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1.
J Nanobiotechnology ; 20(1): 456, 2022 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-36274120

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs with ~ 22 nucleotides, playing important roles in the post-transcriptional regulation of gene expression. The expression profiles of many miRNAs are closely related to the occurrence and progression of cancer and can be used as biomarkers for cancer diagnosis and prognosis. However, their intrinsic properties, such as short length, low abundance and high sequence homology, represent great challenges in miRNA detection of clinical samples. To overcome these challenges, we developed a simple, ultrasensitive detection platform of electrochemical miRNAs chip (e-miRchip) with a novel signal amplification strategy using silver nanoparticle reporters (AgNRs) for multiplexed, direct, electronic profiling of miRNAs. A two-step hybridization strategy was used to detect miRNAs, where the target miRNA hybridizes with a stem-loop probe to unlock the probe first, and the opened stem-loop can further hybridize with AgNRs for signaling amplification. To enhance the detection sensitivity, the gold nanoflower electrodes (GNEs) were constructed in the microaperture arrays of the e-miRchips by electroplating. With the optimal size of the GNEs, the e-miRchip showed excellent performance for miR-21 detection with a detection limit of 0.56 fM and a linear range extended from 1 fM to 10 pM. The e-miRchip also exhibited good specificity in differentiating the 3-base mismatched sequences of the target miRNA. In addition, the e-miRchip was able to directly detect miR-21 expression in the total RNA extracts or cell lysates collected from lung cancer cells and normal cells. This work demonstrated the developed e-miRchip as an efficient and promising miniaturized point-of-care diagnostic device for the early diagnosis and prognosis of cancers.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Prata , MicroRNAs/genética , MicroRNAs/química , Nanopartículas Metálicas/química , Limite de Detecção , Microeletrodos , Ouro/química , Nucleotídeos
2.
Nat Chem Biol ; 15(3): 304-313, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30692685

RESUMO

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.


Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Regulação da Expressão Gênica , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Linfócitos/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/ultraestrutura , NF-kappa B/metabolismo , Proteínas de Neoplasias , Transdução de Sinais
3.
J Allergy Clin Immunol ; 143(5): 1661-1673, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060714

RESUMO

Caspase recruitment domain (CARD) protein-B cell CLL/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) [CBM] complexes are critical signaling adaptors that facilitate immune and inflammatory responses downstream of both cell surface and intracellular receptors. Germline mutations that alter the function of members of this complex (termed CBM-opathies) cause a broad array of clinical phenotypes, ranging from profound combined immunodeficiency to B-cell lymphocytosis. With an increasing number of patients being described in recent years, the clinical spectrum of diseases associated with CBM-opathies is rapidly expanding and becoming unexpectedly heterogeneous. Here we review major discoveries that have shaped our understanding of CBM complex biology, and we provide an overview of the clinical presentation, diagnostic approach, and treatment options for those carrying germline mutations affecting CARD9, CARD11, CARD14, BCL10, and MALT1.


Assuntos
Linfócitos B/fisiologia , Hipersensibilidade Imediata/genética , Síndromes de Imunodeficiência/genética , Mutação/genética , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Hipersensibilidade Imediata/metabolismo , Síndromes de Imunodeficiência/metabolismo , Inflamação , Linfocitose , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Complexos Multiproteicos/metabolismo , Fenótipo , Transdução de Sinais
5.
J Allergy Clin Immunol ; 134(2): 276-84, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25087226

RESUMO

Next-generation DNA sequencing has accelerated the genetic characterization of many human primary immunodeficiency diseases (PIDs). These discoveries can be lifesaving for the affected patients and also provide a unique opportunity to study the effect of specific genes on human immune function. In the past 18 months, a number of independent groups have begun to define novel PIDs caused by defects in the caspase recruitment domain family, member 11 (CARD11)-B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1 [CBM]) signalosome complex. The CBM complex forms an essential molecular link between the triggering of cell-surface antigen receptors and nuclear factor κB activation. Germline mutations affecting the CBM complex are now recognized as the cause of novel combined immunodeficiency phenotypes, which all share abnormal nuclear factor κB activation and dysregulated B-cell development as defining features. For this "Current perspectives" article, we have engaged experts in both basic biology and clinical immunology to capture the worldwide experience in recognizing and managing patients with PIDs caused by CBM complex mutations.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos B/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Caspases/genética , Guanilato Ciclase/genética , Síndromes de Imunodeficiência/genética , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteína 10 de Linfoma CCL de Células B , Linfócitos B/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Caspases/imunologia , Regulação da Expressão Gênica , Mutação em Linhagem Germinativa , Guanilato Ciclase/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais
6.
J Funct Biomater ; 15(3)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38535251

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection caused the COVID-19 pandemic, impacting the global economy and medical system due to its fast spread and extremely high infectivity. Efficient control of the spread of the disease relies on a fast, accurate, and convenient detection system for the early screening of the infected population. Although reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold-standard method for SARS-CoV-2 RNA analysis, it has complex experimental procedures and relies on expensive instruments and professional operators. In this work, we proposed a simple, direct, amplification-free lateral flow immunoassay (LFIA) with dual-mode detection of SARS-CoV-2 RNA via direct visualization as well as fluorescence detection. The viral RNA was detected by the designed DNA probes to specifically hybridize with the conserved open reading frame 1ab (ORF1ab), envelope protein (E), and nucleocapsid (N) regions of the SARS-CoV-2 genome to form DNA-RNA hybrids. These hybrids were then recognized by the dual-mode gold nanoparticles (DMNPs) to produce two different readout signals. The fluorescence characteristics of different sizes of GNPs were explored. Under the optimized conditions, the LFIA presented a linear detection range of 104-106 TU/mL with a limit of detection (LOD) of 0.76, 1.83, and 2.58 × 104 TU/mL for lentiviral particles carrying SARS-CoV-2 ORF1ab, E, and N motifs, respectively, in the fluorescent mode, which was up to 10 times more sensitive than the colorimetric mode. Furthermore, the LFIA exhibited excellent specificity to SARS-CoV-2 in comparison with other respiratory viruses. It could be used to detect SARS-CoV-2 in saliva samples. The developed LFIA represents a promising and convenient point-of-care method for dual-mode, rapid detection of SARS-CoV-2, especially in the periods with high infectivity.

7.
Biomaterials ; 314: 122843, 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39321686

RESUMO

Inflammatory bowel disease (IBD) has become a serious and challenging health problem globally without curative medical treatments. Mounting evidence suggests that intestinal macrophages and their phenotypes are key players in the pathogenesis of IBD. Modulating the phenotypes and functions of intestinal macrophages through targeted interventions could be a promising approach to manage detrimental gut inflammation in IBD. In this study, we rationally design and fabricate a novel class of V-type peptide-decorated nanoparticles, VP-NP, with potent anti-inflammatory activity. Such a design allows two functional motifs FFD in a single peptide molecule to enhance the bioactivity of the nanoparticles. As expected, VP-NP exhibits a strong inhibitory activity on endosomal Toll-like receptor (TLR) signaling. Surprisingly, VP-NP can inhibit M1 polarization while facilitating M2 polarization in mouse bone marrow-derived macrophages through regulating the key transcription factors NF-κB, STAT1 and PPAR-γ. Mechanistically, VP-NP is internalized by macrophages in the endosomes, where it blocks endosomal acidification to inhibit endosomal TLR signaling; the transcriptomic analysis reveals that VP-NP potently down-regulates many genes in TLR, NF-κB, JAK-STAT, and cytokine/chemokine signaling pathways associated with inflammatory responses. In a colitis mouse model, the intraperitoneally administered VP-NP effectively alleviates the disease activities by decreasing colon inflammation and injuries, pro-inflammatory cytokine production, and myeloid cell infiltration in the gut. Furthermore, VP-NP primarily targets intestinal macrophages and alters their phenotypes from inflammatory M1-type toward the anti-inflammatory M2-type. This study provides a new nanotherapeutic strategy to specifically regulate macrophage activation and phenotypes through a dual mechanism to control gut inflammation, which may augment current clinical treatments for IBD.

8.
Comput Struct Biotechnol J ; 21: 3054-3072, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37273853

RESUMO

Crohn's disease (CD) is an inflammatory bowel disease (IBD) with complex clinical manifestations such as chronic diarrhea, weight loss and hematochezia. Despite the increasing incidence worldwide, cure of CD remains extremely difficult. The rapid development of high-throughput sequencing technology with integrated-omics analyses in recent years has provided a new means for exploring the pathogenesis, mining the biomarkers and designing targeted personalized therapeutics of CD. Host genomics and epigenomics unveil heredity-related mechanisms of susceptible individuals, while microbiome and metabolomics map host-microbe interactions in CD patients. Proteomics shows great potential in searching for promising biomarkers. Nonetheless, single omics technology cannot holistically connect the mechanisms with heterogeneity of pathological behavior in CD. The rise of multi-omics analysis integrates genetic/epigenetic profiles with protein/microbial metabolite functionality, providing new hope for comprehensive and in-depth exploration of CD. Herein, we emphasized the different omics features and applications of CD and discussed the current research and limitations of multi-omics in CD. This review will update and deepen our understanding of CD from integration of broad omics spectra and will provide new evidence for targeted individualized therapeutics.

9.
Adv Sci (Weinh) ; 10(19): e2301230, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37078808

RESUMO

Excessive activation of Toll-like receptor (TLR) signaling pathways and the circulating endotoxin are key players in the pathogenesis of many acute and chronic inflammatory diseases. Regulation of TLR-mediated inflammatory responses by bioactive nanodevices represents a promising strategy for treating these diseases. In searching for novel, clinically applicable nanodevices with potent TLR inhibitory activities, three types of hexapeptide-modified nano-hybrids with different cores of phospholipid nanomicelles, liposomes, and poly(lactic-co-glycolic acid) nanoparticles are constructed. Interestingly, only the peptide-modified lipid-core nanomicelles (M-P12) display potent TLR inhibitory activities. Further mechanistic studies disclose that lipid-core nanomicelles have a generic property to bind to and scavenge lipophilic TLR ligands including lipopolysaccharide to block the ligand-receptor interaction and down-regulate the TLR signaling extracellularly. In addition, the peptide modification enables M-P12 a unique capability to modulate endosomal acidification upon being endocytosed into macrophages, which subsequently regulates the endosomal TLR signal transduction. In an acute lung injury mouse model, intratracheal administration of M-P12 can effectively target lung macrophages and reduce lung inflammation and injuries. This work defines a dual mechanism of action of the peptide-modified lipid-core nanomicelles in regulating TLR signaling, and provides new strategies for the development of therapeutic nanodevices for treating inflammatory diseases.


Assuntos
Endotoxinas , Receptores Toll-Like , Animais , Camundongos , Receptores Toll-Like/metabolismo , Lipopolissacarídeos/farmacologia , Peptídeos/química , Concentração de Íons de Hidrogênio
10.
Nanomedicine ; 8(5): 647-54, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21889478

RESUMO

A special class of self-assembling peptide (EAK16-II) has been found to stabilize the hydrophobic anticancer agent ellipticine (EPT) in aqueous solution. In this study, the mechanism of such peptide-EPT complexes to enhance cellular delivery and anticancer activity was evaluated. Results revealed that EAK16-II can form nanoparticles with EPT, having an average size of ∼100 nm. This nanoformulation had cytotoxicity to human lung carcinoma A549 cells that was comparable to EPT dissolved in dimethyl sulfoxide. It enhanced EPT uptake drastically when compared to the microformulation. Such enhanced uptake was significantly reduced by inhibitors specifically for the caveolae-dependent pathway. We also found both protonated and neutral forms of EPT present in the cells. Interestingly, both were found in the cytoplasm, co-localized with LysoTracker, whereas only protonated EPT was seen in the nucleus. The promising therapeutic efficacy, specific delivery pathway, and intracellular distribution pattern discovered in this work may help further develop EPT as a nanoformulation for clinical applications. FROM THE CLINICAL EDITOR: A special class of self-assembling peptide (EAK16-II) has been found to stabilize ellipticine in aqueous solution. The authors demonstrate therapeutic efficacy, describe specific delivery pathways, and effective intracellular distribution pattern, which will aid the development of this technology for future clinical applications.


Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos , Elipticinas , Nanopartículas , Oligopeptídeos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Cavéolas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elipticinas/administração & dosagem , Elipticinas/química , Endocitose , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas/administração & dosagem , Nanopartículas/química , Oligopeptídeos/administração & dosagem , Tamanho da Partícula
11.
Adv Sci (Weinh) ; 9(3): e2104051, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34816630

RESUMO

Toll-like receptor (TLR) activation in macrophages plays a critical role in the pathogenesis of acute lung injury (ALI). While TLR inhibition is a promising strategy to control the overwhelming inflammation in ALI, there still lacks effective TLR inhibitors for clinical uses to date. A unique class of peptide-coated gold nanoparticles (GNPs) is previously discovered, which effectively inhibited TLR signaling and protected mice from lipopolysaccharide (LPS)-induced ALI. To fast translate such a discovery into potential clinical applicable nanotherapeutics, herein an elegant strategy of "nano-enabled drug repurposing" with "nano-targeting" is introduced to empower the existing drugs for new uses. Combining transcriptome sequencing with Connectivity Map analysis, it is identified that the proton pump inhibitors (PPIs) share similar mechanisms of action to the discovered GNP-based TLR inhibitor. It is confirmed that PPIs (including omeprazole) do inhibit endosomal TLR signaling and inflammatory responses in macrophages and human peripheral blood mononuclear cells, and exhibits anti-inflammatory activity in an LPS-induced ALI mouse model. The omeprazole is then formulated into a nanoform with liposomes to enhance its macrophage targeting ability and the therapeutic efficacy in vivo. This research provides a new translational strategy of nano-enabled drug repurposing to translate bioactive nanoparticles into clinically used drugs and targeted nano-therapeutics for ALI.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Nanopartículas Metálicas/administração & dosagem , Nanomedicina/métodos , Inibidores da Bomba de Prótons/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Lesão Pulmonar Aguda/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Bomba de Prótons/metabolismo , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/metabolismo
12.
Front Immunol ; 12: 750128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34659253

RESUMO

Macrophages play an important role in the initiation, progression and resolution of inflammation in many human diseases. Effective regulation of their activation and immune responses could be a promising therapeutic strategy to manage various inflammatory conditions. Nanodevices that naturally target macrophages are ideal agents to regulate immune responses of macrophages. Here we described a special tryptophan (Trp)-containing hexapeptide-coated gold nanoparticle hybrid, PW, which had unique immunomodulatory activities on macrophages. The Trp residues enabled PW higher affinity to cell membranes, and contributed to inducing mild pro-inflammatory responses of NF-κB/AP-1 activation. However, in the presence of TLR stimuli, PW exhibited potent anti-inflammatory activities through inhibiting multiple TLR signaling pathways. Mechanistically, PW was internalized primarily through micropinocytosis pathway into macrophages and attenuated the endosomal acidification process, and hence preferentially affected the endosomal TLR signaling. Interestingly, PW could induce the expression of the TLR negative regulator IRAK-M, which may also contribute to the observed TLR inhibitory activities. In two acute lung injury (ALI) mouse models, PW could effectively ameliorate lung inflammation and protect lung from injuries. This work demonstrated that nanodevices with thoughtful design could serve as novel immunomodulatory agents to manage the dysregulated inflammatory responses for treating many chronic and acute inflammatory conditions, such as ALI.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Ouro/administração & dosagem , Fatores Imunológicos/administração & dosagem , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Peptídeos/administração & dosagem , Triptofano/administração & dosagem , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/imunologia , Humanos , Interleucina-10/imunologia , Lipopolissacarídeos , Lipossomos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Células THP-1 , Transcriptoma/efeitos dos fármacos
13.
Front Immunol ; 12: 749794, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34721419

RESUMO

Nuclear factor kappa B (NF-κB) is a critical transcription factor involved in regulating cell activation, inflammation, and survival. The linear ubiquitin chain assembly complex (LUBAC) which consists of HOIL1, HOIP, and SHARPIN, catalyzes the linear ubiquitination of target proteins-a post-translational modification that is essential for NF-κB activation. Human germline pathogenic variants that dysregulate linear ubiquitination and NF-κB signaling are associated with immunodeficiency and/or autoinflammation including dermatitis, recurrent fevers, systemic inflammation and enteropathy. We previously identified MALT1 paracaspase as a novel negative regulator of LUBAC by proteolytic cleavage of HOIL1. To directly investigate the impact of HOIL1 cleavage activity on the inflammatory response, we employed a stable transduction system to express and directly compare non-cleavable HOIL1 with wild-type HOIL1 in primary HOIL1-deficient patient skin fibroblasts. We discovered that non-cleavable HOIL1 resulted in enhanced NF-κB signaling in response to innate stimuli. Transcriptomics revealed enrichment of inflammation and proinflammatory cytokine-related pathways after stimulation. Multiplexed cytokine assays confirmed a 'hyperinflammatory' phenotype in these cells. This work highlights the physiological importance of MALT1-dependent cleavage and modulation of HOIL1 on NF-κB signaling and inflammation, provides a mechanism for the autoinflammation observed in MALT1-deficient patients, and will inform the development of therapeutics that target MALT1 paracaspase and LUBAC function in treating autoinflammatory skin diseases.


Assuntos
Fibroblastos/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , NF-kappa B/imunologia , Fatores de Transcrição/imunologia , Ubiquitina-Proteína Ligases/imunologia , Citocinas/imunologia , Humanos , Inflamação/imunologia , Transdução de Sinais , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
15.
Cell Death Dis ; 9(2): 162, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29415982

RESUMO

Proteasome inhibitors have emerged as an effective therapy for the treatment of haematological malignancies; however, their efficacy can be limited by the development of tumour resistance mechanisms. Novel combination strategies including the addition of TLR adjuvants to increase cell death and augment immune responses may help enhance their effectiveness. Although generally thought to inhibit inflammatory responses and NF-κB activation, we found that under specific conditions proteasome inhibitors can promote inflammatory responses by mediating IL-1ß maturation and secretion after TLR stimulation. This was dependent on the timing of proteasome inhibition relative to TLR stimulation where reversal of treatment order could alternatively increase or inhibit IL-1ß secretion (P < 0.001). TLR stimulation combined with proteasome inhibition enhanced cell death in vitro and delayed tumour development in vivo in NOD SCID mice (P < 0.01). However, unlike IL-1ß secretion, cell death occurred similarly regardless of treatment order and was only partially caspase dependent, possessing characteristics of both apoptosis and necrosis as indicated by activation of caspase-1, 3, 8 and RIP3 phosphorylation. Although stimulation of various TLRs was capable of driving IL-1ß production, TLR4 stimulation was the most effective at increasing cell death in THP-1 and U937 cells. TLR4 stimulation and proteasome inhibition independently activated the RIP3 necroptotic pathway and ultimately reduced the effectiveness of caspase/necroptosis inhibitors in mitigating overall levels of cell death. This strategy of combining TLR stimulation with proteasome inhibition may improve the ability of proteasome inhibitors to generate immunogenic cell death and increase anti-tumour activity.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Interleucina-1beta/biossíntese , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteassoma/farmacologia , Receptores Toll-Like/agonistas , Animais , Bortezomib/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Camundongos SCID , Necrose , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
16.
J Vis Exp ; (127)2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28994806

RESUMO

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds - PP2, rottlerin, and curcumin - in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.


Assuntos
Aminoácidos/química , Sistemas de Liberação de Medicamentos/métodos , Peptídeos/química , Solventes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas
17.
J Vis Exp ; (125)2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28784964

RESUMO

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Nanopartículas Metálicas , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/antagonistas & inibidores , Fosfatase Alcalina/genética , Linhagem Celular , Genes Reporter , Ouro/química , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Lipopolissacarídeos/farmacologia , Luciferases/genética , Nanopartículas Metálicas/química , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Regiões Promotoras Genéticas , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/genética , Fator de Transcrição AP-1/metabolismo
19.
J Control Release ; 239: 211-22, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27586187

RESUMO

Clinical application of hydrophobic therapeutics is restricted by lack of an efficient vehicle which permits their solubility in aqueous environments. We have previously developed a novel formulation strategy to deliver a hydrophobic Src inhibitor, PP2, involving combinations of one self-assembling peptide (SAP) and one of 4 selected amino acids (AAs). The present study aims to develop a generalized drug delivery platform for intravenous application of hydrophobic drugs by combining self-assembling peptide, amino acid and low concentration of co-solvent. A multi-step screening pipeline is established which includes assessment of drug solubility and physicochemical characteristics, as well as functional efficacy and safety in vitro and in vivo. Using PP2 as an exemplary hydrophobic compound, 480 different combinations of 6 SAPs, 20 naturally existing AAs at 2 concentrations, and 2 co-solvents were evaluated. Among the combinations, 60 formulae dissolved PP2; 10 of which significantly reduced thrombin-induced IL-8 production, a sign of inflammatory response, in normal human lung epithelial BEAS2B cells. These formulations did not show cytotoxicity alone, but 2 reduced cell viability with presence of thrombin. We then performed a double-blinded test in a rat model of pulmonary ischemia-reperfusion. PP2 formulated with EAK16-I peptide plus methionine and 2% ethanol were administrated intravenously, significantly reducing severity of lung injury. The SAP-AA formulation strategy was also successfully applied to other hydrophobic compounds, suggesting this strategy could be applicable to other hydrophobics for a variety of clinical applications.


Assuntos
Aminoácidos/química , Sistemas de Liberação de Medicamentos/métodos , Interações Hidrofóbicas e Hidrofílicas , Oligopeptídeos/química , Células A549 , Administração Intravenosa , Aminoácidos/administração & dosagem , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Composição de Medicamentos , Humanos , Masculino , Oligopeptídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley
20.
Biomaterials ; 111: 90-102, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27728817

RESUMO

Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions. Currently there are no specific TLR inhibitors in clinical use. To overcome this challenge, we have developed a nano-based TLR inhibitor (peptide-gold nanoparticle hybrids) that inhibits a broad spectrum of TLR responses. Through mechanistic studies, we established that specific peptide decorated-gold nanoparticles that display high cellular uptake in phagocytic immune cells modulate endosomal pH, leading to significant attenuation of signaling through multiple TLRs. Using a global transcriptomic approach, we defined the broad anti-inflammatory activity of the nanoparticle in human peripheral blood mononuclear cells. In vivo studies confirmed the beneficial immunomodulatory activity since treatment with the nanoparticle significantly reduced weight loss, improved the disease activity index, and ameliorated colonic inflammation in a murine model of intestinal inflammation. This work enhances our fundamental understanding of the role of peptide coatings on the nanoparticle surface in regulating innate immune signaling, and identifies specific peptide decorated nanoparticles that may represent a novel class of anti-inflammatory therapeutics for human inflammatory diseases.


Assuntos
Endossomos/química , Endossomos/imunologia , Leucócitos Mononucleares/imunologia , Nanocápsulas/química , Peptídeos/administração & dosagem , Fagocitose/imunologia , Receptores Toll-Like/imunologia , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/imunologia , Células Cultivadas , Ouro/química , Humanos , Concentração de Íons de Hidrogênio , Leucócitos Mononucleares/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanocápsulas/administração & dosagem , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Peptídeos/imunologia , Fagocitose/efeitos dos fármacos , Receptores Toll-Like/antagonistas & inibidores
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