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1.
Nano Lett ; 24(5): 1477-1486, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38259198

RESUMO

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.


Assuntos
Barreira Hematoencefálica , Lipossomos , Nanopartículas , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , RNA Mensageiro/genética , Lipídeos , Transfecção , RNA Interferente Pequeno/genética
2.
Adv Healthc Mater ; 11(18): e2200819, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35851855

RESUMO

Emerging diseases require generating new vaccines, which can often be time consuming. An alternate method to boost host defense is by inducing nonspecific innate immune memory, called trained immunity, to develop novel prophylactics. Many molecules, most notably ß-glucan, induce trained immunity, but their effects are often short-lived and uncontrolled. This lack of temporal control limits both the therapeutic ability of training and provides fundamental questions about its nature. To achieve temporal control of trained immunity, controlled release nanoparticles encapsulating only 3.5% of the standard dose of ß-glucan to attain sustained release over a month are engineered. Nanoparticle-trained mice exhibit prolonged training effects and improve resistance to a B16F10 tumor challenge compared to mice that receive an equivalent amount of free ß-glucan. The duration of trained immunity is further fine tuned by synthesizing nanoparticles composed of different molecular weights to modulate the release kinetics. These results demonstrate that dosing and temporal control can substantially alter the trained response to unanticipated levels. As such, this approach using sustained release platforms might lead to a novel prophylactic strategy for improved disease resistance against a wide variety of diseases.


Assuntos
Vacinas , beta-Glucanas , Animais , Preparações de Ação Retardada/farmacologia , Imunidade Inata , Camundongos , beta-Glucanas/farmacologia
3.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34935867

RESUMO

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Axônios/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Degeneração Neural/patologia , Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Canais de Cálcio/metabolismo , ADP-Ribose Cíclica/antagonistas & inibidores , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley
4.
ACS Chem Biol ; 16(2): 380-388, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33523635

RESUMO

We report a mechanistic study comparing the immune activation of conjugated Toll-like receptor (TLR) agonists and their unlinked mixtures. Herein, we synthesized a set of six linked dual agonists with different ligands, molecular structures, receptor locations, and biophysical characteristics. With these dimers, we ran a series of in vitro cell-based assays, comparing initial and overall NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, cytokine expression profiles, as well as time-resolved TNF-α (Tumor Necrosis Factor alpha) expression. We show that initial activation kinetics, ligand specificity, and the dose of the agonist influence the activity of these linked TLR systems. These results can help improve vaccine design by showing how linked TLR agonists can improve their potency with the appropriate selection of key criteria.


Assuntos
Compostos Heterocíclicos com 3 Anéis/farmacologia , Lipopeptídeos/farmacologia , Oligonucleotídeos/farmacologia , Receptores Toll-Like/agonistas , Animais , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 3 Anéis/síntese química , Cinética , Ligantes , Lipopeptídeos/síntese química , Camundongos , NF-kappa B/metabolismo , Oligonucleotídeos/síntese química , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
5.
Cancer Lett ; 423: 71-79, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29526803

RESUMO

PanINs and IPMNs are the two most common precursor lesions that can progress to invasive pancreatic ductal adenocarcinoma (PDA). DCLK1 has been identified as a biomarker of progenitor cells in PDA progressed from PanINs. To explore the potential role of DCLK1-expressing cells in the genesis of IPMNs, we compared the incidence of DCLK1-positive cells in pancreatic tissue samples from genetically-engineered mouse models (GEMMs) for IPMNs, PanINs, and acinar to ductal metaplasia by immunohistochemistry and immunofluorescence. Mouse lineage tracing experiments in the IPMN GEMM showed that DCLK1+ cells originated from a cell lineage distinct from PDX1+ progenitors. The DCLK1+ cells shared the features of tuft cells but were devoid of IPMN tumor biomarkers. The DCLK1+ cells were detected in the earliest proliferative acinar clusters prior to the formation of metaplastic ductal cells, and were enriched in the "IPMN niches". In summary, DCLK1 labels a unique pancreatic cellular lineage in the IPMN GEMM. The clustering of DCLK1+ cells is an early event in Kras-induced pancreatic tumorigenesis and may contribute to IPMN initiation.


Assuntos
Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Intraductais Pancreáticas/genética , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transativadores/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem da Célula , Proliferação de Células , Quinases Semelhantes a Duplacortina , Feminino , Engenharia Genética , Proteínas de Homeodomínio/genética , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Intraductais Pancreáticas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transativadores/genética
6.
Gastroenterology ; 150(1): 218-228.e12, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26408346

RESUMO

BACKGROUND & AIMS: Activin, a member of the transforming growth factor-ß (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. METHODS: We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. RESULTS: Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. CONCLUSIONS: Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/mortalidade , Adenocarcinoma Mucinoso/patologia , Animais , Carcinogênese/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Progressão da Doença , Deleção de Genes , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Taxa de Sobrevida
7.
Pancreas ; 43(2): 245-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24518503

RESUMO

OBJECTIVES: Mucinous cystic neoplasms (MCNs) are rare, potentially curable, mucin-producing neoplasms of the pancreas. We have previously reported PIK3CA (phosphoinositide-3-kinase catalytic subunit, p110α) mutations in intraductal papillary mucinous neoplasms, another mucin-producing neoplasm of the pancreas. In this study, we analyzed the presence of PIK3CA and AKT1/PKB (V-akt murine thymoma viral oncogene homolog 1) hot-spot mutations in MCN specimens. METHODS: Using the genomic DNA sequencing of tumor tissues isolated by laser capture microdissection, we evaluated 15 well-characterized MCNs for the E542K, E545K (exon 9), and H1047R (exon 20) hot-spot mutations in the PIK3CA gene and the E17K mutation in the AKT1 gene. RESULTS: A hot-spot mutation (E545K) of the PIK3CA gene was detected in 1 of the 15 MCNs and further confirmed by a mutant-enriched method. Interestingly, this mutation was found to be present only in the high-grade but not in low-grade dysplastic epithelium obtained from this neoplasm and coexisted with a KRAS mutation. No mutations were identified in the AKT1 gene. CONCLUSIONS: Our data, when combined with previous reports on intraductal papillary mucinous neoplasms, indicate that oncogenic activation of the PI3K pathway involving PIK3CA gene mutations can contribute to the progression of mucin-producing neoplasms but not pancreatic intraepithelial neoplasia. PIK3CA status could be useful for understanding their progression to malignancy.


Assuntos
Cistadenocarcinoma Mucinoso/genética , Mutação , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinases/genética , Adulto , Idoso , Classe I de Fosfatidilinositol 3-Quinases , Cistadenocarcinoma Mucinoso/patologia , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genética
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