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1.
Am J Respir Cell Mol Biol ; 64(5): 557-568, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33556295

RESUMO

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.


Assuntos
Asma/imunologia , Plaquetas/imunologia , Hiper-Reatividade Brônquica/imunologia , Pulmão/imunologia , Ativação Plaquetária/imunologia , Receptores CCR3/imunologia , Adolescente , Adulto , Idoso , Alérgenos/administração & dosagem , Animais , Antígenos de Dermatophagoides/administração & dosagem , Proteínas de Artrópodes/administração & dosagem , Asma/genética , Asma/mortalidade , Asma/patologia , Plaquetas/efeitos dos fármacos , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Criança , Cisteína Endopeptidases/administração & dosagem , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Pyroglyphidae/química , Pyroglyphidae/imunologia , Receptores CCR3/genética , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Transdução de Sinais , Análise de Sobrevida
2.
J Pharmacol Exp Ther ; 376(1): 51-63, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33115824

RESUMO

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Indanos/uso terapêutico , Naftalenos/uso terapêutico , Fenilpropionatos/uso terapêutico , Quinazolinonas/uso terapêutico , Receptores de Detecção de Cálcio/agonistas , Regulação Alostérica , Animais , Antiasmáticos/efeitos adversos , Antiasmáticos/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Indanos/efeitos adversos , Indanos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/efeitos adversos , Naftalenos/farmacologia , Fenilpropionatos/efeitos adversos , Fenilpropionatos/farmacologia , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacologia , Receptores de Detecção de Cálcio/metabolismo
3.
Respir Res ; 21(1): 3, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900184

RESUMO

BACKGROUND: Blood eosinophil count has been proposed as a predictor of response to inhaled corticosteroid (ICS) in the prevention of acute exacerbations of COPD. An optimal threshold of blood eosinophil count for prescribing ICS has not been agreed. Doubt has been cast on the role by observational studies. The role of inhaled corticosteroids in this relationship, independent of long-acting bronchodilators, has not been examined. METHODS: We conducted a systematic review of post-hoc analyses of randomised controlled trials (RCTs) and observational studies examining three blood eosinophil thresholds and the independent role of ICS. Included studies were categorised by the form (relative or absolute count) and cut point of eosinophil threshold used. Thresholds assessed were relative eosinophil count of 2%, and absolute counts of 150 cells/µL and 300 cells/µL. Three meta-analyses of the effect of ICS use in post-hoc analyses of RCTs based on these counts were carried out. Initial analysis included all studies of ICS vs. any non-ICS regimen. Further analysis examined the effect of ICS, independent of the effect of long-acting bronchodilators. RESULTS: Sixteen studies examined the association between blood eosinophil count and response of exacerbation risk to ICS, in COPD patients. Eleven studies (25,881 patients) were post-hoc analyses of RCTs. Five studies (109,704 patients) were retrospective observational studies. The independent effect of ICS on the reduction of exacerbation risk was 20% at ≥2% blood eosinophil threshold (RR, 0.80; 95% CI, 0.74-0.85), 35% at ≥150 cells/µL blood eosinophil threshold (RR, 0.65; 0.52-0.79), and 39% at ≥300 cells/µL blood eosinophil threshold (RR, 0.61; 0.44-0.78). No association was found in four out of five observational studies. CONCLUSION: This is the first systematic review to assess, in post-hoc analyses of RCTs, the independent effect of ICS in reducing the risk of COPD exacerbation across a range of blood eosinophil thresholds. Association between ICS prescription and reduced exacerbation risk at these thresholds was confirmed. The lack of association found in the observational studies questions the relevance of these observations to a "real world" COPD population. To clarify the clinical utility of this biomarker, the association should be tested in prospective effectiveness studies.


Assuntos
Corticosteroides/administração & dosagem , Eosinófilos/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Administração por Inalação , Progressão da Doença , Humanos , Contagem de Leucócitos , Estudos Observacionais como Assunto , Doença Pulmonar Obstrutiva Crônica/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto
4.
Asian Pac J Allergy Immunol ; 38(2): 78-90, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32563233

RESUMO

BACKGROUND: Elucidation of the critical immune pathways involved in allergic inflammation has identified, apart from IgE, therapeutic targets in the cytokine network suitable for intervention by biological therapies. OBJECTIVE: The drugs that target the cytokine networks pertinent to asthma and allergic diseases are reviewed and some illustrative case histories presented. The overview proposes a framework to use when deciding which monoclonal antibody (mAb) to select for treatment of severe asthma based on total IgE concentration, peripheral blood eosinophil count, induced sputum analysis and measurement of fractional exhaled nitric oxide (FENO). METHODS: Internet-based literature search including PubMed for studies on biological therapies targeting IgE and the cytokine network in allergic inflammation focusing on asthma with and without rhinosinusitis and nasal polyposis, eczema, urticaria and food allergies. Lists of pivotal trials published in the peer reviewed literature and pertaining to their own mAb products were also provided by GSK, AstraZeneca and Sanofi. Therapeutic agents licensed or in advanced stages of development (Phase 2b and 3) were selected for discussion. RESULTS: The survey identifies a number of mAbs with substantial potential for the future targeted treatment of asthma with and without rhinosinusitis and nasal polyposis, eczema, urticaria and food allergies uncontrolled by existing therapies. A pragmatic framework is proposed for selecting the optimal mAb for initial use in individual patients with severe asthma. CONCLUSIONS: Launch of these new biologicals may revolutionise the treatment of allergic diseases if employed in an endotype-specific fashion, heralding an unprecedented era of personalised medicine.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Asma/tratamento farmacológico , Terapia Biológica/métodos , Hipersensibilidade/tratamento farmacológico , Animais , Humanos , Medicina de Precisão
5.
Am J Respir Cell Mol Biol ; 54(2): 250-62, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26196219

RESUMO

Exposure to urban particulate matter (UPM) exacerbates asthmatic lung inflammation. Lung dendritic cells (DCs) are critical for stimulating T cell immunity and in maintaining airway tolerance, but they also react to airway UPM. The adjuvant role of UPM in enhancing primary immune responses by naive cells to allergen has been reported, but the direct effects of UPM-activated DCs on the functionality of human memory CD4 T cells (Tms), which constitute the majority of T cells in the lung, has not been investigated. Blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of house dust mite or tetanus toxoid control antigen. 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester-labeled blood Tms were cocultured with autologous DCs, T cell proliferation and effector function were assessed using flow cytometry, and secreted cytokines were measured by combined bead array. UPM-DCs elicited IFN-γ and IL-13 secretion and induced proliferation in Tms isolated from both allergic patients with asthma and healthy control subjects, whereas only IL-13 was produced by Tms from patients with atopic asthma stimulated by house dust mite-loaded DCs. UPM-DCs drove the expansion and differentiation of a mixed population of Th1, Th2, and Th17 cell effectors through a mechanism that was dependent on major histocompatibility class II but not on cytokine-driven expansion. The data suggest that UPM not only has adjuvant properties but is also a source of antigen that stimulates the generation of Th2, Th1, and Th17 effector phenotypes, which have been implicated in both exacerbations of asthma and chronic inflammatory diseases.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Material Particulado/toxicidade , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Saúde da População Urbana , Adulto , Alérgenos/imunologia , Animais , Asma/imunologia , Asma/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Memória Imunológica , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Material Particulado/imunologia , Fenótipo , Pyroglyphidae/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Toxina Tetânica/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adulto Jovem
6.
Eur Respir J ; 47(3): 769-82, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26869672

RESUMO

Activin-A is a pleiotropic cytokine that regulates allergic inflammation. Its role in the regulation of angiogenesis, a key feature of airways remodelling in asthma, remains unexplored. Our objective was to investigate the expression of activin-A in asthma and its effects on angiogenesis in vitro.Expression of soluble/immunoreactive activin-A and its receptors was measured in serum, bronchoalveolar lavage fluid (BALF) and endobronchial biopsies from 16 healthy controls, 19 patients with mild/moderate asthma and 22 severely asthmatic patients. In vitro effects of activin-A on baseline and vascular endothelial growth factor (VEGF)-induced human endothelial cell angiogenesis, signalling and cytokine release were compared with BALF concentrations of these cytokines in vivo.Activin-A expression was significantly elevated in serum, BALF and bronchial tissue of the asthmatics, while expression of its protein receptors was reduced. In vitro, activin-A suppressed VEGF-induced endothelial cell proliferation and angiogenesis, inducing autocrine production of anti-angiogenic soluble VEGF receptor (R)1 and interleukin (IL)-18, while reducing production of pro-angiogenic VEGFR2 and IL-17. In parallel, BALF concentrations of soluble VEGFR1 and IL-18 were significantly reduced in severe asthmatics in vivo and inversely correlated with angiogenesis.Activin-A is overexpressed and has anti-angiogenic effects in vitro that are not propagated in vivo, where reduced basal expression of its receptors is observed particularly in severe asthma.


Assuntos
Ativinas/metabolismo , Asma/metabolismo , Brônquios/patologia , Citocinas/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ativinas/genética , Adulto , Líquido da Lavagem Broncoalveolar , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Índice de Gravidade de Doença , Fator A de Crescimento do Endotélio Vascular/genética
7.
J Allergy Clin Immunol ; 136(3): 628-637.e4, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25772594

RESUMO

BACKGROUND: A small population of patients with severe asthma does not respond to glucocorticoids (steroid resistant [SR]). They have high morbidity, highlighting an urgent need for strategies to enhance glucocorticoid responsiveness. OBJECTIVE: We investigated the immunologic differences between steroid-sensitive (SS) and SR asthmatic patients and the effect on immunophenotype of oral calcitriol treatment because it has been previously shown to beneficially modulate the clinical response to glucocorticoids in patients with SR asthma. METHODS: CD8-depleted PBMCs were isolated from 12 patients with SS and 23 patients with SR asthma and cultured for 7 days with anti-CD3 and IL-2 with or without dexamethasone. Cytokine production was assessed in supernatants by using the Cytometric Bead Array. Patients with SR asthma were subsequently randomized to oral calcitriol or placebo therapy, and identical studies were repeated. RESULTS: Patients with SR asthma produced significantly increased IL-17A and IFN-γ levels compared with those in patients with SS asthma, although it was evident that cells from individual patients might overproduce one or the other of these cytokines. Production of IL-17A was inversely and production of IL-13 was positively associated with the clinical response to prednisolone. Oral calcitriol, compared with placebo, therapy of the patients with SR asthma significantly improved dexamethasone-induced IL-10 production in vitro while suppressing dexamethasone-induced IL-17A production. This effect mirrored the previously demonstrated improvement in clinical response to oral glucocorticoids in calcitriol-treated patients with SR asthma. CONCLUSIONS: IL-17A(high) and IFN-γ(high) immunophenotypes exist in patients with SR asthma. These data identify immunologic pathways that likely underpin the beneficial clinical effects of calcitriol in patients with SR asthma by directing the SR cytokine profile toward a more SS immune phenotype, suggesting strategies for identifying vitamin D responder immunophenotypes.


Assuntos
Asma/tratamento farmacológico , Calcitriol/uso terapêutico , Fatores Imunológicos/uso terapêutico , Interferon gama/biossíntese , Interleucina-17/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Prednisolona/uso terapêutico , Corticosteroides/farmacologia , Adulto , Asma/imunologia , Asma/patologia , Complexo CD3/farmacologia , Dexametasona/farmacologia , Resistência a Medicamentos , Feminino , Humanos , Imunofenotipagem , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-17/imunologia , Interleucina-2/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Índice de Gravidade de Doença
8.
Thorax ; 70(5): 451-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25724847

RESUMO

RATIONALE: Asthma exacerbations are commonly precipitated by viral upper respiratory infections (URIs). Vitamin D insufficiency associates with susceptibility to URI in patients with asthma. Trials of vitamin D in adults with asthma with incidence of exacerbation and URI as primary outcome are lacking. OBJECTIVE: To conduct a randomised controlled trial of vitamin D3 supplementation for the prevention of asthma exacerbation and URI (coprimary outcomes). MEASUREMENTS AND METHODS: 250 adults with asthma in London, UK were allocated to receive six 2-monthly oral doses of 3 mg vitamin D3 (n=125) or placebo (n=125) over 1 year. Secondary outcomes included asthma control test and St George's Respiratory Questionnaire scores, fractional exhaled nitric oxide and concentrations of inflammatory markers in induced sputum. Subgroup analyses were performed to determine whether effects of supplementation were modified by baseline vitamin D status or genotype for 34 single nucleotide polymorphisms in 11 vitamin D pathway genes. MAIN RESULTS: 206/250 participants (82%) were vitamin D insufficient at baseline. Vitamin D3 did not influence time to first severe exacerbation (adjusted HR 1.02, 95% CI 0.69 to 1.53, p=0.91) or first URI (adjusted HR 0.87, 95% CI 0.64 to 1.16, p=0.34). No clinically important effect of vitamin D3 was seen on any of the secondary outcomes listed above. The influence of vitamin D3 on coprimary outcomes was not modified by baseline vitamin D status or genotype. CONCLUSIONS: Bolus-dose vitamin D3 supplementation did not influence time to exacerbation or URI in a population of adults with asthma with a high prevalence of baseline vitamin D insufficiency. TRIAL REGISTRATION NUMBER: NCT00978315 (ClinicalTrials.gov).


Assuntos
Asma/complicações , Asma/prevenção & controle , Colecalciferol/administração & dosagem , Suplementos Nutricionais , Infecções Respiratórias/prevenção & controle , Vitaminas/administração & dosagem , Adulto , Estudos de Coortes , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Infecções Respiratórias/epidemiologia , Fatores de Tempo
10.
J Allergy Clin Immunol ; 133(1): 233-9.e1, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23953710

RESUMO

BACKGROUND: Hitherto, in vivo studies of human granulocyte migration have been based on indiscriminate labeling of total granulocyte populations. We hypothesized that the kinetics of isolated human neutrophil and eosinophil migration through major organs in vivo are fundamentally different, with the corollary that studying unseparated populations distorts measurement of both. METHODS: Blood neutrophils and eosinophils were isolated on 2 separate occasions from human volunteers by using Current Good Manufacturing Practice CD16 CliniMACS isolation, labeled with technetium 99m-hexamethylpropyleneamine oxime, and then reinfused intravenously. The kinetics of cellular efflux were imaged over 4 hours. RESULTS: Neutrophils and eosinophils were isolated to a mean purity of greater than 97% and greater than 95%, respectively. Activation of neutrophils measured as an increase in their CD11b mean fluorescence intensity in whole blood and after isolation and radiolabeling was 25.98 ± 7.59 and 51.82 ± 17.44, respectively, and was not significant (P = .052), but the mean fluorescence intensity of CD69 increased significantly on eosinophils. Analysis of the scintigraphic profile of lung efflux revealed exponential clearance of eosinophils, with a mean half-life of 4.16 ± 0.11 minutes. Neutrophil efflux was at a significantly slower half-life of 13.72 ± 4.14 minutes (P = .009). The migration of neutrophils and eosinophils was significantly different in the spleen at all time points (P = .014), in the liver at 15 minutes (P = .001), and in the bone marrow at 4 hours (P = .003). CONCLUSIONS: The kinetics of migration of neutrophils and eosinophils through the lung, spleen, and bone marrow of human volunteers are significantly different. Study of mixed populations might be misleading.


Assuntos
Medula Óssea/imunologia , Eosinófilos/imunologia , Fígado/imunologia , Neutrófilos/imunologia , Baço/imunologia , Adulto , Movimento Celular , Rastreamento de Células/métodos , Feminino , Humanos , Separação Imunomagnética , Masculino , Oximas , Receptores de IgG/metabolismo , Tecnécio
11.
J Allergy Clin Immunol ; 134(6): 1394-1401.e4, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25112697

RESUMO

BACKGROUND: Naturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood. OBJECTIVE: Address the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation. METHODS: IgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI. RESULTS: IgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen. CONCLUSION: Naturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Asma/imunologia , Autoanticorpos/imunologia , Basófilos/imunologia , Imunoglobulina G/imunologia , Alérgenos/imunologia , Animais , Anticorpos Anti-Idiotípicos/sangue , Antígenos de Plantas/imunologia , Asma/sangue , Autoanticorpos/sangue , Proteínas de Ligação ao Cálcio/imunologia , Linhagem Celular , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Phleum/imunologia , Ratos , Receptores de IgE/imunologia
12.
J Allergy Clin Immunol ; 132(4): 942-50.e1-3, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23731651

RESUMO

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a potent arterial and venous vasodilator. Increased airway epithelial cell expression of CGRP, together with increased CCL17 expression, was previously observed in a model of provoked asthma in atopic human subjects. OBJECTIVE: We sought to determine whether CCL17 induces CCR4-dependent CGRP synthesis and secretion by human airway epithelial cells. METHODS: Human airway epithelial cell lines (BEAS-2B and A549) and human primary airway cells were cultured with CCL17 or various other cytokines, and CGRP expression was measured by using RT-PCR, quantitative immunofluorescence, and enzyme immunoassay. CCR4 expression was determined in cultured cells by using flow cytometry and in bronchial biopsy specimens by using immunohistochemistry. RESULTS: CCL17 induced a several thousand-fold increase in CGRP mRNA expression and released peptide product from BEAS-2B and A549 cells in a time- and concentration-dependent fashion. Concentration-dependent CCL17-induced release of CGRP by primary human airway epithelial cells was also observed. Under comparable conditions, CCL17 induced greater CGRP release from BEAS-2B cells than either IL-13, a cytokine mixture (TNF-α, GM-CSF, and IL-1), or CCL22. CCR4 was expressed by BEAS-2B and A549 cells and internalized after ligation with CCL17. CCL17-induced CGRP release was inhibited by a specific anti-CCR4 blocking antibody. Bronchial biopsy specimens obtained from healthy volunteers and asthmatic patients before and after provoked asthma all exhibited CCR4 staining of equivalent intensity, indicating that the receptor is constitutively expressed. CONCLUSIONS: CCL17-induced, CCR4-dependent release of CGRP by human airway epithelial cells represents a novel inflammatory pathway and a possible target in patients with asthma and allergic disease.


Assuntos
Asma/imunologia , Brônquios/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Quimiocina CCL17/imunologia , Células Epiteliais/metabolismo , Hipersensibilidade Imediata/imunologia , Receptores CCR4/metabolismo , Asma/metabolismo , Brônquios/citologia , Peptídeo Relacionado com Gene de Calcitonina/genética , Linhagem Celular , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Feminino , Humanos , Hipersensibilidade Imediata/metabolismo , Masculino , Receptores CCR4/genética , Receptores CCR4/imunologia , Vasodilatadores
14.
J Allergy Clin Immunol ; 132(2): 297-304.e3, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23683514

RESUMO

BACKGROUND: TH17 cells are proposed to play a role in the pathology of asthma, including steroid-resistant (SR) disease. We previously identified a steroid-enhancing function of vitamin D in patients with SR asthma in restoring the impaired response to steroids for production of the anti-inflammatory cytokine IL-10. OBJECTIVE: We sought to investigate the production of the TH17-associated cytokines IL-17A and IL-22 in culture in patients with moderate-to-severe asthma defined on the basis of their clinical response to steroids and the susceptibility of this response to inhibition by steroids and the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). METHODS: PBMCs were stimulated in culture with or without dexamethasone and 1,25(OH)2D3. A cytometric bead array, ELISA, and intracellular cytokine staining were used to assess cytokine production. The role of CD39 in inhibition of the TH17 response was studied by using quantitative real-time PCR, flow cytometry, and addition of the antagonist POM-1 to culture. RESULTS: Asthmatic patients synthesized much higher levels of IL-17A and IL-22 than nonasthmatic control subjects, with patients with SR asthma expressing the highest levels of IL-17A. Glucocorticoids did not inhibit IL-17A cytokine expression in patients and enhanced production in cultures from control subjects. Treatment with 1,25(OH)2D3 with or without dexamethasone significantly reduced both IL-17A and IL-22 levels. An antagonist of the ectonucleotidase CD39 reversed 1,25(OH)2D3-mediated inhibition of the IL-17A response. CONCLUSION: Patients with severe asthma exhibit increased levels of TH17 cytokines, which are not inhibited by steroids. 1,25(OH)2D3 inhibits TH17 cytokine production in all patients studied, irrespective of their clinical responsiveness to steroids, identifying novel steroid-enhancing properties of vitamin D in asthmatic patients.


Assuntos
Asma/fisiopatologia , Glucocorticoides/uso terapêutico , Interleucina-17/biossíntese , Regulação para Cima , Vitamina D/análogos & derivados , Adulto , Asma/tratamento farmacológico , Asma/imunologia , Células Cultivadas , Resistência a Medicamentos , Feminino , Glucocorticoides/farmacologia , Humanos , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-17/genética , Interleucinas/biossíntese , Interleucinas/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de Doença , Células Th17/imunologia , Células Th17/metabolismo , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Interleucina 22
15.
Nat Commun ; 14(1): 4180, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37491373

RESUMO

All antibodies approved for cancer therapy are monoclonal IgGs but the biology of IgE, supported by comparative preclinical data, offers the potential for enhanced effector cell potency. Here we report a Phase I dose escalation trial (NCT02546921) with the primary objective of exploring the safety and tolerability of MOv18 IgE, a chimeric first-in-class IgE antibody, in patients with tumours expressing the relevant antigen, folate receptor-alpha. The trial incorporated skin prick and basophil activation tests (BAT) to select patients at lowest risk of allergic toxicity. Secondary objectives were exploration of anti-tumour activity, recommended Phase II dose, and pharmacokinetics. Dose escalation ranged from 70 µg-12 mg. The most common toxicity of MOv18 IgE is transient urticaria. A single patient experienced anaphylaxis, likely explained by detection of circulating basophils at baseline that could be activated by MOv18 IgE. The BAT assay was used to avoid enrolling further patients with reactive basophils. The safety profile is tolerable and maximum tolerated dose has not been reached, with evidence of anti-tumour activity observed in a patient with ovarian cancer. These results demonstrate the potential of IgE therapy for cancer.


Assuntos
Imunoglobulina E , Neoplasias Ovarianas , Feminino , Humanos , Anticorpos Monoclonais/efeitos adversos , Basófilos , Ácido Fólico
16.
J Clin Invest ; 119(2): 387-98, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19139565

RESUMO

Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3+ T cells in the presence of 1alpha,25-dihydroxyvitamin D3 (1alpha25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4+CD25+ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1alpha25VitD3 in vitro. Furthermore, ingestion of calcitriol (1alpha25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3+CD4+ T cells analyzed directly ex vivo. Stimulation of 1alpha25VitD3-induced IL-10-secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-gamma synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1alpha25VitD3-induced IL-10-secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.


Assuntos
Calcitriol/farmacologia , Interleucina-10/biossíntese , Linfócitos T Reguladores/fisiologia , Receptor Toll-Like 9/fisiologia , Adulto , Idoso , Dexametasona/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/farmacologia , RNA Mensageiro/análise , Receptores de Calcitriol/fisiologia , Linfócitos T Reguladores/efeitos dos fármacos , Receptor Toll-Like 9/genética
17.
Cancer Immunol Immunother ; 61(9): 1547-64, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22139135

RESUMO

Therapeutic antibodies have revolutionised treatment of some cancers and improved prognosis for many patients. Over half of those available are approved for haematological malignancies, but efficacious antibodies for solid tumours are still urgently needed. Clinically available antibodies belong to the IgG class, the most prevalent antibody class in human blood, while other classes have not been extensively considered. We hypothesised that the unique properties of IgE, a class of tissue-resident antibodies commonly associated with allergies, which can trigger powerful immune responses through strong affinity for their particular receptors on effector cells, could be employed for passive immunotherapy of solid tumours such as ovarian and breast carcinomas. Our laboratory has examined this concept by evaluating two chimaeric antibodies of the same specificity (MOv18) but different isotype, an IgG1 and an IgE against the tumour antigen folate receptor α (FRα). The latter demonstrates the potency of IgE to mount superior immune responses against tumours in disease-relevant models. We identified Fcε receptor-expressing cells, monocytes/macrophages and eosinophils, activated by MOv18 IgE to kill tumour cells by mechanisms such as ADCC and ADCP. We also applied this notion to a marketed therapeutic, the humanised IgG1 antibody trastuzumab and engineered an IgE counterpart, which retained the functions of trastuzumab in restricting proliferation of HER2/neu-expressing tumour cells but also activated effector cells to kill tumour cells by different mechanisms. On-going efficacy, safety evaluations and future first-in-man clinical studies of IgE therapeutics constitute key metrics for this concept, providing new scope for antibody immunotherapies for solid tumours.


Assuntos
Imunização Passiva/métodos , Imunoglobulina E/imunologia , Imunoglobulina E/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos , Receptores Fc/imunologia
18.
Ann Allergy Asthma Immunol ; 109(5): 342-347.e1, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23062390

RESUMO

BACKGROUND: In trials of allergen immunotherapy, allergen exposure is typically assessed by pollen counts, but these may misrepresent exposure if performed remotely from multiple study centers. OBJECTIVE: To assess whether symptomatology in placebo-treated patients is a better measure of local allergen burden at individual centers in such trials. METHODS: Data from a multicenter, placebo-controlled trial of preseasonal grass pollen immunotherapy were reanalyzed to identify the 4 weeks at each center in which the placebo-treated subjects had the highest combined symptom/medication scores (CSMS). The difference in CSMS between active and placebo groups was compared during the 4 peak placebo score weeks (PlSW) and the 4 peak pollen count weeks (PoCW). RESULTS: The benefit of immunotherapy over placebo in the PlSW analysis (18.5%) was greater than in the PoCW analysis (13.6%), with increased statistical significance (P = .0001, .0038, respectively). Similar improved discrimination was observed when analyzing benefits in subgroups of patients with severe symptoms, a high disease burden, and in different geographical locations. CONCLUSION: This novel PlSW analysis results in better discrimination of the effects of allergen immunotherapy compared with placebo and may be widely applicable in similar studies, both prospectively and retrospectively.


Assuntos
Dessensibilização Imunológica/métodos , Lipídeo A/análogos & derivados , Poaceae/imunologia , Pólen/imunologia , Adolescente , Adulto , Alérgenos/administração & dosagem , Alérgenos/imunologia , Asma/diagnóstico , Asma/imunologia , Asma/terapia , Humanos , Lipídeo A/administração & dosagem , Lipídeo A/imunologia , Lipídeo A/uso terapêutico , Pessoa de Meia-Idade , Placebos , Poaceae/efeitos adversos , Pólen/efeitos adversos , Estudos Retrospectivos , Adulto Jovem
19.
Am J Respir Crit Care Med ; 184(7): 771-8, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21719753

RESUMO

RATIONALE: Increased bronchial vascularity is a feature of asthma that can contribute to airflow obstruction and progressive decline in lung function. Angiogenesis is associated with the lung homing and in situ differentiation of endothelial progenitor cells (EPC) in mouse models of asthma. We have previously shown that inhibiting allergen (Ag)-induced recruitment of EPC in sensitized mice attenuated increased bronchial vascularity and development of airway hyperresponsiveness. OBJECTIVES: We investigated the accumulation of EPC and formation of new blood vessels in the lungs of human subjects with asthma after Ag inhalation challenge. METHODS: Consenting patients with mild atopic asthma (n = 13) with FEV1 ≥ 70%, methacholine PC20 ≤ 16 mg/ml, and a dual response to Ag were recruited. Sputum levels of EPC were determined by multigating flow cytometry, and lung vascularity was enumerated by immunostaining with von Willebrand factor. MEASUREMENTS AND MAIN RESULTS: Sputum levels of EPC were determined by multigating flow cytometry and lung vascularity was enumerated by immunostaining with von Willebrand factor. There was a significant increase in sputum EPC levels 24 hours post Ag but not diluent challenge. Similarly, a significant increase in the number and diameter of blood vessels in lung biopsy tissue 24 hours post Ag was observed. In vitro culture of EPC demonstrated the capacity of these cells to differentiate into mature endothelial cells and form tubelike vessel structures. In sputum supernatants, there was a significant increase in CXCR2 agonists, IL-8, and Gro-α 24 hours post Ag. Only Gro-α stimulated a significant EPC migrational response in vitro. CONCLUSIONS: Our data suggest that increased lung homing of EPC may promote bronchial vascularity in allergic asthmatic responses and that the recruitment of these progenitors maybe orchestrated by CXCR2 chemokines.


Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/fisiopatologia , Movimento Celular/imunologia , Células Endoteliais/metabolismo , Neovascularização Patológica/imunologia , Células-Tronco/metabolismo , Adulto , Asma/imunologia , Asma/patologia , Testes de Provocação Brônquica , Células Endoteliais/imunologia , Feminino , Humanos , Masculino , Neovascularização Patológica/patologia , Receptores de Interleucina-8B/metabolismo , Mucosa Respiratória/patologia , Escarro/citologia , Células-Tronco/imunologia , Fator de von Willebrand/metabolismo
20.
Proc Natl Acad Sci U S A ; 106(26): 10775-80, 2009 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-19541629

RESUMO

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Retículo Sarcoplasmático/enzimologia , Asma/patologia , Asma/fisiopatologia , Western Blotting , Brônquios/patologia , Brônquios/fisiopatologia , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL11/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Imuno-Histoquímica , Interleucina-13/farmacologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático
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