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1.
J Immunol ; 207(1): 333-343, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34155069

RESUMO

Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2-expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2-expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.


Assuntos
Transferência Adotiva , Interleucina-2/imunologia , Leucócitos/imunologia , Melanoma/imunologia , Melanoma/terapia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Transdução de Sinais/imunologia
2.
Am J Respir Crit Care Med ; 190(12): 1373-82, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25350863

RESUMO

RATIONALE: Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell-derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation. OBJECTIVES: To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway. METHODS: We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirus-infected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade. MEASUREMENTS AND MAIN RESULTS: IL-4, IL-5, IL-13, and IL-33 are all induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13 levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly induced type 2 cytokines. This induction was entirely dependent on IL-33. CONCLUSIONS: IL-33 and type 2 cytokines are induced during a rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and IL-33-responsive T cells and ILC2s are key mechanistic links between viral infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic approach for asthma exacerbations.


Assuntos
Asma/etiologia , Inflamação/etiologia , Interleucinas/fisiologia , Infecções por Picornaviridae/complicações , Adulto , Asma/fisiopatologia , Asma/virologia , Células Cultivadas , Feminino , Humanos , Inflamação/fisiopatologia , Interleucina-13/fisiologia , Interleucina-33 , Interleucina-4/fisiologia , Interleucina-5/fisiologia , Subpopulações de Linfócitos/fisiologia , Masculino , Infecções por Picornaviridae/fisiopatologia , Rhinovirus , Índice de Gravidade de Doença , Linfócitos T/fisiologia , Células Th2/fisiologia , Carga Viral
3.
Am J Respir Crit Care Med ; 187(1): 78-89, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23087024

RESUMO

RATIONALE: Whether idiopathic, familial, or secondary to another disease, pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, neointimal hyperplasia, medial hypertrophy, and adventitial fibrosis. Imatinib, a potent receptor tyrosine kinase inhibitor, reverses pulmonary remodeling in animal models of PAH and improves hemodynamics and exercise capacity in selected patients with PAH. OBJECTIVES: Here we use both imatinib and knockout animals to determine the relationship between platelet-derived growth factor receptor (PDGFR) and serotonin signaling and investigate the PAH pathologies each mediates. METHODS: We investigated the effects of imatinib (100 mg/kg) on hemodynamics, vascular remodeling, and downstream molecular signatures in the chronic hypoxia/SU5416 murine model of PAH. MEASUREMENTS AND MAIN RESULTS: Treatment with imatinib reduced all measures of PAH pathology observed in hypoxia/SU5416 mice. In addition, 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) expression were reduced compared with the normoxia/SU5416 control group. Imatinib attenuated hypoxia-induced increases in Tph1 expression in pulmonary endothelial cells in vitro via inhibition of the PDGFR-ß pathway. To better understand the consequences of this novel mode of action for imatinib, we examined the development of PAH after hypoxic/SU5416 exposure in Tph1-deficient mice (Tph1(-/-)). The extensive changes in pulmonary vascular remodeling and hemodynamics in response to hypoxia/SU5416 were attenuated in Tph1(-/-) mice and further decreased after imatinib treatment. However, imatinib did not significantly further impact collagen deposition and collagen 3a1 expression in hypoxic Tph1(-/-) mice. Post hoc subgroup analysis suggests that patients with PAH with greater hemodynamic impairment showed significantly reduced 5-HT plasma levels after imatinib treatment compared with placebo. CONCLUSIONS: We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-ß signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Triptofano Hidroxilase/metabolismo , Animais , Benzamidas , Modelos Animais de Doenças , Hemodinâmica/efeitos dos fármacos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Mesilato de Imatinib , Indóis/farmacologia , Camundongos , Camundongos Knockout , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Serotonina/metabolismo
4.
Eur J Immunol ; 42(12): 3394-404, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22930133

RESUMO

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato-poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro-inflammatory cytokine IL-17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL-17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL-17 were not due to an aberrant expansion of IL-17-producing cells. Furthermore, we also identified an increase in IL-17RA expression on PI3Kγ(-/-) CD4(+) T cells, yet these cells exhibited impaired PI3K-dependent signalling in response to IL-17A, and subsequent NF-κB phosphorylation. In vivo, instillation of recombinant IL-17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL-17 was also reduced in PI3Kγ(-/-) lungs. These data demonstrate PI3Kγ-dependent signalling downstream of IL-17RA, which plays a pivotal role in regulating IL-17 production in T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Interleucina-17/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Interleucina-17/genética , Interleucina-17/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Transdução de Sinais/genética
5.
Am J Med Genet A ; 161A(8): 2040-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23824657

RESUMO

The transforming growth factor ß (TGF-ß) family of growth factors are key regulators of mammalian development and their dysregulation is implicated in human disease, notably, heritable vasculopathies including Marfan (MFS, OMIM #154700) and Loeys-Dietz syndromes (LDS, OMIM #609192). We described a syndrome presenting at birth with distal arthrogryposis, hypotonia, bifid uvula, a failure of normal post-natal muscle development but no evidence of vascular disease; some of these features overlap with MFS and LDS. A de novo mutation in TGFB3 was identified by exome sequencing. Several lines of evidence indicate the mutation is hypomorphic suggesting that decreased TGF-ß signaling from a loss of TGFB3 activity is likely responsible for the clinical phenotype. This is the first example of a mutation in the coding portion of TGFB3 implicated in a clinical syndrome suggesting TGFB3 is essential for both human palatogenesis and normal muscle growth.


Assuntos
Artrogripose/genética , Transtornos do Crescimento/genética , Síndrome de Loeys-Dietz/genética , Síndrome de Marfan/genética , Debilidade Muscular/genética , Mutação/genética , Fator de Crescimento Transformador beta3/genética , Adulto , Animais , Artrogripose/diagnóstico , Células Cultivadas , Criança , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Transtornos do Crescimento/diagnóstico , Humanos , Síndrome de Loeys-Dietz/diagnóstico , Masculino , Síndrome de Marfan/diagnóstico , Debilidade Muscular/diagnóstico , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta3/metabolismo , Xenopus laevis/metabolismo
6.
Bioorg Med Chem ; 21(21): 6582-91, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24021582

RESUMO

Optimization of a 7-azaindole-3-acetic acid CRTh2 receptor antagonist chemotype derived from high throughput screening furnished a highly selective compound NVP-QAV680 with low nM functional potency for inhibition of CRTh2 driven human eosinophil and Th2 lymphocyte activation in vitro. The molecule exhibited good oral bioavailability in the rat, combined with efficacy in rodent CRTh2-dependent mechanistic and allergic disease models and was suitable for clinical development.


Assuntos
Indolizinas/química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Dermatite de Contato/tratamento farmacológico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Meia-Vida , Humanos , Hipersensibilidade/tratamento farmacológico , Indolizinas/farmacocinética , Indolizinas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Relação Estrutura-Atividade , Células Th2/imunologia , Células Th2/metabolismo
7.
J Immunol ; 186(8): 4936-45, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21402888

RESUMO

SHIP-1 negatively regulates the PI3K pathway in hematopoietic cells and has an emerging role in T lymphocyte biology. PI3K and SHIP can regulate cell migration in leukocytes, particularly in neutrophils, although their role in T cell migration has been less clear. Therefore, we sought to explore the role of SHIP-1 in human CD4(+) T lymphocyte cell migration responses to chemoattractants using a lentiviral-mediated expression system and a short hairpin RNA approach. Silencing of SHIP-1 leads to increased basal phosphorylation of protein kinase B/Akt and its substrate GSK3ß, as well as an increase in basal levels of polymerized actin, suggesting that SHIP-1 might regulate changes in the cytoskeleton. Accordingly, silencing of SHIP-1 led to loss of microvilli and ezrin/radixin/moesin phosphorylation, which could not be rescued by the PI3K inhibitor Ly294002. There were striking morphological changes, including a loss of microvilli projections, which mirrored changes in wild type cells after stimulation with the chemokine CXCL11. There was no defect in directional T cell migration toward CXCL11 in the SHIP-1-silenced cells but, importantly, there was a defect in the overall basal motility of SHIP-1 knockdown cells. Taken together, these results implicate SHIP-1 as a key regulator of basal PI3K signaling in human CD4(+) T lymphocytes with important phosphatase-independent actions, which together are key for maintaining normal morphology and basal motility.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Movimento Celular , Monoéster Fosfórico Hidrolases/metabolismo , Actinas/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/ultraestrutura , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL11/farmacologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citometria de Fluxo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Inositol Polifosfato 5-Fosfatases , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Varredura , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais
8.
Proc Natl Acad Sci U S A ; 107(20): 9446-51, 2010 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-20439741

RESUMO

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.


Assuntos
Proliferação de Células/efeitos dos fármacos , Modelos Moleculares , Neoplasias/metabolismo , Pirazóis/farmacologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Ativadas por p21/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Pirazóis/química , Pirazóis/metabolismo , Pirróis/química , Pirróis/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho
9.
J Immunol ; 184(11): 6114-23, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20427772

RESUMO

Tissue-resident mast cells (MCs) are important in allergic diseases. In a mouse model of allergic airways inflammation, an increase in peribronchiolar MCs was associated with increased concentrations of the chemokine CCL2 in lung lavage. MC progenitors (MCps) arising in bone marrow (BM) are recruited to tissues by transendothelial migration, and we found that CCL2 is chemotactic for MCps in freshly isolated BM in vitro. Immature, but not mature, BM-derived MCs migrated in response to CCL2 when cultured in IL-3+stem cell factor (SCF) but not when cultured in IL-3 alone. However, the cells under both culture conditions expressed mRNA for CCR2, the receptor for CCL2, and bound the radiolabeled chemokine with similar affinities, highlighting SCF as a key mediator in coupling CCR2 to downstream events, culminating in chemotaxis. Immature BM-derived MCs from IL-3 +SCF cultures, when administered i.v., accumulated at skin sites injected with CCL2 in vivo. MCp recruitment to the allergen-sensitized/challenged lung was significantly reduced in CCR2(-/-) and CCL2(-/-) mouse strains. However, reconstitution studies of sublethally irradiated and BM-reconstituted mice indicated that BM cells and stromal elements could provide CCL2, whereas the CCR2 function resided with stromal elements rather than BM cells. These experiments revealed a new function of SCF in chemokine receptor coupling, but they suggest a complex role of the CCL2/CCR2 axis in recruiting MCps during pulmonary inflammation.


Assuntos
Quimiocina CCL2/imunologia , Quimiotaxia de Leucócito/imunologia , Mastócitos/imunologia , Receptores CCR2/imunologia , Alérgenos/imunologia , Alérgenos/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CCL2/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Ovalbumina/farmacologia , Receptores CCR2/metabolismo , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/imunologia , Fator de Células-Tronco/metabolismo
10.
Am J Respir Crit Care Med ; 184(10): 1171-82, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21868504

RESUMO

RATIONALE: The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. OBJECTIVES: Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. METHODS: Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. MEASUREMENTS AND MAIN RESULTS: The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-ß/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. CONCLUSIONS: These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.


Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/fisiopatologia , Doença Aguda , Animais , Western Blotting , Citocinas/sangue , Ecocardiografia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Indóis/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirróis/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
11.
J Allergy Clin Immunol ; 128(4): 800-807.e9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21719078

RESUMO

BACKGROUND: IL-13 is a key T(H)2 cytokine that is implicated in allergic responses. OBJECTIVE: We evaluated the effects of an anti-IL-13-blocking antibody compared with placebo on repeated nasal allergen challenge responses in hay fever patients out of season. METHODS: We performed a parallel group double-blind study of anti-IL-13 (single dose, 6 mg/kg intravenously, n = 16) and placebo (n = 15), with an additional open label group given a topical nasal corticosteroid (n = 5). Subjects received intranasal timothy grass pollen (Phleum pratense P5 allergen), and serial samples of nasal mucosal lining fluid were taken by using synthetic absorptive matrix and by nasal lavage. RESULTS: Administration of anti-IL-13 on day 1 resulted in a significant decrease in IL-13 levels in synthetic absorptive matrix eluates compared with placebo (area under the curve 0-8 hours, change from baseline) during the late phase after nasal allergen challenge on day 5 (P < .05) and day 7 (P < .01). There were no apparent effects of anti-IL-13 treatment on nasal lavage eosinophil numbers or total nasal symptom scores versus placebo. However, in a subgroup with high late-phase IL-13 levels at screening, there was a trend for a decrease in total nasal symptom scores after nasal allergen challenge on day 5, when compared with subjects with low IL-13 levels (P < .10). Nasal fluticasone caused suppression of IL-13 (P < .05 on day 5) as well as IL-5 (P < .01 on day 5) levels in the late phase compared with placebo. CONCLUSIONS: Anti-IL-13 had specific pharmacodynamic action in this nasal allergen challenge model, causing profound inhibition of nasal lining fluid IL-13 responses. In addition, there was a possible effect of anti-IL-13 treatment on total nasal symptom scores in a subgroup with high late-phase nasal IL-13 levels at screening.


Assuntos
Alérgenos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Interleucina-13/antagonistas & inibidores , Rinite Alérgica Sazonal/tratamento farmacológico , Administração Intranasal , Adolescente , Corticosteroides/administração & dosagem , Adulto , Androstadienos/administração & dosagem , Antialérgicos/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Método Duplo-Cego , Eosinófilos/imunologia , Feminino , Fluticasona , Humanos , Interleucina-13/imunologia , Masculino , Pessoa de Meia-Idade , Phleum/imunologia , Rinite Alérgica Sazonal/sangue , Rinite Alérgica Sazonal/imunologia , Irrigação Terapêutica
12.
JCI Insight ; 7(10)2022 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-35603788

RESUMO

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3-mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell-based immunotherapy.


Assuntos
Linfócitos T CD8-Positivos , Citocinas , Animais , Citocinas/metabolismo , Humanos , Imunoterapia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Citocinas/metabolismo
13.
J Immunol ; 183(8): 5171-9, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19783686

RESUMO

Aspergillus fumigatus is a sporulating fungus found ubiquitously in the environment and is easily cleared from immunocompetent hosts. Invasive aspergillosis develops in immunocompromised patients, and is a leading cause of mortality in hematopoietic stem cell transplant recipients. CCR7 and its ligands, CCL19 and CCL21, are responsible for the migration of dendritic cells from sites of infection and inflammation to secondary lymphoid organs. To investigate the role of CCR7 during invasive aspergillosis, we used a well-characterized neutropenic murine model. During invasive aspergillosis, mice with a CCR7 deficiency in the hematopoietic compartment exhibited increased survival and less pulmonary injury compared with the appropriate wild-type control. Flow cytometric analysis of the chimeric mice revealed an increase in the number of dendritic cells present in the lungs of CCR7-deficient chimeras following infection with Aspergillus conidia. An adoptive transfer of dendritic cells into neutropenic mice provided a protective effect during invasive aspergillosis, which was further enhanced with the adoptive transfer of CCR7-deficient dendritic cells. Additionally, CCR7-deficient dendritic cells activated in vitro with Aspergillus conidia expressed higher TNF-alpha, CXCL10, and CXCL2 levels, indicating a more activated cellular response to the fungus. Our results suggest that the absence of CCR7 is protective during invasive aspergillosis in neutropenic mice. Collectively, these data demonstrate a potential deleterious role for CCR7 during primary immune responses directed against A. fumigatus.


Assuntos
Aspergillus fumigatus , Quimiocina CXCL10/imunologia , Quimiocina CXCL2/imunologia , Células Dendríticas/imunologia , Aspergilose Pulmonar Invasiva/imunologia , Receptores CCR7/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Quimiocina CCL19/imunologia , Quimiocina CCL19/metabolismo , Quimiocina CCL21/imunologia , Quimiocina CCL21/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL2/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Feminino , Aspergilose Pulmonar Invasiva/microbiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/imunologia , Neutropenia/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
14.
Am J Respir Cell Mol Biol ; 43(3): 296-304, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19843708

RESUMO

Chronic obstructive pulmonary disease (COPD) is associated with pulmonary inflammation with increased numbers of macrophages located in the parenchyma. These macrophages have the capacity to mediate the underlying pathophysiology of COPD; therefore, a better understanding of their function in chronic inflammation associated with this disease is vital. Ion channels regulate many cellular functions; however, their role in macrophages is unclear. This study examined the expression and function of transient receptor potential (TRP) channels in human macrophages. Human alveolar macrophages and lung tissue macrophages expressed increased mRNA and protein for TRPC6 when compared with monocytes and monocyte-derived macrophages. Moreover, TRPC6 mRNA expression was significantly elevated in alveolar macrophages from patients with COPD compared with control subjects. There were no differences in mRNA for TRPC3 or TRPC7. Although mRNA for TRPM2 and TRPV1 was detected in these cells, protein expression could not be determined. Fractionation of lung-derived macrophages demonstrated that TRPC6 protein was more highly expressed by smaller macrophages compared with larger macrophages. Using whole-cell patch clamp electrophysiology, TRPC6-like currents were measured in both macrophage subpopulations with appropriate biophysical and basic pharmacological profiles. These currents were active under basal conditions in the small macrophages. These data suggest that TRPC6-like channels are functional on human lung macrophages, and may be associated with COPD.


Assuntos
Macrófagos Alveolares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Adulto , Western Blotting , Estudos de Casos e Controles , Eletrofisiologia , Feminino , Citometria de Fluxo , Humanos , Macrófagos Alveolares/citologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/metabolismo , Técnicas de Patch-Clamp , Doença Pulmonar Obstrutiva Crônica/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Cátion TRPC6 , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
15.
Immunology ; 129(1): 115-24, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20050333

RESUMO

The mechanisms governing the population of tissues by mast cells are not fully understood, but several studies using human mast cells have suggested that expression of the chemokine receptor CCR3 and migration to its ligands may be important. In CCR3-deficient mice, a change in mast cell tissue distribution in the airways following allergen challenge was reported compared with wild-type mice. In addition, there is evidence that CCR3 is important in mast cell maturation in mouse. In this study, bone marrow-derived mast cells (BMMCs) were cultured and CCR3 expression and the migratory response to CCR3 ligands were characterized. In addition, BMMCs were cultured from wild-type and CCR3-deficient mice and their phenotype and migratory responses were compared. CCR3 messenger RNA was detectable in BMMCs, but this was not significantly increased after activation by immunoglobulin E (IgE). CCR3 protein was not detected on BMMCs during maturation and expression could not be enhanced after IgE activation. Resting and IgE-activated immature and mature BMMCs did not migrate in response to the CCR3 ligands eotaxin- 1 and eotaxin-2. Comparing wild-type and CCR3-deficient BMMCs, there were no differences in mast cell phenotype or ability to migrate to the mast cell chemoattractants leukotriene B4 and stem cell factor. The results of this study show that CCR3 may not mediate mast cell migration in mouse BMMCs in vitro. These observations need to be considered in relation to the findings of CCR3 deficiency on mast cells in vivo.


Assuntos
Leucotrieno B4/metabolismo , Mastócitos/metabolismo , Receptores CCR3/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Ensaios de Migração Celular , Células Cultivadas , Quimiocina CCL11/metabolismo , Quimiocina CCL24/metabolismo , Feminino , Imunoglobulina E/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR3/genética , Receptores CCR3/imunologia
16.
Immunology ; 126(3): 413-22, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18754810

RESUMO

We and others have established an important role for phosphoinositide-3 kinase gamma (PI3Kgamma) in the chemotactic responses of macrophages and neutrophils. The involvement of this lipid kinase in allergic inflammatory responses is, however, yet to be fully determined. Here we compare wild-type (WT) and PI3Kgamma(-/-) (KO) mice within a model of ovalbumin (OVA) -specific pulmonary inflammation. Upon OVA aerosol challenge, cell influx into the bronchoalveolar lavage (BAL) fluid consisted of neutrophils, macrophages and, more significantly, eosinophils - which are key effector cells in allergic inflammation. Each population was reduced by up to 80% in KO mice, demonstrating a role for PI3Kgamma in cell infiltration into the airways. The mechanism of reduced eosinophilia was analysed within both development and effector stages of the immune response. Comparable levels of OVA-specific T-cell proliferation and immunoglobulin production were established in both strains. Furthermore, no significant differences between WT and KO chemokine production were observed. Having identified the critical point of PI3Kgamma involvement, KO eosinophil chemotactic dysfunction was confirmed in vitro. These data are the first to demonstrate the vital role of PI3Kgamma in acute allergic inflammation. The profound dependency of eosinophils on PI3Kgamma for pulmonary influx identifies this lipid kinase as an attractive target for the pharmacological intervention of asthma.


Assuntos
Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Pneumonia/imunologia , Doença Aguda , Animais , Asma/imunologia , Linfócitos B/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase , Citocinas/biossíntese , Modelos Animais de Doenças , Eosinofilia/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Isoenzimas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina/imunologia
17.
Cell Signal ; 19(12): 2528-39, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17900864

RESUMO

Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kgamma provides a key migratory signal for T lymphocyte migration. Since PI3Kgamma can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kgamma was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kgamma), class II (PI3KC2alpha and PI3KC2beta) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kgamma. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.


Assuntos
Técnicas de Cultura de Células , Quimiotaxia de Leucócito , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Transfecção/métodos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Fatores de Tempo
18.
Mol Cell Biol ; 22(23): 8175-83, 2002 12.
Artigo em Inglês | MEDLINE | ID: mdl-12417721

RESUMO

Tumor necrosis factor (TNF) signaling through the TNF receptors involves the recruitment of key signaling factors, leading to the activation of both the transcription factor NF-kappaB and the stress-activated Jun kinase (JNK). In most cells, TNF signaling leads to a rapid and transient increase in JNK activity. However, we show that TNF treatment leads to the sustained activation of JNK in cells that are null for the p65/RelA subunit of NF-kappaB as well as in cells expressing the super-repressor form of IkappaB. In addition, the data indicate that the ability of p65/RelA to regulate gene expression is required to suppress the persistent activation of JNK. Interestingly, this suppression occurs upstream of JNK, within the signal transduction cascade leading to JNK activation, without affecting the stress-activated kinase p38. Since NF-kappaB has previously been shown to be involved in the suppression of TNF-induced apoptosis, we were interested in determining the role of deregulated JNK activity, induced by the loss of NF-kappaB, in controlling the cell death response. Through the use of different approaches for inhibition of JNK, we show that the suppression of JNK activity in cells that lack active NF-kappaB enhances the apoptotic response to TNF. These data suggest that the activity of JNK in cells blocked for NF-kappaB function provides an antiapoptotic signal and explains, at least partly, why a significant number of NF-kappaB null cells remain viable following TNF treatment.


Assuntos
Apoptose/fisiologia , MAP Quinase Quinase 4 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antracenos/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Subunidades Proteicas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição RelA , Transcrição Gênica
19.
Methods Mol Biol ; 356: 221-32, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16988406

RESUMO

Mammalian signal transduction occurs in the context of multiprotein complexes, yet currently available drug discovery strategies do not reflect this fact. We present a strategy for screening drugs and targets in living human cells by utilizing high content protein-fragment complementation assays. Synthetic fragments of a mutant fluorescent protein ("Venus" and/or enhanced yellow fluorescent protein) are used for protein-fragment complementation assay construction, allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. Here we describe the utility of this novel strategy for high-throughput screening of known targets, and for screening previously undrugable targets and profiling drug leads for improved selectivity and safety.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Complexos Multiproteicos/metabolismo , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Ligação Proteica , RNA Interferente Pequeno , Sensibilidade e Especificidade , Frações Subcelulares
20.
Oncoimmunology ; 6(2): e1265721, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28344875

RESUMO

The use of high-dose interleukin-2 (IL-2) has fallen out of favor due to severe life-threatening side effects. We have recently described a unique way of directly targeting IL-2 to cytotoxic lymphocytes using a virally encoded immune evasion protein and an IL-2 mutant that avoids off-target side effects such as activation of regulatory T cells and vascular endothelium.

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