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1.
J Allergy Clin Immunol ; 143(5): 1661-1673, 2019 May.
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.

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.
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
4.
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
5.
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
6.
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
7.
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
9.
Nat Commun ; 6: 8777, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26525107

RESUMO

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.


Assuntos
Caspases/genética , Síndromes de Imunodeficiência/genética , Linfócitos/imunologia , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Animais , Células Apresentadoras de Antígenos , Linfócitos B/imunologia , Caspases/imunologia , Caspases/metabolismo , Família , Feminino , Imunofluorescência , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Introdução de Genes , Humanos , Quinase I-kappa B/metabolismo , Immunoblotting , Síndromes de Imunodeficiência/imunologia , Imunoprecipitação , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos Mononucleares , Masculino , Espectrometria de Massas , Camundongos , Microscopia Confocal , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , NF-kappa B/imunologia , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Tonsila Palatina , Proteômica , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/imunologia , Espectrometria de Massas em Tandem , Fatores de Transcrição , Ubiquitinação/imunologia
10.
ACS Nano ; 9(7): 6774-84, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26083966

RESUMO

Manipulation of immune responsiveness using nanodevices provides a potential approach to treat human diseases. 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 of these inflammatory conditions. Here we describe the discovery of a unique class of peptide-gold nanoparticle hybrids that exhibit a broad inhibitory activity on TLR signaling, inhibiting signaling through TLRs 2, 3, 4, and 5. As exemplified using TLR4, the nanoparticles were found to inhibit both arms of TLR4 signaling cascade triggered by the prototypical ligand, lipopolysaccharide (LPS). Through structure-activity relationship studies, we identified the key chemical components of the hybrids that contribute to their immunomodulatory activity. Specifically, the hydrophobicity and aromatic ring structure of the amino acids on the peptides were essential for modulating TLR4 responses. This work enhances our fundamental understanding of the role of nanoparticle surface chemistry in regulating innate immune signaling, and identifies specific nanoparticle hybrids that may represent a unique class of anti-inflammatory therapeutics for human inflammatory diseases.


Assuntos
Aminoácidos Aromáticos/química , Fatores Imunológicos/química , Nanopartículas Metálicas/química , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Aminoácidos Aromáticos/administração & dosagem , Aminoácidos Aromáticos/farmacologia , Linhagem Celular Tumoral , Ouro , Humanos , Fatores Imunológicos/administração & dosagem , Nanoconjugados/química , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/farmacologia
11.
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
13.
ACS Chem Biol ; 9(1): 247-57, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24117378

RESUMO

Toll-like receptors (TLRs) play a critical role in innate immunity, but activation of TLR signaling pathways is also associated with many harmful inflammatory diseases. Identification of novel anti-inflammatory molecules targeting TLR signaling pathways is central to the development of new treatment approaches for acute and chronic inflammation. We performed high-throughput screening from crude marine sponge extracts on TLR5 signaling and identified girolline. We demonstrated that girolline inhibits signaling through both MyD88-dependent and -independent TLRs (i.e., TLR2, 3, 4, 5, and 7) and reduces cytokine (IL-6 and IL-8) production in human peripheral blood mononuclear cells and macrophages. Using a chemical genomics approach, we identified Elongation Factor 2 as the molecular target of girolline, which inhibits protein synthesis at the elongation step. Together these data identify the sponge natural product girolline as a potential anti-inflammatory agent acting through inhibition of protein synthesis.


Assuntos
Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Imidazóis/isolamento & purificação , Imidazóis/farmacologia , Poríferos/química , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Células CHO , Células Cultivadas , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Interleucina-6/imunologia , Interleucina-8/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptores Toll-Like/imunologia
14.
PLoS One ; 7(5): e38049, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22675434

RESUMO

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFß1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFß1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.


Assuntos
Carcinoma/genética , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Fator de Necrose Tumoral alfa/farmacologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Claudina-1 , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Inativação Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Fator de Crescimento Transformador beta1/farmacologia
15.
Intensive Care Med ; 38(5): 894-905, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22349424

RESUMO

PURPOSE: Pulmonary ischemia-reperfusion is a pathological process seen in several clinical conditions, including lung transplantation, cardiopulmonary bypass, resuscitation for circulatory arrest, atherosclerosis, and pulmonary embolism. A better understanding of its molecular mechanisms is very important. METHODS: Rat left lung underwent in situ ischemia for 60 min, followed by 2 h of reperfusion. The gene expression profiles and Src protein tyrosine kinase (PTK) phosphorylation were studied over time, and PP2, an Src PTK inhibitor, was intravenously administered 10 min before lung ischemia to determine the role of Src PTK in lung injury. RESULTS: Reperfusion following ischemia significantly changed the expression of 169 genes, with Mmp8, Mmp9, S100a9, and S100a8 being the most upregulated genes. Ischemia alone only affected expression of 9 genes in the lung. However, Src PTK phosphorylation (activation) was increased in the ischemic lung, mainly on the alveolar wall. Src PTK inhibitor pretreatment decreased phosphorylation of Src PTKs, total protein tyrosine phosphorylation, and STAT3 phosphorylation. It increased phosphorylation of the p85α subunit of PI3 kinase, a signal pathway that can inhibit coagulation and inflammation. PP2 reduced leukocyte infiltration in the lung, apoptotic cell death, fibrin deposition, and severity of acute lung injury after reperfusion. Src inhibition also significantly reduced CXCL1 (GRO/KI) and CCL2 (MCP-1) chemokine levels in the serum. CONCLUSION: During pulmonary ischemia, Src PTK activation, rather than alteration in gene expression, may play a critical role in reperfusion-induced lung injury. Src PTK inhibition presents a new prophylactic treatment for pulmonary ischemia-reperfusion-induced acute lung injury.


Assuntos
Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/prevenção & controle , Isquemia/prevenção & controle , Traumatismo por Reperfusão/prevenção & controle , Reperfusão/efeitos adversos , Quinases da Família src/antagonistas & inibidores , Animais , Expressão Gênica/genética , Expressão Gênica/fisiologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Traumatismo por Reperfusão/fisiopatologia , Quinases da Família src/farmacologia
16.
Nanomedicine ; 8(5): 647-54, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21889478

RESUMO

UNLABELLED: 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
18.
Biomaterials ; 32(16): 4000-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21376387

RESUMO

Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.


Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Aminoácidos/uso terapêutico , Lipopolissacarídeos/toxicidade , Oligopeptídeos/uso terapêutico , Pirimidinas/uso terapêutico , Aminoácidos/química , Animais , Western Blotting , Linhagem Celular , Hemólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Pirimidinas/química , Ratos , Ratos Sprague-Dawley
19.
Langmuir ; 25(14): 7773-7, 2009 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-19594172

RESUMO

A novel electrochemical biosensing platform is described using biocompatible, self-assembled ionic-complementary peptide nanofibers. The compatibility of a graphite electrode modified by these peptide nanofibers with enzymes is demonstrated using a model enzyme glucose oxidase (GOx). A glucose biosensor has been successfully fabricated by incorporating this enzyme into the modified electrode. From measurement of its electrode response and sensitivity, this nanofiber-modified electrode shows promise as an enzyme-based biosensor. The findings presented here demonstrate excellent potential of the use of ionic-complementary peptides to modify electrode surfaces for biomolecular sensing and diagnostics.


Assuntos
Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Enzimas Imobilizadas/química , Peptídeos/química , Eletrodos , Microscopia de Força Atômica , Modelos Teóricos
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