RESUMO
Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.
Assuntos
Antineoplásicos , Imunoconjugados , Camundongos , Animais , Humanos , Imunoconjugados/metabolismo , Acetais , Ado-Trastuzumab Emtansina , Linhagem Celular Tumoral , Antineoplásicos/metabolismo , Hidrazonas , Proteínas de TransporteRESUMO
We previously reported that the nonsteroidal compound CpdX, which was initially characterized 20 y ago as a possible gestagen and, shortly afterward, as a possible drug for treatments of inflammatory diseases, selectively triggers the NFκB/AP1-mediated tethered indirect transrepression function of the glucocorticoid receptor (GR), and could therefore be a selective glucocorticoid receptor agonistic modulator (SEGRAM). We now demonstrate that, upon administration to the mouse, CpdX and one of its deuterated derivatives, CpdX-D3, repress as efficiently as a synthetic glucocorticoid (e.g., Dexamethasone) an induced skin atopic dermatitis, an induced psoriasis-like inflammation, a house dust mite (HDM)-induced asthma-like allergic lung inflammation, a collagen-induced arthritis, an induced ulcerative colitis, and an ovalbumin-induced allergic conjunctivitis. Interestingly, in the cases of an HDM-induced asthma-like allergic lung inflammation and of a collagen-induced arthritis, the CpdX antiinflammatory activity was selectively exerted by one of the two CpdX enantiomers, namely, CpdX(eA) or CpdX-D3(eA).
Assuntos
Anti-Inflamatórios/farmacologia , Glucocorticoides/farmacologia , Inflamação/tratamento farmacológico , Receptores de Glucocorticoides/genética , Animais , Anti-Inflamatórios/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/genética , Artrite Experimental/patologia , Asma/tratamento farmacológico , Asma/genética , Asma/patologia , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Alérgica/genética , Conjuntivite Alérgica/patologia , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/patologia , Dexametasona/farmacologia , Modelos Animais de Doenças , Glucocorticoides/genética , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , NF-kappa B/genética , Ovalbumina/toxicidade , Progestinas/química , Progestinas/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/química , Pele/efeitos dos fármacos , Pele/patologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase, taking part in the activation pathway of the pro-inflammatory transcription factor NF-kB and is demonstrating a therapeutic target potential in inflammatory diseases such as asthma, psoriasis and atherosclerosis. To date, few MSK1 inhibitors were reported. In order to identify new MSK1 inhibitors, a screening of a library of low molecular weight compounds was performed, and the results highlighted the 6-phenylpyridin-2-yl guanidine (compound 1a, IC50~18 µM) as a starting hit for structure-activity relationship study. Derivatives, homologues and rigid mimetics of 1a were designed, and all synthesized compounds were evaluated for their inhibitory activity towards MSK1. Among them, the non-cytotoxic 2-aminobenzimidazole 49d was the most potent at inhibiting significantly: (i) MSK1 activity, (ii) the release of IL-6 in inflammatory conditions in vitro (IC50~2 µM) and (iii) the inflammatory cell recruitment to the airways in a mouse model of asthma.
Assuntos
Desenho de Fármacos , Guanidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Células Cultivadas , Guanidinas/síntese química , Guanidinas/química , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy. Thus, to increase efficacy and avoid tumor recurrence, therapies need to target not only the differentiated cells of the tumor mass, but also the cancer stem-like cells. These therapies need to be effective on cells present in the hypoxic, slightly acidic microenvironment found within tumors. Such a microenvironment is known to favor more aggressive undifferentiated phenotypes and a slow-growing "quiescent state" that preserves the cells from chemotherapeutic agents, which mostly target proliferating cells. Based on these considerations, we performed a differential screening of the Prestwick Chemical Library of approved drugs on both proliferating and quiescent glioblastoma stem-like cells and identified bisacodyl as a cytotoxic agent with selectivity for quiescent glioblastoma stem-like cells. In the present study we further characterize bisacodyl activity and show its efficacy in vitro on clonal macro-tumorospheres, as well as in vivo in glioblastoma mouse models. Our work further suggests that bisacodyl acts through inhibition of Ca2+ release from the InsP3 receptors.
Assuntos
Bisacodil/farmacologia , Neoplasias Encefálicas/patologia , Sinalização do Cálcio , Glioblastoma/patologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismoRESUMO
OBJECTIVE: The chemokine CXCL12 interacting with the CXC receptor 4 (CXCR4) has been reported to play a role in the development and progression of bronchial asthma, but its mechanism of action is still unknown. The objective of this study was to assess the effect of the CXCL12 neutraligand chalcone 4 on the migration of dendritic cells (DCs) in a murine model of allergic airway inflammation. METHODS: A 21-day ovalbumin (OVA)-induced allergic-airway TH2 inflammation model in BALB/c mice was used. Four groups were sensitized with OVA adsorbed on alum and challenged either with OVA or saline for 4 days. Mice were treated intranasally with chalcone 4 (300 nmol/kg body weight) or solvent 2 h before each OVA or saline challenge; 24 h after the last challenge, CD11c+F4/80- DCs were counted in the bronchoalveolar lavage. Jugular-nodose ganglion complex (JNC) sections were sampled, and for immunofluorescence staining, cryocut sections were prepared. MHC II+F4/80- DCs as well as calcitonin gene-related peptide (CGRP)- and substance P (SP)-positive neuronal cell bodies were analyzed. RESULTS: In OVA-challenged mice, chalcone 4 caused a significantly decreased DC/neuron ratio in the JNC from 51.7% in solvent-treated to 32.6% in chalcone 4-treated mice. In parallel, chalcone 4 also decreased the DC population in BALF from 11.5 × 103 cells in solvent to 4.5 × 103 cells in chalcone 4-treated mice. By contrast, chalcone 4 had no effect on the expression of the neuropeptides CGRP and SP in JNC. CONCLUSION: This study reported the CXCL12 neutraligand chalcone 4 to affect DC infiltration into the airways and airway ganglia as well as to decrease airway eosinophilic inflammation and, therefore, validated CXCL12 as a new target in allergic disease models of asthma.
Assuntos
Asma/imunologia , Movimento Celular/imunologia , Chalcona/farmacologia , Quimiocina CXCL12/farmacologia , Células Dendríticas/imunologia , Gânglio Nodoso/imunologia , Animais , Asma/induzido quimicamente , Asma/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Chalcona/uso terapêutico , Quimiocina CXCL12/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Gânglio Nodoso/citologia , Gânglio Nodoso/efeitos dos fármacos , Ovalbumina/toxicidadeRESUMO
OBJECTIVES: Mast cells (MCs) and nerves play an important role in allergic rhinitis (AR), but little is known about their crosstalk in AR. The aim of this study was to investigate MC-nerve interaction in the human nasal mucosa during AR. METHODS: The association between MCs and nerves, the expression of neuropeptide receptors (neurokinin 1 receptor [NK1R], neurokinin 2 receptor [NK2R], calcitonin gene-related peptide receptor [CGRPR], and MrgX2) on MCs, and protease-activated receptor 2 (PAR2) and tyrosine receptor kinase A (TrkA) on nerve fibres in the human nasal mucosa were investigated with immunofluorescence and real-time PCR. RESULTS: The association between MCs and nerves was found to be significantly increased, although the numbers of MCs and nerve fibres were unchanged during AR. MCs expressing tryptase-chymase (MCtc) were frequently associated with nerve fibres and these contacts increased significantly in AR. Neuropeptide receptors NK1R, NK2R, and CGRPR were firstly found to be largely localised on MCs. The number of MCs expressing NK1R and NK2R, but not CGRPR, was significantly increased in AR. Interestingly, MCtc mostly expressed these neuropeptide receptors. The newly discovered tachykinin receptor MrgX2 was not expressed on nasal MCs, but was expressed on gland cells and increased in AR. Additionally, tachykinergic nerve fibres were found to express PAR2 or TrkA as receptors for MCs. CONCLUSIONS: This study revealed for the first time an increase of MC-nerve association and neuropeptide receptor expression on MCs during AR as well as nerve fibres containing receptors for MCs. These results suggest that targeting or controlling airway sensory nerve function as a modulator of MCs may prevent allergic airway inflammation such as AR.
Assuntos
Mastócitos/metabolismo , Mucosa Nasal/inervação , Fibras Nervosas/metabolismo , Receptores de Neuropeptídeos/metabolismo , Rinite Alérgica/patologia , Adolescente , Adulto , Quimases/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/metabolismo , Receptores de Neuropeptídeos/genética , Substância P/metabolismo , Fatores de Transcrição/metabolismo , Triptases/metabolismo , Ubiquitina Tiolesterase/metabolismo , Adulto JovemRESUMO
Mast cells (MCs) are found in large numbers in lungs of patients with pulmonary fibrosis. However, the functions of MCs in lung fibrosis remain largely unknown. We assessed the role of MCs and MC protease 4 (MCPT4), the mouse counterpart of human MC chymase, in a mouse model of bleomycin (BLM)-induced lung injury. We found that levels of inflammation in the bronchoalveolar lavage and the lung, as well as levels of lung fibrosis, were reduced 7 d after intranasal delivery of BLM MC-deficient Kit(W-sh/W-sh) mice compared with wild-type (WT) mice. Confirming the implication of MCs in these processes, we report that the levels of inflammation and fibrosis observed in Kit(W-sh/W-sh) mice can be restored to those observed in WT mice after the adoptive transfer of bone marrow-derived cultured MCs into Kit(W-sh/W-sh) mice. Additionally, we show that levels of inflammation and fibrosis are also reduced in MC chymase MCPT4-deficient mice as compared with WT mice at day 7, suggesting a role for MC-derived MCPT4 in these processes. Our results support the conclusion that MCs can contribute to the initial lung injury induced by BLM through release of the MCPT4 chymase.
Assuntos
Quimases/metabolismo , Mastócitos/imunologia , Pneumonia/imunologia , Fibrose Pulmonar/imunologia , Serina Endopeptidases/metabolismo , Transferência Adotiva , Animais , Bleomicina , Células da Medula Óssea/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Pulmão/imunologia , Pulmão/patologia , Mastócitos/metabolismo , Mastócitos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/induzido quimicamente , Serina Endopeptidases/genéticaRESUMO
BACKGROUND: Potential involvement of the CCR10/CCL28 axis was recently reported in murine models of allergic asthma. If confirmed, blockade of the CCR10 receptor would represent an alternative to current asthma therapies. We evaluated the effect of a novel Protein Epitope Mimetic CCR10 antagonist, POL7085, in a murine model of allergic eosinophilic airway inflammation. METHODS: Mice were sensitized and challenged to ovalbumin. POL7085, a CCR10 antagonist (7.5 and 15 mg/kg), dexamethasone (1 mg/kg) or vehicle were administered intranasally once daily 1h before each allergen challenge. On day 21, airway hyperresponsiveness, bronchoalveolar lavage inflammatory cells and Th2 cytokines, and lung tissue mucus and collagen were measured. RESULTS: Allergen challenge induced airway hyperresponsiveness in vehicle-treated animals as measured by whole body barometric plethysmography, and eosinophilia in bronchoalveolar lavage. POL7085 dose-dependently and significantly decreased airway hyperresponsiveness (34 ± 16 %) and eosinophil numbers in bronchoalveolar lavage (66 ± 6 %). In addition, the highest dose of POL7085 used significantly inhibited lung IL-4 (85 ± 4 %), IL-5 (87 ± 2 %) and IL-13 (190 ± 19 %) levels, and lung collagen (43 ± 11 %). CONCLUSIONS: The Protein Epitope Mimetic CCR10 antagonist, POL7085, significantly and dose-dependently decreased allergen-induced airway hyperresponsiveness and airway inflammation after once daily local treatment. Our data give strong support for further investigations with CCR10 antagonists in asthmatic disease.
Assuntos
Asma/prevenção & controle , Hiper-Reatividade Brônquica/prevenção & controle , Epitopos/uso terapêutico , Eosinofilia Pulmonar/prevenção & controle , Receptores CCR10/antagonistas & inibidores , Animais , Asma/patologia , Hiper-Reatividade Brônquica/patologia , Relação Dose-Resposta a Droga , Epitopos/química , Epitopos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eosinofilia Pulmonar/patologia , Receptores CCR10/químicaRESUMO
The chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair, in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue and should not diffuse in the systemic circulation, where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by the intranasal route, but not when administered systemically by the intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands although the target protein is widely distributed in the organism.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Chalconas/farmacologia , Quimiocina CXCL12/antagonistas & inibidores , Animais , Antiasmáticos/química , Asma/metabolismo , Asma/patologia , Chalconas/química , Quimiocina CXCL12/metabolismo , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/metabolismo , Eosinófilos/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores CXCR4/metabolismoRESUMO
PURPOSE: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies. METHODS: The experiments were conducted on intact lung biopsy samples of pig, rat, mouse, and human using a Bruker Advance III 500 spectrometer. Thirty-five to 39 metabolites were identified and 23 metabolites were quantified. Principal component analysis, partial least-squares discriminant analysis, and analysis of variance tests were performed in order to compare the metabolic profiles of each animal lung biopsies to those of the human lung. RESULTS: The metabolic composition between human and pig lung was similar. However, human lung was distinguishable from mouse and rat regarding: Trimethylamine N-oxide and betaïne which were present in rodents but not in human lung, carnitine, and glycerophosphocholine which were present in mouse but not in human lung. Conversely, succinic acid was undetected in rat lung. Furthermore, fatty acids concentration was significantly higher in rodent lungs compared to human lung. CONCLUSION: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy on lung biopsy, samples allowed to highlight that pig lung seems to be close to human lung as regarding its metabolite composition with more similarities than dissimilarities.
Assuntos
Pulmão/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metaboloma/fisiologia , Camundongos/metabolismo , Ratos/metabolismo , Suínos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Feminino , Humanos , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4-induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.
Assuntos
Antiasmáticos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Compostos de Amônio Quaternário/uso terapêutico , Receptores de Glucocorticoides/efeitos dos fármacos , Acetatos , Animais , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/biossíntese , Citocinas/genética , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fosfatase 1 de Especificidade Dupla/biossíntese , Fosfatase 1 de Especificidade Dupla/genética , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/patologia , Inflamação , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/patologia , Mastócitos/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/toxicidade , Compostos de Amônio Quaternário/farmacologia , Receptores de Glucocorticoides/fisiologia , Fator de Transcrição STAT6/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tiramina/análogos & derivadosRESUMO
The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.
Assuntos
Receptores de Neuropeptídeos , Receptores Opioides mu , Animais , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Camundongos , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/metabolismo , Masculino , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/uso terapêutico , Analgésicos/síntese química , Humanos , Relação Estrutura-Atividade , Analgésicos Opioides/farmacologia , Analgésicos Opioides/químicaRESUMO
Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies.
RESUMO
Atopic diseases, including atopic dermatitis (AD) and asthma, affect a large proportion of the population, with increasing prevalence worldwide. AD often precedes the development of asthma, known as the atopic march. Allergen sensitization developed through the barrier-defective skin of AD has been recognized to be a critical step leading to asthma, in which thymic stromal lymphopoietin (TSLP) was previously shown to be critical. In this study, using a laser-assistant microporation system to disrupt targeted skin layers for generating micropores at a precise anatomic depth of mouse skin, we model allergen exposure superficially or deeply in the skin, leading to epicutaneous sensitization or dermacutaneous sensitization that is associated with a different cytokine microenvironment. Our work shows a differential requirement for TSLP in these two contexts, and identifies an important function for IL-1ß, which is independent of TSLP, in promoting allergen sensitization and subsequent allergic asthma.
Assuntos
Asma , Citocinas , Dermatite Atópica , Interleucina-1beta , Alérgenos , Animais , Asma/complicações , Citocinas/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Pele , Linfopoietina do Estroma do TimoRESUMO
Murine models of asthma are developed to better understand the mechanisms of asthma including eosinophil recruitment in the airways with the aim of evaluating new therapeutic strategies. They are intended to model the typical features of human disease, in particular airway inflammation, hyperresponsiveness (AHR), and remodeling. The phenotype of inflammatory cells recovered from the bronchoalveolar lavage fluid (BAL) is studied with innovative flow cytometry techniques while airway obstruction is measured using the forced oscillation technique, and airway responsiveness approached by barometric plethysmography in awake and unconstrained animals. We here describe models of asthma of house dust mite (HDM) as a clinically relevant allergen: a short study design (8 days) model of hypereosinophilic asthma and a chronic (31 days) asthma model, both suitable to evaluate the potential of new drug candidates to prevent allergic asthma.
Assuntos
Desenvolvimento de Medicamentos/métodos , Eosinófilos/citologia , Pyroglyphidae/imunologia , Alérgenos/imunologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas , Modelos Animais de Doenças , Eosinófilos/metabolismo , Hipersensibilidade/imunologia , Contagem de Leucócitos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos/imunologia , Camundongos Transgênicos , Transtornos Respiratórios/imunologia , Hipersensibilidade Respiratória/imunologia , Células Th2/imunologiaRESUMO
The involvement of autophagy and its dysfunction in asthma is still poorly documented. By using a murine model of chronic house dust mite (HDM)-induced airway inflammation, we tested the expression of several autophagy markers in the lung and spleen of asthma-like animals. Compared to control mice, in HDM-sensitized and challenged mice, the expression of sequestosome-1/p62, a multifunctional adaptor protein that plays an important role in the autophagy machinery, was raised in the splenocytes. In contrast, its expression was decreased in the neutrophils recovered from the bronchoalveolar fluid, indicating that autophagy was independently regulated in these two compartments. In a strategy of drug repositioning, we treated allergen-sensitized mice with the therapeutic peptide P140 known to target chaperone-mediated autophagy. A single intravenous administration of P140 in these mice resulted in a significant reduction in airway resistance and elastance, and a reduction in the number of neutrophils and eosinophils present in the bronchoalveolar fluid. It corrected the autophagic alteration without showing any suppressive effect in the production of IgG1 and IgE. Collectively, these findings show that autophagy processes are altered in allergic airway inflammation. This cellular pathway may represent a potential therapeutic target for treating selected patients with asthma.
Assuntos
Asma/complicações , Hipersensibilidade/complicações , Inflamação/prevenção & controle , Pulmão/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Pyroglyphidae/patogenicidade , Animais , Asma/patologia , Autofagia , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Hipersensibilidade/patologia , Imunoglobulina E/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/química , Proteína Sequestossoma-1/metabolismoRESUMO
Dysregulation of CXCL12/SDF-1-CXCR4/CD184 signaling is associated with inflammatory diseases and notably with systemic lupus erythematosus. Issued from the lead molecule chalcone-4, the first neutraligand of the CXCL12 chemokine, LIT-927 was recently described as a potent analogue with improved solubility and stability. We aimed to investigate the capacity of LIT-927 to correct immune alterations in lupus-prone MRL/lpr mice and to explore the mechanism of action implemented by this small molecule in this model. We found that in contrast to AMD3100, an antagonist of CXCR4 and agonist of CXCR7, LIT-927 reduces the excessive number of several B/T lymphocyte subsets occurring in the blood of sick MRL/lpr mice (including CD3+/CD4-/CD8-/B220+ double negative T cells). In vitro, LIT-927 downregulated the overexpression of several activation markers on splenic MRL/lpr lymphocytes. It exerted effects on the CXCR4 pathway in MRL/lpr CD4+ T spleen cells. The results underline the importance of the CXCL12/CXCR4 axis in lupus pathophysiology. They indicate that neutralizing CXCL12 by the neutraligand LIT-927 can attenuate hyperactive lymphocytes in lupus. This mode of intervention might represent a novel strategy to control a common pathophysiological mechanism occurring in inflammatory diseases.
RESUMO
BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.
Assuntos
Bronquiolite Obliterante/enzimologia , Fibroblastos/enzimologia , Transplante de Pulmão/efeitos adversos , Pulmão/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Adolescente , Adulto , Idoso , Animais , Bronquiolite Obliterante/tratamento farmacológico , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/patologia , Proliferação de Células , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , França , Humanos , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/cirurgia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Reepitelização , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima , Adulto JovemRESUMO
Fluorescent probes are commonly used in studying G protein-coupled receptors in living cells; however their application to the whole animal receptor imaging is still challenging. To address this problem, we report the design and the synthesis of the first near-infrared emitting fluorogenic dimer with environment-sensitive folding. Due to the formation of non-fluorescent H-aggregates in an aqueous medium, the near-infrared fluorogenic dimer displays a strong turn-on response (up to 140-fold) in an apolar environment and exceptional brightness: 56% quantum yield and ≈444 000 M-1 cm-1 extinction coefficient. Grafted on a ligand of the oxytocin receptor, it allows the unprecedented background-free and target-specific imaging of the naturally expressed receptor in living mice.
RESUMO
Opioid analgesics, such as morphine, oxycodone, and fentanyl, are the cornerstones for treating moderate to severe pain. However, on chronic administration, their efficiency is limited by prominent side effects such as analgesic tolerance and dependence liability. Neuropeptide FF (NPFF) and its receptors (NPFF1R and NPFF2R) are recognized as an important pronociceptive system involved in opioid-induced hyperalgesia and analgesic tolerance. In this article, we report the design of multitarget peptidomimetic compounds that show high-affinity binding to the mu-opioid receptor (MOPr) and NPFFRs. In vitro characterization of these compounds led to identification of KGFF03 and KGFF09 as G-protein-biased MOPr agonists with full agonist or antagonist activity at NPFFRs, respectively. In agreement with their biased MOPr agonism, KGFF03/09 showed reduced respiratory depression in mice, as compared to the unbiased parent opioid agonist KGOP01. Chronic subcutaneous administration of KGOP01 and KGFF03 in mice rapidly induced hyperalgesia and analgesic tolerance, effects that were not observed on chronic treatment with KGFF09. This favorable profile was further confirmed in a model of persistent inflammatory pain. In addition, we showed that KGFF09 induced less physical dependence compared with KGOP01 and KGFF03. Altogether, our data establish that combining, within a single molecule, the G-protein-biased MOPr agonism and NPFFR antagonism have beneficial effects on both acute and chronic side effects of conventional opioid analgesics. This strategy can lead to the development of novel and potent antinociceptive drugs with limited side effects on acute and chronic administration.