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1.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468674

RESUMO

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.


Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas Conjugadas/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Vacina BCG/farmacologia , Proteínas de Bactérias , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Receptor 2 Toll-Like/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia
2.
PLoS One ; 15(9): e0239161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915926

RESUMO

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.


Assuntos
Colesteatoma da Orelha Média/genética , Orelha Média/patologia , Membrana Mucosa/patologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Colesteatoma da Orelha Média/imunologia , Colesteatoma da Orelha Média/patologia , Colesteatoma da Orelha Média/cirurgia , Orelha Média/imunologia , Orelha Média/cirurgia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/imunologia , Membrana Mucosa/cirurgia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Padrões de Referência , Reprodutibilidade dos Testes , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Adulto Jovem
3.
Front Immunol ; 11: 1663, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754160

RESUMO

A recent pandemic caused by a single-stranded RNA virus, COVID-19, initially discovered in China, is now spreading globally. This poses a serious threat that needs to be addressed immediately. Genome analysis of SARS-CoV-2 has revealed its close relation to SARS-coronavirus along with few changes in its spike protein. The spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. In the current study, the spike protein of SARS-CoV-2 was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. The S1 and S2 domains of spike proteins were analyzed, and two vaccine constructs were prioritized with T-cell and B-cell epitopes. We adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against COVID-19. Prioritized epitopes were then modeled using linkers and adjuvants, and respective 3D models were constructed to evaluate their physiochemical properties and their possible interactions with ACE2, HLA Superfamily alleles, TLR2, and TLR4.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Modelos Químicos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/virologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/imunologia , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/imunologia , Vacinas Virais/química
4.
Anim Sci J ; 91(1): e13439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32779289

RESUMO

Lactobacillus rhamnosus GG (LGG) is increasingly applied in functional food products and acts as a probiotic model in nutritious and clinical studies. Increasing evidences have revealed the immune modulation of LGG on macrophages. The aim of this study is to investigate the effect of LGG on macrophage polarization of murine bone marrow-derived macrophages (BMDMs). BMDMs were treated with 108 colony-forming units (CFU)/ml LGG for 1.5, 3, and 6 hr. Results showed that LGG obviously upregulated the mRNA expression of M1-associated cytokines (p < .05), including interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS), whereas had no effect on the expression of M2-associated markers (p > .05), including arginase 1 (Arg1), mannose receptor, and chitinase-like protein 3 (YM1). Furthermore, LGG markedly increased the expression of pro-inflammatory cytokines (IL-12p40, cyclooxygenase-2 [COX-2], and interferon-γ [IFN-γ]) (p < .05) and anti-inflammatory cytokines (IL-10, IL-4, and transforming growth factor-ß [TGF-ß]) (p < .05). In addition, we also found that TLR2/MyD88/MAPK signaling pathway was required for LGG-induced M1 macrophage polarization and M1-related cytokines expression. Together, these findings demonstrate that probiotic LGG facilitates M1 polarization of BMDMs, suggesting that LGG may have an immunotherapeutic potential in regulating the host defense against pathogen invasion.


Assuntos
Células da Medula Óssea , Lactobacillus rhamnosus/química , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Camundongos
5.
PLoS Pathog ; 16(7): e1008622, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634175

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Listeriose/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Endossomos/metabolismo , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
6.
Sci Rep ; 10(1): 10895, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616763

RESUMO

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Vacinas Virais/metabolismo
7.
Am J Med Sci ; 360(2): 176-191, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32553747

RESUMO

BACKGROUND: This study aimed to investigate the role of Clostridium butyricum (C. butyricum) in conjunction with the Toll-like receptor2 (TLR2) signaling pathway and T helper 17 (Th17) cells in dextran sodium sulfate (DSS)-induced colitis in mice. METHODS: Forty 8-week-old BALB/c mice were randomly divided into 5 groups of 8 mice for 7 days: control, DSS (5% DSS), DSS+C. butyricum (1 × 109 CFU), DSS+C. butyricum (1 × 108 CFU) and DSS+C. butyricum (1 × 107 CFU) groups. We assessed the disease activity index (DAI) and histological damage scores. The expression levels of TLR2, myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B p65 (NF-κBp65), interleukin (IL) 17 (IL17), IL23 and retineic acid receptor related orphan nuclear receptor gamma t (RORγt) were determined through immunohistochemical staining, western blot and quantitative real-time PCR (qRT-PCR). The expression levels of CD3+CD4+IL17+ cells in peripheral blood were measured by flow cytometry. RESULTS: C. butyricum dose-dependently decreased DAI and histological damage scores in DSS mice and down-regulated the mRNA and protein levels of TLR2, MyD88 and NF-κBp65 in mouse colon tissue (all P < 0.05). In addition, C. butyricum dose-dependently decreased the levels of CD3+CD4+IL17+ cells in peripheral blood and down-regulated the mRNA and protein levels of IL17, IL23 and RORγt in mouse colon tissue (all P < 0.05). Moreover, the effect of C. butyricum on TLR2 was positively correlated with IL17, IL23 and RORγt. CONCLUSIONS: C. butyricum exerts a dose-dependently protective effect on acute intestinal inflammation induced by DSS in mice, by inhibiting the TLR2 signaling pathway, down-regulating the expression of IL23 and RORγt, and inhibiting the secretion of IL17.


Assuntos
Clostridium butyricum , Colite/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Probióticos , Células Th17/imunologia , Receptor 2 Toll-Like/imunologia , Fator de Transcrição RelA/imunologia , Animais , Peso Corporal , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Comportamento de Ingestão de Líquido , Comportamento Alimentar , Feminino , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-23/genética , Interleucina-23/imunologia , Interleucina-23/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
8.
Int J Mol Sci ; 21(6)2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32183481

RESUMO

The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-κB was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-κB. Furthermore, inhibition of TLR2, MyD88, and NF-κB in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-κB signaling pathway.


Assuntos
Proteínas Aviárias/imunologia , Proteínas de Bactérias/imunologia , Chlamydophila psittaci/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Células Th2/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Linhagem Celular , Galinhas , Macrófagos/microbiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Psitacose/imunologia , Psitacose/microbiologia , Psitacose/veterinária
9.
Cell Rep ; 30(4): 1052-1062.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995749

RESUMO

Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1ß2) to activate the LT beta receptor (LTßR) on the lymphatic endothelial cells (LECs), modulating LEC adhesion molecules, intercellular junctions, and chemokines. We demonstrate a role for Tregs through this pathway to condition the permissiveness of lymphatic endothelia for transendothelial migration (TEM), thus gating leukocyte traffic. Human Tregs share the same property with murine Tregs. Activation of TLR2 on Tregs during inflammation specifically augments LTα1ß2-LTßR signaling, which further enhances the permissiveness of LECs to facilitate TEM. The conditioning of endothelia may promote the resolution of inflammation by directing leukocytes out of tissues to lymphatic vessels and draining lymph nodes (dLNs). Thus, Tregs interact with lymphatic endothelia under homeostasis and inflammation and dictate endothelial permissiveness and gating mechanisms for subsequent leukocyte migration through endothelial barriers.


Assuntos
Movimento Celular/imunologia , Endotélio Linfático/metabolismo , Inflamação/metabolismo , Linfócitos T Reguladores/metabolismo , Receptor 2 Toll-Like/metabolismo , Migração Transendotelial e Transepitelial/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Caderinas/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CCL21/metabolismo , Endotélio Linfático/efeitos dos fármacos , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-2/farmacologia , Ilhotas Pancreáticas/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Receptor beta de Linfotoxina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Receptor 2 Toll-Like/imunologia , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo
10.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31907194

RESUMO

Yersinia pestis is the causative agent of bubonic, pneumonic, and septicemic plague. We demonstrate that Toll-like receptor 2-deficient (TLR2-/-) mice are resistant to septicemic infection by the KIM5 strain of Y. pestis but not to infection by the CO92 Δpgm strain. This resistance is dependent on TLR2, the route of infection, and the isoform of YopJ. Elevated bacterial burdens were found in the spleens of CO92 Δpgm-infected animals by 24 h postinfection and in the livers by 4 days. The YopJ isoform present contributed directly to cytotoxicity and inflammatory cytokine production of bone marrow-derived macrophages from TLR2-/- mice. Immune cell trafficking is altered in CO92 Δpgm infections, with an increased neutrophil infiltration to the spleen 5 days postinfection. Immune cell infiltration to the liver was greater and earlier in KIM5-infected TLR2-/- mice. The functionality of the immune cells was assessed by the ability to develop reactive oxygen and nitrogen species. Our data suggest an inhibition of granulocytes in forming these species in CO92 Δpgm-infected TLR2-/- mice. These findings suggest that resistance to KIM5 in TLR2-/- mice is dependent on early immune cell trafficking and functionality.


Assuntos
Peste/imunologia , Receptor 2 Toll-Like/deficiência , Yersinia pestis/patogenicidade , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Granulócitos/metabolismo , Fígado/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peste/metabolismo , Peste/microbiologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Baço/imunologia , Baço/microbiologia , Receptor 2 Toll-Like/imunologia , Virulência/genética , Yersinia pestis/genética
11.
Clin Exp Immunol ; 199(2): 230-243, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31631328

RESUMO

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.


Assuntos
Células Epiteliais Alveolares/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Nicotina/farmacologia , Tuberculose Pulmonar/imunologia , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Tuberculose Pulmonar/patologia
12.
Autoimmun Rev ; 19(1): 102430, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31734400

RESUMO

The pathophysiology of autoimmune diseases such as Multiple Sclerosis (MS) involves a complex interaction between genetic and environmental factors. Studies of monozygotic twins suggest a significant role for environmental factors in susceptibility to MS. Numerous studies, driven by the "Hygiene Hypothesis," have focused on the role of environmental factors in allergic and autoimmune diseases. The hygiene hypothesis postulates that individuals living in environments that are too "clean" lack the requisite exposure to "immune-tolerizing" microbial products, resulting in poorly regulated immune systems and increased immune-mediated diseases. Interestingly, few studies have linked MS with the hygiene hypothesis. Similarly, although numerous studies have examined the role of the microbiome in autoimmune diseases, there has been no consistent documentation of disease-specific alterations in the MS microbiome. In this review, we present evidence that integrating the hygiene hypothesis and the microbiome allows for the identification of novel pathophysiologic mechanisms in MS. Our central hypothesis is that the microbiome in MS represents a "defective environment" that fails to provide normal levels of "TLR2-tolerizing" bacterial products to the systemic immune system. Consistent with the hygiene hypothesis, we posit that this defective microbiome function results in abnormally regulated systemic innate immune TLR2 responses that play a critical role in both the inflammatory and defective remyelinative aspects of MS. We have completed proof of concept studies that support the inflammatory, remyelinating, and human immune response components of this paradigm. Our studies suggest that induction of TLR2 tolerance may represent a novel approach to treating MS, inhibiting autoimmune inflammation while simultaneously facilitating remyelination.


Assuntos
Hipótese da Higiene , Microbiota , Esclerose Múltipla/imunologia , Esclerose Múltipla/microbiologia , Receptor 2 Toll-Like/imunologia , Humanos
13.
Int Immunol ; 32(2): 89-104, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31713625

RESUMO

Staphylococcus aureus is a main pathogen of osteomyelitis and protein A is a virulence factor with high affinity for IgG. In this study, we investigated whether S. aureus affects the differentiation and bone resorption of osteoclasts through the IgG-binding capacity of protein A. Staphylococcus aureus pre-treated with serum or IgG showed marked enhancement in osteoclastogenesis and bone resorption compared to non-treated S. aureus or a protein A-deficient mutant. Blocking of the Fc receptor and deletion of the Fcγ receptor gene in osteoclast precursor cells showed that enhanced osteoclastogenesis stimulated by S. aureus IgG immune complexes (ICs) was mediated by the Fc receptor on osteoclast precursor cells. In addition, osteoclastogenesis stimulated by S. aureus ICs but not the protein A-deficient mutant was markedly reduced in osteoclast precursor cells of Myd88-knockout mice. Moreover, NFATc1, Syk and NF-κB signals were necessary for osteoclastogenesis stimulated by S. aureus ICs. The results suggest the contribution of a of Toll-like receptor 2 (TLR2)-Myd88 signal to the activity of S. aureus ICs. We further examined the expression of pro-inflammatory cytokines that is known to be enhanced by FcγR-TLR cross-talk. Osteoclasts induced by S. aureus ICs showed higher expression of TNF-α and IL-1ß, and marked stimulation of proton secretion of osteoclasts activated by pro-inflammatory cytokines. Finally, injection of S. aureus, but not the protein A-deficient mutant, exacerbated bone loss in implantation and intra-peritoneal administration mouse models. Our results provide a novel mechanistic aspect of bone loss induced by S. aureus in which ICs and both Fc receptors and TLR pathways are involved.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Diferenciação Celular , Imunoglobulina G/imunologia , Receptores Fc/imunologia , Proteína Estafilocócica A/imunologia , Staphylococcus aureus/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/efeitos dos fármacos , Osteoclastos/imunologia , Osteogênese/efeitos dos fármacos , Ligante RANK/antagonistas & inibidores , Ligante RANK/farmacologia , Receptores Fc/deficiência , Receptores Fc/genética , Proteína Estafilocócica A/genética , Staphylococcus aureus/citologia , Ácidos Teicoicos/farmacologia
14.
Nat Rev Gastroenterol Hepatol ; 17(1): 53-64, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31811279

RESUMO

Diseases intrinsic to the pancreas such as pancreatitis, pancreatic cancer and type 1 diabetes mellitus impart substantial health and financial burdens on society but identification of novel mechanisms contributing to these pathologies are slow to emerge. A novel area of research suggests that pancreatic-specific disorders might be modulated by the gut microbiota, either through a local (direct pancreatic influence) or in a remote (nonpancreatic) fashion. In this Perspectives, we examine literature implicating microorganisms in diseases of the pancreas, specifically pancreatitis, type 1 diabetes mellitus and pancreatic ductal adenocarcinoma. We also discuss evidence of an inherent pancreatic microbiota and the influence of the intestinal microbiota as it relates to disease association and development. In doing so, we address pitfalls in the current literature and areas of investigation that are needed to advance a developing field of research that has clinical potential to reduce the societal burden of pancreatic diseases.


Assuntos
Carcinoma Ductal Pancreático/microbiologia , Diabetes Mellitus Tipo 1/microbiologia , Microbioma Gastrointestinal/fisiologia , Pâncreas/microbiologia , Neoplasias Pancreáticas/microbiologia , Pancreatite/microbiologia , Translocação Bacteriana , Diabetes Mellitus Tipo 1/imunologia , Microbioma Gastrointestinal/imunologia , Humanos , Microbiota , Pâncreas/fisiologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia
15.
Front Immunol ; 10: 2691, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803195

RESUMO

Host defense peptides (HDPs) have antimicrobial and immunoregulatory activities and are involved in epithelial innate immune defense. Dietary modulation of endogenous HDP synthesis is an effective way to boost the host innate immune system. This study aimed to investigate the role of the probiotic Lactobacillus plantarum strain ZLP001 in porcine HDP induction and the underlying mechanism. To this end, we evaluated the stimulatory effect of L. plantarum ZLP001 on HDP expression in piglet intestinal tissue in vivo and porcine IPEC-J2 cells and 3D4/31 cells in vitro, and we examined the underlying intracellular signaling pathway in IPEC-J2 cells. L. plantarum ZLP001 treatment increased the mRNA expression of jejunal and ileal HDPs in weaned piglets. In IPEC-J2 and 3D4/31 cells, L. plantarum ZLP001 stimulated HDP expression, but different HDP induction patterns were observed, with the various HDPs exhibiting different relative mRNA levels in each cell line. L. plantarum ZLP001 induced porcine HDP expression through toll-like receptor (TLR)2 recognition as evidenced by the fact that HDP expression was suppressed in TLR2-knockdown IPEC-J2 cells. Furthermore, we found that L. plantarum ZLP001 activated the extracellular signal-regulated kinase (ERK)1/2 and c-jun N-terminal kinase (JNK) signaling pathways, as indicated by enhanced phosphorylation of both ERK1/2 and JNK and the fact that HDP expression was suppressed upon inhibition of ERK1/2 and JNK. Furthermore, L. plantarum ZLP001 activated c-fos and c-jun transcription factor phosphorylation and activity. We conclude that L. plantarum ZLP001 induces porcine HDP expression in vivo and in vitro, and the induction seems to be regulated via TLR2 as well as the ERK1/2/JNK and c-jun/c-fos signaling pathways. Modulation of endogenous HDPs mediated by L. plantarum ZLP001 might be a promising approach to improving intestinal health and enhancing diarrhea resistance in weaning piglets.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Intestinos/imunologia , Lactobacillus plantarum , Sistema de Sinalização das MAP Quinases/imunologia , Probióticos , Receptor 2 Toll-Like/imunologia , Fator de Transcrição AP-1/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular , Suínos , Receptor 2 Toll-Like/genética
16.
Front Immunol ; 10: 2673, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824484

RESUMO

It is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes. However, the intracellular signaling pathways leading to the production of these cytokines still remain unknown. In this paper, we have investigated the innate receptors and intracellular signaling pathways that are associated with T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-α by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MyD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our results provide insight into T. congolense-induced activation of the immune system that leads to the production of proinflammatory cytokines and resistance to the infection.


Assuntos
Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/metabolismo , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/metabolismo , Adenilato Quinase/imunologia , Adenilato Quinase/metabolismo , Animais , Citocinas/biossíntese , Ativação Enzimática/imunologia , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Fatores de Transcrição STAT/imunologia , Fatores de Transcrição STAT/metabolismo , Receptor 2 Toll-Like/imunologia , Trypanosoma congolense/imunologia
17.
Int J Mol Sci ; 20(24)2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31817424

RESUMO

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly (p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1ß up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.


Assuntos
Proliferação de Células , Fibroblastos/imunologia , Gengiva/imunologia , Gengivite/imunologia , Linfócitos T/imunologia , Receptor 2 Toll-Like/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Fibroblastos/patologia , Gengiva/patologia , Gengivite/patologia , Humanos , Interleucina-1beta/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Monócitos/imunologia , Monócitos/patologia , Linfócitos T/patologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
18.
J Immunol Res ; 2019: 1462098, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815150

RESUMO

Diabetes mellitus, a metabolic disease characterized by hyperglycemia and poor glucose control, is a risk factor for Mycobacterium tuberculosis (M. tuberculosis) infection and the development of active tuberculosis. To evaluate whether M. tuberculosis infection susceptibility is associated with an intrinsic factor in monocytes from type 2 diabetes (T2D) patients or it is associated with hyperglycemia per se, we analyzed TLR-2 and TLR-4 expression by flow cytometry and the cytokines IL-1ß, IL-6, IL-8, IL-10, and TNF-α by cytometric bead array assays, either stimulated with TLR-2 and TLR-4 ligands or infected with M. tuberculosis in the whole blood from T2D patients (n = 43) and healthy subjects (n = 26) or in CD14+ monocytes from healthy subjects cultured in high glucose (HG) (30 mM). The intracellular growth of M. tuberculosis was evaluated by CFU counts at 0, 1, and 3 days in both monocytes from T2D patients and monocytes from healthy subjects cultured in HG. We did not find significant differences in TLR expression, cytokine production, or growth of M. tuberculosis in monocytes from T2D patients compared with those in monocytes from healthy subjects. Despite these results, in vitro assays of monocytes cultured with 30 mM glucose led to significantly increased TLR-2 and TLR-4 basal expression compared to those of monocytes cultured with 11 mM glucose (P < 0.05). Conversely, the production of IL-6 by TLR-2 ligand stimulation, of IL-1ß, IL-6, and IL-8 by TLR-4 ligand stimulation, and of IL-8 by M. tuberculosis infection significantly decreased in monocytes cultured in HG (P < 0.05). Additionally, the intracellular survival of M. tuberculosis increased in monocytes in HG after day 3 of culture (P < 0.05). In conclusion, HG decreased IL-8 production and the intracellular growth control of M. tuberculosis by monocytes, supporting the hypothesis that hyperglycemia plays an important role in the impaired immune responses to M. tuberculosis in patients with T2D.


Assuntos
Diabetes Mellitus Tipo 2/imunologia , Glucose/farmacologia , Hiperglicemia/imunologia , Monócitos/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose Pulmonar/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/microbiologia , Hiperglicemia/patologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Cultura Primária de Células , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
19.
J Immunol Res ; 2019: 3562672, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815153

RESUMO

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H. contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L3. 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L3, and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1ß expression levels (P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed (P < 0.05). Higher levels (P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels (P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H. contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response.


Assuntos
Aminopeptidases/genética , Resistência à Doença/genética , Hemoncose/imunologia , Imunidade Inata , Doenças dos Ovinos/imunologia , Receptor 2 Toll-Like/genética , Aminopeptidases/imunologia , Animais , Fezes/parasitologia , Feminino , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Expressão Gênica , Hemoncose/genética , Hemoncose/parasitologia , Hemoncose/patologia , Haemonchus/imunologia , Haemonchus/patogenicidade , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-33/genética , Interleucina-33/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Ovos de Parasitas , Fenótipo , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/patologia , Células Th2/imunologia , Células Th2/parasitologia , Células Th2/patologia , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
20.
Parasit Vectors ; 12(1): 575, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31806038

RESUMO

BACKGROUND: Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin. METHODS: We determined and compared the transcription of toll like receptors (TLRs) 2, 4 and 7, interferon gamma (IFN-γ), interleukin (IL) 10 and programmed cell death protein ligand (PD-L) 1 by real-time PCR in paired clinically-lesioned and normal-looking skin from 25 diseased dogs (mild disease-stage I (n = 11) and moderate to severe disease-stages II and III (n = 14) as well as in normal-looking skin from healthy dogs (n = 10) from a non-endemic area. We also assessed the association between the transcripts in clinically-lesioned and normal-looking skin of dogs with leishmaniosis with clinicopathological, immunological and parasitological findings. RESULTS: Clinically-lesioned skin from mildly affected dogs was characterized by a significant upregulation of TLR2 (P < 0.0001) and IL-10 (P = 0.021) and downregulation of TLR7 (P = 0.004) when compared with more severely affected dogs. Normal-looking skin of mildly affected dogs was characterized by a significant lower expression of TLR7 (P = 0.003), IFN-γ (P < 0.0001) and PD-L1 (P = 0.001) when compared with more severely affected dogs. TLR2, TLR4, IL-10 and IFN-γ upregulation in clinically-lesioned skin was correlated with lower disease severity while TLR7 upregulation was correlated with markers of disease severity. Upregulation of TLR7, IL-10, IFN-γ and PD-L1 in normal-looking skin was correlated with disease severity. CONCLUSIONS: This study demonstrated different expression profiles of immune genes in clinically-lesioned and normal-looking skin among mildly and more severely affected dogs. These immunological conditions might favor the maintenance and replication of the parasite in the skin of more severely affected dogs.


Assuntos
Antígeno B7-H1/genética , Interferon gama/genética , Interleucina-10/genética , Leishmaniose/veterinária , Dermatopatias Parasitárias/veterinária , Receptores Toll-Like/genética , Animais , Antígeno B7-H1/imunologia , Biópsia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Regulação para Baixo , Feminino , Interferon gama/imunologia , Interleucina-10/imunologia , Leishmania infantum , Leishmaniose/imunologia , Masculino , Pele/imunologia , Pele/parasitologia , Pele/patologia , Dermatopatias Parasitárias/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Regulação para Cima
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