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1.
Biomed Res Int ; 2021: 3725316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34414234

RESUMO

Dexmedetomidine is an α2 adrenergic receptor agonist that has been reported to modulate the polarization of CD4+ T cells. However, the underlying mechanisms by which dexmedetomidine induces T-helper 1 (Th1) cell differentiation remain poorly understood. The aim of this study was to explore the potential mechanisms through which dexmedetomidine can induce Th1 cell differentiation. Purified CD4+ T cells were stimulated with anti-CD3/anti-CD28 and then treated with dexmedetomidine. Flow cytometry analysis was adopted to measure the concentration of Th1 cells. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qPCR) were performed to detect protein levels and mRNA expression, respectively, of IFN-γ and IL-4. Western blotting was used to determine the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and T-bet expression. The Th1 cell subset and IFN-γ levels were elevated in the dexmedetomidine-induced CD4+ T cells. Dexmedetomidine enhanced the phosphorylation of STAT1 and the expression of T-bet in the CD4+ T cells. Atipamezole (an α2 adrenergic antagonist) and fludarabine (a STAT1 inhibitor) reversed the dexmedetomidine-induced Th1 cell differentiation. These results suggested that dexmedetomidine induced Th1 cell differentiation via the STAT1-T-bet signaling pathway.


Assuntos
Linfócitos T CD4-Positivos/citologia , Dexmedetomidina/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th1/citologia , Animais , Anticorpos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo
2.
Commun Biol ; 4(1): 960, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381153

RESUMO

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI's evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.


Assuntos
Anticorpos/farmacologia , Materiais Biomiméticos/química , Clostridioides difficile/fisiologia , Ácido Clorídrico/farmacologia , Pepsina A/farmacologia , Inibidor da Tripsina de Soja de Kunitz/química , Animais , Anticorpos/química , Materiais Biomiméticos/farmacologia , Galinhas , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Inibidor da Tripsina de Soja de Kunitz/farmacologia
3.
Exp Cell Res ; 405(2): 112705, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34166678

RESUMO

The interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) pathway modulates immune response and inflammation, associated with allograft dysfunction and rejection. We hypothesized that IL-33/ST2 is a marker of renal allograft rejection and IL-33/ST2 expression may differ according to rejection type. IL-33/ST2 expression was measured in sera and kidney tissues from recipients with acute antibody-mediated rejection (AAMR), acute cell-mediated rejection (ACMR), chronic antibody-mediated rejection (CAMR), and healthy controls. The soluble ST2 and IL-33/ST2 expression levels were higher in participants with all three rejection types than in controls. Although the expression levels in recipients with AAMR and ACMR were significantly higher than those with CAMR, there was no significant difference between the expression levels in AAMR and ACMR. Although IL-33, IL-8, and fibronectin expression were significantly increased after the addition of the recipients' serum in primary cultured human renal proximal tubular epithelial cells, the levels decreased after treatment with an anti-ST2 antibody. Furthermore, the anti-ST2 antibody specifically suppressed the upregulation of the mixed lymphocyte reaction. Boyden chamber assays demonstrated that anti-ST2 antibody abrogated chemotaxis induced by recombinant IL-33. Thus, IL-33 and ST2 are potent mediators of rejection. Treatment with an anti-ST2 antibody ameliorates rejection and could be a potential therapeutic strategy for renal allograft rejection.


Assuntos
Aloenxertos/imunologia , Rejeição de Enxerto/imunologia , Interleucina-33/metabolismo , Transplante de Rim , Adulto , Anticorpos/farmacologia , Biomarcadores/análise , Feminino , Humanos , Rim/imunologia , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade , Transplante Homólogo/métodos
4.
Eur J Med Chem ; 223: 113654, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34175537

RESUMO

Niemann-Pick C1 (NPC1) receptor is an intracellular protein located in late endosomes and lysosomes whose main function is to regulate intracellular cholesterol trafficking. Besides being postulated as necessary for the infection of highly pathogenic viruses in which the integrity of cholesterol transport is required, this protein also allows the entry of the Ebola virus (EBOV) into the host cells acting as an intracellular receptor. EBOV glycoprotein (EBOV-GP) interaction with NPC1 at the endosomal membrane triggers the release of the viral material into the host cell, starting the infective cycle. Disruption of the NPC1/EBOV-GP interaction could represent an attractive strategy for the development of drugs aimed at inhibiting viral entry and thus infection. Some of the today available EBOV inhibitors were proposed to interrupt this interaction, but molecular and structural details about their mode of action are still preliminary thus more efforts are needed to properly address these points. Here, we provide a critical discussion of the potential of NPC1 and its interaction with EBOV-GP as a therapeutic target for viral infections.


Assuntos
Glicoproteínas/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Ebolavirus/metabolismo , Glicoproteínas/química , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/patologia , Humanos , Simulação de Acoplamento Molecular , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/imunologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Internalização do Vírus/efeitos dos fármacos
5.
Methods Mol Biol ; 2329: 205-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085225

RESUMO

Reversible protein phosphorylation regulates the transitions between different phases of the cell cycle ensuring proper segregation of the duplicated genome into two daughter cells. Protein kinases and protein phosphatases establish the appropriate phosphorylation stoichiometries in diverse substrates maintaining genomic stability as a cell undergoes this complex process. Along with regulating common substrates, these opposing enzymes regulate one another by fine-tuning each other's activity both spatially and temporally throughout mitosis. Protein phosphatase catalytic subunits work together with regulatory proteins, which control their localization, activity, and specificity. Protein phosphatase 1 (PP1) recognizes its regulatory proteins via a short linear interaction motif (SLIM) called the "RVxF" motif. A subset of proteins with these "RVxF" motifs contain a phosphorylatable amino acid (S/T) at the 'x' position.Here, we describe methods to generate, affinity purify and utilize phospho-specific antibodies to monitor phosphorylation sites during the cell cycle and the appropriate use of mitotic kinase inhibitors. More specifically, we employ phospho-specific antibodies, which recognize phosphorylated RVp[S/T]F motif-containing proteins, to monitor the phosphorylation status of these motifs throughout the cell cycle. Furthermore, we use mitotic kinase inhibitors to examine the effect of kinase inhibition on the phosphorylation status of multiple RV[S/T]F motifs using these phospho-specific antibodies.


Assuntos
Anticorpos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 1/metabolismo , Proteínas/química , Motivos de Aminoácidos/efeitos dos fármacos , Sítios de Ligação , Ciclo Celular , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Proteínas/efeitos dos fármacos , Proteínas/metabolismo
6.
Int Immunopharmacol ; 96: 107797, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34162159

RESUMO

Specific antibodies against SARS-CoV-2 structural protein have a wide range of effects in the diagnose, prevention and treatment of the COVID-19 epidemic. Among them, egg yolk immunoglobulin Y (IgY), which has high safety, high yield, and without inducing antibody-dependent enhancement, is an important biological candidate. In this study, specific IgY against the conservative nucleocapsid protein (NP) of SARS-CoV-2 was obtained by immunizing hens. Through a series of optimized precipitation and ultrafiltration extraction schemes, its purity was increased to 98%. The hyperimmune IgY against NP (N-IgY) at a titer of 1:50,000 showed strong NP binding ability, which laid the foundation of N-IgY's application targeting NP. In an in vitro immunoregulatory study, N-IgY (1 mg/mL) modulated NP-induced immune response by alleviating type II interferon secretion stimulated by NP (20 µg/mL). In summary, N-IgY can be mass produced by achievable method, which endows it with potential value against the current COVID-19 pandemic.


Assuntos
Anticorpos/imunologia , Antivirais/imunologia , COVID-19/imunologia , Imunoglobulinas/imunologia , Fatores Imunológicos/imunologia , Interferon gama/metabolismo , SARS-CoV-2/imunologia , Animais , Anticorpos/farmacologia , Antivirais/farmacologia , COVID-19/terapia , Galinhas , Desenvolvimento de Medicamentos , Gema de Ovo/química , Gema de Ovo/metabolismo , Humanos , Imunidade , Imunoglobulinas/farmacologia , Fatores Imunológicos/farmacologia , Imunomodulação , Técnicas In Vitro , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/metabolismo , SARS-CoV-2/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071276

RESUMO

Cardiovascular disease is the leading cause of death worldwide, and its prevalence is increasing due to the aging of societies. Atherosclerosis, a type of chronic inflammatory disease that occurs in arteries, is considered to be the main cause of cardiovascular diseases such as ischemic heart disease or stroke. In addition, the inflammatory response caused by atherosclerosis confers a significant effect on chronic inflammatory diseases such as psoriasis and rheumatic arthritis. Here, we review the mechanism of action of the main causes of atherosclerosis such as plasma LDL level and inflammation; furthermore, we review the recent findings on the preclinical and clinical effects of antibodies that reduce the LDL level and those that neutralize the cytokines involved in inflammation. The apolipoprotein B autoantibody and anti-PCSK9 antibody reduced the level of LDL and plaques in animal studies, but failed to significantly reduce carotid inflammation plaques in clinical trials. The monoclonal antibodies against PCSK9 (alirocumab, evolocumab), which are used as a treatment for hyperlipidemia, lowered cholesterol levels and the incidence of cardiovascular diseases. Antibodies that neutralize inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-17, and IL-12/23) have shown promising but contradictory results and thus warrant further research.


Assuntos
Anticorpos/farmacologia , Aterosclerose/tratamento farmacológico , Doenças Cardiovasculares/tratamento farmacológico , Imunização Passiva/métodos , Animais , Anticorpos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticolesterolemiantes/uso terapêutico , Apolipoproteínas B , Autoanticorpos , LDL-Colesterol/sangue , Citocinas/imunologia , Humanos , Inflamação/tratamento farmacológico , Fragmentos de Peptídeos , Pró-Proteína Convertase 9/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico
8.
Nat Immunol ; 22(7): 851-864, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099918

RESUMO

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.


Assuntos
Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-33/farmacologia , Linfócitos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo
9.
Am J Respir Cell Mol Biol ; 65(4): 403-412, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34014798

RESUMO

Mechanical ventilation is a known risk factor for delirium, a cognitive impairment characterized by dysfunction of the frontal cortex and hippocampus. Although IL-6 is upregulated in mechanical ventilation-induced lung injury (VILI) and may contribute to delirium, it is not known whether the inhibition of systemic IL-6 mitigates delirium-relevant neuropathology. To histologically define neuropathological effects of IL-6 inhibition in an experimental VILI model, VILI was simulated in anesthetized adult mice using a 35 cc/kg tidal volume mechanical ventilation model. There were two control groups, as follow: 1) spontaneously breathing or 2) anesthetized and mechanically ventilated with 10 cc/kg tidal volume to distinguish effects of anesthesia from VILI. Two hours before inducing VILI, mice were treated with either anti-IL-6 antibody, anti-IL-6 receptor antibody, or saline. Neuronal injury, stress, and inflammation were assessed using immunohistochemistry. CC3 (cleaved caspase-3), a neuronal apoptosis marker, was significantly increased in the frontal (P < 0.001) and hippocampal (P < 0.0001) brain regions and accompanied by significant increases in c-Fos and heat shock protein-90 in the frontal cortices of VILI mice compared with control mice (P < 0.001). These findings were not related to cerebral hypoxia, and there was no evidence of irreversible neuronal death. Frontal and hippocampal neuronal CC3 were significantly reduced with anti-IL-6 antibody (P < 0.01 and P < 0.0001, respectively) and anti-IL-6 receptor antibody (P < 0.05 and P < 0.0001, respectively) compared with saline VILI mice. In summary, VILI induces potentially reversible neuronal injury and inflammation in the frontal cortex and hippocampus, which is mitigated with systemic IL-6 inhibition. These data suggest a potentially novel neuroprotective role of systemic IL-6 inhibition that justifies further investigation.


Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Delírio/metabolismo , Interleucina-6/antagonistas & inibidores , Neurônios/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Animais , Delírio/tratamento farmacológico , Delírio/patologia , Modelos Animais de Doenças , Feminino , Lobo Frontal/lesões , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Hipocampo/lesões , Hipocampo/metabolismo , Hipocampo/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Camundongos , Neurônios/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
10.
Nat Commun ; 12(1): 2547, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953162

RESUMO

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.


Assuntos
Anticorpos/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de Interleucina-8B/metabolismo , Animais , Sítios de Ligação , Quimiocinas , Quimiotaxia , Encefalomielite Autoimune Experimental/imunologia , Endocitose , Epitopos , Humanos , Imunoglobulina G , Interleucina-8/metabolismo , Ligantes , Camundongos , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa
11.
Int J Biol Macromol ; 182: 1292-1300, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000307

RESUMO

Astragalus membranaceus (A. membranaceus) is commonly used in various herbal formulations to treat several human and animal diseases. Polysaccharides, which are the major bioactive components in the A. membranaceus, exhibit various bioactive properties. However, the ability of A. membranaceus polysaccharides (APS) to activate the mucosal immune response has not been examined. We examined the effect of intranasal administration of APS on mucosal immune cell activation and the growth-inhibitory activity against pulmonary metastatic melanoma in mice by combination treatment with immune checkpoint blockade. The intranasal treatment of APS increased the number of lineage-CD11c+ dendritic cell (DCs) in the mesenteric lymph nodes (mLN) through the upregulation of CC-chemokine receptor 7 expression. Moreover, intranasal treatment of APS activated DCs, which further stimulated natural killer (NK) and T cells in the mLN. The APS/anti-PD-L1 antibody combination inhibited the pulmonary infiltration of B16 melanoma cells. The depletion of NK cells and CD8 T cells in mice mitigated the anti-cancer effect of this combination, thereby highlighting the critical role of NK cells and CD8 T cells in mediating anti-cancer immunity. These findings demonstrated that APS could be used as a topical mucosal adjuvant to enhance the immune check point inhibitor anti-cancer effect.


Assuntos
Astragalus propinquus/química , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/secundário , Melanoma/patologia , Polissacarídeos/farmacologia , Administração Intranasal , Animais , Anticorpos/farmacologia , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Sinergismo Farmacológico , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Melanoma/imunologia , Camundongos Endogâmicos C57BL , Peso Molecular , Monossacarídeos/análise , Polissacarídeos/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
12.
Int J Biol Macromol ; 182: 1455-1462, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015405

RESUMO

CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy. Indeed, a conceivable approach to tackle disease is to interfere with CD55-mediated complement regulation with the use of CD55-targeting antibodies. However, the large size and poor tissue penetration together with to the high costs of antibodies often limits their widespread therapeutic use. Here, we employed bioinformatic and chemical approaches to design and synthesize molecules of small dimensions able to mimic a CD55 blocking antibody. As a result, a bicyclic peptide, named as miniAB55, proved to bind CD55 with nanomolar affinity. This molecule represents an attracting chemical scaffold for CD55-directed diagnostic tools in diseases associated with CD55 overproduction. To further support the applicative potential of miniAB55, we prove that the miniAB55 binds CD55 on the same region involved in inactivation of the complement C3 and C5 convertases, thus opening promising scenarios for the development of complement-modulating tools.


Assuntos
Anticorpos/farmacologia , Antígenos CD55/imunologia , Miniaturização , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD55/química , Ciclização , Humanos , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular
13.
Mol Biol Rep ; 48(4): 3475-3484, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33904141

RESUMO

Ischemia-reperfusion frequently occurs in ischemic cerebral vascular disease, during which the inflammatory signaling plays essential roles. The aim of this study was to discover the efficacy of the antibody to a key immune cytokine IL-23 (anti-IL-23) for the therapy of cerebral ischemia-reperfusion injury. We established the cerebral ischemia-reperfusion injury model by middle cerebral artery occlusion (MCAO). Anti-IL-23 injection attenuated lesions indicated by histology study. RT-PCR and Western blot were employed to detect the mRNA and protein expression of JAK2 and STAT3 after anti-IL-23 treatment. ELISA was utilized to measure the levels of MDA (malondialdehyde) and superoxide dismutase (SOD). Moreover, curcumin and IL-6 were implicated in the endogenous intervention of IL-23 signaling in vivo. Our data demonstrated that the treatment of anti-IL-23 might transcriptionally activate the classic immune pathway in the brain. Anti-IL-23 augmented phosphorylation levels of both JAK2 and STAT3, suggesting the amplification signaling of JAK/STAT after exogenous IL-23 intervention. Anti-IL-23 reduced ROS molecules of STAT downstream in the serum and brain. It also alleviated the injury by bringing down levels of MDA and SOD in the serum. JAK2 inhibitor could abolish the effect of anti-IL-23 whereas JAK3 ameliorated the injury. The combination of anti-IL-23 and JAK3i could reduce infarct volume more effectively. In summary, this study indicated that anti-IL-23 had protective effects against cerebral ischemia-reperfusion injury by targeting the immune specific JAK2-STAT3 in JAK/STAT pathway.


Assuntos
Anticorpos/farmacologia , Interleucina-23/imunologia , Janus Quinase 2/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Encéfalo/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Modelos Animais de Doenças , Masculino , Fármacos Neuroprotetores/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo
14.
Methods Mol Biol ; 2285: 65-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928543

RESUMO

CD4+ T helper (TH) cells are key mediators of immunity, and according to their effector functions, they can be divided into different subsets, namely, TH1, TH2, TH17, and TH22. In order to maintain systemic homeostasis and peripheral tolerance, CD4+ TH cells are counterbalanced by CD4+ T cells with regulatory properties, namely, Foxp3+ regulatory T cells (Foxp3+TREG) and TR1 cells. Here, we describe how to in vitro differentiate murine naïve CD4+ T cells toward helper (TH1, TH2, TH17, and TH22) and regulatory (Foxp3+TREG and TR1) cells.


Assuntos
Diferenciação Celular , Plasticidade Celular , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Plasticidade Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/farmacologia , Citometria de Fluxo , Separação Imunomagnética , Camundongos Endogâmicos C57BL , Fenótipo , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fluxo de Trabalho
15.
Front Immunol ; 12: 650864, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767714

RESUMO

Triggering receptor expressed on myeloid cell-1 (TREM-1) signaling is expressed on neutrophils and monocytes that is necessary for the successful antimicrobial response and resolution of inflammation in the gut. In this study, we determined the effect of an anti-TREM-1 agonistic antibody (α-TREM-1) on colitis and identify its underlying mechanism of action. Administration of α-TREM-1 alleviated colitis in mice and resolved dysbiosis, which required TLR4/Myd88 signaling. α-TREM-1 increased the production of neutrophil extracellular traps and interleukin-22 by CD177+ neutrophils, which led to pathogen clearance and protection of the intestinal barrier. TREM-1 activation using an α-TREM-1 antibody protects against colitis by rebalancing the microbiota and protecting the epithelium against the immune response as well as modulates the function of neutrophils and macrophages. These results highlight the importance of the TREM-1 pathway in intestinal homeostasis and suggest that α-TREM-1 treatment may be an effective therapeutic strategy for inflammatory bowel disease.


Assuntos
Anticorpos/farmacologia , Proteínas Ligadas por GPI/imunologia , Inflamação/prevenção & controle , Intestinos/efeitos dos fármacos , Isoantígenos/imunologia , Neutrófilos/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Receptor Gatilho 1 Expresso em Células Mieloides/agonistas , Animais , Anticorpos/imunologia , Colite/imunologia , Colite/metabolismo , Colite/prevenção & controle , Disbiose/microbiologia , Disbiose/prevenção & controle , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Proteínas Ligadas por GPI/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/prevenção & controle , Interleucinas/imunologia , Interleucinas/metabolismo , Intestinos/imunologia , Intestinos/patologia , Isoantígenos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/imunologia
16.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33651716

RESUMO

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.


Assuntos
Fator Ativador de Células B/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Adolescente , Adulto , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Fator Ativador de Células B/genética , Inibidores dos Fatores de Coagulação Sanguínea/genética , Criança , Pré-Escolar , Fator VIII/antagonistas & inibidores , Fator VIII/genética , Fator VIII/uso terapêutico , Feminino , Células HEK293 , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Humanos , Tolerância Imunológica/efeitos dos fármacos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pessoa de Meia-Idade
17.
Front Immunol ; 12: 614320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33708208

RESUMO

Heat shock proteins (Hsp) are constitutive and stress-induced molecules which have been reported to impact innate and adaptive immune responses. Here, we evaluated the role of Hsp70 as a treatment target in the imiquimod-induced, psoriasis-like skin inflammation mouse model and related in vitro assays. We found that immunization of mice with Hsp70 resulted in decreased clinical and histological disease severity associated with expansion of T cells in favor of regulatory subtypes (CD4+FoxP3+/CD4+CD25+ cells). Similarly, anti-Hsp70 antibody treatment led to lowered disease activity associated with down-regulation of pro-inflammatory Th17 cells. A direct stimulating action of Hsp70 on regulatory T cells and its anti-proliferative effects on keratinocytes were confirmed in cell culture experiments. Our observations suggest that Hsp70 may be a promising therapeutic target in psoriasis and potentially other autoimmune dermatoses.


Assuntos
Anticorpos/imunologia , Dermatite/etiologia , Dermatite/metabolismo , Suscetibilidade a Doenças , Proteínas de Choque Térmico HSP70/imunologia , Animais , Anticorpos/farmacologia , Biomarcadores , Biópsia , Citocinas/metabolismo , Dermatite/diagnóstico , Dermatite/tratamento farmacológico , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Feminino , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Imunização , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Proteínas Recombinantes , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo
18.
Front Immunol ; 12: 586521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717067

RESUMO

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.


Assuntos
Anticorpos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Receptores de AMPA/antagonistas & inibidores , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Complemento C1q/imunologia , Imunofluorescência , Masculino , Camundongos , Receptores de AMPA/química , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
19.
Theranostics ; 11(7): 3527-3539, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537102

RESUMO

To investigate the utility of noninvasive µPET-CT with 64Cu-DOTA-anti-CD11b (64Cu-αCD11b) in assessing bone marrow status after anticancer therapies, and the protective role of anti-CSF-1 (αCSF-1) against bone marrow suppression induced by Abraxane. Methods: MDA-MB-435 tumor-bearing mice were treated with Abraxane, αCSF-1, or αCSF-1 plus Abraxane. µPET-CT and biodistribution of 64Cu-αCD11b were performed after intravenous injection of the radiotracer. Cells from mouse bone marrow and MDA-MB-435 tumor were analyzed by flow cytometry. A humanized αCSF-1 was investigated for its role in protecting bone marrow cells, using a transgenic mouse model that expresses functional human CSF-1. Results: µPET-CT showed that 64Cu-αCD11b had high uptake in the bone marrow and spleen of both normal and tumor-bearing mice. Abraxane significantly reduced 64Cu-αCD11b uptake in the bone marrow and spleen of treated mice compared to untreated mice. Interestingly, 64Cu-αCD11b µPET-CT revealed that αCSF-1 alleviated the depletion of bone marrow cells by Abraxane. These changes in the bone marrow population of CD11b+ myeloid cells were confirmed by flow cytometry. Moreover, αCSF-1 potently enhanced tolerance of bone marrow granulocytic myeloid cells to Abraxane, decreased cell migration, and suppressed recruitment of myeloid cells to the tumor microenvironment. The humanized αCSF-1 also alleviated the effects of Abraxane on bone marrow cells in transgenic mice expressing human CSF-1, suggesting clinical relevance of αCSF-1 in prevention of bone marrow suppression in addition to its role in reducing tumor-infiltrating myeloid cells. Conclusions: Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice could be noninvasively assessed by µPET-CT with 64Cu-αCD11b and prevented by αCSF-1.


Assuntos
Paclitaxel Ligado a Albumina/toxicidade , Anticorpos/farmacologia , Antineoplásicos/toxicidade , Medula Óssea/diagnóstico por imagem , Neoplasias/diagnóstico por imagem , Baço/diagnóstico por imagem , Paclitaxel Ligado a Albumina/antagonistas & inibidores , Animais , Anticorpos/química , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Medula Óssea/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Linhagem Celular Tumoral , Radioisótopos de Cobre , Feminino , Expressão Gênica , Compostos Heterocíclicos com 1 Anel/química , Xenoenxertos , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/imunologia , Camundongos , Camundongos Nus , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/patologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Microambiente Tumoral/efeitos dos fármacos
20.
Int Immunopharmacol ; 94: 107444, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33578263

RESUMO

Toluene diisocyanate (TDI) exhibits an ability to induce steroid insensitive asthma with the involvement of Th17 cells. And emerging evidence has indicated that DLL4 signaling promotes Th17 differentiation through directly upregulating Rorc and IL-17 transcription. Thus, we sought to evaluate the effects of DLL4 blocking antibody on TDI-induced asthma model. Female BALB/c mice were sensitized and challenged with TDI to generate an asthma model. TDI-exposed mice were intraperitoneally injected with anti-DLL4 antibody and then analyzed for various parameters of the airway inflammatory responses. Increased expression of DLL4 in spleen and lung was detected in TDI-exposed mice. Furthermore, anti-DLL4 treatment alleviated TDI-induced airway hyperreactivity (AHR), airway inflammation, airway epithelial injury and airway smooth muscle (ASM) thickening. In the meantime, neutralizing DLL4 also blunted Th17 response via downregulation of ROR-γt expression, while had no effect on Th2 cells and regulatory T (Treg) cells. Overall, anti-DLL4 ameliorated TDI-induced experimental asthma by inhibiting Th17 response, implying the feasibility of targeting DLL4 for therapy of Th17-predominant severe asthma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anticorpos/uso terapêutico , Asma/tratamento farmacológico , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Células Th17/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos/farmacologia , Asma/sangue , Asma/induzido quimicamente , Asma/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina E/sangue , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Células Th17/imunologia , Tolueno 2,4-Di-Isocianato
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