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1.
Adv Exp Med Biol ; 1172: 79-96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628652

RESUMO

The Interleukin (IL)-10 cytokine family includes IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26, which are considered as Class 2α-helical cytokines. IL-10 is the most important cytokine in suppressing pro-inflammatory responses in all kinds of autoimmune diseases and limiting excessive immune responses. Due to protein structure homology and shared usage of receptor complexes as well as downstream signaling pathway, other IL-10 family cytokines also show indispensable functions in immune regulation, tissue homeostasis, and host defense. In this review, we focus on immune functions and structures of different cytokines in this family and try to better understand how their molecular mechanisms connect to their biological functions. The molecular details regarding their actions also provide useful information in developing candidate immune therapy reagents for a variety of diseases.


Assuntos
Interleucina-10 , Doenças Autoimunes/imunologia , Humanos , Imunoterapia , Interleucina-10/química , Interleucina-10/imunologia , Transdução de Sinais/imunologia , Relação Estrutura-Atividade
2.
Fish Shellfish Immunol ; 89: 149-157, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30926478

RESUMO

Cyprinid herpesvirus 3 (CyHV-3), a virus that encodes an interleukin10 (IL-10) homologue, causes severe economic losses to the common carp and koi culture industry. The present study was devoted to this IL-10 homologue. Recombinant viral IL-10 (vIL-10) protein encoded by CyHV-3 ORF134 gene using prokaryotic expression system was obtained successfully. Bioinformatics analysis revealed that the amino acid sequence of CyHV-3 vIL-10 has low homology with other host IL-10 or viruses encoded IL-10s. However, their tertiary structure is quite similar, suggesting conservative biological functions between IL-10s and vIL-10s. The biological activity of CyHV-3 vIL-10 was detected by using CCK-8 kit and real time quantitative PCR. The results showed that CyHV-3 vIL-10 down regulate epithelioma papulosum cyprini (EPC) cellular activity at 72 h. Moreover, CyHV-3 vIL-10 inhibits the LPS-induced expression of proinflammatory genes, similar to common carp IL-10. Altogether, the results of this study demonstrate that a clear biological activity of CyHV-3 vIL-10 on its host cells and indicates CyHV-3 vIL-10 may play an important role in viral immune evasion.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Herpesviridae/genética , Herpesviridae/imunologia , Interleucina-10/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Carpas/microbiologia , Linhagem Celular , Evasão da Resposta Imune , Interleucina-10/química , Interleucina-10/genética , Macrófagos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência/veterinária , Proteínas Virais/química , Proteínas Virais/genética
3.
N Z Vet J ; 67(3): 138-142, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30753790

RESUMO

AIM: To compare the concentration of faecal cytokines interleukin (IL)-6, -8, -10, and tumour necrosis factor (TNF)-α in dogs with acute diarrhoea with clinically normal (non-diarrhoeic) dogs. METHODS: A total of 14 dogs presenting with acute diarrhoea, and 25 dogs with no history of gastrointestinal signs in the 2 months prior to enrolment, were recruited from two veterinary hospitals in Melbourne, Australia. Concentrations of IL-6, -8, -10, and TNF-α were measured in faecal samples using canine-specific ELISA. RESULTS: The diarrhoeic dogs were diagnosed with or managed for acute gastroenteritis (n = 6), extra-intestinal neoplasia (n = 2), parvoviral enteritis (n = 1), hepatopathy (n = 1), acute pancreatitis (n = 1), hypoadrenocorticism (n = 1), gastric dilatation volvulus (n = 1) and myelopathy (n = 1). IL-6 was detectable in the faeces of 10/14 (71%) diarrhoeic and 7/25 (28%) non-diarrhoeic dogs, and median concentrations were 10.8 (min 0.0, max 54.0) and 2.0 (min 0.0, max15.0) pg/mL, respectively (p = 0.01). IL-8 was detectable in the faeces of all diarrhoeic and 11 non-diarrhoeic dogs, and median concentrations were 149.7 (min 3.72, max 730.1) and 3.4 (min 0.0, max 22.5) pg/mL, respectively (p < 0.001). TNF-α was detected in the faeces of two of the diarrhoeic dogs (3.4 and 15.6 pg/mL) and none of the non-diarrhoeic dogs. IL-10 was not detected in the faeces of any dog. CONCLUSIONS: Faecal concentrations of IL-6 and -8 were higher in diarrhoeic compared to non-diarrhoeic dogs, and are therefore potential candidates for non-invasive biomarkers to assess the severity and resolution of acute intestinal disease in dogs. However their correlation with disease progression and severity needs to be further investigated before their full clinical application can be determined.


Assuntos
Citocinas/metabolismo , Diarreia/veterinária , Doenças do Cão/metabolismo , Fezes/química , Gastroenteropatias/veterinária , Doença Aguda , Animais , Biomarcadores , Citocinas/química , Citocinas/genética , Diarreia/metabolismo , Cães , Gastroenteropatias/diagnóstico , Gastroenteropatias/metabolismo , Regulação da Expressão Gênica , Interleucina-10/química , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/química , Interleucina-8/genética , Interleucina-8/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
4.
Life Sci ; 209: 78-84, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30075176

RESUMO

AIMS: The interleukin-10 (IL-10) is an immuno-regulatory cytokine that plays a protective effect in the vasculature. IL-10 binding to its receptor, activating the IL-10/JAK1/STAT3 cascade to exert its effects. Therefore, STAT3 phosphorylation is essential for IL-10 actions. O-Glycosylation with linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification able to regulate many proteins by interfering with protein on a phosphorylation level. Our aim was to determine whether O-GlcNAc promotes the inhibition of IL-10-pathway (JAK1/STAT3/IL-10), inactivationg its action in the vasculature. MAIN METHODS: Mice (C57BL/6) aortic segments were incubated with vehicle or Thiamet G (0.1 mM, for 24 h) to increase global O-GlcNAc levels. Aortas from knockout mice for IL-10 were also used. Vascular reactivity and western blot tests were performed to evaluate protein expression. KEY FINDINGS: High levels of O-GlcNAc, induced by Thiamet G incubation, increased vascular expression of JAK1, but decreased expression and activity of STAT3. In addition, IL-10 levels were diminished in arteries treated with Thiamet G. Absence of IL-10, as well as augmented O-GlcNAcylation, increased vascular reactivity to constrictor stimuli, an effect that was abolished by ERK 1/2 inhibitor. High levels of O-GlcNAc and the absence of IL-10 also leads to increased vascular expression of ERK1/2. SIGNIFICANCE: Our data suggest that O-GlcNAc modification seems to (dys)regulate IL-10 signaling pathway and consequently, compromise the protective effect of this cytokine in vasculature. It is possible that there is a promising relationship in pathophysiological conditions where changes in O-GlcNAcylation and IL-10 levels are observed, such as hypertension and diabetes.


Assuntos
Acetilglucosamina/química , Interleucina-10/química , Interleucina-10/metabolismo , Processamento de Proteína Pós-Traducional , Vasoconstrição , Animais , Glicosilação , Transdução de Sinais
5.
Cytometry A ; 91(9): 901-907, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28700121

RESUMO

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R2 > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry.


Assuntos
Bioensaio/métodos , Citocinas/química , Animais , Anticorpos Monoclonais/química , Bovinos , Reações Cruzadas/imunologia , Citometria de Fluxo/métodos , Interferon gama/química , Interleucina-10/química , Interleucina-12/química , Interleucina-4/química , Fator de Necrose Tumoral alfa/química
6.
Fish Shellfish Immunol ; 65: 244-255, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28442416

RESUMO

In the present study, members of the interleukin (IL)-10 family of cytokines, including IL-10 (TOIL-10) and IL-22 (TOIL-22) of golden pompano (Trachinotus ovatus), were cloned for the first time, and their expression patterns and 3D structures analyzed. The full-length cDNA sequences of TOIL-10 and TOIL-22 contained open reading frames of 564 and 567 bp, respectively. TOIL-10 and TOIL-22 shared higher homology (78%-89%) with the corresponding genes from various fish relative to other species (25%-34%) and contained the IL-10 family signature and four cysteine residues that are well conserved in other vertebrate IL-10 members. Phylogenetic tree analysis of our sequences alongside other IL-10 family proteins revealed that TOIL-10 and TOIL-22 cluster together with other teleost IL-10 and IL-22 molecules. Expression of TOIL-10 and TOIL-22 genes was ubiquitous in all tissues examined. The TOIL-10 gene was also highly expressed in skin, heart, gill, spleen, kidney, brain and liver, and lower levels were detected in intestine and muscle. High expression of the TOIL-22 gene was observed in gill, intestine, kidney, spleen, with the lowest levels in liver. TOIL-10 and TOIL-22 were rapidly activated after SAΔphoB immunization and significantly increased to peak levels at 12 h and 4 d in golden pompano kidney and spleen respectively following challenge. Expression in the brain reached peak levels at 4 d and 3 d respectively after post-immunization. Our results collectively indicate that TOIL-10 and TOIL-22 participate in the host immune response to bacterial infection. Moreover, TOIL-22 plays a potentially important role in mucosal immunity.


Assuntos
Doenças dos Peixes/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/imunologia , Interleucina-10/genética , Interleucinas/genética , Perciformes , Infecções Estreptocócicas/veterinária , Sequência de Aminoácidos , Animais , Vacinas Bacterianas/administração & dosagem , Sequência de Bases , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Interleucina-10/química , Interleucina-10/metabolismo , Interleucinas/química , Interleucinas/metabolismo , Perciformes/classificação , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/fisiologia , Vacinas Atenuadas/administração & dosagem
7.
Biosens Bioelectron ; 93: 170-175, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27660015

RESUMO

Interleukin-1b (IL-1b) and interleukin-10 (IL-10) biomarkers are one of many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). In the present study, we have developed a fully integrated electrochemical biosensor platform for cytokine detection at minute concentrations. Using eight gold working microelectrodes (WEs) the design will increase the sensitivity of detection, decrease the time of measurements, and allow a simultaneous detection of varying cytokine biomarkers. The biosensor platform was fabricated onto silicon substrates using silicon technology. Monoclonal antibodies (mAb) of anti-human IL-1b and anti-human IL-10 were electroaddressed onto the gold WEs through functionalization with 4-carboxymethyl aryl diazonium (CMA). Cyclic voltammetry (CV) was applied during the WE functionalization process to characterize the gold WE surface properties. Finally, electrochemical impedance spectroscopy (EIS) characterized the modified gold WE. The biosensor platform was highly sensitive to the corresponding cytokines and no interference with other cytokines was observed. Both cytokines: IL-10 and IL-1b were detected within the range of 1pgmL-1 to 15pgmL-1. The present electrochemical biosensor platform is very promising for multi-detection of biomolecules which can dramatically decrease the time of analysis. This can provide data to clinicians and doctors concerning cytokines secretion at minute concentrations and the prediction of the first signs of inflammation after LVAD implantation.


Assuntos
Técnicas Biossensoriais/métodos , Inflamação/metabolismo , Interleucina-10/isolamento & purificação , Interleucina-1beta/isolamento & purificação , Anticorpos Imobilizados/química , Citocinas/química , Citocinas/isolamento & purificação , Espectroscopia Dielétrica , Ouro/química , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Inflamação/patologia , Interleucina-10/química , Interleucina-1beta/química , Silício/química
8.
Crit Rev Immunol ; 36(2): 99-129, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27910763

RESUMO

IL10 was discovered in 1989, and since then it has been the subject of intense investigation, which has revealed its potent anti-inflammatory and regulatory activities in most immune processes during infection and disease. In 2003, the first non-mammalian IL10 sequence was identified in teleost fish, followed in 2004 by the chicken IL10 sequence. In this review, we summarize the work performed in non-mammalian vertebrates in which the IL10, IL10 receptors (IL10Rs), and their signaling components have been identified. We review the genomic organization, genes, and protein structure of IL10(Rs); we focus on studies providing a functional characterization of their biological activities. In addition, we describe the activities of viral IL10s identified in viruses infecting non-mammalian hosts. Altogether, our analysis reveals remarkable conservation of the anti-inflammatory and regulatory activities of (viral) IL10 across vertebrates, confirming the crucial role of IL10 throughout evolution. Interestingly, in some teleost fish, the presence of multiple copies of IL10(Rs) adds an additional degree of complexity. In fact, the evidence suggests that gene duplication does not necessarily imply functional redundancy, and leaves teleosts with additional possibilities to fine tune IL10 activities. Finally, we discuss the use of zebrafish (Danio rerio) as a complementary animal model for the study of IL10 activities in non-mammalian vertebrates.


Assuntos
Sequência Conservada , Evolução Molecular , Interleucina-10/genética , Animais , Humanos , Interleucina-10/química , Interleucina-10/imunologia , Modelos Moleculares , Filogenia
9.
J Microbiol ; 54(11): 761-767, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27796930

RESUMO

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.


Assuntos
Proteínas de Bactérias/química , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Escarro/química , Tuberculose Pulmonar/microbiologia , Antígenos de Diferenciação de Linfócitos T/química , Antígenos de Diferenciação de Linfócitos T/imunologia , Biomarcadores/química , Western Blotting , Feminino , Humanos , Interferon gama/química , Interferon gama/imunologia , Interleucina-10/química , Interleucina-10/imunologia , Interleucina-17/química , Interleucina-17/imunologia , Masculino , Mycobacterium tuberculosis/fisiologia , Proteínas/química , Proteínas/imunologia , Sensibilidade e Especificidade , Transferrina/química , Urease/química
10.
Appl Microbiol Biotechnol ; 100(24): 10479-10493, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27430741

RESUMO

Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.


Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Interleucina-10/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Interleucina-10/química , Interleucina-10/genética , Peso Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
BMC Pulm Med ; 16(1): 90, 2016 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-27260506

RESUMO

BACKGROUND: Ventilation-induced lung injury (VILI) is a health problem for patients with acute respiratory dysfunction syndrome. The aim of this study was to investigate the effectiveness of budesonide in treating VILI. METHODS: Twenty-four rats were randomized to three groups: a ventilation group, ventilation/budesonide group, and sham group were ventilated with 30 ml/kg tidal volume or only anesthesia for 4 hor saline or budesonide airway instillation immediately after ventilation. The PaO2/FiO2and wet-to-dry weight ratios, protein concentration, neutrophil count, and neutrophil elastase levels in bronchoalveolar lavage fluid (BALF) and the levels of inflammation-related factors were examined. Histological evaluation of and apoptosis measurement inthe lung were conducted. RESULTS: Compared with that in the ventilation group, the PaO2/FiO2 ratio was significantly increased by treatment with budesonide. The lung wet-to-dry weight ratio, total protein, neutrophil elastase level, and neutrophilcount in BALF were decreased in the budesonide group. The BALF and plasma tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, intercellular adhesion molecule (ICAM)-1, and macrophage inflammatory protein (MIP)-2 levels were decreased, whereas the IL-10 level was increased in the budesonide group. The phosphorylated nuclear factor (NF)-kBlevels in lung tissue were inhibited by budesonide. The histological changes in the lung and apoptosis were reduced by budesonide treatment. Bax, caspase-3, and cleaved caspase-3 were down-regulated, and Bcl-2 was up-regulated by budesonide. CONCLUSIONS: Budesonide ameliorated lung injury induced by large volume ventilation, likely by improving epithelial permeability, decreasing edema, inhibiting local and systemic inflammation, and reducing apoptosis in VILI.


Assuntos
Budesonida/uso terapêutico , Glucocorticoides/uso terapêutico , Pulmão/fisiopatologia , Respiração Artificial/efeitos adversos , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Caspase 3/sangue , Caspase 3/química , Quimiocina CXCL2/sangue , Quimiocina CXCL2/química , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/química , Interleucina-10/sangue , Interleucina-10/química , Interleucina-1beta/sangue , Interleucina-1beta/química , Interleucina-6/sangue , Interleucina-6/química , Contagem de Leucócitos , Masculino , NF-kappa B/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Distribuição Aleatória , Ratos , Ratos Wistar , Volume de Ventilação Pulmonar , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/química , Proteína X Associada a bcl-2/química
12.
PLoS One ; 11(6): e0156229, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27299860

RESUMO

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10's dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10's increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10's cholesterol regulating biology is consistent between mice and humans.


Assuntos
Colesterol/sangue , Hipercolesterolemia/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Interleucina-10/uso terapêutico , Macrófagos do Fígado/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Colesterol/imunologia , Feminino , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Interleucina-10/química , Interleucina-10/farmacologia , Macrófagos do Fígado/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Adulto Jovem
13.
PLoS One ; 11(4): e0154046, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27123592

RESUMO

Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.


Assuntos
Desenho de Drogas , Subunidade alfa de Receptor de Interleucina-10/metabolismo , Interleucina-10/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Sítios de Ligação , Projeto Auxiliado por Computador , Humanos , Interleucina-10/química , Subunidade alfa de Receptor de Interleucina-10/química , Ligantes , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica
14.
Chemistry ; 22(16): 5563-74, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-26918733

RESUMO

Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan-ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex.


Assuntos
Materiais Biocompatíveis/química , Glicosaminoglicanos/química , Ácido Hialurônico/química , Ácido Hialurônico/síntese química , Interleucina-10/química , Glicosaminoglicanos/metabolismo , Ligantes , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica
15.
Drug Deliv ; 23(6): 2058-64, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26673899

RESUMO

The oral delivery of peptides is a highly attractive treatment approach. However, the harsh environment of the gastrointestinal tract limits its application. Here, we utilize Bifidobacterium as a delivery system to orally deliver a potent anti-inflammatory but short duration peptide alpha-melanocyte-stimulating hormone (α-MSH) against experimental colitis. The aim of our study was to facilitate the efficient oral delivery of α-MSH. We designed a vector of pBDMSH and used it to construct a Bifidobacterium longum expressing α-MSH. We then determined the bioactivity of recombinant Bifidobacterium in lipopolysaccharide-induced inflammatory models of HT-29 cells. Finally, we used Bifidobacterium expressing α-MSH against dextran sulfate sodium (DSS)-induced ulcerative colitis mice. Results based on the myeloperoxidase activity, the levels of inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-10 and the histological injury of colon tissue reveal recombinant Bifidobacterium was efficient in attenuating DSS-induced ulcerative colitis, suggesting an alternative way to use Bifidobacterium as a delivery system to deliver α-MSH for DSS-induced ulcerative colitis therapy.


Assuntos
Colo/química , Sulfato de Dextrana/química , Interleucina-10/química , Fator de Necrose Tumoral alfa/química , alfa-MSH/química , alfa-MSH/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Bifidobacterium longum , Colo/efeitos dos fármacos , Células HT29 , Humanos , Interleucina-10/farmacologia , Camundongos , alfa-MSH/metabolismo
16.
J Biol Chem ; 291(6): 3100-13, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26677224

RESUMO

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.


Assuntos
Glicosaminoglicanos/química , Interleucina-10/química , Modelos Moleculares , Animais , Sítios de Ligação , Cristalografia por Raios X , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular
17.
Inhal Toxicol ; 27(14): 802-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26572172

RESUMO

OBJECTIVE: The purpose of this study was to investigate the effects of cigaret smoke (CS) on a mouse model of emphysema and examine the protective role of N-acetylcysteine (NAC) in the CS-induced exacerbation of pulmonary damage in the mice. METHOD: Particulate matter (PM) in sidestream cigaret smoke aerosol was analyzed by a scanning mobility particle sizer spectrometer. A mouse model of emphysema was established by an injection of porcine pancreatic elastase (PPE) into the trachea. Mice with emphysema were then exposed to filtered air, or sidestream CS with intragastric administration of NAC or normal saline. Mouse body weight, survival, pulmonary tissue histology, total antioxidant capacity (T-AOC) and malonaldehyde (MDA) contents in lung tissue, and inflammatory responses were examined. RESULTS: Particles with a size of ≤346 nm constituted 99.06% of CS PM. Mice exhibited ruptured alveolar septal, alveolar fusion, significantly increased mean lining interval, and reduced mean alveolar number (all p < 0.05), 21 d after PPE injection. Exposure of mice with emphysema to CS exacerbated the pulmonary tissue damage, caused weight loss, significantly increased mortality, decreased T-AOC, elevated MDA contents in lung tissue, and increased interleukin (IL)-1ß levels in bronchoalveolar lavage (BAL) fluids (all p < 0.05). Administration of NAC attenuated those CS-induced adverse effects in the mice and increased anti-inflammatory factor IL-10 levels in BAL fluids significantly (all p < 0.05). CONCLUSIONS: Exposure of mice with emphysema to CS exacerbated the pulmonary damage, and NAC reduced the CS-mediated pulmonary damage by preventing oxidative damage and reducing inflammatory responses.


Assuntos
Acetilcisteína/uso terapêutico , Enfisema/induzido quimicamente , Enfisema/tratamento farmacológico , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/química , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-10/química , Interleucina-10/metabolismo , Interleucina-1beta/química , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
18.
J Mol Graph Model ; 62: 97-104, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26409190

RESUMO

The biological function of the pleiotropic cytokine interleukin-10 (IL-10), which has an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are essential constituents of the extracellular matrix with an important role in modulating the biological function of many proteins. The molecular mechanisms governing the IL-10-GAG interaction, though, are unclear so far. In particular, detailed knowledge about GAG binding sites and recognition mode on IL-10 is lacking, despite of its imminent importance for understanding the functional consequences of IL-10-GAG interaction. In the present work, we report a GAG binding site on IL-10 identified by applying computational methods based on Coulomb potential calculations and specialized molecular dynamics simulations. The identified GAG binding site is constituted of several positively charged residues, which are conserved among species. Exhaustive conformational space sampling of a series of GAG ligands binding to IL-10 led to the observation of two GAG binding modes in the predicted binding site, and to the identification of IL-10 residues R104, R106, R107, and K119 as being most important for molecular GAG recognition. In silico mutation as well as single-residue energy decomposition and detailed analysis of hydrogen-bonding behavior led to the conclusion that R107 is most essential and assumes a unique role in IL-10-GAG interaction. This structural and dynamic characterization of GAG-binding to IL-10 represents an important step for further understanding the modulation of the biological function of IL-10.


Assuntos
Glicosaminoglicanos/química , Interleucina-10/química , Sítios de Ligação , Humanos , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína
19.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 31(2): 107-10, 2015 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-26248411

RESUMO

OBJECTIVE: To study the effects of Volatile Oil of Radix Angelicae Sinensis (VOA) on experimental asthma in rat model based on abnormal immune functions of Treg cells. METHODS: After grouping, the asthmatic rats were developed through injecting OVA and AI(OH)3 for sensitization and then administering OVA aerosol for challenge, and the respiratory functions, asthmatic behaviors, IL-10 levels in bronchoalveolar lavage fluid (BALF) (ELISA) and Foxp3 expression (immunohistochemistry) in lung of asthmatic rats were observed. RESULTS: VOA at the doses of 40-160 mg/kg could improve the respiratory functions and the asthmatic behaviors, and upgrade IL-10 levels in BALF and Foxp3 expression in lung of asthmatic rats. CONCLUSION: VOA has some effects of anti-asthma and one of the mechanisms is to improving the lower immune functions of Treg cells.


Assuntos
Angelica sinensis/química , Asma/tratamento farmacológico , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Interleucina-10/química , Pulmão/metabolismo , Ratos , Linfócitos T Reguladores/citologia
20.
J Immunol Methods ; 420: 38-49, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25837415

RESUMO

Macrophages play a key role in tissue regeneration following peripheral nerve injury by preparing the surrounding parenchyma for regeneration, however, they can be damaging if the response is excessive. Interleukin 10 (IL-10) is a cytokine that promotes macrophages toward an anti-inflammatory/wound healing state (M2 phenotype). The bioactive half-life of IL-10 is dependent on the cellular microenvironment and ranges from minutes to hours in vivo. Our objective was to extend the in vivo bioavailability and bioactivity of IL-10 by attaching the protein onto nanofibrous scaffolds and demonstrating increased expression levels of M2 macrophages when placed around healthy intact peripheral nerves. IL-10 was adsorbed and covalently bound to electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffolds. In vivo bioavailability and bioactivity of IL-10 was confirmed by wrapping IL-10 conjugated nanofibres around the sciatic nerves of Wistar rats and quantifying M2 macrophages immunohistochemically double labelled with ED1 and either arginase 1 or CD206. IL-10 remained immobilised to PCL scaffolds for more than 120 days when stored in phosphate buffered saline at room temperature and for up to 14d ays when implanted around the sciatic nerve. IL-10 conjugated nanofibres successfully induced macrophage polarisation towards the M2 activated state within the scaffold material as well as the adjacent tissue surrounding the nerve. PCL biofunctionalised nanofibres are useful for manipulating the cellular microenvironment. Materials such as these could potentially lead to new therapeutic strategies for nervous tissue injuries as well as provide novel investigative tools for biological research.


Assuntos
Interleucina-10/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Nanofibras/química , Nervos Periféricos/imunologia , Poliésteres/farmacologia , Tecidos Suporte , Animais , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Interleucina-10/química , Macrófagos/patologia , Masculino , Nervos Periféricos/patologia , Poliésteres/química , Ratos , Ratos Wistar
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