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1.
Front Immunol ; 9: 1895, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30174671

RESUMO

Interleukin- 33 (IL-33) is an epithelial-derived cytokine that initiates type 2 immune responses to allergens, though whether IL-33 has the ability to modify responses to respiratory viral infections remains unclear. This study aimed to investigate the effects of IL-33 on rhinovirus (RV)-induced immune responses by circulating leukocytes from people with allergic asthma, and how this response may differ from non-allergic controls. Our experimental approach involved co-exposing peripheral blood mononuclear cells to IL-33 and RV in order to model how the functions of virus-responsive lymphocytes could be modified after recruitment to an airway environment enriched in IL-33. In the current study, IL-33 enhanced RV-induced IL-5 and IL-13 release by cells from people with allergic asthma, but had no effect on IL-5 and IL-13 production by cells from healthy donors. In asthmatic individuals, IL-33 also enhanced mRNA and surface protein expression of ST2 (the IL-33 receptor IL1RL1), while soluble ST2 concentrations were low. In contrast, IL-33 had no effect on mRNA and surface expression of ST2 in healthy individuals. In people with allergic asthma, RV-activated ST2+ innate lymphoid cells (ST2+ILC) were the predominant source of IL-33 augmented IL-13 release. In contrast, RV-activated natural killer cells (NK cells) were the predominant source of IL-33 augmented IFNγ release in healthy individuals. This suggests that the effects of IL-33 on the cellular immune response to RV differ between asthmatic and healthy individuals. These findings provide a mechanism by which RV infections and IL-33 might interact in asthmatic individuals to exacerbate type 2 immune responses and allergic airway inflammation.


Assuntos
Asma/etiologia , Asma/metabolismo , Imunidade , Interleucina-33/metabolismo , Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/imunologia , Rhinovirus/imunologia , Adolescente , Adulto , Estudos de Casos e Controles , Citocinas/biossíntese , Feminino , Humanos , Imunidade Inata , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
Biol Chem ; 397(12): 1299-1305, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27533117

RESUMO

Kallikrein-related peptidase (KLK) 14 is a serine protease linked to several pathologies including prostate cancer. We show that KLK14 has biphasic effects in vitro on activating and inhibiting components of the prostate cancer associated hepatocyte growth factor (HGF)/Met system. At 5-10 nm, KLK14 converts pro-HGF to the two-chain heterodimer required for Met activation, while higher concentrations degrade the HGF α-chain. HGF activator-inhibitor (HAI)-1A and HAI-1B, which inhibit pro-HGF activators, are degraded by KLK14 when protease:inhibitor stoichiometry is 1:1 or the protease is in excess. When inhibitors are in excess, KLK14 generates HAI-1A and HAI-1B fragments known to inhibit pro-HGF activating serine proteases. These in vitro data suggest that increased KLK14 activity could contribute at multiple levels to HGF/Met-mediated processes in prostate and other cancers.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Células Sf9 , Spodoptera
3.
Clin Transl Immunology ; 4(10): e46, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26682054

RESUMO

Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-α and IFN-ß) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracellular flow cytometry, >90% of the IFN-α-producing cells in human blood mononuclear cells following HRV16 exposure are plasmacytoid dendritic cells, whereas monocytes and myeloid dendritic cells contribute only 10% and <1%, respectively, of the IFN-α production. Bafilomycin and chloroquine, agents that inhibit the function of endosomal toll-like receptors (TLRs), significantly reduced the capacity of TLR3-, TLR7- and TLR-9-stimulated cells to produce IFN-α and the IFN-induced chemokine CXCL10 (IP-10). In contrast, only bafilomycin (but not chloroquine) effectively suppressed HRV16-stimulated IFN-α and IP-10 production, whereas neither bafilomycin or chloroquine inhibited HRV16-stimulated interleukin-6 release. Attempts to block IFN-α production with commercially available TLR-specific oligonucleotides were unsuccessful due to major 'off-target' effects. These findings suggest that among circulating haemopoietic cells, plasmacytoid dendritic cells and TLRs located within endosomes are critical for inducing efficient IFN-I production in response to HRVs.

4.
PLoS One ; 9(9): e106501, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25203745

RESUMO

BACKGROUND: Human rhinovirus (HRV) infection is a major trigger for asthma exacerbations. Anti-viral immunity appears to be abnormal in asthma, with immune dysfunction reported in both airway structural cells and migratory, bone marrow derived cells. Though decreased capacity to produce anti-viral interferons (IFNs) has been reported in asthma, a detailed analysis of the molecular events involved has not been undertaken. OBJECTIVE: To compare the molecular pathway controlling type I IFN synthesis in HRV-stimulated peripheral blood mononuclear cells (PBMC) from asthmatic and healthy subjects. METHODS: PBMC from 22 allergic asthmatics and 20 healthy donors were cultured with HRV for 24 hours. Multiple components of the Toll-like receptor (TLR), IFN regulatory and NFκß pathways were compared at the mRNA and protein level. RESULTS: Multiple deficiencies in the innate immune response to HRV were identified in asthma, with significantly lower expression of IFNα, IFNß and interferon stimulated genes than in healthy subjects. This was accompanied by reduced expression of intra-cellular signalling molecules including interferon regulatory factors (IRF1, IRF7), NF-κB family members (p50, p52, p65 and IκKα) and STAT1, and by reduced responsiveness to TLR7/TLR8 activation. These observations could not be attributed to alterations in the numbers of dendritic cell (DC) subsets in asthma or baseline expression of the viral RNA sensing receptors TLR7/TLR8. In healthy subjects, blocking the activity of type-I IFN or depleting plasmacytoid DC recapitulated many of the abnormalities observed in asthma. CONCLUSIONS: Multiple abnormalities in innate anti-viral signalling pathways were identified in asthma, with deficiencies in both IFN-dependent and IFN-independent molecules identified.


Assuntos
Asma/imunologia , Asma/virologia , Imunidade Inata , Rhinovirus/fisiologia , Transdução de Sinais/imunologia , Adulto , Animais , Asma/genética , Células Dendríticas/imunologia , Feminino , Humanos , Interferon-alfa/biossíntese , Interferon-alfa/genética , Interferon beta/biossíntese , Interferon beta/genética , Masculino , RNA Viral/metabolismo , Rhinovirus/genética , Receptores Toll-Like/metabolismo , Ativação Transcricional/imunologia
5.
BMC Pulm Med ; 12: 37, 2012 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-22849333

RESUMO

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are often linked to respiratory infections. However, it is unknown if COPD patients who experience frequent exacerbations have impaired humoral immunity. The aim of this study was to determine if antibodies specific for common respiratory pathogens are associated with AECOPD. METHODS: Plasma was obtained from COPD patients when clinically stable. AECOPD requiring hospitalisation were recorded. IgG1 antibodies to H. Influenzae outer membrane protein 6 (P6), pneumococcal surface protein C (PspC) and the VP1 viral capsid protein of rhinovirus were measured. RESULTS: COPD patients who had an AECOPD (n = 32) had significantly lower anti-VP1 IgG1 antibody levels when stable compared to COPD patients who did not have an AECOPD (n = 28, p = 0.024). Furthermore, the number of hospitalisations was inversely proportional to anti-VP1 antibody levels (r = -0.331, p = 0.011). In contrast, antibodies specific for P6 and PspC were present at similar concentrations between groups. Plasma IL-21, a cytokine important for B-cell development and antibody synthesis, was also lower in COPD patients who had an AECOPD, than in stable COPD patients (p = 0.046). CONCLUSION: Deficient humoral immunity specific for rhinoviruses is associated with AECOPD requiring hospitalisation, and may partly explain why some COPD patients have an increased exacerbation risk following respiratory viral infections.


Assuntos
Anticorpos Antivirais/sangue , Progressão da Doença , Hospitalização , Imunidade Humoral/fisiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Rhinovirus/imunologia , Índice de Gravidade de Doença , Idoso , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Feminino , Vacinas Anti-Haemophilus/imunologia , Humanos , Imunoglobulina G/sangue , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Infecções Respiratórias/complicações , Proteínas Virais/imunologia
6.
J Immunol ; 188(12): 5898-905, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22611238

RESUMO

Human rhinoviruses (RV) cause only minor illness in healthy individuals, but can have deleterious consequences in people with asthma. This study sought to examine normal homeostatic mechanisms regulating adaptive immunity to RV in healthy humans, focusing on effects of IFN-αß and plasmacytoid dendritic cells (pDC) on Th2 immune responses. PBMC were isolated from 27 healthy individuals and cultured with RV16 for up to 5 d. In some experiments, IFN-αß was neutralized using a decoy receptor that blocks IFN signaling, whereas specific dendritic cell subsets were depleted from cultures with immune-magnetic beads. RV16 induced robust expression of IFN-α, IFN-ß, multiple IFN-stimulated genes, and T cell-polarizing factors within the first 24 h. At 5 d, the production of memory T cell-derived IFN-γ, IL-10, and IL-13, but not IL-17A, was significantly elevated. Neutralizing the effects of type-I IFN with the decoy receptor B18R led to a significant increase in IL-13 synthesis, but had no effect on IFN-γ synthesis. Depletion of pDC from RV-stimulated cultures markedly inhibited IFN-α secretion, and led to a significant increase in expression and production of the Th2 cytokines IL-5 (p = 0.02), IL-9 (p < 0.01), and IL-13 (p < 0.01), but had no effect on IFN-γ synthesis. Depletion of CD1c(+) dendritic cells did not alter cytokine synthesis. In healthy humans, pDC and the IFN-αß they secrete selectively constrain Th2 cytokine synthesis following RV exposure in vitro. This important regulatory mechanism may be lost in asthma; deficient IFN-αß synthesis and/or pDC dysfunction have the potential to contribute to asthma exacerbations during RV infections.


Assuntos
Imunidade Adaptativa/imunologia , Asma/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Interferon-alfa/imunologia , Infecções por Picornaviridae/imunologia , Células Th2/imunologia , Adulto , Asma/metabolismo , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Separação Imunomagnética , Masculino , Infecções por Picornaviridae/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus , Células Th2/metabolismo
7.
J Pediatr ; 161(4): 621-5.e1, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22575255

RESUMO

OBJECTIVE: To determine bronchoalveolar lavage (BAL) levels of 3 innate immunity components (human ß-defensin-2 [hBD2], mannose-binding lectin [MBL], and surfactant protein-A [SP-A]), the relationship with airway neutrophilia and infection, and cytokine production of stimulated BAL cells in children with current protracted bacterial bronchitis (PBB), children with resolved PBB (PBB well), and controls. STUDY DESIGN: BAL of 102 children (mean age 2.8 years) fulfilling predefined criteria of current PBB (n = 61), PBB well (n = 20), and controls (n = 21) was cultured (quantitative bacteriology) and viruses examined by polymerase chain reaction. hBD2, MBL, and SP-A were measured, and cytokine production by lipopolysaccharide-stimulated BAL cells was determined. RESULTS: Median hBD2 and MBL levels were significantly higher in the current PBB group (hBD2 = 164.4, IQR 0-435.5 pg/mL; MBL = 1.7, 0.4-4 ng/mL) than in the PBB well group (hBD2 = 0, IQR 0-85.2; MBL = 0.6, IQR 0.03-2.9) and controls (hBD2 = 3.6, IQR 0-126; MBL = 0.4, IQR 0.02-79). hBD2 was significantly higher in children with airway infection (n = 54; median 76.9, IQR 0-397.3) compared with those without (n = 48; 0, IQR 0-236.3), P= .04. SP-A levels and cytokine production of stimulated BAL cells were similar between groups. CONCLUSION: In children's airways, hBD2, but not MBL and SP-A, relates to inflammation and infection. In children with PBB, mechanisms involving airway hBD2 and MBL are augmented. These pulmonary innate immunity components and the ability of BAL cells to respond to stimuli are unlikely to be deficient.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Lectina de Ligação a Manose/análise , Proteína A Associada a Surfactante Pulmonar/análise , beta-Defensinas/análise , Bronquite Crônica , Broncoscopia , Pré-Escolar , Feminino , Humanos , Imunidade Inata , Lactente , Masculino
8.
Respirology ; 17(5): 841-6, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22452564

RESUMO

BACKGROUND AND OBJECTIVE: The soluble receptor for advanced glycation end products (sRAGE) plays an important role in inflammation. Few studies have looked at sRAGE levels in human lungs, and there is no information in children. Therefore, this study aimed to compare bronchoalveolar lavage fluid (BALF) and plasma sRAGE concentrations in children in relation to age and inflammation. METHODS: BAL was performed in 76 children, and BALF and plasma sRAGE levels were determined by enzyme-linked immunosorbent assay. RESULTS: sRAGE levels were fourfold higher in BALF than in plasma (P < 0.001). BALF sRAGE was inversely proportional to age (r = -0.333, P = 0.008) and serum immunoglobulin A (r = -0.283, P = 0.028). Plasma sRAGE showed a positive correlation to the percentage of BAL macrophages and negative correlation to the percentage of neutrophils and lymphocytes (P < 0.05). Multivariate linear regression analysis identified that the percentage of BAL lymphocytes and neutrophils were significant independent predictors of plasma sRAGE levels, while age and the percentage of BAL macrophages independently predicted BALF sRAGE levels. CONCLUSIONS: In children, sRAGE is present at higher concentrations in the lung compared with blood. It appears that sRAGE varies with age, and hence future studies of sRAGE in paediatric lung disease require age matching. The significant relationship between sRAGE and lung inflammation warrants further research.


Assuntos
Envelhecimento/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Receptores Imunológicos/metabolismo , Adolescente , Adulto , Líquido da Lavagem Broncoalveolar/química , Broncoscopia , Criança , Feminino , Humanos , Imunidade Humoral/fisiologia , Imunoglobulina A/sangue , Masculino , Análise Multivariada , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/análise , Estudos Retrospectivos , Adulto Jovem
9.
PLoS One ; 6(11): e27898, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22125636

RESUMO

Asthma is a chronic inflammatory airways disease in which respiratory viral infections frequently trigger exacerbations. Current treatment of asthma with combinations of inhaled corticosteroids and long acting beta2 agonists improves asthma control and reduces exacerbations but what impact this might have on innate anti-viral immunity is unclear. We investigated the in vitro effects of asthma drugs on innate anti-viral immunity. Peripheral blood mononuclear cells (PBMC) from healthy and asthmatic donors were cultured for 24 hours with the Toll-like receptor 7 agonist, imiquimod, or rhinovirus 16 (RV16) in the presence of budesonide and/or formoterol. Production of proinflammatory cytokines and expression of anti-viral intracellular signalling molecules were measured by ELISA and RT-PCR respectively. In PBMC from healthy donors, budesonide alone inhibited IP-10 and IL-6 production induced by imiquimod in a concentration-dependent manner and the degree of inhibition was amplified when budesonide and formoterol were used in combination. Formoterol alone had little effect on these parameters, except at high concentrations (10⁻6 M) when IL-6 production increased. In RV16 stimulated PBMC, the combination of budesonide and formoterol inhibited IFNα and IP-10 production in asthmatic as well as healthy donors. Combination of budesonide and formoterol also inhibited RV16-stimulated expression of the type I IFN induced genes myxovirus protein A and 2', 5' oligoadenylate synthetise. Notably, RV16 stimulated lower levels of type Myxovirus A and oligoadenylate synthase in PBMC of asthmatics than control donors. These in vitro studies demonstrate that combinations of drugs commonly used in asthma therapy inhibit both early pro-inflammatory cytokines and key aspects of the type I IFN pathway. These findings suggest that budesonide and formoterol curtail excessive inflammation induced by rhinovirus infections in patients with asthma, but whether this inhibits viral clearance in vivo remains to be determined.


Assuntos
Budesonida/farmacologia , Etanolaminas/farmacologia , Imunidade Inata/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Rhinovirus/imunologia , Adjuvantes Imunológicos/farmacologia , Adulto , Aminoquinolinas/farmacologia , Asma/imunologia , Asma/patologia , Broncodilatadores/farmacologia , Células Cultivadas , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática , Feminino , Fumarato de Formoterol , Humanos , Imiquimode , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Receptor 7 Toll-Like/agonistas
10.
Respir Res ; 11: 184, 2010 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-21194432

RESUMO

BACKGROUND: Rhinoviruses (RV) are key triggers in acute asthma exacerbations. Previous studies suggest that men suffer from infectious diseases more frequently and with greater severity than women. Additionally, the immune response to most infections and vaccinations decreases with age. Most immune function studies do not account for such differences, therefore the aim of this study was to determine if the immune response to rhinovirus varies with sex or age. METHODS: Blood mononuclear cells were isolated from 63 healthy individuals and grouped by sex and age (≤50 years old and ≥52 years old). Cells were cultured with rhinovirus 16 at a multiplicity of infection of 1. The chemokine IP-10 was measured at 24 h as an index of innate immunity while IFNγ and IL-13 were measured at 5 days as an index of adaptive immunity. RESULTS: Rhinovirus induced IFNγ and IL-13 was significantly higher in ≤50 year old women than in age matched men (p < 0.02 and p < 0.05) and ≥52 year old women (p < 0.02 and p > 0.005). There was no sex or age based difference in rhinovirus induced IP-10 expression. Both IFNγ and IL-13 were negatively correlated with age in women but not in men. CONCLUSIONS: This study suggests that pre-menopausal women have a stronger adaptive immune response to rhinovirus infection than men and older people, though the mechanisms responsible for these differences remain to be determined. Our findings highlight the importance of gender and age balance in clinical studies and in the development of new treatments and vaccines.


Assuntos
Imunidade Adaptativa/imunologia , Envelhecimento/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Rhinovirus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Adulto Jovem
11.
J Biol Chem ; 283(35): 24000-10, 2008 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-18577513

RESUMO

The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease.


Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Células CHO , Membrana Celular/genética , Membrana Celular/metabolismo , Canais de Cloreto/genética , Cricetinae , Cricetulus , Complexos Endossomais de Distribuição Requeridos para Transporte , Glicosilação , Humanos , Proteínas de Membrana/genética , Ubiquitina-Proteína Ligases Nedd4 , Proteínas de Neoplasias/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/fisiologia
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