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1.
Nat Immunol ; 21(2): 135-144, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932813

RESUMO

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.


Assuntos
Ritmo Circadiano/imunologia , Inflamação/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Animais , Degranulação Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação/imunologia , Camundongos , Neutrófilos/imunologia , Pneumonia/complicações , Pneumonia/imunologia , Proteoma/imunologia , Proteoma/metabolismo , Síndrome do Desconforto Respiratório do Adulto/imunologia
2.
PLoS One ; 15(1): e0227314, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31951638

RESUMO

Acute lymphoblastic leukemia (ALL) is the most common cancer in children around the globe. Mexico City has one of the highest incidence rates of childhood leukemia worldwide with 49.5 cases per million children under the age of 15 which is similar to that reported for Hispanic populations living in the United States. In addition, it has been noted a dismal prognosis in Mexican and Hispanic ALL pediatric population. Although ALL, like cancer in general, has its origins in endogenous, exogenous, and genetic factors, several studies have shown that the immune system also plays a deterministic role in cancer development. Among various elements of the immune system, T lymphocytes and NK cells seem to dominate the immune response against leukemia. The aim of the present study was to perform a phenotypic and functional characterization of NK cells in ALL Mexican children at the moment of diagnosis and before treatment initiation. A case-control study was conducted by the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia (MIGICCL). 41 cases were incident ALL children younger than 17 years old and residents of Mexico City. 14 controls were children without leukemia, matched by age and sex with cases. NK cell function was evaluated by degranulation assays towards K562 cells and SLAM-associated protein (SAP) expression was measured by intracellular staining. All assays were performed using peripheral blood mononuclear cells from controls and patients. The results indicate that NK mediated cytotoxicity, measured by CD107a degranulation assays in response to K562 cells, was reduced in ALL patients compared to controls. Interestingly, an impaired NK cell killing of target cells was not equally distributed among ALL patients. In contrast to patients classified as high-risk, standard-risk patients did not display a significant reduction in NK cell-mediated cytotoxicity. Moreover, patients presenting a leukocyte count ≥ 50,000xmm3 displayed a reduction in NK-cell mediated cytotoxicity and a reduction in SAP expression, indicating a positive correlation between a reduced SAP expression and an impaired NK cell-mediated citotoxicity. In the present study it was observed that unlike patients with standard-risk, NK cells from children presenting high-risk ALL, harbor an impaired cytotoxicity towards K562 at diagnosis. In addition, NK cell function was observed to be compromised in patients with a leukocyte count ≥50,000xmm3, where also it was noticed a decreased expression of SAP compared to patients with a leukocyte count <50,000xmm3. These data indicate NK cell-mediated cytotoxicity is not equally affected in ALL patients, nevertheless a positive correlation between low SAP expression and decreased NK cell-mediated cytotoxicity was observed in ALL patients with a leukocyte count ≥50,000xmm3. Finally, an abnormal NK cell-mediated cytotoxicity may represent a prognostic factor for high-risk acute lymphoblastic leukemia.


Assuntos
Células Matadoras Naturais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Linfócitos T Citotóxicos/metabolismo , Adolescente , Estudos de Casos e Controles , Degranulação Celular/genética , Degranulação Celular/imunologia , Criança , Pré-Escolar , Citotoxicidade Imunológica/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células K562 , Células Matadoras Naturais/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Masculino , México , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Linfócitos T Citotóxicos/patologia
3.
Basic Res Cardiol ; 114(5): 37, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31418072

RESUMO

In response to myocardial infarction (MI), neutrophils (PMNs) are early responders that initiate the inflammatory reaction. Because macrophages and fibroblasts show polarization states after MI, we hypothesized PMNs also undergo phenotypic changes over the MI time course. The objective of the current study was to map the continuum of polarization phenotypes in cardiac neutrophils over the first week of MI. C57BL/6J male mice (3-6 months old) underwent permanent coronary artery ligation to induce MI, and PMNs were isolated from the infarct region at days 1, 3, 5, and 7 after MI. Day 0 served as a no MI negative control. Aptamer proteomics was performed on biological replicates (n = 10-12) for each time point. Day (D)1 MI neutrophils had a high degranulation profile with increased matrix metalloproteinase (MMP) activity. D3 MI neutrophil profiles showed upregulation of apoptosis and induction of extracellular matrix (ECM) organization. D5 MI neutrophils further increased their ECM reorganization profile. D7 MI neutrophils had a reparative signature that included expression of fibronectin, galectin-3, and fibrinogen to contribute to scar formation by stimulating ECM reorganization. Of note, fibronectin was a key modulator of degranulation, as it amplified MMP-9 release in the presence of an inflammatory stimulus. Our results indicate that neutrophils selectively degranulate over the MI time course, reflective of both their intrinsic protein profiles as well as the ECM environment in which they reside. MMPs, cathepsins, and ECM proteins were prominent neutrophil degranulation indicators.


Assuntos
Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Neutrófilos , Animais , Degranulação Celular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteoma , Remodelação Ventricular/fisiologia
4.
J Surg Res ; 244: 241-250, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31301480

RESUMO

BACKGROUND: Ischemic preconditioning (IPC) can provide a defense against ischemia-reperfusion (IR)-induced acute inflammation and barrier dysfunction in many organs. Because nitric oxide (NO) has been implicated as a trigger or mediator in the IPC mechanism and because neuronal NO synthase (nNOS) is a dominant isoform of NOS in the gastrointestinal tract, our aim was to investigate the role of nNOS in IPC-induced protection after mesenteric IR. MATERIALS AND METHODS: Intestinal IR was induced in sodium pentobarbital-anesthetized dogs by clamping the superior mesenteric artery for 60 min followed by 2 h of reperfusion (IR group; n = 7). In further groups, IPC was used (three cycles of 5-min ischemia/5-min reperfusion periods) before IR in the presence or absence of selective inhibition of nNOS with 7-nitroindazole (5 mg/kg, intravenously, in a bolus 15 min before IPC, n = 6 each). Changes in mesenteric vascular resistance, intramucosal pH (pHi), and small bowel motility were monitored. Plasma nitrite/nitrate levels, intestinal NO synthase activity, leukocyte accumulation, mast cell degranulation, and histologic injury were also determined. RESULTS: Ischemia significantly decreased mesenteric vascular resistance and pHi, whereas IR induced a temporary bowel hypermotility and acute inflammatory reaction. IPC facilitated pHi recovery, attenuated motility dysfunction, elevated NOS-dependent NO production, and reduced leukocyte accumulation, mast cell degranulation, and mucosal injury. Pretreatment with 7-nitroindazole halted the IPC-induced increase in NO availability, pHi recovery, and the anti-inflammatory and morphologic effects. CONCLUSIONS: Our data demonstrate that NO generated by intestinal nNOS plays a pivotal role in IPC-linked tissue protection by inhibiting an IR-related acute inflammatory response.


Assuntos
Mucosa Intestinal/imunologia , Precondicionamento Isquêmico/métodos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/imunologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Degranulação Celular/imunologia , Modelos Animais de Doenças , Cães , Feminino , Humanos , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/metabolismo , Masculino , Mastócitos/imunologia , Artéria Mesentérica Superior/cirurgia , Óxido Nítrico/metabolismo , Traumatismo por Reperfusão/etiologia
5.
Nat Commun ; 10(1): 2735, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227713

RESUMO

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.


Assuntos
Interleucina-33/imunologia , Macrófagos/imunologia , Mastócitos/imunologia , Neoplasias Gástricas/imunologia , Aminopiridinas/administração & dosagem , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Cromolina Sódica/administração & dosagem , Modelos Animais de Doenças , Epitélio/imunologia , Epitélio/patologia , Feminino , Mucosa Gástrica/citologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pirróis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Microambiente Tumoral/imunologia
6.
Nat Commun ; 10(1): 2218, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101811

RESUMO

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.


Assuntos
Infecções Bacterianas/imunologia , Neutrófilos/imunologia , Mucosa Respiratória/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Infecções Bacterianas/microbiologia , Degranulação Celular/imunologia , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Quênia , Metagenômica/métodos , Microbiota/imunologia , Proteômica/métodos , Mucosa Respiratória/citologia , Mucosa Respiratória/microbiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Streptococcus/imunologia , Streptococcus/isolamento & purificação
7.
Int Arch Allergy Immunol ; 179(2): 102-107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30965334

RESUMO

BACKGROUND: MicroRNAs (miRNAs) represent important post-transcriptional regulators with a dynamic expression profile during health and disease. OBJECTIVES: We explored the miRNA profile of human mast cells (MCs) during sen-sitization with IgE, during activation through IgE, and relat ed it to prostaglandin D2 synthesis and histamine release. METHOD: We investigated the expression pattern of 762 miRNAs during the IgE-mediated sensitization and activation of MCs cultured from CD133+ stem cells that were isolated from allergic asthmatic patients and nonatopic controls. RESULTS: IgE-mediated sensitization increased the expression of miRNA-210 eight-fold. This increase was sustained during IgE-mediated MC activation. Furthermore, we confirmed the increase of the miRNA-132/212 cluster after MC activation. Predicted target genes of miRNA-210/132/212 were enriched in several pathways known to be involved in MC activation. Histamine release was significantly higher in MCs from allergic patients when compared to controls, and a number of miRNAs correlated with histamine release and prostaglandin D2 synthesis during MC activation. CONCLUSION: The miRNAs and analysis presented here can help to elucidate the role of miRNAs in mediator release during MC activation. We speculate that miRNA-210 could be important in MC sensitization that leads to allergic symptoms.


Assuntos
Regulação da Expressão Gênica , Imunoglobulina E/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , MicroRNAs/genética , Biomarcadores , Degranulação Celular/imunologia , Perfilação da Expressão Gênica , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo
9.
J Immunol Res ; 2019: 9542656, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31011586

RESUMO

Anaphylaxis is a notorious type 2 immune response which may result in a systemic response and lead to death. A precondition for the unfolding of the anaphylactic shock is the secretion of inflammatory mediators from mast cells in response to an allergen, mostly through activation of the cells via the IgE-dependent pathway. While mast cells are specialized secretory cells that can secrete through a variety of exocytic modes, the most predominant mode exerted by the mast cell during anaphylaxis is compound exocytosis-a specialized form of regulated exocytosis where secretory granules fuse to one another. Here, we review the modes of regulated exocytosis in the mast cell and focus on compound exocytosis. We review historical landmarks in the research of compound exocytosis in mast cells and the methods available for investigating compound exocytosis. We also review the molecular mechanisms reported to underlie compound exocytosis in mast cells and expand further with reviewing key findings from other cell types. Finally, we discuss the possible reasons for the mast cell to utilize compound exocytosis during anaphylaxis, the conflicting evidence in different mast cell models, and the open questions in the field which remain to be answered.


Assuntos
Anafilaxia/imunologia , Degranulação Celular/imunologia , Exocitose , Mastócitos/patologia , Animais , Contagem de Células , Regulação da Expressão Gênica , Humanos , Camundongos
10.
Front Immunol ; 10: 205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809227

RESUMO

Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory. Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA. Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized. Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum. Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans.


Assuntos
Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Microbiota , Animais , Biomarcadores , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Hipersensibilidade Alimentar/patologia , Microbioma Gastrointestinal , Vida Livre de Germes , Metagenoma , Metagenômica/métodos , Camundongos , Microbiota/imunologia , RNA Ribossômico 16S
11.
J Cancer Res Clin Oncol ; 145(5): 1179-1190, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30778749

RESUMO

PURPOSE: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). METHODS: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. RESULTS: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. CONCLUSION: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CD24/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Receptores Fc/metabolismo , Transdução de Sinais , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Part Fibre Toxicol ; 16(1): 3, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621720

RESUMO

BACKGROUND: The effects of carbon nanotubes on skin toxicity have not been extensively studied; however, our lab has previously shown that a carboxylated multi-walled carbon nanotube (MWCNT) exacerbates the 2, 4-dinitrofluorobenzene induced contact hypersensitivity response in mice. Here we examine the role of carboxylation in MWCNT skin toxicity. RESULTS: MWCNTs were analyzed by transmission electron microscopy, zetasizer, and x-ray photoelectron spectroscopy to fully characterize the physical properties. Two MWCNTs with different levels of surface carboxylation were chosen for further testing. The MWCNTs with a high level of carboxylation displayed increased cytotoxicity in a HaCaT keratinocyte cell line, compared to the MWCNTs with intermediate levels of carboxylation. However, neither functionalized MWCNT increased the level of in vitro reactive oxygen species suggesting an alternative mechanism of cytotoxicity. Each MWCNT was tested in the contact hypersensitivity model, and only the MWCNTs with greater than 20% surface carboxylation exacerbated the ear swelling responses. Analysis of the skin after MWCNT exposure reveals that the same MWCNTs with a high level of carboxylation increase epidermal thickness, mast cell and basophil degranulation, and lead to increases in polymorphonuclear cell recruitment when co-administered with 2, 4-dinitrofluorobenzene. CONCLUSIONS: The data presented here suggest that acute, topical application of low doses of MWCNTs can induce keratinocyte cytotoxicity and exacerbation of allergic skin conditions in a carboxylation dependent manner.


Assuntos
Dermatite de Contato/etiologia , Queratinócitos/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Pele/efeitos dos fármacos , Animais , Ácidos Carboxílicos/química , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Dinitrofluorbenzeno/toxicidade , Edema/induzido quimicamente , Edema/imunologia , Edema/patologia , Humanos , Queratinócitos/imunologia , Queratinócitos/patologia , Camundongos Pelados , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/química , Infiltração de Neutrófilos/efeitos dos fármacos , Oxirredução , Pele/imunologia , Pele/patologia
13.
J Dermatol Sci ; 93(1): 58-64, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30658871

RESUMO

BACKGROUND: Rosacea is a chronic inflammatory skin condition whose etiology has been linked to mast cells and the antimicrobial peptide cathelicidin LL-37. Individuals with refractory disease have demonstrated clinical benefit with periodic injections of onabotulinum toxin, but the mechanism of action is unknown. OBJECTIVES: To investigate the molecular mechanism by which botulinum toxin improves rosacea lesions. METHODS: Primary human and murine mast cells were pretreated with onabotulinum toxin A or B or control. Mast cell degranulation was evaluated by ß-hexosaminidase activity. Expression of botulinum toxin receptor Sv2 was measured by qPCR. The presence of SNAP-25 and VAMP2 was established by immunofluorescence. In vivo rosacea model was established by intradermally injecting LL-37 with or without onabotulinum toxin A pretreatment. Mast cell degranulation was assessed in vivo by histologic counts. Rosacea biomarkers were analyzed by qPCR of mouse skin sections. RESULTS: Onabotulinum toxin A and B inhibited compound 48/80-induced degranulation of both human and murine mast cells. Expression of Sv2 was established in mouse mast cells. Onabotulinum toxin A and B increased cleaved SNAP-25 and decreased VAMP2 staining in mast cells respectively. In mice, injection of onabotulinum toxin A significantly reduced LL-37-induced skin erythema, mast cell degranulation, and mRNA expression of rosacea biomarkers. CONCLUSIONS: These findings suggest that onabotulinum toxin reduces rosacea-associated skin inflammation by directly inhibiting mast cell degranulation. Periodic applications of onabotulinum toxin may be an effective therapy for refractory rosacea and deserves further study.


Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Degranulação Celular/efeitos dos fármacos , Eritema/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Rosácea/tratamento farmacológico , Inibidores da Liberação da Acetilcolina , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/imunologia , Biópsia , Toxinas Botulínicas Tipo A/uso terapêutico , Degranulação Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Eritema/imunologia , Eritema/patologia , Humanos , Injeções Intradérmicas , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Rosácea/imunologia , Rosácea/patologia , Pele/citologia , Pele/imunologia , Pele/patologia , p-Metoxi-N-metilfenetilamina/farmacologia
14.
Ann Otol Rhinol Laryngol ; 128(4): 330-337, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30678469

RESUMO

OBJECTIVE:: IgA-dependent degranulation of eosinophils and positive correlation between IgA and eosinophil cytotoxic protein levels in nasal secretions have been reported. However, the association between IgA and allergic reactions remains unclear. Therefore, we evaluated the changes in Japanese cedar-specific IgA levels and allergy symptoms after Japanese cedar pollen scattering in symptomatic and asymptomatic individuals sensitized to Japanese cedar pollen. METHODS:: Nasal secretion and serum samples were collected from 31 participants (21 symptomatic and 10 asymptomatic participants) in January (preseason) and March (peak season). Japanese cedar-specific IgA or IgE levels were measured using ELISA with diamond-like carbon-coated chips. RESULTS:: The ratio of Japanese cedar pollen-specific IgA to total IgA (rIgA) in the nasal secretions of symptomatic participants increased significantly in March compared with that in January ( P < .01); however, the ratio of specific IgE to total IgE (rIgE) in nasal secretions did not. rIgA in nasal secretions among asymptomatic participants also did not increase during pollen season. rIgA in nasal secretions was significantly correlated with nasal allergic symptoms (r = 0.82; P < .0001) with no significant correlation between rIgE and symptoms. CONCLUSIONS:: To our knowledge, this is the first study to show an association between nasal symptoms and rIgA in nasal secretions, suggesting that rIgA is useful as an antigen-specific biomarker for allergic rhinitis or pollinosis. Furthermore, rIgA values in nasal secretions do not increase in asymptomatic participants sensitized to Japanese cedar during the pollen season.


Assuntos
Alérgenos , Cryptomeria , Imunoglobulina A , Pólen , Rinite Alérgica Sazonal , Avaliação de Sintomas/métodos , Adulto , Degranulação Celular/imunologia , Correlação de Dados , Eosinófilos/fisiologia , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Japão , Masculino , Pessoa de Meia-Idade , Líquido da Lavagem Nasal/imunologia , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/etiologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/fisiopatologia , Inquéritos e Questionários
15.
Int Immunol ; 31(1): 33-40, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30239772

RESUMO

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) that is characterized by intractable nasal polyp formation. Eosinophil-derived neurotoxin (EDN) is an eosinophil granule protein that is closely related to allergic inflammation, but the pathological implications of EDN in ECRS remain unknown. In this study, we evaluated the function of EDN in ECRS pathogenesis and assessed its potential as a disease activity marker. Serum EDN levels were significantly higher in patients with ECRS than in those with other nasal and paranasal diseases, and were positively correlated with clinical disease activity. Production of EDN from isolated human eosinophils was induced by stimulation with IL-5 in vitro. Human nasal epithelial cells were stimulated with EDN, and the resultant changes in gene expression were detected by RNA sequencing. Pathway analysis revealed that the major canonical pathway affected by EDN stimulation was 'regulation of the epithelial-mesenchymal transition pathway'; the only gene in this pathway to be up-regulated was matrix metalloproteinase 9 (MMP-9). Consistent with this, immunostaining analysis revealed intense staining of both EDN and MMP-9 in nasal polyps from patients with ECRS. In conclusion, our data demonstrate that serum EDN level is a useful marker for the evaluation of ECRS severity. Furthermore, EDN induces production of MMP-9 from the nasal epithelium, which may be involved in the pathogenesis of ECRS.


Assuntos
Remodelação das Vias Aéreas , Neurotoxina Derivada de Eosinófilo/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Rinite/etiologia , Rinite/metabolismo , Sinusite/etiologia , Sinusite/metabolismo , Adulto , Idoso , Biomarcadores , Estudos de Casos e Controles , Degranulação Celular/imunologia , Doença Crônica , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Contagem de Leucócitos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Rinite/diagnóstico , Índice de Gravidade de Doença , Sinusite/diagnóstico
16.
Immunol Invest ; 48(3): 303-320, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30335529

RESUMO

Allergic diseases have increased in the last three decades. Mast cells play a critical role in allergic diseases along with allergen-specific immunoglobulin E (IgE). Following mast cell degranulation elicited by ligation of the IgE-FcεRI receptor complex with allergen, allergic reactions are followed by various symptoms such as vascular hyperpermeability, mucous secretion, itching, sneezing, wheezing, rashes, fever, and anaphylactic shock. Susceptibility or inclination to allergy varies depending on individual genetic traits and living environment, and it has long been believed that such an inclination is determined by an immunologic balance of T helper cell types. Mouse strains also have different susceptibilities to allergy. Similar to T helper cells and macrophages, it is not known whether mast cells can also be divided into two different types between mouse strains. In this study, we prepared bone marrow-derived mast cells from BALB/c and C57BL/6 mice and examined their cellular properties. Cellular response to IL-3 and the process of mast cell differentiation from bone marrow cells were different on the basis of cell surface marker molecules. BALB/c-derived cells more efficiently exhibited degranulation than did C57BL/6-derived cells following both calcium ionophore and receptor crosslinking. These functional differences persisted even after a longer cell culture for 8 weeks, suggesting a difference in cell-autonomous characteristics. These results support the concept that mast cells also have different cell types dependent on their genetic background. Abbreviations: Ab: antibody; BMMC: bone marrow-derived mast cell; DNP: dinitrophenyl; FACS: fluorescence-activated cell sorter; FCS: fetal calf serum; FITC: fluorescein isothiocyanate; FSC: forward scatter; HRP: horseradish peroxidase; HSA: human serum albumin; Ig: immunoglobulin; IL: interleukin; MIP-2: macrophage inflammatory protein-2; MCP: mast cell protease; PE: phycoerythrin; PerCP: Peridinin chlorophyll protein complex; SNP: single nucleotide polymorphisms; SSC: side scatter; Th: T helper; TNF-α: tumor necrosis factor alpha.


Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/imunologia , Mastócitos/fisiologia , Camundongos Endogâmicos BALB C/imunologia , Camundongos Endogâmicos C57BL/imunologia , Animais , Degranulação Celular/imunologia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Hipersensibilidade/imunologia , Camundongos , Cultura Primária de Células
17.
Methods Mol Biol ; 1894: 31-45, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30547453

RESUMO

Mast cells are key effector cells in inflammatory and allergic immune responses such as asthma, rhinitis, and atopic dermatitis. Activation of mast cells leads to immediate release of preformed mediators such as histamine and proteases, which can regulate vascular permeability and the function of a number of immune and nonimmune cells. Engineered nanomaterials (ENM) have been utilized for a wide range of applications and introduced into a number of consumer products; yet the consequent increase in human exposure and any potential adverse effects have not been fully evaluated. Modulation of the immune system function has been shown to be a major toxicological consequence of ENM exposure. The implication of mast cells in ENM-mediated toxicity, including the most widely utilized carbon and metal-based ENMs, has been previously demonstrated; and therefore, understanding direct ENM interaction with mast cells at the cellular and molecular level is of critical importance for the safe implementation of ENMs into consumer products.


Assuntos
Mastócitos/imunologia , Nanoestruturas/toxicidade , Cultura Primária de Células/métodos , Animais , Carbono/toxicidade , Degranulação Celular/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Qualidade de Produtos para o Consumidor , Humanos , Hipersensibilidade , Metais/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Cultura Primária de Células/instrumentação , Testes de Toxicidade/instrumentação , Testes de Toxicidade/métodos
18.
Immunol Cell Biol ; 97(3): 317-325, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536991

RESUMO

Human genetic studies demonstrate a link between the 32-bp deletion that produces a nonfunctional CCR5 receptor and enhanced recovery from acute hepatitis B virus (HBV) infection. To investigate the role of CCR5 in immune responses to acute HBV, we intravenously infected Ccr5+/+ (WT) and Ccr5-/- (KO) mice with a replication-incompetent adenovirus containing the overlapping HBV1.3 construct (AdHBV), or vector control. At day 3 following AdHBV infection, analysis of intrahepatic leukocytes (IHL) showed KO mice had increased CD11b+ NK cells compared to WT (18.2% versus 7.6% of live IHL, P < 0.01). These CD11b+ NK cells were nonresident (CD49a- ) and had capacity to degranulate and produce IFN-γ following stimulation. At day 3, plasma CXCL10 was significantly increased in KO, but not WT, mice receiving AdHBV as compared to vector control, while CXCR3 expression on hepatic CD11b+ NK cells in AdHBV-treated KO mice was significantly lower than that in uninfected mice, suggesting these NK cells are recruited along the CXCL10-CXCR3 axis. At days 7 and 14, no differences between genotypes were observed in number, or HBV-specific function, of intrahepatic CD8+ T cells. Instead, at day 14, KO mice had increased intrahepatic proinflammatory monocytes compared to WT mice (17.56% versus 6.57% of live IHL, P = 0.014), corresponding with an increase in plasma alanine aminotransferase and intrahepatic IL-1ß observed in KO mice. Taken together, these findings demonstrate that loss of CCR5 signaling drives a more robust inflammatory liver microenvironment early in acute HBV infection via enrichment of hepatic innate immune cells.


Assuntos
Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Vírus da Hepatite B , Hepatite B/etiologia , Imunidade Inata/genética , Receptores CCR5/deficiência , Animais , Biomarcadores , Degranulação Celular/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Expressão Gênica , Hepatite B/metabolismo , Hepatite B/patologia , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Baço/citologia , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
19.
Histol Histopathol ; 34(2): 191-200, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30211433

RESUMO

The alteration of intestinal mucosal barrier is considered to be the central pathophysiological process in response to gastrointestinal infections, and mucosal microstructural damage is a major factor for enhancing epithelial permeability in persistent bacterial infections. However, the mechanism involved in hyperpermeability in the early stage of acute bacterial infections is not fully understood. In the present study, fluorescein isothiocyanate-dextran across and transepithelial resistance measured in Ussing chambers were used to assess the intestinal paracellular permeability. Mast cell activation was evaluated by western blotting for the presence of tryptase released from mast cells. Serum levels of interleukin-6 were evaluated using enzyme-linked immunosorbent assay. Our results indicated that mast cells played a pivotal role in colonic mucosal hyperpermeability in wild type mice treated with lipopolysaccharide (LPS) for 2 h. And the effect of LPS was mainly dependent on mast cell degranulation, while no change in permeability was observed in the mast cell-deficient mice (Wads⁻/⁻) after LPS administration. No obvious changes of the mucosal structure including histomorphological architecture and expression of intercellular junction proteins were obtained in either wild type or Wads⁻/⁻ mice after LPS stimulation by hematoxylin and eosin staining, immunofluorescence staining and western blot analysis. Furthermore, the self-renewal of intestinal epithelia, detected by using proliferation marker 5'-bromo-2'-deoxyuridine, was not involved in increased permeability. Collectively, activation of mast cells induced by LPS mediated intestinal hyperpermeability in the initial stage, and played a crucial role in barrier dysfunction rather than mucosal microstructural damage in acute enterogenous bacterial infection.


Assuntos
Junções Intercelulares/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/toxicidade , Mastócitos/imunologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Colo , Modelos Animais de Doenças , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade
20.
Methods Mol Biol ; 1884: 119-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465198

RESUMO

Cancer development is under surveillance by the immune system of the host. Tumor cells can be recognized and killed by cytotoxic lymphocytes- such as CD8+ T lymphocytes and natural killer (NK) cells-mainly through the immune secretion of lytic granules that kill target cells. This process involves the fusion of the granule membrane with the cytoplasmic membrane of the immune effector cell, resulting in surface exposure of lysosomal-associated proteins that are typically present on the lipid bilayer surrounding lytic granules, such as CD107a. Therefore, membrane expression of CD107a constitutes a marker of immune cell activation and cytotoxic degranulation. In this chapter, we detail the steps required to isolate peripheral blood mononuclear cells (PBMCs), coculture them with target tumor cell lines, and evaluate the cytotoxic immune function by means of flow cytometry evaluation of CD107a expression on the surface of NK cells.


Assuntos
Degranulação Celular/imunologia , Separação Celular/métodos , Citometria de Fluxo/métodos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Neoplasias/imunologia , Buffy Coat/citologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Separação Celular/instrumentação , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Citometria de Fluxo/instrumentação , Corantes Fluorescentes/química , Voluntários Saudáveis , Humanos , Vigilância Imunológica , Células K562 , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos
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