. The score for each target gene obtained after normalization, is aimed to be predictive of targeted therapies efficiency. We performed this qPCR analysis on human colorectal cancers to demonstrate the importance of reference samples to obtain accurate data and on a collection of patient-derived xenografted (PDX) colon tumors treated with Cetuximab (anti-EGFR) to demonstrate that the calculated EGFR score is predictive of Cetuximab efficacy. Interestingly, the score allowed to select an efficient treatment in a PDX model refractory to standard of care. This method is opening a novel way to predict targeted therapy efficiency which could be extended to several tumor types, and to unlimited target genes.
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Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.
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Current treatments in multiple sclerosis (MS) are modulating the inflammatory component of the disease, but no drugs are currently available to repair lesions. Our study identifies in MS patients the overexpression of Plexin-A1, the signalling receptor of the oligodendrocyte inhibitor Semaphorin 3A. Using a novel type of peptidic antagonist, we showed the possibility to counteract the Sema3A inhibitory effect on oligodendrocyte migration and differentiation in vitro when antagonizing Plexin-A1. The use of this compound in vivo demonstrated a myelin protective effect as shown with DTI-MRI and confirmed at the histological level in the mouse cuprizone model of induced demyelination/remyelination. This effect correlated with locomotor performances fully preserved in chronically treated animals. The administration of the peptide also showed protective effects, leading to a reduced severity of demyelination in the context of experimental autoimmune encephalitis (EAE). Hence, the disruption of the inhibitory microenvironmental molecular barriers allows normal myelinating cells to exert their spontaneous remyelinating capacity. This opens unprecedented therapeutic opportunity for patients suffering a disease for which no curative options are yet available.
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Esclerosis Múltiple/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/fisiología , Receptores de Superficie Celular/metabolismo , Remielinización , Semaforina-3A/metabolismo , Transducción de Señal , Animales , Encéfalo/diagnóstico por imagen , Línea Celular , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidoresRESUMEN
Components with self-assembly properties derived from plant viruses provide the opportunity to design biological nanoscaffolds for the ordered display of agents of diverse nature and with complementing functions. With the aim of designing a functionalized nanoscaffold to target cancer, the coat protein (CP) of Tobacco mosaic virus (TMV) was tested as nanocarrier for an insoluble, highly hydrophobic peptide that targets the transmembrane domain of the Neuropilin-1 (NRP1) receptor in cancer cells. The resulting construct CPL-K (CP-linker-"Kill") binds to NRP1 in cancer cells and disrupts NRP1 complex formation with PlexA1 as well as downstream Akt survival signaling. The application of CPL-K also inhibits angiogenesis and cell migration. CP was also fused to a peptide that targets the extracellular domain of NRP1 and this fusion protein (CPL-F, CP-Linker-"Find") is shown to bind to cultured cancer cells and to inhibit NRP1-dependent angiogenesis as well. CPL-K and CPL-F maintain their anti-angiogenic properties upon co-assembly to oligomers/nanoparticles together with CPL. The observations show that the CP of TMV can be employed to generate a functionalized nanoparticle with biological activity. Remarkably, fusion to CPL allowed us to solubilize the highly insoluble transmembrane NRP1 peptide and to retain its anti-angiogenic effect.
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The sensory innervation of the dental pulp is essential for tooth function and protection. It is mediated by axons originating from the trigeminal ganglia and is spatio-temporally regulated. We have previously shown that the innervation of bioengineered teeth can be achieved only under immunosuppressive conditions. The aim of this study was to develop a model to determine the role of Semaphorin 3A (Sema3A) in the innervation of bioengineered teeth. We first analysed innervation of the dental pulp of mandibular first molars in newborn (postnatal day 0: PN0) mice deficient for Sema3A (Sema3A-/- ), a strong inhibitor of axon growth. While at PN0, axons detected by immunostaining for peripherin and NF200 were restricted to the peridental mesenchyme in Sema3A+/+ mice, they entered the dental pulp in Sema3A-/- mice. Then, we have implanted cultured teeth obtained from embryonic day-14 (E14) molar germs of Sema3A-/- mice together with trigeminal ganglia. The dental pulps of E14 cultured and implanted Sema3A-/- teeth were innervated, whereas the axons did not enter the pulp of E14 Sema3A+/+ cultured and implanted teeth. A "Membrane Targeting Peptide NRP1," suppressing the inhibitory effect of Sema3A, has been previously identified. The injection of this peptide at the site of implantation allowed the innervation of the dental pulp of bioengineered teeth obtained from E14 dental dissociated mesenchymal and epithelial cells reassociations of ICR mice. In conclusion, these data show that inhibition of only one axon repellent molecule, Sema3A, allows for pulp innervation of bioengineered teeth.
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Pulpa Dental , Diente Molar , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Ingeniería de Tejidos , Ganglio del Trigémino , Animales , Pulpa Dental/inervación , Pulpa Dental/metabolismo , Pulpa Dental/patología , Mandíbula/inervación , Mandíbula/metabolismo , Mandíbula/patología , Ratones , Ratones Endogámicos ICR , Ratones Mutantes , Diente Molar/inervación , Diente Molar/metabolismo , Diente Molar/patología , Receptores de Superficie Celular/genética , Semaforina-3A/genética , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/patologíaRESUMEN
Recently, it has been shown that the efficiency of antitumoral drugs can be enhanced when combined with therapeutic siRNAs. In the present study, an original platform based on polydiacetylenic micelles containing a cationic head group able to efficiently deliver a small interfering RNA (siRNA) targeting the PLK-1 gene while offering a hydrophobic environment for encapsulation of lipophilic drugs such as camptothecin is developed. We demonstrate that the co-delivery of these two agents with our micellar system results in a synergistic tumor cell killing of cervical and breast cancer cell lines in vitro. The combined drugs are active in a subcutaneous in vivo cancer model. Altogether, the results show that our nanometric micellar delivery system can be used for the development of new drug-siRNA combo-therapies.
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The axonal growth cone is a specialized structure enabling axon extension and proper guidance to its target by sensing the extracellular environment. A growth cone collapse assay is a popular approach designed to characterize the inhibitory effect of secreted guidance cues in vitro. However, the actin cytoskeleton of the growth cone is very sensitive to various factors like physical impact, temperature, and acidity of environment that may also induce responses resembling those of guidance signals. Herein, we provide an easy and reproducible method to analyze growth cone sensitivity to the prototypic guidance molecule family class 3 semaphorin. This protocol is intended to present a systematic approach that is easy to apply to any soluble factors with a potential to impact axon elongation.
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Axones/fisiología , Conos de Crecimiento/fisiología , Semaforina-3A/fisiología , Citoesqueleto/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Neuronas/fisiología , Reproducibilidad de los ResultadosRESUMEN
The Semaphorin/Neuropilin/Plexin (SNP) complexes control a wide range of biological processes. Consistently, activity deregulation of these complexes is associated with many diseases. The increasing knowledge on SNP had in turn validated these molecular complexes as novel therapeutic targets. Targeting SNP activities by small molecules, antibodies and peptides or by soluble semaphorins have been proposed as new therapeutic approach. This review is focusing on the latest demonstration of this potential and discusses some of the key questions that need to be addressed before translating SNP targeting into clinically relevant approaches.